US 201603.1931.3A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0319313 A1 SUN et al. (43) Pub. Date: Nov. 3, 2016

(54) MICROORGANISMS AND METHODS FOR continuation of application No. 12/940,021, filed on THE COPRODUCTION 1,4-BUTANEDIOL Nov. 4, 2010, now Pat. No. 8,530,210. AND GAMMA-BUTYROLACTONE (60) Provisional application No. 61/264,598, filed on Nov. (71) Applicant: Genomatica, Inc., San Diego, CA (US) 25, 2009. Publication Classification (72) Inventors: Jun SUN, San Diego, CA (US); Mark J. BURK, San Diego, CA (US); (51) Int. Cl. Anthony P. BURGARD, Bellefonte, CI2P I 7/04 (2006.01) CA (US); Robin E. OSTERHOUT, CI2N 15/52 (2006.01) San Diego, CA (US); Wei NIU, CI2P 7/18 (2006.01) Lincoln, NE (US); John D. (52) U.S. Cl. TRAWICK, La Mesa, CA (US); CPC ...... CI2P 17/04 (2013.01); C12P 7/18 Robert HASELBECK, San Diego, CA (2013.01); C12N 15/52 (2013.01) (US) (57) ABSTRACT (21) Appl. No.: 14/954,487 The invention provides non-naturally occurring microbial organisms comprising 1,4-butanediol (14-BDO) and (22) Filed: Nov. 30, 2015 gamma-butyrolactone (GBL) pathways comprising at least one exogenous nucleic acid encoding a 14-BDO and GBL pathway expressed in a Sufficient amount to produce Related U.S. Application Data 14-BDO and GBL. The invention additionally provides (63) Continuation of application No. 13/936,878, filed on methods of using Such microbial organisms to produce Jul. 8, 2013, now Pat. No. 9,222,113, which is a 14-BDO and GBL. Patent Application Publication Nov. 3, 2016 Sheet 1 of 8 US 2016/0319313 A1

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MCROORGANISMS AND METHODS FOR ments includes the non-enzymatic lactonization of THE COPRODUCTION 14-BUTANEDIOL 4-hydroxybutyryl-phosphate or 4-hydroxybutyryl-CoA to AND GAMMA-BUTYROLACTONE form GBL.

0001. This application is a continuation of U.S. Non BRIEF DESCRIPTION OF THE DRAWINGS provisional application Ser. No. 13/936,878, filed Jul. 8, 2013, which is a continuation of U.S. Non-provisional 0008 FIGS. 1A and 1B show exemplary pathways for application Ser. No. 12/940,021, filed Nov. 4, 2010, now co-production of 14-BDO and GBL. for transfor issued U.S. Pat. No. 8,530,210, issued Sep. 10, 2013, which mation of identified Substrates to products include: A) Suc claims the benefit of priority of U.S. Provisional Application cinyl-CoA reductase (aldehyde forming), B) alpha-ketoglu No. 61/264,598, filed Nov. 25, 2009, the entire contents of tarate decarboxylase, C) 4-hydroxybutyrate dehydrogenase, which are each incorporated herein by reference. D) 4-hydroxybutyrate reductase, E) 1,4-butanediol dehydro genase, F) Succinate reductase, G) Succinyl-CoA , BACKGROUND OF THE INVENTION H) Succinyl-CoA , I) succinyl-CoA synthetase (or Succinyl-CoA ), J) glutamate dehydrogenase, K) glu 0002 The instant application contains a Sequence Listing tamate transaminase, L) glutamate decarboxylase, M) which has been submitted in ASCII format via EFS-Web and 4-aminobutyrate dehydrogenase, N) 4-aminobutyrate is hereby incorporated by reference in its entirety. Said transaminase, O) 4-hydroxybutyrate kinase, P) phospho ASCII copy, created on Nov. 30, 2015, is named 12956 trans-4-hydroxybutyrylase, Q) 4-hydroxybutyryl-CoA 386-999 Sequence Listing and is 5,588 bytes in size. reductase (aldehyde forming), R) 4-hydroxybutyryl-phos 0003. The present invention relates generally to biosyn phate reductase, S) Succinyl-CoA reductase (alcohol form thetic processes, and more specifically to organisms having ing), T) 4-hydroxybutyryl-CoA transferase, U) 4-hydroxy 1,4-butanediol and gamma-butyrolactone biosynthetic capa butyryl-CoA hydrolase, V) 4-hydroxybutyryl-CoA bility. synthetase (or 4-hydroxybutyryl-CoA ligase), W) 4-hy 0004 1,4-Butanediol (14-BDO) is a polymer intermedi droxybutyryl-CoA reductase (alcohol forming), X) alpha ate and industrial solvent with a global market of about 3 ketoglutarate reductase, Y) 5-hydroxy-2-oxopentanoate billion lb/year. 14-BDO is currently produced from petro dehydrogenase, Z) 5-hydroxy-2-oxopentanoate decarboxy chemical precursors, primarily acetylene, maleic anhydride, lase, AA) 5-hydroxy-2-oxopentanoate dehydrogenase (de and propylene oxide. For example, acetylene is reacted with carboxylation), AB) 4-hydroxybutyrate lactonase, AC) non 2 molecules of formaldehyde in the Reppe synthesis reaction enzymatic 4-hydroxybutyryl-phosphate lactonization, and (Kroschwitz and Grant, Encyclopedia of Chem. Tech. John AD) non-enzymatic 4-hydroxybutyryl-CoA lactonization. Wiley and Sons, Inc., New York (1999)), followed by 0009 FIG. 2 shows the production of 4-HB in glucose catalytic hydrogenation to form 1,4-butanediol. Down minimal medium using E. coli Strains harboring plasmids stream, 14-BDO can be further transformed; for example, by expressing various combinations of 4-HB pathway genes. oxidation to gamma-butyrolactone (GBL) or hydrogenolysis (a) 4-HB concentration in culture broth; (b) succinate con to tetrahydrofuran (THF). These compounds have varied centration in culture broth; (c) culture OD, measured at 600 uses as polymer intermediates, solvents, and additives, and nm. Clusters of bars represent the 24 hour, 48 hour, and 72 have a combined market of nearly 2 billion lb/year. hour (if measured) timepoints. The codes along the X-axis 0005 Gamma-butyrolactone (GBL) is a chemical inter indicate the strain/plasmid combination used. The first index mediate and common solvent with a global market of about refers to the host strain: 1, MG1655 lacI’: 2, MG1655 0.5 billion lb/year. GBL has wide applications. GBL serves Agabl) lacI: 3, MG 1655 AgabD AaldA lacI. The second as a chemical intermediate in the manufacture of pyrroli index refers to the plasmid combination used: 1, pZE13 dones that are widely used in the pharmaceutical industry. It 0004-0035 and pZA33-0036: 2, p7E13-0004-0035 and can be used in the production of pesticides, herbicides and pZA33-0010n: 3, p.ZE13-0004-0008 and pZA33-0036; 4, plant growth regulators. GBL is also used as an aroma pZE13-0004-0008 and pZA33-0010n; 5, Control vectors compound, as a stain remover, as a Superglue remover, as a pZE13 and pZA33. paint stripper, and as a solvent in some wet aluminum 0010 FIG. 3 shows the production of 4-HB from glucose electrolytic capacitors. Currently, GBL is mainly synthe in E. coli Strains expressing O-ketoglutarate decarboxylase sized through 14-BDO from crude oil and/or natural gas from Mycobacterium tuberculosis. Strains 1-3 contain derived feedstocks by the Reppe acetylene chemistry. pZE13-0032 and pZA33-0036. Strain 4 expresses only the empty vectors pZE13 and pZA33. Host strains are as fol 0006 Microbial organisms and methods for effectively lows: 1 and 4, MG1655 lacI’: 2, MG1655 AgabD lacI: 3, co-producing commercial quantities of 14-BDO and GBL MG1655 Agabd AaldA lacI. The bars refer to concentra are described herein and include related advantages. tion at 24 and 48 hours. SUMMARY OF INVENTION (0011 FIG. 4 shows the production of BDO from 10 mM 4-HB in recombinant E. coli strains. Numbered positions 0007. The invention provides non-naturally occurring correspond to experiments with MG1655 lacI containing microbial organisms containing 1,4-butanediol (14-BDO) pZA33-0024, expressing cat? from P gingivalis, and the and gamma-butyrolactone (GBL) pathways comprising at following genes expressed on pZE13: 1. none (control); 2. least one exogenous nucleic acid encoding a 14-BDO path 0002; 3,0003: 4,0003n; 5, 0011; 6, 0013; 7, 0023; 8, 0025; way enzyme and at least one exogenous nucleic acid encod 9,0008n; 10, 0035. Gene numbers are defined in Table 56. ing a GBL pathway enzyme each expressed in a Sufficient For each position, the bars refer to aerobic, microaerobic, amount to produce 14-BDO and GBL. The invention addi and anaerobic conditions, respectively. Microaerobic condi tionally provides methods of using Such microbial organ tions were created by sealing the culture tubes but not isms to produce 14-BDO and GBL, which in some embodi evacuating them. US 2016/03193 13 A1 Nov. 3, 2016

0012 FIG. 5 is a schematic process flow diagram of bioprocesses. Separate strain design strategies were identi bioprocesses for the production of Y-butyrolactone. Panel (a) fied with incorporation of different non-native or heterolo illustrates fed-batch fermentation with batch separation and gous reaction capabilities into E. coli or other host organ panel (b) illustrates fed-batch fermentation with continuous isms leading to 14-BDO and GBL producing metabolic separation. pathways from either Succinate. Succinyl-CoA or alpha 0013 FIG. 6 shows the mass spectrum of 4-HB and BDO ketoglutarate. In silico metabolic designs were identified produced by MG1655 lacI pZE13-0004-0035-0002 that resulted in the biosynthesis of 14-BDO and GBL in pZA33-0034-0036 grown in M9 minimal medium supple microorganisms from each of these metabolic intermediates. mented with 4 g/L unlabeled glucose (a, c, e, and g) Embodiments of the invention include the controlled pro uniformly labeled 'C-glucose (b, d, f, and h). (a) and (b). duction of 14-BDO and GBL at a predetermined ratio. As mass 116 characteristic fragment of derivatized BDO, con described herein, this ratio can be regulated by, for example, taining 2 carbon atoms; (c) and (d), mass 177 characteristic introducing an exogenous nucleic acid encoding an enzyme fragment of derivatized BDO, containing 1 carbon atom; (e) or protein with an increased or decreased activity or speci and (f), mass 117 characteristic fragment of derivatized ficity for one or more intermediates in the respective 4-HB, containing 2 carbon atoms; (g) and (h), mass 233 14-BDO or GBL pathways or modulating the expression of characteristic fragment of derivatized 4-HB, containing 4 an enzyme or protein in a 14-BDO or GBL pathway, thereby carbon atoms. regulating the production of 14-BDO and GBL. 0014 FIG. 7A shows the nucleotide sequence (SEQ ID 0020 Strains identified via the computational component NO: 1) of the adh1 gene from Geobacillus thermoglucosi of the platform can be put into actual production by geneti dasius, and FIG.7B shows the encoded amino acid sequence cally engineering any of the predicted metabolic alterations (SEQ ID NO: 2). which lead to the biosynthetic production of 14-BDO and 0015 FIG. 8A shows the expression of the Geobacillus GBL or other intermediate and/or downstream products. In thermoglucosidasius adh1 gene in E. coli. Either whole cell yet a further embodiment, strains exhibiting biosynthetic lysates or supernatants were analyzed by SDS-PAGE and production of these compounds can be further subjected to stained with Coomassie blue for plasmid with no insert, adaptive evolution to further augment biosynthesis. plasmid with 083 (Geotrichum capitatum N-benzyl-3-pyr The levels of product biosynthesis yield following adaptive rolidinol dehydrogenase) and plasmid with 084 (Geobacil evolution also can be predicted by the computational com lus thermoglucosidasius adh1) inserts. FIG. 8B shows the ponent of the system. activity of 084 with butyraldehyde (diamonds) or 4-hy droxybutyraldehyde (squares) as Substrates. 0021 Embodiments of the invention also provide meth 0016 FIG.9 shows the production of 14-BDO in various ods for co-production of 14-BDO and GBL. Co-production strains: plasmid with no insert: 025B, 025B-026n: 025B of 14-BDO and GBL is attractive due to the following 026A: 025B-026B: 025B-026C: 025B-050; 025B-052: advantages: 1) co-production will satisfy the needs of both 025B-053; 025B-055; 025B-057; 025B-058; 025B-071; chemicals and reduce the cost and environmental impact of 025B-083; 025B-084; PTSlacO-025B; PTSlacO-025B the transformation of 14-BDO to GBL.; 2) construction of a O26n. single large regional facility for co-production will save capital investment and reduce fixed cost comparing to DETAILED DESCRIPTION OF THE separate productions; 3) separating the two co-products from INVENTION fermentation broth can be easily achieve through distillation due to their boiling points (14-BDO at 230° C. and GBL at 0017. The present invention is directed to the design and production of cells and organisms having biosynthetic pro 204° C.). duction capabilities for 1,4-butanediol (14-BDO) and 0022. As used herein, the term “non-naturally occurring gamma-butyrolactone (GBL). The invention, in particular, when used in reference to a microbial organism or micro relates to the design of microbial organisms capable of organism of the invention is intended to mean that the producing 14-BDO and GBL by introducing one or more microbial organism has at least one genetic alteration not nucleic acids encoding a 14-BDO and GBL pathway normally found in a naturally occurring strain of the refer enzyme. enced species, including wild-type strains of the referenced 0.018. In one embodiment, the invention utilizes in silico species. Genetic alterations include, for example, modifica stoichiometric models of Escherichia coli metabolism that tions introducing expressible nucleic acids encoding meta identify metabolic designs for biosynthetic production of bolic polypeptides, other nucleic acid additions, nucleic acid 14-BDO and GBL. The results described herein indicate that deletions and/or other functional disruption of the microbial metabolic pathways can be designed and recombinantly organism's genetic material. Such modifications include, for engineered to achieve the biosynthesis of 14-BDO and GBL example, coding regions and functional fragments thereof, in Escherichia coli and other cells or organisms. Biosyn for heterologous, homologous or both heterologous and thetic production of 14-BDO and GBL, for example, for the homologous polypeptides for the referenced species. Addi in silico designs can be confirmed by construction of strains tional modifications include, for example, non-coding regu having the designed metabolic genotype. These metaboli latory regions in which the modifications alter expression of cally engineered cells or organisms also can be subjected to a gene or operon. Exemplary metabolic polypeptides include adaptive evolution to further augment 14-BDO and GBL enzymes or proteins within a 14-BDO and GBL biosynthetic biosynthesis, including under conditions approaching theo pathway. retical maximum growth. 0023. A metabolic modification refers to a biochemical 0019. In certain embodiments, the 14-BDO and GBL reaction that is altered from its naturally occurring state. biosynthesis characteristics of the designed strains make Therefore, non-naturally occurring microorganisms can them genetically stable and particularly useful in continuous have genetic modifications to nucleic acids encoding meta US 2016/03193 13 A1 Nov. 3, 2016 bolic polypeptides or, functional fragments thereof. Exem certain condensing enzymes, acts in acetyl or other acyl plary metabolic modifications are disclosed herein. group transfer and in fatty acid synthesis and oxidation, 0024. As used herein, the term "isolated when used in pyruvate oxidation and in other acetylation. reference to a microbial organism is intended to mean an can also function as an activating group for a spontaneous organism that is Substantially free of at least one component intramolecular , Such as, circulazation or as the referenced microbial organism is found in nature. The lactonization. term includes a microbial organism that is removed from 0030. As used herein, the term “phosphate' is intended to Some or all components as it is found in its natural envi mean an inorganic chemical ion of the empirical formula ronment. The term also includes a microbial organism that PO and a molar mass of 94.973 g/mol. A phosphate can is removed from some or all components as the microbial act as a functional group when added to a compound. A organism is found in non-naturally occurring environments. phosphate can function as an activating group for a spon Therefore, an isolated microbial organism is partly or com taneous intramolecular chemical reaction, such as, cirulaza pletely separated from other Substances as it is found in tion or lactonization. nature or as it is grown, stored or Subsisted in non-naturally 0031. As used herein, the term “substantially anaerobic' occurring environments. Specific examples of isolated when used in reference to a culture or growth condition is microbial organisms include partially pure microbes, Sub intended to mean that the amount of oxygen is less than stantially pure microbes and microbes cultured in a medium about 10% of saturation for dissolved oxygen in liquid that is non-naturally occurring. media. The term also is intended to include sealed chambers 0025. As used herein, the terms “microbial,” “microbial of liquid or Solid medium maintained with an atmosphere of organism' or “microorganism' is intended to mean any less than about 1% oxygen. organism that exists as a microscopic cell that is included 0032. As used herein, the term “predetermined ratio” within the domains of archaea, bacteria or eukarya. There when used in reference to the amount of one compound fore, the term is intended to encompass prokaryotic or relative to the amount of another compound is intended to eukaryotic cells or organisms having a microscopic size and mean a ratio of compounds that is established in advance includes bacteria, archaea and eubacteria of all species as that can be produced by a non-naturally occurring microor well as eukaryotic microorganisms such as yeast and fungi. ganism described herein through prior experimental deter The term also includes cell cultures of any species that can mination for each compound separately or in combination. be cultured for the production of a biochemical. A predetermined ratio can also be established in advance by 0026. As used herein, the term “1,4-butanediol is determining the maximum theoretical yield for a non-natu intended to mean an alcohol derivative of the alkane butane, rally occurring microorganism to produce 14-BDO and GBL carrying two hydroxyl groups which has the chemical for as described herein. Embodiments described herein include mula CHO and a molecular mass of 90.12 g/mol. The that the predetermined ratio intends there is at least some chemical compound 1,4-butanediol also is known in the art detectable amount of one product relative to the amount of as 14-BDO or BDO and is a chemical intermediate or a second, third, or fourth product up to any number of precursor for a family of compounds referred to herein as products or intermediate compounds in a 14-BDO or GBL BDO family of compounds pathway produced by a non-naturally occurring microorgan 0027. As used herein, the term “gamma-butyrolactone' is ism described herein. Embodiments described herein intended to mean a lactone having the chemical formula include the amount of intracellular 14-BDO or GBL pro CHO and a molecular mass of 86.089 g/mol. The chemi duced by the microorganisms is at least 0.001 mM, or cal compound gamma-butyrolactone also is know in the art alternatively at least 0.01 mM, or alternatively at least 0.1 as GBL, butyrolactone, 1.4-lactone, 4-butyrolactone, 4-hy mM, or alternatively at least 1.0 mM or alternatively at least droxybutyric acid lactone, and gamma-hydroxybutyric acid 10 mM or alternatively at least 100 mM, or alternatively at lactone. The term as it is used herein is intended to include least 1M. Embodiments described herein also include the any of the compounds various salt forms. amount of intracellular 14-BDO or GBL produced by the 0028. As used herein, the term “4-hydroxybutanoic acid microorganisms is at least 1%, or alternatively at least 5%, is intended to mean a 4-hydroxy derivative of or alternatively at least 10%, or alternatively at least 15%, or having the chemical formula CHO and a molecular mass alternatively at least 20%, or alternatively at least 25%, or of 104.11 g/mol (126.09 g/mol for its sodium salt). The alternatively at least 30%, or alternatively at least 35%, or chemical compound 4-hydroxybutanoic acid also is known alternatively at least 40%, or alternatively at least 45%, or in the art as 4-HB, 4-hydroxybutyrate, gamma-hydroxybu alternatively at least 50%, or alternatively at least 55%, or tyric acid or GHB. The term as it is used herein is intended alternatively at least 60%, or alternatively at least 65%, or to include any of the compounds various salt forms and alternatively at least 70%, or alternatively at least 75%, or include, for example, 4-hydroxybutanoate and 4-hydroxy alternatively at least 80%, or alternatively at least 85%, or butyrate. Specific examples of salt forms for 4-HB include alternatively at least 90%, or alternatively at least 95%, or sodium 4-HB and potassium 4-HB. Therefore, the terms alternatively at least 98%, or alternatively at least 100% of 4-hydroxybutanoic acid, 4-HB, 4-hydroxybutyrate, 4-hy the maximum theoretical yield. Embodiments described droxybutanoate, gamma-hydroxybutyric acid and GHB as herein include the predetermined ratio of intracellular well as other art recognized names are used synonymously 14-BDO to GLB produced by the microorganisms is 1:1, or herein. alternatively 1:2, or alternatively 1:3, or alternatively 1:4, or 0029. As used herein, the term “CoA' or “coenzyme A” alternatively 1:5, or alternatively 1:6, or alternatively 1:7, or is intended to mean an organic or prosthetic group alternatively 1:8, or alternatively 1:9, or alternatively 1:10, (nonprotein portion of an enzyme) whose presence is or alternatively 1:15, or alternatively about 1:20, or alter required for the activity of many enzymes (the apoenzyme) natively 1:25, or alternatively 1:30, or alternatively 1:35, or to form an active enzyme system. Coenzyme A functions in alternatively 1:40, or alternatively 1:45, or alternatively US 2016/03193 13 A1 Nov. 3, 2016

about 1:50, or alternatively 1:60, or alternatively 1:70, or multiple sites, and still be considered as two or more alternatively 1:80, or alternatively 1:90, or alternatively exogenous nucleic acids, for example three exogenous 1:100. Embodiments described herein include the predeter nucleic acids. Thus, the number of referenced exogenous mined ratio of intracellular 14-BDO to GLB produced by the nucleic acids or biosynthetic activities refers to the number microorganisms is 2:1, or alternatively 3:1, or alternatively of encoding nucleic acids or the number of biosynthetic 4:1, or alternatively 5:1, or alternatively 6:1, or alternatively activities, not the number of separate nucleic acids intro 7:1, or alternatively 8:1, or alternatively 9:1, or alternatively duced into the host organism. 10:1, or alternatively 15:1, or alternatively about 20:1, or 0035. The non-naturally occurring microbal organisms of alternatively 25:1, or alternatively 30:1, or alternatively the invention can contain stable genetic alterations, which 35:1, or alternatively 40:1, or alternatively 45:1, or alterna refers to microorganisms that can be cultured for greater tively about 50:1, or alternatively 60:1, or alternatively 70:1, than five generations without loss of the alteration. Gener or alternatively 80:1, or alternatively 90:1, or alternatively ally, stable genetic alterations include modifications that 100:1. persist greater than 10 generations, particularly stable modi 0033) “Exogenous” as it is used herein is intended to fications will persist more than about 25 generations, and mean that the referenced molecule or the referenced activity more particularly, stable genetic modifications will be is introduced into the host microbial organism. The molecule greater than 50 generations, including indefinitely. can be introduced, for example, by introduction of an 0036 Those skilled in the art will understand that the encoding nucleic acid into the host genetic material Such as genetic alterations, including metabolic modifications exem by integration into a host chromosome or as non-chromo plified herein, are described with reference to a suitable host Somal genetic material Such as a plasmid. Therefore, the organism such as E. coli and their corresponding metabolic term as it is used in reference to expression of an encoding reactions or a suitable source organism for desired genetic nucleic acid refers to introduction of the encoding nucleic material Such as genes for a desired metabolic pathway. acid in an expressible form into the microbial organism. However, given the complete genome sequencing of a wide When used in reference to a biosynthetic activity, the term variety of organisms and the high level of skill in the area of refers to an activity that is introduced into the host reference genomics, those skilled in the art will readily be able to organism. The Source can be, for example, a homologous or apply the teachings and guidance provided herein to essen heterologous encoding nucleic acid that expresses the ref tially all other organisms. For example, the E. coli metabolic erenced activity following introduction into the host micro alterations exemplified herein can readily be applied to other bial organism. Therefore, the term “endogenous” refers to a species by incorporating the same or analogous encoding referenced molecule or activity that is present in the host. nucleic acid from species other than the referenced species. Similarly, the term when used in reference to expression of Such genetic alterations include, for example, genetic altera an encoding nucleic acid refers to expression of an encoding tions of species homologs, in general, and in particular, nucleic acid contained within the microbial organism. The orthologs, paralogs or nonorthologous gene displacements. term "heterologous” refers to a molecule or activity derived 0037. An ortholog is a gene or genes that are related by from a source other than the referenced species whereas vertical descent and are responsible for substantially the “homologous” refers to a molecule or activity derived from same or identical functions in different organisms. For the host microbial organism. Accordingly, exogenous example, mouse epoxide hydrolase and human epoxide expression of an encoding nucleic acid of the invention can hydrolase can be considered orthologs for the biological utilize either or both a heterologous or homologous encod function of hydrolysis of epoxides. Genes are related by ing nucleic acid. vertical descent when, for example, they share sequence 0034. It is understood that when more than one exog similarity of Sufficient amount to indicate they are homolo enous nucleic acid is included in a microbial organism that gous, or related by evolution from a common ancestor. the more than one exogenous nucleic acids refers to the Genes can also be considered orthologs if they share three referenced encoding nucleic acid or biosynthetic activity, as dimensional structure but not necessarily sequence similar discussed above. It is further understood, as disclosed ity, of a sufficient amount to indicate that they have evolved herein, that such more than one exogenous nucleic acids can from a common ancestor to the extent that the primary be introduced into the host microbial organism on separate sequence similarity is not identifiable. Genes that are nucleic acid molecules, on polycistronic nucleic acid mol orthologous can encode proteins with sequence similarity of ecules, or a combination thereof, and still be considered as about 25% to 100% amino acid sequence identity. Genes more than one exogenous nucleic acid. For example, as encoding proteins sharing an amino acid similarity less that disclosed herein a microbial organism can be engineered to 25% can also be considered to have arisen by vertical express two or more exogenous nucleic acids encoding a descent if their three-dimensional structure also shows simi desired pathway enzyme or protein. In the case where two larities. Members of the serine protease family of enzymes, exogenous nucleic acids encoding a desired activity are including tissue plasminogen activator and elastase, are introduced into a host microbial organism, it is understood considered to have arisen by Vertical descent from a com that the two exogenous nucleic acids can be introduced as a mon anceStor. single nucleic acid, for example, on a single plasmid, on 0038 Orthologs include genes or their encoded gene separate plasmids, can be integrated into the host chromo products that through, for example, evolution, have diverged Some at a single site or multiple sites, and still be considered in structure or overall activity. For example, where one as two exogenous nucleic acids. Similarly, it is understood species encodes a gene product exhibiting two functions and that more than two exogenous nucleic acids can be intro where such functions have been separated into distinct genes duced into a host organism in any desired combination, for in a second species, the three genes and their corresponding example, on a single plasmid, on separate plasmids, can be products are considered to be orthologs. For the production integrated into the host chromosome at a single site or of a biochemical product, those skilled in the art will US 2016/03193 13 A1 Nov. 3, 2016

understand that the orthologous gene harboring the meta pared sequences. Based on Such similarities, one skilled in bolic activity to be introduced or disrupted is to be chosen the art can determine if the similarity is sufficiently high to for construction of the non-naturally occurring microorgan indicate the proteins are related through evolution from a ism. An example of orthologs exhibiting separable activities common ancestor. Algorithms well known to those skilled in is where distinct activities have been separated into distinct the art, such as Align, BLAST, Clustal W and others gene products between two or more species or within a compare and determine a raw sequence similarity or identity, single species. A specific example is the separation of and also determine the presence or significance of gaps in elastase proteolysis and plasminogen proteolysis, two types the sequence which can be assigned a weight or score. Such of serine protease activity, into distinct molecules as plas algorithms also are known in the art and are similarly minogen activator and elastase. A second example is the applicable for determining nucleotide sequence similarity or separation of mycoplasma 5'-3' exonuclease and Drosophila identity. Parameters for sufficient similarity to determine DNA polymerase III activity. The DNA polymerase from the relatedness are computed based on well known methods for first species can be considered an ortholog to either or both calculating statistical similarity, or the chance of finding a of the exonuclease or the polymerase from the second similar match in a random polypeptide, and the significance species and vice versa. of the match determined. A computer comparison of two or 0039. In contrast, paralogs are homologs related by, for more sequences can, if desired, also be optimized visually example, duplication followed by evolutionary divergence by those skilled in the art. Related gene products or proteins and have similar or common, but not identical functions. can be expected to have a high similarity, for example, 25% Paralogs can originate or derive from, for example, the same to 100% sequence identity. Proteins that are unrelated can species or from a different species. For example, microsomal have an identity which is essentially the same as would be epoxide hydrolase (epoxide hydrolase I) and soluble epoxide expected to occur by chance, if a database of Sufficient size hydrolase (epoxide hydrolase II) can be considered paralogs is scanned (about 5%). Sequences between 5% and 24% because they represent two distinct enzymes, co-evolved may or may not represent Sufficient homology to conclude from a common ancestor, that catalyze distinct reactions and that the compared sequences are related. Additional statis have distinct functions in the same species. Paralogs are tical analysis to determine the significance of Such matches proteins from the same species with significant sequence given the size of the data set can be carried out to determine similarity to each other Suggesting that they are homolo the relevance of these sequences. gous, or related through co-evolution from a common ances 0043. Exemplary parameters for determining relatedness tor. Groups of paralogous protein families include HipA of two or more sequences using the BLAST algorithm, for homologs, luciferase genes, peptidases, and others. example, can be as set forth below. Briefly, amino acid 0040. A nonorthologous gene displacement is a nonor sequence alignments can be performed using BLASTP thologous gene from one species that can Substitute for a version 2.0.8 (Jan.-05-1999) and the following parameters: referenced gene function in a different species. Substitution Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; includes, for example, being able to perform Substantially X dropoff 50; expect: 10.0; wordsize: 3; filter: on. Nucleic the same or a similar function in the species of origin acid sequence alignments can be performed using BLASTN compared to the referenced function in the different species. version 2.0.6 (Sept-16-1998) and the following parameters: Although generally, a nonorthologous gene displacement Match: 1; mismatch: -2, gap open: 5; gap extension: 2; will be identifiable as structurally related to a known gene X dropoff 50; expect: 10.0; wordsize: 11; filter: off. Those encoding the referenced function, less structurally related skilled in the art will know what modifications can be made but functionally similar genes and their corresponding gene to the above parameters to either increase or decrease the products nevertheless will still fall within the meaning of the stringency of the comparison, for example, and determine term as it is used herein. Functional similarity requires, for the relatedness of two or more sequences. example, at least Some structural similarity in the 0044. In one embodiment, the invention provides a non or binding region of a nonorthologous gene product com naturally occurring microbial organism, comprising a micro pared to a gene encoding the function sought to be substi bial organism having a 1,4-butanediol (14-BDO) pathway tuted. Therefore, a nonorthologous gene includes, for and a gamma-butyrolactone (GBL) pathway, said 14-BDO example, a paralog or an unrelated gene. pathway comprising at least one exogenous nucleic acid 0041. Therefore, in identifying and constructing the non encoding a 14-BDO pathway enzyme expressed in a Sufi naturally occurring microbial organisms of the invention cient amount to produce 14-BDO, said 14-BDO pathway having 14-BDO and GBL biosynthetic capability, those comprising a succinyl-CoA reductase (aldehyde forming), skilled in the art will understand with applying the teaching an alpha-ketoglutarate decarboxylase, a 4-hydroxybutyrate and guidance provided herein to a particular species that the dehydrogenase, a 4-hydroxybutyrate reductase, a 1,4-bu identification of metabolic modifications can include iden tanediol dehydrogenase, a Succinate reductase, a Succinyl tification and inclusion or inactivation of orthologs. To the CoA transferase, a Succinyl-CoA hydrolase, a Succinyl-CoA extent that paralogs and/or nonorthologous gene displace synthetase, a glutamate dehydrogenase, a glutamate ments are present in the referenced microorganism that transaminase, a glutamate decarboxylase, an 4-aminobu encode an enzyme catalyzing a similar or Substantially tyrate dehydrogenase, an 4-aminobutyrate transaminase, a similar metabolic reaction, those skilled in the art also can 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybu utilize these evolutionally related genes. tyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde 0.042 Orthologs, paralogs and nonorthologous gene dis forming), a 4-hydroxybutyryl-phosphate reductase, a Succi placements can be determined by methods well known to nyl-CoA reductase (alcohol forming), a 4-hydroxybutyryl those skilled in the art. For example, inspection of nucleic CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a acid or amino acid sequences for two polypeptides will 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reveal sequence identity and similarities between the com reductase (alcohol forming), an alpha-ketoglutarate reduc US 2016/03193 13 A1 Nov. 3, 2016 tase, a 5-hydroxy-2-oxopentanoate dehydrogenase, a 5-hy Succinyl-CoA synthetase; a Succinyl-CoA reductase (alcohol droxy-2-oxopentanoate decarboxylase or a 5-hydroxy-2- forming); a 4-hydroxybutyrate reductase; and 1,4-butane oXopentanoate dehydrogenase (decarboxylation) (see FIG. diol dehydrogenase (see FIG. 1, steps G/H/I, S, D, E). 1, steps A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, 0051. In a further embodiment, the non-naturally occur S, T, U, V, W, X, Y, Z. AA), said GBL pathway comprising ring microbial organism has a 14-BDO pathway comprising at least one exogenous nucleic acid encoding an GBL a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a pathway enzyme expressed in a sufficient amount to produce Succinyl-CoA synthetase; a Succinyl-CoA reductase (alcohol GBL, said GBL pathway comprising a Succinyl-CoA reduc forming); a 4-hydroxybutyrate kinase; a phosphotrans-4- tase (aldehyde forming), an alpha-ketoglutarate decarboxy hydroxybutyrylase; a 4-hydroxybutyryl-CoA reductase (al lase, a 4-hydroxybutyrate dehydrogenase, a Succinate reduc dehyde forming); and a 1,4-butanediol dehydrogenase (see tase, a Succinyl-CoA transferase, a Succinyl-CoA hydrolase, FIG. 1, steps G/H/I, S, O, P, Q, E). a Succinyl-CoA synthetase, a glutamate dehydrogenase, a 0052. In a further embodiment, the non-naturally occur glutamate transaminase, a glutamate decarboxylase, an ring microbial organism has a 14-BDO pathway comprising 4-aminobutyrate dehydrogenase, an 4-aminobutyrate a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or transaminase, a Succinyl-CoA reductase (alcohol forming) Succinyl-CoA synthetase; a Succinyl-CoA reductase (alcohol or a 4-hydroxybutyrate lactonase (see FIG. 1, steps A, B, C, forming), a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl F, G, H, I, J, K, L, M, N, S, AB). phosphate reductase; and a 1,4-butanediol dehydrogenase 0045. In a further embodiment, the non-naturally occur (see FIG. 1, steps G/H/I, S, O, R, E). ring microbial organism has a 14-BDO pathway comprising 0053. In a further embodiment, the non-naturally occur a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a ring microbial organism has a 14-BDO pathway comprising Succinyl-CoA synthetase; a Succinyl-CoA reductase (alde a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a hyde forming); a 4-hydroxybutyrate dehydrogenase; a 4-hy Succinyl-CoA synthetase; a Succinyl-CoA reductase (alcohol droxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; forming); a 4-hydroxybutyryl-CoA transferase, a 4-hy a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA a 1,4-butanediol dehydrogenase (see FIG. 1, steps G/H/I, A. synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde C, O, P, Q, E). forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, 0046. In a further embodiment, the non-naturally occur steps G/H/I, S, T/U/V, Q, E). ring microbial organism has a 14-BDO pathway comprising 0054. In a further embodiment, the non-naturally occur a succinyl-CoA transferase, a succinyl-CoA hydrolase or a ring microbial organism has a 14-BDO pathway comprising Succinyl-CoA synthetase; a Succinyl-CoA reductase (alde a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a hyde forming); a 4-hydroxybutyrate dehydrogenase; a 4-hy Succinyl-CoA synthetase; a Succinyl-CoA reductase (alcohol droxybutyrate kinase; a 4-hydroxybutyryl-phosphate reduc forming); a 4-hydroxybutyryl-CoA transferase, a 4-hy tase; and a 1,4-butanediol dehydrogenase (see FIG. 1, steps droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA G/H/I, A, C, O, R, E). synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol 0047. In a further embodiment, the non-naturally occur forming) (see FIG. 1, steps G/H/I, S, T/U/V, W). ring microbial organism has a 14-BDO pathway comprising a Succinyl-CoA transferase, a Succinyl-CoA hydrolase, or a 0055. In a further embodiment, the non-naturally occur Succinyl-CoA synthetase; a Succinyl-CoA reductase (alde ring microbial organism has a 14-BDO pathway comprising hyde forming); a 4-hydroxybutyrate dehydrogenase; a 4-hy a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a droxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA Succinyl-CoA synthetase; a Succinyl-CoA reductase (alcohol hydrolase or a 4-hydroxybutyryl-CoA synthetase; a 4-hy forming); a 4-hydroxybutyrate kinase; a phosphotrans-4- droxybutyryl-CoA reductase (aldehyde forming); and a 1,4- hydroxybutyrylase; and a 4-hydroxybutyryl-CoA reductase butanediol dehydrogenase (see FIG. 1, steps G/H/I, A, C, (alcohol forming) (see FIG. 1, steps G/H/I, S. O. P. W). T/U/V, Q, E). 0056. In a further embodiment, the non-naturally occur 0048. In a further embodiment, the non-naturally occur ring microbial organism has a 14-BDO pathway comprising ring microbial organism has a 14-BDO pathway comprising a Succinate reductase; a 4-hydroxybutyrate dehydrogenase; a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a 4-hydroxybutyrate reductase; and 1,4-butanediol dehydro Succinyl-CoA synthetase; a Succinyl-CoA reductase (alde genase (see FIG. 1, steps F, C, D, E). hyde forming); a 4-hydroxybutyrate dehydrogenase; a 4-hy 0057. In a further embodiment, the non-naturally occur droxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA ring microbial organism has a 14-BDO pathway comprising hydrolase or a 4-hydroxybutyryl-CoA synthetase; and a a Succinate reductase; a 4-hydroxybutyrate dehydrogenase; 4-hydroxybutyryl-CoA reductase (alcohol forming (see FIG. a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu 1, steps G/H/I, A, C, T/U/V, W). tyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde 0049. In a further embodiment, the non-naturally occur forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, ring microbial organism has a 14-BDO pathway comprising steps F. C., O, P, Q, E). a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a 0058. In a further embodiment, the non-naturally occur Succinyl-CoA synthetase; a Succinyl-CoA reductase (alde ring microbial organism has a 14-BDO pathway comprising hyde forming); a 4-hydroxybutyrate dehydrogenase; a 4-hy a Succinate reductase; a 4-hydroxybutyrate dehydrogenase; droxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu and a 4-hydroxybutyryl-CoA reductase (alcohol forming) tyrylase; and a 4-hydroxybutyryl-CoA reductase (alcohol (see FIG. 1, steps G/H/I, A, C, O, P, W). forming) (see FIG. 1, steps F. C., O, P, W). 0050. In a further embodiment, the non-naturally occur 0059. In a further embodiment, the non-naturally occur ring microbial organism has a 14-BDO pathway comprising ring microbial organism has a 14-BDO pathway comprising a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a a Succinate reductase; a 4-hydroxybutyrate dehydrogenase; US 2016/03193 13 A1 Nov. 3, 2016 a 4-hydroxybutyrate kinase; a 4-hydroxybutyryl-phosphate dehydrogenase; a 4-hydroxybutyrate reductase; and a 1,4- reductase; and 1,4-butanediol dehydrogenase (see FIG. 1, butanediol dehydrogenase (see FIG. 1, steps J/K, L, M/N. C. steps F. C. O. R. E). D, E). 0060. In a further embodiment, the non-naturally occur 0069. In a further embodiment, the non-naturally occur ring microbial organism has a 14-BDO pathway comprising ring microbial organism has a 14-BDO pathway comprising a Succinate reductase; a 4-hydroxybutyrate dehydrogenase; a glutamate dehydrogenase or a glutamate transaminase; a a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl glutamate decarboxylase; a 4-aminobutyrate dehydrogenase CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; a or a 4-aminobutyrate transaminase; a 4-hydroxybutyrate 4-hydroxybutyryl-CoA reductase (aldehyde forming); and dehydrogenase; a 4-hydroxybutyrate kinase; a phospho 1,4-butanediol dehydrogenase (see FIG. 1, steps F.C, T/U/V. trans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA reduc Q, E). tase (aldehyde forming); and E) 1,4-butanediol dehydroge 0061. In a further embodiment, the non-naturally occur nase (see FIG. 1, steps J/K, L, M/N, C, O, P, Q, E). ring microbial organism has a 14-BDO pathway comprising 0070. In a further embodiment, the non-naturally occur a Succinate reductase; a 4-hydroxybutyrate dehydrogenase; ring microbial organism has a 14-BDO pathway comprising a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl a glutamate dehydrogenase or glutamate transaminase; a CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and glutamate decarboxylase; a 4-aminobutyrate dehydrogenase a 4-hydroxybutyryl-CoA reductase (alcohol forming) (see or a 4-aminobutyrate transaminase; a 4-hydroxybutyrate FIG. 1, steps F, C, T/U/V, W). dehydrogenase; a 4-hydroxybutyrate kinase; a phospho 0062. In a further embodiment, the non-naturally occur trans-4-hydroxybutyrylase; and a 4-hydroxybutyryl-CoA ring microbial organism has a 14-BDO pathway comprising reductase (alcohol forming) (see FIG. 1, steps J/K, L, M/N. an alpha-ketoglutarate decarboxylase; a 4-hydroxybutyrate C, O, P, W). dehydrogenase; a 4-hydroxybutyrate reductase; and 1,4- 0071. In a further embodiment, the non-naturally occur butanediol dehydrogenase (see FIG. 1, steps B, C, D, E). ring microbial organism has a 14-BDO pathway comprising 0063. In a further embodiment, the non-naturally occur a glutamate dehydrogenase or a glutamate transaminase; a ring microbial organism has a 14-BDO pathway comprising glutamate decarboxylase; a 4-aminobutyrate dehydrogenase an alpha-ketoglutarate decarboxylase; a 4-hydroxybutyrate or a 4-aminobutyrate transaminase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phospho dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxy trans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA reduc butyryl-phosphate reductase; and a 1,4-butanediol dehydro tase (aldehyde forming); and a 1,4-butanediol dehydroge genase (see FIG. 1, steps J/K, L, M/N, C, O, R, E). nase (see FIG. 1, steps B, C, O, P, Q, E). 0072. In a further embodiment, the non-naturally occur 0064. In a further embodiment, the non-naturally occur ring microbial organism has a 14-BDO pathway comprising ring microbial organism has a 14-BDO pathway comprising a glutamate dehydrogenase or a glutamate transaminase; a an alpha-ketoglutarate decarboxylase; a 4-hydroxybutyrate glutamate decarboxylase; a 4-aminobutyrate dehydrogenase dehydrogenase; a 4-hydroxybutyrate kinase; a phospho or a 4-aminobutyrate transaminase; a 4-hydroxybutyrate trans-4-hydroxybutyrylase and 4-hydroxybutyryl-CoA dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a reductase (alcohol forming) (see FIG. 1, steps B, C, O. P. 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl W). CoA synthetase; a 4-hydroxybutyryl-CoA reductase (alde 0065. In a further embodiment, the non-naturally occur hyde forming); and a 1,4-butanediol dehydrogenase (see ring microbial organism has a 14-BDO pathway comprising. FIG. 1, steps J/K, L, M/N, C, T/U/V, Q, E). an alpha-ketoglutarate decarboxylase; a 4-hydroxybutyrate 0073. In a further embodiment, the non-naturally occur dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxy ring microbial organism has a 14-BDO pathway comprising butyryl-phosphate reductase; and a 1,4-butanediol dehydro a glutamate dehydrogenase or a glutamate transaminase; a genase (see FIG. 1, steps B, C, O. R. E). glutamate decarboxylase; a 4-aminobutyrate dehydrogenase 0066. In a further embodiment, the non-naturally occur or a 4-aminobutyrate transaminase; a 4-hydroxybutyrate ring microbial organism has a 14-BDO pathway comprising dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a an alpha-ketoglutarate decarboxylase; a 4-hydroxybutyrate 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a CoA synthetase; and a 4-hydroxybutyryl-CoA reductase 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA (alcohol forming) (see FIG. 1, steps J/K, L, M/N, C, T/U/V. synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde W). forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, 0074. In a further embodiment, the non-naturally occur steps B, C, T/U/V, Q, E). ring microbial organism has a 14-BDO pathway comprising 0067. In a further embodiment, the non-naturally occur an alpha-ketoglutarate reductase; a 5-hydroxy-2-oxopen ring microbial organism has a 14-BDO pathway comprising tanoate dehydrogenase; a 5-hydroxy-2-oxopentanoate dehy an alpha-ketoglutarate decarboxylase; a 4-hydroxybutyrate drogenase (decarboxylation); a 4-hydroxybutyryl-CoA dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a reductase (aldehyde forming); and a 1,4-butanediol dehy 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl drogenase (see FIG. 1, steps X, Y, AA, Q, E). CoA synthetase; and 4-hydroxybutyryl-CoA reductase (al 0075. In a further embodiment, the non-naturally occur cohol forming) (see FIG. 1, steps B, C, T/U/V. W). ring microbial organism has a 14-BDO pathway comprising 0068. In a further embodiment, the non-naturally occur an alpha-ketoglutarate reductase; a 5-hydroxy-2-oxopen ring microbial organism has a 14-BDO pathway comprising tanoate dehydrogenase; a 5-hydroxy-2-oxopentanoate dehy a glutamate dehydrogenase or a glutamate transaminase; a drogenase (decarboxylation); and a 4-hydroxybutyryl-CoA glutamate decarboxylase; a 4-aminobutyrate dehydrogenase reductase (alcohol forming) (see FIG. 1, steps X, Y, AA, and or 4-aminobutyrate transaminase; a 4-hydroxybutyrate W). US 2016/03193 13 A1 Nov. 3, 2016

0076. In a further embodiment, the non-naturally occur ferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxy ring microbial organism has a 14-BDO pathway comprising butyryl-CoA synthetase; a 4-hydroxybutyryl-CoA reductase an alpha-ketoglutarate reductase; a 5-hydroxy-2-oxopen (aldehyde forming); and a 1,4-butanediol dehydrogenase tanoate dehydrogenase; a 5-hydroxy-2-oxopentanoate (see FIG. 1, steps G/H/I, A, C, T/U/V, Q, E); a succinyl decarboxylase; and a 1,4-butanediol dehydrogenase (see CoA transferase, a Succinyl-CoA hydrolase or Succinyl-CoA FIG. 1, steps X, Y, Z, E). synthetase; a Succinyl-CoA reductase (aldehyde forming); a 0077. In a further embodiment, the non-naturally occur 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl ring microbial organism has a GBL pathway comprising a CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and a 4-hydroxybutyryl Succinyl-CoA synthetase; a Succinyl-CoA reductase (alde CoA reductase (alcohol forming) (see FIG. 1, steps G/H/I, hyde forming); a 4-hydroxybutyrate dehydrogenase; and a A, C, T/U/V, WI; a succinyl-CoA transferase, a succinyl 4-hydroxybutyrate lactonase (see FIG. 1, steps G/H/I, A, C, CoA hydrolase or a succinyl-CoA synthetase; a Succinyl AB). CoA reductase (aldehyde forming); a 4-hydroxybutyrate 0078. In a further embodiment, the non-naturally occur dehydrogenase; a 4-hydroxybutyrate kinase; a phospho ring microbial organism has a GBL pathway comprising a trans-4-hydroxybutyrylase; and a 4-hydroxybutyryl-CoA Succinyl-CoA transferase, a Succinyl-CoA hydrolase and a reductase (alcohol forming) (see FIG. 1, steps G/H/I, A, C, Succinyl-CoA synthetase; a Succinyl-CoA reductase (alcohol O. P. W); a succinyl-CoA transferase, a succinyl-CoA forming); and a 4-hydroxybutyrate lactonase (see FIG. 1, hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA steps G/H/I, S, AB). reductase (alcohol forming); a 4-hydroxybutyrate reductase; 0079. In a further embodiment, the non-naturally occur and 1,4-butanediol dehydrogenase (see FIG. 1, steps G/H/I, ring microbial organism has a GBL pathway comprising a S, D, E); a Succinyl-CoA transferase, a Succinyl-CoA Succinate reductase; a 4-hydroxybutyrate dehydrogenase; hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA and a 4-hydroxybutyrate lactonase (see FIG. 1, steps F. C. reductase (alcohol forming); a 4-hydroxybutyrate kinase; a AB). phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl 0080. In a further embodiment, the non-naturally occur CoA reductase (aldehyde forming); and a 1,4-butanediol ring microbial organism has a GBL pathway comprising an dehydrogenase (see FIG. 1, steps G/H/I, S, O, P, Q, E); a alpha-ketoglutarate decarboxylase, a 4-hydroxybutyrate Succinyl-CoA transferase, a Succinyl-CoA hydrolase or Suc dehydrogenase and a 4-hydroxybutyrate lactonase (see FIG. cinyl-CoA synthetase; a Succinyl-CoA reductase (alcohol 1, steps B, C, AB). forming), a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl 0081. In a further embodiment, the non-naturally occur phosphate reductase; and a 1,4-butanediol dehydrogenase ring microbial organism has a GBL pathway comprising a (see FIG. 1, steps G/H/I, S. O. R. E); a succinyl-CoA glutamate dehydrogenase or a glutamate transaminase; a transferase, a Succinyl-CoA hydrolase or a Succinyl-CoA glutamate decarboxylase; a 4-aminobutyrate dehydrogenase synthetase; a Succinyl-CoA reductase (alcohol forming); a or a 4-aminobutyrate transaminase; a 4-hydroxybutyrate 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl dehydrogenase; and a 4-hydroxybutyrate lactonase (see FIG. CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; a 1, steps J/K, L, M/N, C, AB). 4-hydroxybutyryl-CoA reductase (aldehyde forming); and a 0082 In one embodiment, the invention provides a non 1,4-butanediol dehydrogenase (see FIG. 1, steps G/H/I, S, naturally occurring microbial organism, comprising a micro T/U/V. Q, E); a succinyl-CoA transferase, a succinyl-CoA bial organism having a 1,4-butanediol (14-BDO) pathway hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA and a gamma-butyrolactone (GBL) pathway, said 14-BDO reductase (alcohol forming); a 4-hydroxybutyryl-CoA trans pathway comprising a first set of exogenous nucleic acids ferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxy encoding 14-BDO pathway enzymes expressed in a Sufi butyryl-CoA synthetase; and a 4-hydroxybutyryl-CoA cient amount to produce 14-BDO, wherein said first set is reductase (alcohol forming) (see FIG. 1, steps G/H/I, S. selected from the group consisting of a Succinyl-CoA T/U/V. W); a succinyl-CoA transferase, a succinyl-CoA transferase, a Succinyl-CoA hydrolase or a Succinyl-CoA hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA synthetase; a Succinyl-CoA reductase (aldehyde forming); a reductase (alcohol forming); a 4-hydroxybutyrate kinase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate phosphotrans-4-hydroxybutyrylase; and a 4-hydroxybu reductase; and a 1,4-butanediol dehydrogenase) (see FIG. tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps 1, steps G/H/I, A, C, D, E); a succinyl-CoA transferase, a G/H/I, S. O. P. W); a succinate reductase; a 4-hydroxybu Succinyl-CoA hydrolase or a Succinyl-CoA synthetase; a tyrate dehydrogenase; a 4-hydroxybutyrate reductase; and Succinyl-CoA reductase (aldehyde forming); a 4-hydroxy 1,4-butanediol dehydrogenase; a Succinate reductase; a butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxy CoA reductase (aldehyde forming); and a 1,4-butanediol butyryl-CoA reductase (aldehyde forming); and a 1,4-bu dehydrogenase (see FIG. 1, steps G/H/I, A, C, O, P, Q, E): tanediol dehydrogenase (see FIG. 1, steps F.C., O, P, Q, E): a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a a Succinate reductase; a 4-hydroxybutyrate dehydrogenase; Succinyl-CoA synthetase; a Succinyl-CoA reductase (alde a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu hyde forming); a 4-hydroxybutyrate dehydrogenase; a 4-hy tyrylase; and a 4-hydroxybutyryl-CoA reductase (alcohol droxybutyrate kinase; a 4-hydroxybutyryl-phosphate reduc forming) (see FIG. 1, steps F. C., O, P, W); a succinate tase; and a 1,4-butanediol dehydrogenase (see FIG. 1, steps reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hy G/H/I, A, C, O. R. E); a succinyl-CoA transferase, a droxybutyrate kinase; a 4-hydroxybutyryl-phosphate reduc Succinyl-CoA hydrolase, or a Succinyl-CoA synthetase; a tase; and 1,4-butanediol dehydrogenase (see FIG. 1, Steps F. Succinyl-CoA reductase (aldehyde forming); a 4-hydroxy C, O. R. E); a Succinate reductase; a 4-hydroxybutyrate butyrate dehydrogenase; a 4-hydroxybutyryl-CoA trans dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a US 2016/03193 13 A1 Nov. 3, 2016

4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl droxybutyryl-CoA reductase (aldehyde forming); and a 1,4- CoA synthetase; a 4-hydroxybutyryl-CoA reductase (alde butanediol dehydrogenase (see FIG. 1, steps J/K, L, M/N. hyde forming); and 1,4-butanediol dehydrogenase (see C, T/U/V, Q, E); a glutamate dehydrogenase or a glutamate FIG. 1, steps F. C. T/U/V, Q, E); a succinate reductase; a transaminase; a glutamate decarboxylase; a 4-aminobutyrate 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl dehydrogenase or a 4-aminobutyrate transaminase; a 4-hy CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a droxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA 4-hydroxybutyryl-CoA synthetase; and a 4-hydroxybutyryl transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hy CoA reductase (alcohol forming) (see FIG. 1, steps F. C. droxybutyryl-CoA synthetase; and a 4-hydroxybutyryl-CoA T/U/V. W); an alpha-ketoglutarate decarboxylase; a 4-hy reductase (alcohol forming) (see FIG. 1, steps J/K, L, M/N. C, T/U/V. W); an alpha-ketoglutarate reductase; a 5-hy droxybutyrate dehydrogenase; a 4-hydroxybutyrate reduc droxy-2-oxopentanoate dehydrogenase; a 5-hydroxy-2-oxo tase; and 1,4-butanediol dehydrogenase;an alpha-ketoglu pentanoate dehydrogenase (decarboxylation); a 4-hydroxy tarate decarboxylase; a 4-hydroxybutyrate dehydrogenase; a butyryl-CoA reductase (aldehyde forming); and a 1,4- 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu butanediol dehydrogenase (see FIG. 1, steps X, Y, AA, Q, tyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde E); an alpha-ketoglutarate reductase; a 5-hydroxy-2-oxo forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, pentanoate dehydrogenase; a 5-hydroxy-2-oxopentanoate steps B, C, O, P, Q, E); an alpha-ketoglutarate decarboxy dehydrogenase (decarboxylation); and a 4-hydroxybutyryl lase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu CoA reductase (alcohol forming) (see FIG. 1, steps X, Y, tyrate kinase; a phosphotrans-4-hydroxybutyrylase and AA, and W); and an alpha-ketoglutarate reductase; a 5-hy 4-hydroxybutyryl-CoA reductase (alcohol forming) (see droxy-2-oxopentanoate dehydrogenase; a 5-hydroxy-2-oxo FIG. 1, steps B, C, O, P, W); an alpha-ketoglutarate pentanoate decarboxylase; and a 1,4-butanediol dehydroge decarboxylase; a 4-hydroxybutyrate dehydrogenase; a 4-hy nase (see FIG. 1, steps X, Y, Z, E), said GBL pathway droxybutyrate kinase; a 4-hydroxybutyryl-phosphate reduc comprising a second set of exogenous nucleic acid encoding tase; and a 1,4-butanediol dehydrogenase (see FIG. 1, steps an GBL pathway enzyme expressed in a sufficient amount to B, C, O. R. E); an alpha-ketoglutarate decarboxylase; a produce GBL, said second set is selected from the group 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl consisting of a Succinyl-CoA transferase, a Succinyl-CoA CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde forming); a 4-hydroxybutyrate dehy reductase (aldehyde forming); and a 1,4-butanediol dehy drogenase; and a 4-hydroxybutyrate lactonase (see FIG. 1, drogenase (see FIG. 1, steps B, C, T/U/V. W); an alpha steps G/H/I, A, C, AB); a succinyl-CoA transferase, a ketoglutarate decarboxylase; a 4-hydroxybutyrate dehydro Succinyl-CoA hydrolase and a Succinyl-CoA synthetase; a genase; a 4-hydroxybutyryl-CoA transferase, a Succinyl-CoA reductase (alcohol forming); and a 4-hydroxy 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl butyrate lactonase (see FIG. 1, steps G/H/I, S, AB); a CoA synthetase; and 4-hydroxybutyryl-CoA reductase (al Succinate reductase; a 4-hydroxybutyrate dehydrogenase; cohol forming) (see FIG. 1, steps B, C, T/U/V. W); a and a 4-hydroxybutyrate lactonase (see FIG. 1, steps F. C. glutamate dehydrogenase or a glutamate transaminase; a AB); an alpha-ketoglutarate decarboxylase, a 4-hydroxy glutamate decarboxylase; a 4-aminobutyrate dehydrogenase butyrate dehydrogenase and a 4-hydroxybutyrate lactonase or 4-aminobutyrate transaminase; a 4-hydroxybutyrate (see FIG. 1, steps B, C, AB); and a glutamate dehydroge dehydrogenase; a 4-hydroxybutyrate reductase; and a 1,4- nase or a glutamate transaminase; a glutamate decarboxy butanediol dehydrogenase (see FIG. 1, steps J/K, L, M/N. lase; a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate C, D, E); a glutamate dehydrogenase or a glutamate transaminase; a glutamate decarboxylase; a 4-aminobutyrate transaminase; a 4-hydroxybutyrate dehydrogenase; and a dehydrogenase or a 4-aminobutyrate transaminase; a 4-hy 4-hydroxybutyrate lactonase (see FIG. 1, steps J/K, L, M/N. droxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; C, AB). a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl I0083. In a further aspect of each of the above embodi CoA reductase (aldehyde forming); and E) 1,4-butanediol ments, the exogenous nucleic acid is a heterologous nucleic dehydrogenase (see FIG. 1, steps J/K, L, M/N, C, O, P, Q, acid. E); a glutamate dehydrogenase or glutamate transaminase; I0084. In a further aspect of each of the above embodi a glutamate decarboxylase; a 4-aminobutyrate dehydroge ments, the non-naturally occurring microbial organism is in nase or a 4-aminobutyrate transaminase; a 4-hydroxybu a Substantially anaerobic culture medium. tyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phos I0085. In a further aspect of each of the above embodi photrans-4-hydroxybutyrylase; and a 4-hydroxybutyryl ments, the 14-BDO and GBL products are produced at a CoA reductase (alcohol forming) (see FIG. 1, steps J/K, L, predetermined ratio. In some aspects of the invention, the M/N, C, O, P, W); a glutamate dehydrogenase or a gluta predetermined ratio of 14-BDO to GBL is 1:1, or alterna mate transaminase; a glutamate decarboxylase; a 4-amin tively 1:2, or alternatively 1:3, or alternatively 1:4, or obutyrate dehydrogenase or a 4-aminobutyrate transami alternatively 1:5, or alternatively 1:6, or alternatively 1:7, or nase, a 4-hydroxybutyrate dehydrogenase; a alternatively 1:8, or alternatively 1:9, or alternatively 1:10, 4-hydroxybutyrate kinase; a 4-hydroxybutyryl-phosphate or alternatively 1:15, or alternatively about 1:20, or alter reductase; and a 1,4-butanediol dehydrogenase (see FIG. 1, natively 1:25, or alternatively 1:30, or alternatively 1:35, or steps J/K, L, M/N, C, O. R. E); a glutamate dehydrogenase alternatively 1:40, or alternatively 1:45, or alternatively or a glutamate transaminase; a glutamate decarboxylase; a about 1:50, or alternatively 1:60, or alternatively 1:70, or 4-aminobutyrate dehydrogenase or a 4-aminobutyrate alternatively 1:80, or alternatively 1:90, or alternatively transaminase; a 4-hydroxybutyrate dehydrogenase; a 4-hy 1:100. In some aspects of the invention, the predetermined droxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA ratio of 14-BDO to GBL is 2:1, or alternatively 3:1, or hydrolase or a 4-hydroxybutyryl-CoA synthetase; a 4-hy alternatively 4:1 or alternatively 5:1, or alternatively 6:1, or US 2016/03193 13 A1 Nov. 3, 2016

alternatively 7:1, or alternatively 8:1, or alternatively 9:1, or plified in FIG. 1. Therefore, in addition to a microbial alternatively 10:1, or alternatively 15:1, or alternatively organism containing a 14-BDO and GBL pathway that about 20:1, or alternatively 25:1, or alternatively 30:1, or produces 14-BDO and GBL, the invention additionally alternatively 35:1, or alternatively 40:1, or alternatively provides a non-naturally occurring microbial organism com 45:1, or alternatively about 50:1, or alternatively 60:1, or prising at least one exogenous nucleic acid encoding a alternatively 70:1, or alternatively 80:1, or alternatively 14-BDO or GBL pathway enzyme, where the microbial 90:1, or alternatively 100:1. organism produces a 14-BDO or GBL pathway intermedi I0086. It is understood by one of skill in the art that the ate, for example, Succinyl-CoA, succinic semialdehyde, non-naturally occurring microorganisms described herein, 4-hydroxybutyrate, 4-hydroxybutyryl-phosphate, 4-hy having a 14-BDO and a GBL pathway, can have exogenous droxybutyryl-CoA, 4-hydroxybutanal, glutamate, 4-amin nucleic acids encoding enzymes or proteins of more than obutyrate. Succinic semialdehyde, 2,5-dioxopentanoic acid one pathway described herein. For example, a microorgan or 5-hydroxy-2OXopentanoic acid. ism can have exogenous nucleic acids encoding enzymes or I0089. It is understood that any of the pathways disclosed proteins for steps G/H/I, A, C, O, P, Q, and E of FIG. 1 and herein, as described in the Examples and exemplified in the steps J/K, L, M/N, C, O, P and W of FIG. 1 for production Figures, including the pathways of FIGS. 1A and 1B, can be of 14-BDO. It is also understood that exogenous nucleic utilized to generate a non-naturally occurring microbial acids encoding enzymes or proteins of the 14-BDO pathway organism that produces any pathway intermediate or prod can also satisfy the enzymes or proteins of the GBL pathway, uct, as desired. As disclosed herein, such a microbial organ and vice versa. For example, an exogenous nucleic acid ism that produces an intermediate can be used in combina encoding the enzyme succinate reductase in the 14-BDO tion with another microbial organism expressing pathway is also encoding the Succinate reductase for the downstream pathway enzymes to produce a desired product. GBL pathway for co-production of 14-BDO and GBL. However, it is understood that a non-naturally occurring 0087. In an additional embodiment, the invention pro microbial organism that produces a 14-BDO or GBL path vides a non-naturally occurring microbial organism having way intermediate can be utilized to produce the intermediate a 14-BDO and GBL pathway, wherein the non-naturally as a desired product. occurring microbial organism comprises at least one exog 0090 The invention is described herein with general enous nucleic acid encoding an enzyme or protein that reference to the metabolic reaction, reactant or product converts a to a product selected from the group thereof, or with specific reference to one or more nucleic consisting of Succinate to Succinyl-CoA. Succinate to Suc acids or genes encoding an enzyme associated with or cinic semialdehyde. Succinyl-CoA to Succinic semialdehyde, catalyzing, or a protein associated with, the referenced Succinyl-CoA to 4-hydroxybutyrate. Succinic semialdehyde metabolic reaction, reactant or product. Unless otherwise to 4-hydroxybutyrate, alpha-ketoglutarate to Succinic semi expressly stated herein, those skilled in the art will under aldehyde, alpha-ketoglutarate to glutamate, glutamate to stand that reference to a reaction also constitutes reference 4-aminobutyrate, 4-aminobutyrate to Succinic semialde to the reactants and products of the reaction. Similarly, hyde, alpha-ketoglutarate to 2,5-dioxopentanoic acid, 2.5- unless otherwise expressly stated herein, reference to a dioxopentanoic acid to 5-hydroxy-2-oxopentanoic acid, reactant or product also references the reaction, and refer 5-hydroxy-2-oxopentanoic acid to 4-hydroxybutanal, 4-hy ence to any of these metabolic constituents also references droxybutyrate to 4-hydroxybutanal, 4-hydroxybutyrate to the gene or genes encoding the enzymes that catalyze or 4-hydroxybutyryl-phosphate, 4-hydroxybutyryl-phosphate proteins involved in the referenced reaction, reactant or to 4-hydroxybutyryl-CoA, 4-hydroxybutyryl-phosphate to product. Likewise, given the well known fields of metabolic 4-hydroxybutanal, 5-hydroxy-2OXopentanoic acid to 4-hy biochemistry, enzymology and genomics, reference herein droxybutyryl-CoA, 4-hydroxybutyryl-CoA to 4-hydroxybu to a gene or encoding nucleic acid also constitutes a refer tanal, 4-hydroxybutyrate to 4-hydroxybutyryl-CoA, 4-hy ence to the corresponding encoded enzyme and the reaction droxybutyryl-CoA to 1,4-butanediol, 4-hydroxybutanal to it catalyzes or a protein associated with the reaction as well 1,4-butanediol and 4-hydroxybutyrate to gamma-butyrolac as the reactants and products of the reaction. tone. One skilled in the art will understand that these are 0091 Exemplary alcohol and aldehyde dehydrogenases merely exemplary and that any of the Substrate-product pairs that can be used for in vivo conversions described herein disclosed herein suitable to produce a desired product and from 4-HB to 14-BDO are listed below in Table 1. for which an appropriate activity is available for the con version of the substrate to the product can be readily TABLE 1. determined by one skilled in the art based on the teachings Alcohol and Aldehyde Dehydrogenases for herein. Thus, the invention provides a non-naturally occur Conversion of 4-HB to 14-BDO. ring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the ALCOHOL DEHYDROGENASES enzyme or protein converts the Substrates and products of a ec: 1.1.1.1 alcohol dehydrogenase 14-BDO and GBL pathway, such as that shown in FIG. 1. ec: 1.1.1.2 alcohol dehydrogenase (NADP+) ec: 1.1.1.4 (R,R)-butanediol dehydrogenase 0088 While generally described herein as a microbial ec: 1.1.1.5 acetoin dehydrogenase organism that contains a 14-BDO and GBL pathway, it is ec: 1.1.1.6 glycerol dehydrogenase understood that the invention additionally provides a non ec: 1.1.1.7 propanediol-phosphate naturally occurring microbial organism comprising at least dehydrogenase ec: 1.1.1.8 glycerol-3-phosphate dehydrogenase one exogenous nucleic acid encoding a 14-BDO or GBL (NAD+) pathway enzyme expressed in a sufficient amount to produce ec: 1.1.1.11 D-arabinitol 4-dehydrogenase an intermediate of a 14-BDO or GBL pathway. For example, ec: 1.1.1.12 L-arabinitol 4-dehydrogenase as disclosed herein, 14-BDO and GBL pathways are exem US 2016/03193 13 A1 Nov. 3, 2016 11

TABLE 1-continued TABLE 1-continued Alcohol and Aldehyde Dehydrogenases for Alcohol and Aldehyde Dehydrogenases for Conversion of 4-HB to 14-BDO. Conversion of 4-HB to 14-BDO.

(C: 1.13 L-arabinitol 2-dehydrogenase (C: 1129 L-threonate 3-dehydrogenase (C: .1.14 L-iditol 2-dehydrogenase (C: .1.1.37 ribitol-5-phosphate 2-dehydrogenase (C: 1.15 D-iditol 2-dehydrogenase (C: 1.138 mannitol 2-dehydrogenase (NADP+) (C: .1.16 galactitol 2-dehydrogenase (C: 140 sorbitol-6-phosphate 2 (C: 17 mannitol-1-phosphate 5 dehydrogenase dehydrogenase (C: .1.142 D-pinitol dehydrogenase (C: 1.18 inositol 2-dehydrogenase (C: .1.143 Sequoyitol dehydrogenase (C: .1.21 aldehyde reductase (C: .1.144 perillyl-alcohol dehydrogenase (C: 1.23 histidinol dehydrogenase (C: 1.156 glycerol 2-dehydrogenase (NADP+) (C: .1.26 glyoxylate reductase (C: 157 3-hydroxybutyryl-CoA (C: 1.27 L-lactate dehydrogenase dehydrogenase (C: 1.28 D-lactate dehydrogenase (C: 1.163 cyclopentanol dehydrogenase (C: 1.29 glycerate dehydrogenase (C: .1.164 hexadecanol dehydrogenase (C: 1.30 3-hydroxybutyrate dehydrogenase (C: 1165 2-alkyn-1-oldehydrogenase (C: 1.31 3-hydroxyisobutyrate dehydrogenase (C: 166 hydroxycyclohexanecarboxylate (C: 1.35 3-hydroxyacyl-CoA dehydrogenase dehydrogenase (C: 1.36 acetoacetyl-CoA reductase (C: 1.167 hydroxymalonate dehydrogenase (C: 1.37 malate dehydrogenase (C: 1.174 cyclohexane-1,2-diol dehydrogenase (C: 38 malate dehydrogenase (oxaloacetate (C: 177 glycerol-3-phosphate 1 decarboxylating) dehydrogenase (NADP+) (C: 39 malate dehydrogenase (C: 178 3-hydroxy-2-methylbutyryl-CoA (decarboxylating) dehydrogenase (C: .1.40 malate dehydrogenase (oxaloacetate (C: 1.185 L-glycol dehydrogenase decarboxylating) (NADP+) (C: 190 indole-3-acetaldehyde reductase (C: .1.1.41 isocitrate dehydrogenase (NAD+) (NADH) (C: .1.42 isocitrate dehydrogenase (NADP+) (C: 191 indole-3-acetaldehyde reductase (C: 1.54 allyl-alcohol dehydrogenase (NADPH) (C: 1.55 lactaldehyde reductase (NADPH) (C: 1.192 long-chain-alcohol dehydrogenase (C: 1.56 ribitol 2-dehydrogenase (C: 1.194 coniferyl-alcohol dehydrogenase (C: 1.59 3-hydroxypropionate dehydrogenase (C: 1.195 cinnamyl-alcohol dehydrogenase (C: 60 2-hydroxy-3-oxopropionate (C: 1.198. (+)-borneol dehydrogenase reductase (C: 1.2O2 1,3-propanediol dehydrogenase (C: 1.61 4-hydroxybutyrate dehydrogenase (C: 1.2O7 (-)-menthol dehydrogenase (C: 66 omega-hydroxydecanoate (C: 1208 (+)-neomenthol dehydrogenase dehydrogenase (C: .1.216 farnesol dehydrogenase (C: 1.67 mannitol 2-dehydrogenase (C: 217 benzyl-2-methyl-hydroxybutyrate (C: 1.71 alcohol dehydrogenase NAD(P)+ dehydrogenase (C: 1.72 glycerol dehydrogenase (NADP+) (C: 222 (R)-4-hydroxyphenylactate (C: 1.73 octanol dehydrogenase dehydrogenase (C: 1.75 (R)-aminopropanol dehydrogenase (C: 1.223 isopiperitenol dehydrogenase (C: 1.76 (S,S)-butanediol dehydrogenase (C: 226 4-hydroxycyclohexanecarboxylate (C: 1.77 lactaldehyde reductase dehydrogenase (C: .78 methylglyoxal reductase (NADH (C: 229 diethyl 2-methyl-3-oxoSuccinate dependent) reductase (C: 1.79 glyoxylate reductase (NADP+) (C: 1.237 hydroxyphenylpyruvate reductase (C: 8O isopropanol dehydrogenase (C: .1.244 methanol dehydrogenase (NADP+) (C: 1.245 cyclohexanol dehydrogenase (C: 1.81 hydroxypyruvate reductase (C: 1.2SO D-arabinitol 2-dehydrogenase (C: 1.82 malate dehydrogenase (NADP+) (C: 251 galactitol 1-phosphate 5 (C: .83 D-malate dehydrogenase dehydrogenase (decarboxylating) (C: 1.255 mannitol dehydrogenase (C: 1.84 dimethylmalate dehydrogenase (C: 1.256 fluoren-9-ol dehydrogenase (C: 1.85 3-isopropylmalate dehydrogenase (C: 257 4-(hydroxymethyl)benzenesulfonate (C: 186 ketol-acid reductoisomerase dehydrogenase (C: 1.87 homoisocitrate dehydrogenase (C: 1.258 6-hydroxyhexanoate dehydrogenase (C: 88 hydroxymethylglutaryl-CoA (C: 259 3-hydroxypimeloyl-CoA reductase dehydrogenase (C: 1.90 aryl-alcohol dehydrogenase (C: 261 glycerol-1-phosphate dehydrogenase (C: 91 aryl-alcohol dehydrogenase NAD(P)+ (NADP+) (C: 1.265 3-methylbutanal reductase (C: 92 oxaloglycolate reductase (C: .283 methylglyoxal reductase (NADPH (decarboxylating) dependent) (C: .94 glycerol-3-phosphate dehydrogenase (C: 286 isocitrate-homoisocitrate NAD(P)+ dehydrogenase (C: 1.9S phosphoglycerate dehydrogenase (C: 287 (C: 97 3-hydroxybenzyl-alcohol D-arabinitol dehydrogenase dehydrogenase (NADP+) (C: .1.101 acylglycerone-phosphate reductase butanol dehydrogenase (C: 1.103 L-threonine 3-dehydrogenase ALDEHYDE DEHYDROGENASES (C: .1.104 4-oxoproline reductase (C: 1105 retinol dehydrogenase ec: 1.2.1.2 formate dehydrogenase (C: .1.110 indolelactate dehydrogenase ec: 1.2.1.3 (NAD+) (C: .1.112 indanol dehydrogenase ec: 1.2.1.4 aldehyde dehydrogenase (NADP+) (C: 113 L-xylose 1-dehydrogenase ec: 1.2.1.5 aldehyde dehydrogenase NAD(P)+ US 2016/03193 13 A1 Nov. 3, 2016

TABLE 1-continued TABLE 1-continued Alcohol and Aldehyde Dehydrogenases for Alcohol and Aldehyde Dehydrogenases for Conversion of 4-HB to 14-BDO. Conversion of 4-HB to 14-BDO.

(C: benzaldehyde dehydrogenase ec: 1.2.1.66 mycothiol-dependent formaldehyde (NADP+) dehydrogenase (C: 8 betaine-aldehyde dehydrogenase ec: 1.2.1.67 vanillin dehydrogenase (C: glyceraldehyde-3-phosphate ec: 1.2.1.68 coniferyl-aldehyde dehydrogenase ehydrogenase (NADP+) ec: 1.2.1.69 fluoroacetaldehyde dehydrogenase (C: acetaldehyde dehydrogenase ec: 1.2.1.71 Succinylglutamate-semialdehyde (acetylating) dehydrogenase (C: aspartate-semialdehyde enydrogenase (C: glyceraldehyde-3-phosphate 0092. Other exemplary enzymes and pathways are dis ehydrogenase (phosphorylating) (C: glyceraldehyde-3-phosphate closed herein (see Examples). Furthermore, it is understood ehydrogenase (NADP+) (phosphorylating) that enzymes can be utilized for carry out reactions for (C: malonate-semialdehyde which the substrate is not the natural substrate. While the enydrogenase activity for the non-natural substrate may be lower than the (C: Succinate-semialdehyde ehydrogenase NAD(P)+ natural Substrate, it is understood that such enzymes can be (C: glyoxylate dehydrogenase (acylating) utilized, either as naturally occurring or modified using the (C: malonate-semialdehyde directed evolution or adaptive evolution, as disclosed herein. ehydrogenase (acetylating) (0093 14-BDO and GBL production through any of the (C: 2. aminobutyraldehyde dehydrogenase (C: glutarate-semialdehyde pathways disclosed herein are based, in part, on the identi enydrogenase fication of the appropriate enzymes for conversion of pre (C: .21 glycolaldehyde dehydrogenase cursors to 14-BDO and GBL. A number of specific enzymes (C: 22 lactaldehyde dehydrogenase (C: 23 2-oxoaldehyde dehydrogenase for several of the reaction steps have been identified. For (NAD+) those transformations where enzymes specific to the reaction (C: .24 Succinate-semialdehyde precursors have not been identified, enzyme candidates have dehydrogenase been identified that are best suited for catalyzing the reaction (C: 25 2-oxoisovalerate dehydrogenase (acylating) steps. Enzymes have been shown to operate on a broad range (C: 26 2,5-dioxovalerate dehydrogenase of substrates, as discussed below. In addition, advances in (C: 27 methylmalonate-semialdehyde the field of protein engineering also make it feasible to alter dehydrogenase (acylating) enzymes to act efficiently on Substrates, even if not a natural (C: 28 benzaldehyde dehydrogenase (NAD+) substrate. Described below are several examples of broad (C: 29 aryl-aldehyde dehydrogenase specificity enzymes from diverse classes Suitable for a (C: 30 aryl-aldehyde dehydrogenase 14-BDO and GBL pathway as well as methods that have (NADP+) been used for evolving enzymes to act on non-natural (C: 31 L-aminoadipate-semialdehyde dehydrogenase Substrates. (C: 32 aminomuconate-semialdehyde (0094. A key class of enzymes in 14-BDO and GBL dehydrogenase pathways is the that interconvert ketones or (C: 36 retinal dehydrogenase aldehydes to alcohols (1.1.1). Numerous exemplary (C: 39 phenylacetaldehyde dehydrogenase (C: 41 glutamate-5-semialdehyde enzymes in this class can operate on a wide range of dehydrogenase substrates. An alcohol dehydrogenase (1.1.1.1) purified from (C: 42 hexadecanal dehydrogenase the soil bacterium Brevibacterium sp. KU 1309 (Hirano et (acylating) al., J. Biosc. Bioeng. 100:318-322 (2005)) was shown to (C: 43 ormate dehydrogenase (NADP+) (C: 45 4-carboxy-2-hydroxymuconate-6- operate on a plethora of aliphatic as well as aromatic semialdehyde dehydrogenase alcohols with high activities. Table 2 shows the activity of (C: 46 ormaldehyde dehydrogenase the enzyme and its K. on different alcohols. The enzyme is (C: 47 4-trimethylammoniobutyraldehyde dehydrogenase reversible and has very high activity on several aldehydes (C: 48 ong-chain-aldehyde dehydrogenase also (Table 3). (C: 49 2-oxoaldehyde dehydrogenase (NADP+) TABLE 2 (C: S1 (NADP+) (C: 52 oxoglutarate dehydrogenase Relative activities of an alcohol dehydrogenase from Brevibacterium sp (NADP+) KU to oxidize various alcohols. (C: 53 4-hydroxyphenylacetaldehyde dehydrogenase Relative Activity K. (C: 57 butanal dehydrogenase Substrate (0%) (mM) (C: S8 phenylglyoxylate dehydrogenase (acylating) 2-Phenylethanol 100% O.O2S (C: 59 glyceraldehyde-3-phosphate (S)-2-Phenylpropanol 156 0.157 dehydrogenase (NAD(P)+) (phosphorylating) (R)-2-Phenylpropanol 63 O.O2O (C: 62 4-formylbenzenesulfonate Bynzyl alcohol 199 O.O12 dehydrogenase 3-Phenylpropanol 135 O.O33 (C: 63 6-oxohexanoate dehydrogenase Ethanol 76 (C: .64 4-hydroxybenzaldehyde 1-Butanol 111 dehydrogenase 1-Octanol 101 (C: .65 Salicylaldehyde dehydrogenase 1-Dodecanol 68 US 2016/03193 13 A1 Nov. 3, 2016

TABLE 2-continued evisiae (Sinclair et al., Biochem. Mol Biol. Int. 32:911-922 (1993), this complex has been shown to have a broad Relative activities of an alcohol dehydrogenase from Brevibacterium sp Substrate range that includes linear oxo-acids such as KU to oxidize various alcohols. 2-oxobutanoate and alpha-ketoglutarate, in addition to the Relative Activity K. branched-chain amino acid precursors. Substrate (0%) (mM) 0097 Members of yet another class of enzymes, namely 1-Phenylethanol 46 aminotransferases (2.6.1), have been reported to act on 2-Propanol S4 multiple Substrates. Aspartate aminotransferase (aspAT) from Pyrococcus firsious has been identified, expressed in *The activity of 2-phenylethanol, corresponding to 19.2 Umg, was taken as 100%. E. coli and the recombinant protein characterized to dem onstrate that the enzyme has the highest activities towards TABLE 3 aspartate and alpha-ketoglutarate but lower, yet significant activities towards alanine, glutamate and the aromatic amino Relative activities of an alcohol dehydrogenase from Brevibacterium sp acids (Ward et al., Archaea 133-141 (2002)). In another KU 1309 to reduce various carbonyl compounds. instance, an aminotransferase identified from Leishmania Relative Activity K mexicana and expressed in E. coli (Vernal et al., FEMS Substrate (%) (mM) Microbiol. Lett. 229:217-222 (2003)) was reported to have a broad Substrate specificity towards tyrosine (activity con Phenylacetaldehyde 100 O.261 2-Phenylpropionaldehyde 188 O.864 sidered 100% on tyrosine), phenylalanine (90%), tryptophan 1-Octylaldehyde 87 (85%), aspartate (30%), leucine (25%) and methionine Acetophenone O (25%), respectively (Vernal et al., Mol. Biochem. Parasitol 96:83-92 (1998)). Similar broad specificity has been reported for a tyrosine aminotransferase from Trypanosoma 0095 Lactate dehydrogenase (1.1.1.27) from Ralstonia cruzi, even though both of these enzymes have a sequence eutropha is another enzyme that has been demonstrated to homology of only 6%. The latter enzyme can accept leucine, have high activities on several 2-oxoacids such as 2-oxobu methionine as well as tyrosine, phenylalanine, tryptophan tyrate, 2-oxopentanoate and 2-oxoglutarate (a C5 compound and alanine as efficient amino donors (Nowicki et al., analogous to 2-oxoadipate) (Steinbuchel and Schlegel, Eur: Biochim. Biophys. Acta 1546: 268-281 (2001)). J. Biochem. 130:329-334 (1983)). Column 2 in Table 4 I0098 CoA (2.8.3) have been demonstrated demonstrates the activities of laha from R. eutropha (for to have the ability to act on more than one substrate. merly A. eutrophus) on different substrates (Steinbuchel and Specifically, a CoA transferase was purified from Schlegel, supra, 1983). Clostridium acetobutylicum and was reported to have the highest activities on acetate, propionate, and butyrate. It also TABLE 4 had significant activities with Valerate, isobutyrate, and The in vitro activity of R. eutropha laha (Steinbuchel and Schlegel, Supra, crotonate (Wiesenborn et al., Appl. Environ. Microbiol. 1983) on different substrates and compared with that on pyruvate. 55:323-329 (1989)). In another study, the E. coli enzyme acyl-CoA:acetate-CoA transferase, also known as acetate Activity (%) of CoA transferase (EC 2.8.3.8), has been shown to transfer the L(+)-lactate D(-)-lactate CoA moiety to acetate from a variety of branched and linear L(+)-lactate dehydrogenase dehydrogenase acyl-CoA substrates, including isobutyrate (Matthies and dehydrogenase from rabbit from Schink, App. Environm. Microbiol. 58:1435-1439 (1992)), Substrate from A. eutrophus muscle L. ieichmani Valerate (Vanderwinkel et al., Biochem. Biophys. Res Com Glyoxylate 8.7 23.9 S.O mun. 33:902-908 (1968b)) and butanoate (Vanderwinkel et Pyruvate 1OOO 1OOO 1OO.O al., Biochem. Biophys. Res Commun. 33:902-908(1968a). 2-Oxobutyrate 107.0 18.6 1.1 2-Oxovalerate 12SO 0.7 O.O (0099. Another class of enzymes useful in 14-BDO and 3-Methyl-2- 28.5 O.O O.O GBL pathways is the acid-thiol (6.2.1). Like Oxobutyrate enzymes in other classes, certain enzymes in this class have 3-Methyl-2- 5.3 O.O O.O oxovalerate been determined to have broad substrate specificity. For 4-Methyl-2- 39.0 1.4 1.1 example, acyl CoA ligase from Pseudomonas putida has oXopentanoate been demonstrated to work on several aliphatic substrates Oxaloacetate O.O 33.1 23.1 including acetic, propionic, butyric, Valeric, hexanoic, hep 2-Oxoglutarate 79.6 O.O O.O tanoic, and octanoic acids and on aromatic compounds such 3-Fluoropyruvate 33.6 74.3 40.O as phenylacetic and phenoxyacetic acids (Fernandez Valverde et al., Appl. Environ. Microbiol. 59:1149-1154 0096. Oxidoreductases that can convert 2-oxoacids to (1993)). A related enzyme, malonyl CoA synthetase (6.3.4. their acyl-CoA counterparts (1.2.1) have been shown to 9) from Rhizobium trifolii could convert several diacids, accept multiple Substrates as well. For example, branched namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-, cyclo chain 2-keto-acid dehydrogenase complex (BCKAD), also propyl-, cyclopropylmethylene-, cyclobutyl-, and benzyl known as 2-oxoisovalerate dehydrogenase (1.2.1.25), par malonate into their corresponding monothioesters (Pohl et ticipates in branched-chain amino acid degradation path al., J. Am. Chem. Soc. 123:5822-5823 (2001)). Similarly, ways, converting 2-keto acids derivatives of valine, leucine decarboxylases (4.1.1) have also been found with broad and isoleucine to their acyl-CoA derivatives and CO. In substrate ranges. Pyruvate decarboxylase (PDC), also Some organisms including Rattus norvegicus (Paxton et al., termed keto-acid decarboxylase, is a key enzyme in alco Biochem. J. 234:295-303 (1986)) and Saccharomyces cer holic fermentation, catalyzing the decarboxylation of pyru US 2016/03193 13 A1 Nov. 3, 2016 vate to acetaldehyde. The enzyme isolated from Saccharo tarum, Streptomyces coelicolor, Clostridium acetobutyli myces cerevisiae has a broad Substrate range for aliphatic cum, Pseudomonas fluorescens, and Pseudomonas putida. 2-keto acids including 2-ketobutyrate, 2-ketovalerate, and Exemplary yeasts or fungi include species selected from 2-phenylpyruvate (Li and Jordan, Biochemistry 38:10004 Saccharomyces cerevisiae, Schizosaccharomyces pombe, 10012 (1999)). Similarly, benzoylformate decarboxylase has Kluyveromyces lactis, Kluyveromyces marxianus, Aspergil a broad Substrate range and has been the target of enzyme lus terreus, Aspergillus niger; Pichia pastoris, Rhizopus engineering studies. The enzyme from Pseudomonas putida arrhizus, Rhizopus Oryzae, and the like. E. coli is a particu has been extensively studied and crystal structures of this larly useful host organisms since it is a well characterized enzyme are available (Polovnikova et al., Biochemistry microbial organism Suitable for genetic engineering. Other 42:1820-1830 (2003); Hasson et al., Biochemistry 37:9918 particularly useful host organisms include yeast Such as 9930 (1998)). Branched chain alpha-ketoacid decarboxylase Saccharomyces cerevisiae. It is understood that any Suitable (BCKA) has been shown to act on a variety of compounds microbial host organism can be used to introduce metabolic varying in chain length from 3 to 6 carbons (Oku and and/or genetic modifications to produce a desired product. Kaneda, J. Biol. Chem. 263:18386-18396 (1998); Smit et al., (0103) Depending on the 14-BDO and GBL biosynthetic Appl. Environ. Microbiol. 71:303-311 (2005b)). The enzyme pathway constituents of a selected host microbial organism, in Lactococcus lactis has been characterized on a variety of the non-naturally occurring microbial organisms of the branched and linear Substrates including 2-oxobutanoate, invention will include at least one exogenously expressed 2-oxohexanoate, 2-oxopentanoate, 3-methyl-2-oxobutano 14-BDO or GBL pathway-encoding nucleic acid and up to ate, 4-methyl-2-oxobutanoate and isocaproate (Smit et al., all encoding nucleic acids for one or more 14-BDO and GBL Appl. Environ. Microbiol. 71:303-311 (2005a). biosynthetic pathway. For example, 14-BDO and GBL bio 0100 Interestingly, enzymes known to have one domi synthesis can be established in a host deficient in a pathway nant activity have also been reported to catalyze a very enzyme or protein through exogenous expression of the different function. For example, the cofactor-dependent corresponding encoding nucleic acid. In a host deficient in phosphoglycerate mutase (5.4.2.1) from Bacillus Stearother all enzymes or proteins of a 14-BDO or GBL pathway, mophilus and Bacillus subtilis is known to function as a exogenous expression of all enzymes or proteins in the phosphatase as well (Rigden et al., Protein Sci. 10:1835 pathway can be included, although it is understood that all 1846 (2001)). The enzyme from B. Stearothermophilus is enzymes or proteins of a pathway can be expressed even if known to have activity on several Substrates, including the host contains at least one of the pathway enzymes or 3-phosphoglycerate, alpha-napthylphosphate, p-nitrophe proteins. For example, exogenous expression of all enzymes nylphosphate, AMP fructose-6-phosphate, ribose-5-phos or proteins in a pathway for production of 14-BDO and GBL phate and CMP. can be included. Such as a Succinyl-CoA reductase (aldehyde 0101 The non-naturally occurring microbial organisms forming), an alpha-ketoglutarate decarboxylase, a 4-hy of the invention can be produced by introducing expressible droxybutyrate dehydrogenase, a 4-hydroxybutyrate reduc nucleic acids encoding one or more of the enzymes or tase, a 1,4-butanediol dehydrogenase, a Succinate reductase, proteins participating in one or more 14-BDO or GBL a Succinyl-CoA transferase, a Succinyl-CoA hydrolase, a biosynthetic pathways. Depending on the host microbial Succinyl-CoA synthetase (or Succinyl-CoA ligase), a gluta organism chosen for biosynthesis, nucleic acids for some or mate dehydrogenase, a glutamate transaminase, a glutamate all of a particular 14-BDO or GBL biosynthetic pathway can decarboxylase, a 4-aminobutyrate dehydrogenase, a 4-amin be expressed. For example, if a chosen host is deficient in obutyrate transaminase, a 4-hydroxybutyrate kinase, a phos one or more enzymes or proteins for a desired biosynthetic photrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA pathway, then expressible nucleic acids for the deficient reductase (aldehyde forming), a 4-hydroxybutyryl-phos enzyme(s) or protein(s) are introduced into the host for phate reductase, a Succinyl-CoA reductase (alcohol form Subsequent exogenous expression. Alternatively, if the cho ing), a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybu Sen host exhibits endogenous expression of some pathway tyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase genes, but is deficient in others, then an encoding nucleic (or 4-hydroxybutyryl-CoA ligase), a 4-hydroxybutyryl-CoA acid is needed for the deficient enzyme(s) or protein(s) to reductase (alcohol forming), an alpha-ketoglutarate reduc achieve 14-BDO and GBL biosynthesis. Thus, a non-natu tase, a 5-hydroxy-2-oxopentanoate dehydrogenase, a 5-hy rally occurring microbial organism of the invention can be droxy-2-oxopentanoate decarboxylase, a 5-hydroxy-2-oxo produced by introducing exogenous enzyme or protein pentanoate dehydrogenase (decarboxylation) or a activities to obtain a desired biosynthetic pathway or a 4-hydroxybutyrate lactonase. desired biosynthetic pathway can be obtained by introducing 0104. Given the teachings and guidance provided herein, one or more exogenous enzyme or protein activities that, those skilled in the art will understand that the number of together with one or more endogenous enzymes or proteins, encoding nucleic acids to introduce in an expressible form produces a desired product such as 14-BDO or GBL. will, at least, parallel the 14-BDO and GBL pathway defi 0102 Host microbial organisms can be selected from, ciencies of the selected host microbial organism. Therefore, and the non-naturally occurring microbial organisms gener a non-naturally occurring microbial organism of the inven ated in, for example, bacteria, yeast, fungus or any of a tion can have one, two, three, four, five, six, seven or eight variety of other microorganisms applicable to fermentation up to all nucleic acids encoding the enzymes or proteins processes. Exemplary bacteria include species selected from constituting a 14-BDO or GBL biosynthetic pathway dis sp. Escherichia coli, Klebsiella Oxytoca, Anaerobiospirillum closed herein. In some embodiments, the non-naturally succiniciproducens, Actinobacillus succinogenes, Man occurring microbial organisms also can include other inheimia succiniciproducens, Rhizobium etli, Bacillus subti genetic modifications that facilitate or optimize 14-BDO and lis, Corynebacterium glutamicum, Gluconobacter Oxydans, GBL biosynthesis or that confer other useful functions onto Zymomonas mobilis, Lactococcus lactis, Lactobacillus plan the host microbial organism. One Such other functionality US 2016/03193 13 A1 Nov. 3, 2016 can include, for example, augmentation of the synthesis of produced by the non-naturally occurring microorganisms one or more of the 14-BDO or GBL pathway precursors described herein by enzymatically converting one or more Such as Succinate, Succinyl-CoA or alpha-ketoglutarate. pathway intermediates, which are shared between the 0105 Generally, a host microbial organism is selected 14-BDO and GBL pathways, towards the production of one such that it produces the precursor of a 14-BDO or GBL of the products. In this embodiment, the pool of intermedi pathway, either as a naturally produced molecule or as an ates for production of one product can be shifted toward the engineered product that either provides de novo production production of another product ultimately resulting in change of a desired precursor or increased production of a precursor in the ratio of 14-BDO and GBL produced by the microor naturally produced by the host microbial organism. For ganisms or methods described herein. example, Succinate is produced naturally in a host organism 0108. It is understood that, in methods of the invention, Such as E. coli. A host organism can be engineered to any of the one or more exogenous nucleic acids can be increase production of a precursor, as disclosed herein. In introduced into a microbial organism to produce a non addition, a microbial organism that has been engineered to naturally occurring microbial organism of the invention. The produce a desired precursor can be used as a host organism nucleic acids can be introduced so as to confer, for example, and further engineered to express enzymes or proteins of a a 14-BDO and GBL biosynthetic pathway onto the microbial 14-BDO or GBL pathway. organism. Alternatively, encoding nucleic acids can be intro 0106. In some embodiments, a non-naturally occurring duced to produce an intermediate microbial organism having microbial organism of the invention is generated from a host the biosynthetic capability to catalyze some of the required that contains the enzymatic capability to synthesize 14-BDO reactions to confer 14-BDO and GBL biosynthetic capabil and GBL and in some embodiments synthesizing 14-BDO ity. For example, a non-naturally occurring microbial organ and GBL at a predetermined ratio. In this specific embodi ism having 14-BDO and GBL biosynthetic pathways can ment it can be useful to increase the synthesis or accumu comprise at least two exogenous nucleic acids encoding lation of a 14-BDO or GBL pathway product to, for desired enzymes or proteins, such as the combination of a example, drive 14-BDO and GBL pathway reactions toward Succinyl-CoA transferase and a 4-hydroxybutyrate kinase, or 14-BDO and GBL production. Increased synthesis or accu alternatively a 4-hydroxybutyrate lactonase and a 1,4-bu mulation can be accomplished by, for example, overexpres tanediol dehydrogenase, or alternatively a glutamate decar sion of nucleic acids encoding one or more of the above boxylase and a 5-hydroxy-2-oxopentanoate decarboxylase, described 14-BDO or GBL pathway enzymes or proteins. and the like. Thus, it is understood that any combination of Over expression of the enzyme or enzymes and/or protein or two or more enzymes or proteins of a biosynthetic pathway proteins of the 14-BDO or GBL pathway can occur, for can be included in a non-naturally occurring microbial example, through exogenous expression of the endogenous organism of the invention. Similarly, it is understood that gene or genes, or through exogenous expression of the any combination of three or more enzymes or proteins of a heterologous gene or genes. Therefore, naturally occurring biosynthetic pathway can be included in a non-naturally organisms can be readily generated to be non-naturally occurring microbial organism of the invention, for example, occurring microbial organisms of the invention, for a Succinyl-CoA reductase (alcohol forming), a 4-hydroxy example, producing 14-BDO and GBL, through overexpres butyryl-CoA transferase and a 4-hydroxybutyrate lactonase, sion of one, two, three, four, five, six, seven or eight, that is, or alternatively a 5-hydroxy-2-oxopentanoate dehydroge up to all nucleic acids encoding 14-BDO or GBL biosyn nase (decarboxylation), a Succinate reductase and a phos thetic pathway enzymes or proteins. In addition, a non photrans-4-hydroxybutyrylase, or alternatively an alpha naturally occurring organism can be generated by mutagen ketoglutarate reductase, a 1,4-butanediol dehydrogenase and esis of an endogenous gene that results in an increase in a 4-hydroxybutyrate lactonase, and so forth, as desired, so activity of an enzyme in the 14-BDO or GBL biosynthetic long as the combination of enzymes and/or proteins of the pathway. desired biosynthetic pathway results in production of the 0107. In particularly useful embodiments, exogenous corresponding desired product. Similarly, any combination expression of the encoding nucleic acids is employed. Exog of four, a Succinate reductase, a 4-hydroxybutyrate dehy enous expression confers the ability to custom tailor the drogenase, a 4-hydroxybutyrate kinase and a 4-hydroxybu expression and/or regulatory elements to the host and appli tyryl-CoA reductase (alcohol forming), or alternatively a cation to achieve a desired expression level that is controlled 4-hydroxybutyrate lactonase, a Succinyl-CoA reductase (al by the user, which can be used to produce a predetermined cohol forming), a 4-hydroxybutyryl-CoA synthetase and a ratio of 14-BDO to GBL. However, endogenous expression 4-hydroxybutyryl-CoA reductase (alcohol forming), or alter also can be utilized in other embodiments such as by natively a 4-aminobutyrate dehydrogenase, a 4-hydroxybu removing a negative regulatory effector or induction of the tyrate dehydrogenase, a 4-hydroxybutyrate lactonase and a gene's promoter when linked to an inducible promoter or 4-hydroxybutyryl-CoA reductase (aldehyde forming), or other regulatory element. Thus, an endogenous gene having more enzymes or proteins of a biosynthetic pathway as a naturally occurring inducible promoter can be up-regulated disclosed herein can be included in a non-naturally occur by providing the appropriate inducing agent, or the regula ring microbial organism of the invention, as desired, so long tory region of an endogenous gene can be engineered to as the combination of enzymes and/or proteins of the desired incorporate an inducible regulatory element, thereby allow biosynthetic pathway results in production of the corre ing the regulation of increased expression of an endogenous sponding desired product. gene at a desired time. Similarly, an inducible promoter can 0109. In addition to the biosynthesis of 14-BDO and be included as a regulatory element for an exogenous gene GBL as described herein, the non-naturally occurring micro introduced into a non-naturally occurring microbial organ bial organisms and methods of the invention also can be ism. It is also understood that increasing the expression of an utilized in various combinations with each other and with enzyme or protein can alter the ratio of 14-BDO to GBL other microbial organisms and methods well known in the US 2016/03193 13 A1 Nov. 3, 2016

art to achieve product biosynthesis by other routes. For Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Bos example, one alternative to produce 14-BDO and GBL other taurus, Bradyrhizobium japonicum, butyrate producing bac than use of the 14-BDO and GBL producers is through terium L2-50, Campylobacter jejuni, Candida albicans, addition of another microbial organism capable of convert Candida Antarctica, Chloroflexus aurantiacus, Clostridium ing a 14-BDO or GBL pathway intermediate to 14-BDO or acetobutyllicum, Clostridium acetobutyllicum ATCC 824, GBL. One such procedure includes, for example, the fer Clostridium aminobutyricum, Clostridium beijerinckii, mentation of a microbial organism that produces a 14-BDO Clostridium beijerinckii, Clostridium difficile, Clostridium or GBL pathway intermediate. The 14-BDO or GBL path Kluyveri, Clostridium kluyveri DSM 555, Clostridium per way intermediate can then be used as a Substrate for a second fringens, Clostridium saccharoperbutylacetonicum, microbial organism that converts the 14-BDO or GBL Clostridium tetani, Clostridium thermocellum, Corynebac pathway intermediate to 14-BDO and GBL. The 14-BDO or terium glutamicum, Erythrobacter sp. NAP1, Escherichia GBL pathway intermediate can be added directly to another coli Str. K12, Euglena gracilis, Flavobacterium lutescens, culture of the second organism or the original culture of the Fusarium oxysporum, Fusobacterium nucleatum, Geobacil 14-BDO or GBL pathway intermediate producers can be lus Stearothermophilus, Geobacillus thermoglucosidasius depleted of these microbial organisms by, for example, cell M10EXG. Haemophilus influenzae, Haloarcula marismor separation, and then Subsequent addition of the second tui ATCC 43049, Halobacterium salinarum, Haloferax organism to the fermentation broth can be utilized to pro mediterranei, Heliobacter pylori, Homo sapiens, isolated duce the final product without intermediate purification from metalibrary of anaerobic sewage digester microbial steps. consortia, Klebsiella pneumonia MGH78578, Kluyveromy 0110. In other embodiments, the non-naturally occurring ces lactis, Lachancea kluyveri, Lactococcus lactis, Leu microbial organisms and methods of the invention can be conostoc mesenteroides, marine gamma proteobacterium assembled in a wide variety of subpathways to achieve HTCC2080, Mesorhizobium loti, Metallosphaera sedula, biosynthesis of, for example, 14-BDO and GBL. In these Mus musculus, Mycobacterium bovis BCG, Mycobacterium embodiments, biosynthetic pathways for a desired product tuberculosis, Myxococcus xanthus, Nicotiana tabacum, of the invention can be segregated into different microbial Nocardia farcinica IFM 10152, Nocardia iowensis (sp. organisms, and the different microbial organisms can be NRRL 5646), Oryctolagus cuniculus, Oryza sativa, Penicil co-cultured to produce the final product. In Such a biosyn lium chrysogenium, Porphyromonas gingivalis, Porphy thetic scheme, the product of one microbial organism is the romonas gingivalis W83, Pseudomonas aeruginosa, Substrate for a second microbial organism until the final Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens, products are synthesized. For example, the biosynthesis of Pseudomonas putida, Pseudomonas putida E23. Pseudomo 14-BDO and GBL can be accomplished by constructing a nas putida KT2440, Pseudomonas sp., Pseudomonas microbial organism that contains biosynthetic pathways for Stutzeri, Pyrobaculum aerophilum Str. IM2, Ralstonia eutro conversion of one pathway intermediate to another pathway pha, Ralstonia eutropha H16, Rattus norvegicus, Rhodo intermediate or the product. Alternatively, 14-BDO and bacter spaeroides, Roseiflexus castenholzii, Saccharomyces GBL also can be biosynthetically produced from microbial cerevisiae, Salmonella typhimurium, Schizosaccharomyces organisms through co-culture or co-fermentation using two pombe, Simmondsia chinensis, Streptomyces clavuligenus, organisms in the same vessel, where the first microbial Streptomyces griseus subsp. griseus NBRC 13350, Sulfolo organism produces a 14-BDO or GBL intermediate and the bus acidocaldarius, Sulfolobus solfataricus, Sulfolobus second microbial organism converts the intermediate to tokodai, Sulfolobus tokodai 7, Sus scrofa, Thermotoga 14-BDO and GBL. maritime, Thermus thermophilus, Trichomonas vaginalis 0111. Given the teachings and guidance provided herein, G3. Trypanosoma brucei, Uncultured bacterium, Vibrio those skilled in the art will understand that a wide variety of cholera, Xanthomonas campestris, Zymomonas mobilus, as combinations and permutations exist for the non-naturally well as other exemplary species disclosed herein or available occurring microbial organisms and methods of the invention as Source organisms for corresponding genes. However, with together with other microbial organisms, with the co-culture the complete genome sequence available for now more than of other non-naturally occurring microbial organisms having 550 species (with more than half of these available on public Subpathways and with combinations of other chemical and/ databases such as the NCBI), including 395 microorganism or biochemical procedures well known in the art to produce genomes and a variety of yeast, fungi, plant, and mammalian 14-BDO and GBL. genomes, the identification of genes encoding the requisite 0112 Sources of encoding nucleic acids for a 14-BDO or 14-BDO or GBL biosynthetic activity for one or more genes GBL pathway enzyme or protein can include, for example, in related or distant species, including for example, homo any species where the encoded gene product is capable of logues, orthologs, paralogs and nonorthologous gene dis catalyzing the referenced reaction. Such species include placements of known genes, and the interchange of genetic both prokaryotic and eukaryotic organisms including, but alterations between organisms is routine and well known in not limited to, bacteria, including archaea and eubacteria, the art. Accordingly, the metabolic alterations allowing and eukaryotes, including yeast, plant, insect, animal, and biosynthesis of 14-BDO and GBL described herein with mammal, including human. Exemplary species for Such reference to a particular organism Such as E. coli can be Sources include, for example, sp. Escherichia coli, Acineto readily applied to other microorganisms, including prokary bacter sp. Strain M-1, Acetobacter pasteurians, Achromo otic and eukaryotic organisms alike. Given the teachings and bacter denitrificans, Acidaminococcus fermentans, Acineto guidance provided herein, those skilled in the art will know bacter baumanii, Acinetobacter baylvi, Acinetobacter that a metabolic alteration exemplified in one organism can calcoaceticus, Acinetobacter sp. Strain M-1, Aeropyrum be applied equally to other organisms. pernix K1, Agrobacterium tumefaciens, Arabidopsis thali 0113. In some instances, such as when an alternative ana, Archaeoglobus fulgidus DSM 4304, Aspergillus niger, 14-BDO or GBL biosynthetic pathway exists in an unrelated US 2016/03193 13 A1 Nov. 3, 2016

species, 14-BDO or GBL biosynthesis can be conferred onto sion control sequences can include constitutive and induc the host species by, for example, exogenous expression of a ible promoters, transcription enhancers, transcription termi paralog or paralogs from the unrelated species that catalyzes nators, and the like which are well known in the art. When a similar, yet non-identical metabolic reaction to replace the two or more exogenous encoding nucleic acids are to be referenced reaction. Because certain differences among co-expressed, both nucleic acids can be inserted, for metabolic networks exist between different organisms, those example, into a single expression vector or in separate skilled in the art will understand that the actual gene usage expression vectors. For single vector expression, the encod between different organisms may differ. However, given the ing nucleic acids can be operationally linked to one common teachings and guidance provided herein, those skilled in the expression control sequence or linked to different expression art also will understand that the teachings and methods of the control sequences, such as one inducible promoter and one invention can be applied to all microbial organisms using the constitutive promoter. The transformation of exogenous cognate metabolic alterations to those exemplified herein to nucleic acid sequences involved in a metabolic or synthetic construct a microbial organism in a species of interest that pathway can be confirmed using methods well known in the will synthesize 14-BDO and GBL. art. Such methods include, for example, nucleic acid analy 0114 Methods for constructing and testing the expres sis Such as Northern blots or polymerase chain reaction sion levels of a non-naturally occurring 14-BDO and GBL (PCR) amplification of mRNA, or immunoblotting for co-producing host can be performed, for example, by recom expression of gene products, or other suitable analytical binant and detection methods well known in the art. Such methods to test the expression of an introduced nucleic acid methods can be found described in, for example, Sambrook sequence or its corresponding gene product. It is understood et al., Molecular Cloning: A Laboratory Manual. Third Ed., by those skilled in the art that the exogenous nucleic acid is Cold Spring Harbor Laboratory, New York (2001); and expressed in a Sufficient amount to produce the desired Ausubel et al., Current Protocols in Molecular Biology, product, and it is further understood that expression levels John Wiley and Sons, Baltimore, Md. (1999). can be optimized to obtain Sufficient expression using meth 0115 Exogenous nucleic acid sequences involved in a ods well known in the art and as disclosed herein. pathway for production of 14-BDO and GBL can be intro 0117. In one embodiment, the invention provides a duced stably or transiently into a host cell using techniques method for producing 14-BDO and GBL that includes well known in the art including, but not limited to, conju culturing a non-naturally occurring microbial organism, gation, electroporation, chemical transformation, transduc including a microbial organism having a 14-BDO pathway tion, transfection, and ultrasound transformation. For exog and a GBL pathway, said 14-BDO pathway comprising at enous expression in E. coli or other prokaryotic cells, some least one exogenous nucleic acid encoding a 14-BDO path nucleic acid sequences in the genes or cDNAs of eukaryotic way enzyme expressed in a Sufficient amount to produce nucleic acids can encode targeting signals such as an N-ter 14-BDO, said 14-BDO pathway comprising a succinyl-CoA minal mitochondrial or other targeting signal, which can be reductase (aldehyde forming), an alpha-ketoglutarate decar removed before transformation into prokaryotic host cells, if boxylase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxy desired. For example, removal of a mitochondrial leader butyrate reductase, a 1,4-butanediol dehydrogenase, a Suc sequence led to increased expression in E. coli (Hoffmeister cinate reductase, a Succinyl-CoA transferase, a Succinyl et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous CoA hydrolase, a Succinyl-CoA synthetase, a glutamate expression in yeast or other eukaryotic cells, genes can be dehydrogenase, a glutamate transaminase, a glutamate expressed in the cytosol without the addition of leader decarboxylase, an 4-aminobutyrate dehydrogenase, an sequence, or can be targeted to mitochondrion or other 4-aminobutyrate transaminase, a 4-hydroxybutyrate kinase, organelles, or targeted for secretion, by the addition of a a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl Suitable targeting sequence Such as a mitochondrial targeting CoA reductase (aldehyde forming), a 4-hydroxybutyryl or secretion signal suitable for the host cells. Thus, it is phosphate reductase, a Succinyl-CoA reductase (alcohol understood that appropriate modifications to a nucleic acid forming), a 4-hydroxybutyryl-CoA transferase, a 4-hydroxy sequence to remove or include a targeting sequence can be butyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA syn incorporated into an exogenous nucleic acid sequence to thetase, a 4-hydroxybutyryl-CoA reductase (alcohol form impart desirable properties. Furthermore, genes can be Sub ing), an alpha-ketoglutarate reductase, a 5-hydroxy-2- jected to codon optimization with techniques well known in oXopentanoate dehydrogenase, a 5-hydroxy-2- the art to achieve optimized expression of the proteins. oXopentanoate decarboxylase or a 5-hydroxy-2- 0116. An expression vector or vectors can be constructed oXopentanoate dehydrogenase (decarboxylation) (see FIG. to include one or more 14-BDO or GBL biosynthetic path 1, steps A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, way encoding nucleic acids as exemplified herein operably S, T, U, V, W, X, Y, Z. AA), said GBL pathway comprising linked to expression control sequences functional in the host at least one exogenous nucleic acid encoding an GBL organism. Expression vectors applicable for use in the pathway enzyme expressed in a Sufficient amount to produce microbial host organisms of the invention include, for GBL, said GBL pathway comprising a Succinyl-CoA reduc example, plasmids, phage vectors, viral vectors, episomes tase (aldehyde forming), an alpha-ketoglutarate decarboxy and artificial chromosomes, including vectors and selection lase, a 4-hydroxybutyrate dehydrogenase, a Succinate reduc sequences or markers operable for stable integration into a tase, a Succinyl-CoA transferase, a Succinyl-CoA hydrolase, host chromosome. Additionally, the expression vectors can a Succinyl-CoA synthetase, a glutamate dehydrogenase, a include one or more selectable marker genes and appropriate glutamate transaminase, a glutamate decarboxylase, an expression control sequences. Selectable marker genes also 4-aminobutyrate dehydrogenase, an 4-aminobutyrate can be included that, for example, provide resistance to transaminase, a Succinyl-CoA reductase (alcohol forming) antibiotics or toxins, complement auxotrophic deficiencies, or a 4-hydroxybutyrate lactonase (see FIG. 1, steps A, B, C, or Supply critical nutrients not in the culture media. Expres F, G, H, I, J, K, L, M, N, S, AB). US 2016/03193 13 A1 Nov. 3, 2016

0118. In a further aspect of the above embodiment, the CoA hydrolase or Succinyl-CoA synthetase; a Succinyl-CoA method includes a microbial organism having a 14-BDO reductase (alcohol forming), a 4-hydroxybutyrate kinase, a pathway comprising a Succinyl-CoA transferase, a Succinyl 4-hydroxybutyryl-phosphate reductase; and a 1,4-butanediol CoA hydrolase or a succinyl-CoA synthetase; a Succinyl dehydrogenase (see FIG. 1, steps G/H/I, S. O. R. E). CoA reductase (aldehyde forming); a 4-hydroxybutyrate I0126. In a further aspect of the above embodiment, the dehydrogenase; a 4-hydroxybutyrate kinase; a phospho method includes a microbial organism having a 14-BDO trans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA reduc pathway comprising a Succinyl-CoA transferase, a Succinyl tase (aldehyde forming); and a 1,4-butanediol dehydroge CoA hydrolase or a succinyl-CoA synthetase; a Succinyl nase (see FIG. 1, steps G/H/I, A, C, O, P, Q, E). CoA reductase (alcohol forming); a 4-hydroxybutyryl-CoA 0119. In a further aspect of the above embodiment, the transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hy method includes a microbial organism having a 14-BDO droxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA pathway comprising a Succinyl-CoA transferase, a Succinyl reductase (aldehyde forming); and a 1,4-butanediol dehy CoA hydrolase or a succinyl-CoA synthetase; a Succinyl drogenase (see FIG. 1, steps G/H/I, S, T/U/V, Q, E). CoA reductase (aldehyde forming); a 4-hydroxybutyrate I0127. In a further aspect of the above embodiment, the dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxy method includes a microbial organism having a 14-BDO butyryl-phosphate reductase; and a 1,4-butanediol dehydro pathway comprising a Succinyl-CoA transferase, a Succinyl genase (see FIG. 1, steps G/H/I, A, C, O. R. E). CoA hydrolase or a succinyl-CoA synthetase; a Succinyl 0120 In a further aspect of the above embodiment, the CoA reductase (alcohol forming); a 4-hydroxybutyryl-CoA method includes a microbial organism having a 14-BDO transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hy pathway comprising a Succinyl-CoA transferase, a Succinyl droxybutyryl-CoA synthetase; and a 4-hydroxybutyryl-CoA CoA hydrolase, or a Succinyl-CoA synthetase; a Succinyl CoA reductase (aldehyde forming); a 4-hydroxybutyrate reductase (alcohol forming) (see FIG. 1, steps G/H/I, S. dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a T/U/V, W). 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl I0128. In a further aspect of the above embodiment, the method includes a microbial organism having a 14-BDO CoA synthetase; a 4-hydroxybutyryl-CoA reductase (alde pathway comprising a Succinyl-CoA transferase, a Succinyl hyde forming); and a 1,4-butanediol dehydrogenase (see CoA hydrolase or a succinyl-CoA synthetase; a Succinyl FIG. 1, steps G/H/I, A, C, T/U/V, Q, E). CoA reductase (alcohol forming); a 4-hydroxybutyrate 0121. In a further aspect of the above embodiment, the kinase; a phosphotrans-4-hydroxybutyrylase; and a 4-hy method includes a microbial organism having a 14-BDO pathway comprising a Succinyl-CoA transferase, a Succinyl droxybutyryl-CoA reductase (alcohol forming) (see FIG. 1, CoA hydrolase or Succinyl-CoA synthetase; a Succinyl-CoA steps G/H/I, S, O, P, W). reductase (aldehyde forming); a 4-hydroxybutyrate dehy I0129. In a further aspect of the above embodiment, the drogenase; a 4-hydroxybutyryl-CoA transferase, a 4-hy method includes a microbial organism having a 14-BDO droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA pathway comprising a Succinate reductase; a 4-hydroxybu synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol tyrate dehydrogenase; a 4-hydroxybutyrate reductase; and forming) (see FIG. 1, steps G/H/I, A, C, T/U/V, W). 1,4-butanediol dehydrogenase (see FIG. 1, steps F.C, D, E). 0122. In a further aspect of the above embodiment, the 0.130. In a further aspect of the above embodiment, the method includes a microbial organism having a 1,4-BDO method includes a microbial organism having a 14-BDO pathway comprising a Succinyl-CoA transferase, a Succinyl pathway comprising a Succinate reductase; a 4-hydroxybu CoA hydrolase or a succinyl-CoA synthetase; a Succinyl tyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phos CoA reductase (aldehyde forming); a 4-hydroxybutyrate photrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA dehydrogenase; a 4-hydroxybutyrate kinase; a phospho reductase (aldehyde forming); and a 1,4-butanediol dehy trans-4-hydroxybutyrylase; and a 4-hydroxybutyryl-CoA drogenase (see FIG. 1, steps F. C., O, P, Q, E). reductase (alcohol forming) (see FIG. 1, steps G/H/I, A, C, I0131. In a further aspect of the above embodiment, the O, P, W). method includes a microbial organism having a 14-BDO 0123. In a further aspect of the above embodiment, the pathway comprising a Succinate reductase; a 4-hydroxybu method includes a microbial organism having a 14-BDO tyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phos pathway comprising a Succinyl-CoA transferase, a Succinyl photrans-4-hydroxybutyrylase; and a 4-hydroxybutyryl CoA hydrolase or a succinyl-CoA synthetase; a Succinyl CoA reductase (alcohol forming) (see FIG. 1, steps F. C., O. CoA reductase (alcohol forming); a 4-hydroxybutyrate P. W). reductase; and 1,4-butanediol dehydrogenase (see FIG. 1, 0.132. In a further aspect of the above embodiment, the steps G/H/I, S, D, E). method includes a microbial organism having a 14-BDO 0.124. In a further aspect of the above embodiment, the pathway comprising a Succinate reductase; a 4-hydroxybu method includes a microbial organism having a 14-BDO tyrate dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hy pathway comprising a Succinyl-CoA transferase, a Succinyl droxybutyryl-phosphate reductase; and 1,4-butanediol dehy CoA hydrolase or a succinyl-CoA synthetase; a Succinyl drogenase (see FIG. 1, steps F. C. O. R. E). CoA reductase (alcohol forming); a 4-hydroxybutyrate I0133. In a further aspect of the above embodiment, the kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxy method includes a microbial organism having a 14-BDO butyryl-CoA reductase (aldehyde forming); and a 1,4-bu pathway comprising a Succinate reductase; a 4-hydroxybu tanediol dehydrogenase (see FIG. 1, steps G/H/I, S, O, P, Q, tyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase, E). a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl 0.125. In a further aspect of the above embodiment, the CoA synthetase; a 4-hydroxybutyryl-CoA reductase (alde method includes a microbial organism having a 14-BDO hyde forming); and 1,4-butanediol dehydrogenase (see FIG. pathway comprising a Succinyl-CoA transferase, a Succinyl 1, steps F, C, T/U/V, Q, E). US 2016/03193 13 A1 Nov. 3, 2016

0134. In a further aspect of the above embodiment, the tyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde method includes a microbial organism having a 14-BDO forming); and E) 1,4-butanediol dehydrogenase (see FIG. 1, pathway comprising a Succinate reductase; a 4-hydroxybu steps J/K, L, M/N, C, O, P, Q, E). tyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase, 0143. In a further aspect of the above embodiment, the a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl method includes a microbial organism having a 14-BDO CoA synthetase; and a 4-hydroxybutyryl-CoA reductase pathway comprising a glutamate dehydrogenase or gluta (alcohol forming) (see FIG. 1, steps F. C. T/U/V, W). mate transaminase; a glutamate decarboxylase; a 4-amin 0135) In a further aspect of the above embodiment, the obutyrate dehydrogenase or a 4-aminobutyrate transami method includes a microbial organism having a 14-BDO nase, a 4-hydroxybutyrate dehydrogenase; a pathway comprising an alpha-ketoglutarate decarboxylase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate tyrylase; and a 4-hydroxybutyryl-CoA reductase (alcohol reductase; and 1,4-butanediol dehydrogenase (see FIG. 1, forming), (see FIG. 1, steps J/K, L, M/N, C, O, P, W) steps B, C, D, E). 0144. In a further aspect of the above embodiment, the 0136. In a further aspect of the above embodiment, the method includes a microbial organism having a 14-BDO method includes a microbial organism having a 14-BDO pathway comprising a glutamate dehydrogenase or a gluta pathway comprising an alpha-ketoglutarate decarboxylase; a mate transaminase; a glutamate decarboxylase; a 4-amin 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate obutyrate dehydrogenase or a 4-aminobutyrate transami kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxy nase, a 4-hydroxybutyrate dehydrogenase; a butyryl-CoA reductase (aldehyde forming); and a 1,4-bu 4-hydroxybutyrate kinase; a 4-hydroxybutyryl-phosphate tanediol dehydrogenase (see FIG. 1, steps B, C, O, P, Q, E). reductase; and a 1,4-butanediol dehydrogenase (see FIG. 1, 0.137 In a further aspect of the above embodiment, the steps J/K, L, M/N, C, O, R, E). method includes a microbial organism having a 14-BDO 0145. In a further aspect of the above embodiment, the pathway comprising an alpha-ketoglutarate decarboxylase; a method includes a microbial organism having a 14-BDO 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate pathway comprising a glutamate dehydrogenase or a gluta kinase; a phosphotrans-4-hydroxybutyrylase and 4-hy mate transaminase; a glutamate decarboxylase; a 4-amin droxybutyryl-CoA reductase (alcohol forming) (see FIG. 1, obutyrate dehydrogenase or a 4-aminobutyrate transami steps B, C, O, P, W). nase, a 4-hydroxybutyrate dehydrogenase; a 0.138. In a further aspect of the above embodiment, the 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl method includes a microbial organism having a 14-BDO pathway comprising. an alpha-ketoglutarate decarboxylase; CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; a a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate 4-hydroxybutyryl-CoA reductase (aldehyde forming); and a kinase; a 4-hydroxybutyryl-phosphate reductase; and a 1,4- 1,4-butanediol dehydrogenase (see FIG. 1, steps J/K, L, butanediol dehydrogenase (see FIG. 1, steps B, C, O. R. E). M/N, C, T/U/V, Q, E). 0.139. In a further aspect of the above embodiment, the 0146 In a further aspect of the above embodiment, the method includes a microbial organism having a 14-BDO method includes a microbial organism having a 14-BDO pathway comprising an alpha-ketoglutarate decarboxylase; a pathway comprising a glutamate dehydrogenase or a gluta mate transaminase; a glutamate decarboxylase; a 4-amin 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl obutyrate dehydrogenase or a 4-aminobutyrate transami CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or nase, a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl reductase (aldehyde forming); and a 1,4-butanediol dehy CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and drogenase (see FIG. 1, steps B, C, T/U/V, Q, E). a 4-hydroxybutyryl-CoA reductase (alcohol forming) (see 0140. In a further aspect of the above embodiment, the method includes a microbial organism having a 14-BDO FIG. 1, steps J/K, L, M/N, C, T/U/V, W). pathway comprising an alpha-ketoglutarate decarboxylase; a 0.147. In a further aspect of the above embodiment, the 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl method includes a microbial organism having a 14-BDO CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a pathway comprising an alpha-ketoglutarate reductase; a 4-hydroxybutyryl-CoA synthetase; and 4-hydroxybutyryl 5-hydroxy-2-oxopentanoate dehydrogenase; a 5-hydroxy-2- CoA reductase (alcohol forming) (see FIG. 1, steps B, C, oXopentanoate dehydrogenase (decarboxylation); a 4-hy T/U/V, W). droxybutyryl-CoA reductase (aldehyde forming); and a 1,4- 0141. In a further aspect of the above embodiment, the butanediol dehydrogenase (see FIG. 1, steps X, Y, AA, Q, method includes a microbial organism having a 14-BDO E). pathway comprising a glutamate dehydrogenase or a gluta 0.148. In a further aspect of the above embodiment, the mate transaminase; a glutamate decarboxylase; a 4-amin method includes a microbial organism having a 14-BDO obutyrate dehydrogenase or 4-aminobutyrate transaminase; pathway comprising an alpha-ketoglutarate reductase; a a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate 5-hydroxy-2-oxopentanoate dehydrogenase; a 5-hydroxy-2- reductase; and a 1,4-butanediol dehydrogenase (see FIG. 1, oXopentanoate dehydrogenase (decarboxylation); and a steps J/K, L, M/N, C, D, E). 4-hydroxybutyryl-CoA reductase (alcohol forming) (FIG. 1, 0142. In a further aspect of the above embodiment, the steps X, Y, AA, and W). method includes a microbial organism having a 14-BDO 0149. In a further aspect of the above embodiment, the pathway comprising a glutamate dehydrogenase or a gluta method includes a microbial organism having a 14-BDO mate transaminase; a glutamate decarboxylase; a 4-amin pathway comprising an alpha-ketoglutarate reductase; a obutyrate dehydrogenase or a 4-aminobutyrate transami 5-hydroxy-2-oxopentanoate dehydrogenase; a 5-hydroxy-2- nase, a 4-hydroxybutyrate dehydrogenase; a oXopentanoate decarboxylase; and a 1,4-butanediol dehy 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu drogenase (see FIG. 1, steps X, Y, Z. E). US 2016/03193 13 A1 Nov. 3, 2016 20

0150. In a further aspect of the above embodiment, the cinyl-CoA synthetase; a Succinyl-CoA reductase (aldehyde method includes a microbial organism having a GBL path forming); a 4-hydroxybutyrate dehydrogenase; a 4-hydroxy way comprising a Succinyl-CoA transferase, a Succinyl-CoA butyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA or a 4-hydroxybutyryl-CoA synthetase; and a 4-hydroxybu reductase (aldehyde forming); a 4-hydroxybutyrate dehy tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps drogenase; and a 4-hydroxybutyrate lactonase (see FIG. 1, G/H/I, A, C, T/U/V. W); a succinyl-CoA transferase, a steps G/H/I, A, C, AB). Succinyl-CoA hydrolase or a Succinyl-CoA synthetase; a 0151. In a further aspect of the above embodiment, the Succinyl-CoA reductase (aldehyde forming); a 4-hydroxy method includes a microbial organism having a GBL path butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a way comprising a Succinyl-CoA transferase, a Succinyl-CoA phosphotrans-4-hydroxybutyrylase; and a 4-hydroxybu hydrolase and a Succinyl-CoA synthetase; a Succinyl-CoA tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps reductase (alcohol forming); and a 4-hydroxybutyrate lac G/H/I, A, C, O, P, W); a succinyl-CoA transferase, a tonase, (see FIG. 1, steps G/H/I, S, AB) Succinyl-CoA hydrolase or a Succinyl-CoA synthetase; a 0152. In a further aspect of the above embodiment, the Succinyl-CoA reductase (alcohol forming); a 4-hydroxybu method includes a microbial organism having a GBL path tyrate reductase; and 1,4-butanediol dehydrogenase (see way comprising a Succinate reductase; a 4-hydroxybutyrate FIG. 1, steps G/H/I, S, D, E); a succinyl-CoA transferase, dehydrogenase; and a 4-hydroxybutyrate lactonase (see FIG. a Succinyl-CoA hydrolase or a Succinyl-CoA synthetase; a 1, steps F. C. AB). Succinyl-CoA reductase (alcohol forming); a 4-hydroxybu 0153. In a further aspect of the above embodiment, the tyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hy method includes a microbial organism having a GBL path droxybutyryl-CoA reductase (aldehyde forming); and a 1,4- way comprising an alpha-ketoglutarate decarboxylase, a butanediol dehydrogenase (see FIG. 1, steps G/H/I, S. O. P. 4-hydroxybutyrate dehydrogenase and a 4-hydroxybutyrate Q, E); a Succinyl-CoA transferase, a Succinyl-CoA hydro lactonase (see FIG. 1, steps B, C, AB). lase or Succinyl-CoA synthetase; a Succinyl-CoA reductase 0154) In a further aspect of the above embodiment, the (alcohol forming), a 4-hydroxybutyrate kinase, a 4-hydroxy method includes a microbial organism having a GBL path butyryl-phosphate reductase; and a 1,4-butanediol dehydro way comprising a glutamate dehydrogenase or a glutamate genase (see FIG. 1, steps G/H/I, S. O. R. E); a succinyl transaminase; a glutamate decarboxylase; a 4-aminobutyrate CoA transferase, a Succinyl-CoA hydrolase or a Succinyl dehydrogenase or a 4-aminobutyrate transaminase; a 4-hy CoA synthetase; a Succinyl-CoA reductase (alcohol droxybutyrate dehydrogenase; and a 4-hydroxybutyrate lac forming); a 4-hydroxybutyryl-CoA transferase, a 4-hy tonase (see FIG. 1, steps J/K, L, M/N, C, AB). droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA 0155. In one embodiment, the invention provides a synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde method for producing 14-BDO and GBL that includes forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, culturing a non-naturally occurring microbial organism, steps G/H/I, S, T/U/V. Q, E); a succinyl-CoA transferase, a including a microbial organism having a 1,4-butanediol Succinyl-CoA hydrolase or a Succinyl-CoA synthetase; a (14-BDO) pathway and a gamma-butyrolactone (GBL) Succinyl-CoA reductase (alcohol forming); a 4-hydroxybu pathway, said 14-BDO pathway comprising a first set of tyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase exogenous nucleic acids encoding 14-BDO pathway or a 4-hydroxybutyryl-CoA synthetase; and a 4-hydroxybu enzymes expressed in a Sufficient amount to produce tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps 14-BDO, wherein said first set is selected from the group G/H/I, S, T/U/V. W); a succinyl-CoA transferase, a succi consisting of a Succinyl-CoA transferase, a Succinyl-CoA nyl-CoA hydrolase or a succinyl-CoA synthetase; a Succi hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA nyl-CoA reductase (alcohol forming); a 4-hydroxybutyrate reductase (aldehyde forming); a 4-hydroxybutyrate dehy kinase; a phosphotrans-4-hydroxybutyrylase; and a 4-hy drogenase; a 4-hydroxybutyrate reductase; and a 1,4-butane droxybutyryl-CoA reductase (alcohol forming) (see FIG. 1, diol dehydrogenase) (see FIG. 1, steps G/H/I, A, C, D, E): steps G/H/I, S, O, P, W); a succinate reductase; a 4-hy a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a droxybutyrate dehydrogenase; a 4-hydroxybutyrate reduc Succinyl-CoA synthetase; a Succinyl-CoA reductase (alde tase; and 1,4-butanediol dehydrogenase (see FIG. 1, Steps F. hyde forming); a 4-hydroxybutyrate dehydrogenase; a 4-hy C, D, E); a Succinate reductase; a 4-hydroxybutyrate dehy droxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; drogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4- a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and hydroxybutyrylase; a 4-hydroxybutyryl-CoA reductase (al a 1,4-butanediol dehydrogenase (see FIG. 1, steps G/H/I, A. dehyde forming); and a 1,4-butanediol dehydrogenase (see C, O, P, Q, E); a succinyl-CoA transferase, a succinyl-CoA FIG. 1, steps F. C., O, P, Q, E); a succinate reductase; a hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate reductase (aldehyde forming); a 4-hydroxybutyrate dehy kinase; a phosphotrans-4-hydroxybutyrylase; and a 4-hy drogenase; a 4-hydroxybutyrate kinase; a 4-hydroxybutyryl droxybutyryl-CoA reductase (alcohol forming) (see FIG. 1, phosphate reductase; and a 1,4-butanediol dehydrogenase steps F. C., O, P, W); a succinate reductase; a 4-hydroxy (see FIG. 1, steps G/H/I, A, C, O. R. E); a succinyl-CoA butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a transferase, a Succinyl-CoA hydrolase, or a Succinyl-CoA 4-hydroxybutyryl-phosphate reductase; and 1,4-butanediol synthetase; a Succinyl-CoA reductase (aldehyde forming); a dehydrogenase (see FIG. 1, steps F.C., O. R. E); a succinate 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hy CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a droxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; a 4-hy reductase (aldehyde forming); and a 1,4-butanediol dehy droxybutyryl-CoA reductase (aldehyde forming); and 1,4- drogenase (see FIG. 1, steps G/H/I, A, C, T/U/V. Q, E); a butanediol dehydrogenase (see FIG. 1, steps F.C, T/U/V. Q. Succinyl-CoA transferase, a Succinyl-CoA hydrolase or Suc E); a Succinate reductase; a 4-hydroxybutyrate dehydroge US 2016/03193 13 A1 Nov. 3, 2016 nase; a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybu 4-aminobutyrate transaminase; a 4-hydroxybutyrate dehy tyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase: drogenase; a 4-hydroxybutyryl-CoA transferase, a 4-hy and a 4-hydroxybutyryl-CoA reductase (alcohol forming) droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA (see FIG. 1, steps F. C. T/U/V. W); an alpha-ketoglutarate synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol decarboxylase; a 4-hydroxybutyrate dehydrogenase; a 4-hy forming) (see FIG. 1, steps J/K, L, M/N, C, T/U/V. W); an droxybutyrate reductase; and 1,4-butanediol dehydroge alpha-ketoglutarate reductase; a 5-hydroxy-2-oxopentanoate nase (see FIG. 1, Steps B, C, D, E); an alpha-ketoglutarate dehydrogenase; a 5-hydroxy-2-oxopentanoate dehydroge decarboxylase; a 4-hydroxybutyrate dehydrogenase; a 4-hy nase (decarboxylation); a 4-hydroxybutyryl-CoA reductase droxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; (aldehyde forming); and a 1,4-butanediol dehydrogenase a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and (see FIG. 1, steps X, Y, AA, Q, E); an alpha-ketoglutarate a 1,4-butanediol dehydrogenase (see FIG. 1, steps B, C, O. reductase; a 5-hydroxy-2-oxopentanoate dehydrogenase; a P. Q., E); an alpha-ketoglutarate decarboxylase; a 4-hy 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxy droxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; lation); and a 4-hydroxybutyryl-CoA reductase (alcohol a phosphotrans-4-hydroxybutyrylase and 4-hydroxybutyryl forming) (see FIG. 1, steps X, Y, AA, and W); and an CoA reductase (alcohol forming) (see FIG. 1, steps B, C, O. alpha-ketoglutarate reductase; a 5-hydroxy-2-oxopentanoate P. W); an alpha-ketoglutarate decarboxylase; a 4-hydroxy dehydrogenase; a 5-hydroxy-2-oxopentanoate decarboxy butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a lase; and a 1,4-butanediol dehydrogenase (see FIG. 1, steps 4-hydroxybutyryl-phosphate reductase; and a 1,4-butanediol X, Y, Z, E), said GBL pathway comprising a second set of dehydrogenase (see FIG. 1, steps B, C, O. R. E); an exogenous nucleic acid encoding an GBL pathway enzyme alpha-ketoglutarate decarboxylase; a 4-hydroxybutyrate expressed in a Sufficient amount to produce GBL, said dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a second set is selected from the group consisting of a 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde Succinyl-CoA synthetase; a Succinyl-CoA reductase (alde forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, hyde forming); a 4-hydroxybutyrate dehydrogenase; and a steps B, C, T/U/V, Q, E); an alpha-ketoglutarate decarboxy 4-hydroxybutyrate lactonase (see FIG. 1, steps G/H/I, A, C, lase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu AB); a Succinyl-CoA transferase, a Succinyl-CoA hydrolase tyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase and a Succinyl-CoA synthetase; a Succinyl-CoA reductase or a 4-hydroxybutyryl-CoA synthetase; and 4-hydroxybu (alcohol forming); and a 4-hydroxybutyrate lactonase (see tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps B, FIG. 1, steps G/H/I, S, AB); a succinate reductase; a C, T/U/V. W); a glutamate dehydrogenase or a glutamate 4-hydroxybutyrate dehydrogenase; and a 4-hydroxybutyrate transaminase; a glutamate decarboxylase; a 4-aminobutyrate lactonase (see FIG. 1, steps F. C. AB); an alpha-ketoglu dehydrogenase or 4-aminobutyrate transaminase; a 4-hy tarate decarboxylase, a 4-hydroxybutyrate dehydrogenase droxybutyrate dehydrogenase; a 4-hydroxybutyrate reduc and a 4-hydroxybutyrate lactonase (see FIG. 1, steps B, C, tase; and a 1,4-butanediol dehydrogenase (see FIG. 1, steps AB); and a glutamate dehydrogenase or a glutamate J/K, L, M/N, C, D, E); a glutamate dehydrogenase or a transaminase; a glutamate decarboxylase; a 4-aminobutyrate glutamate transaminase; a glutamate decarboxylase; a dehydrogenase or a 4-aminobutyrate transaminase; a 4-hy 4-aminobutyrate dehydrogenase or a 4-aminobutyrate droxybutyrate dehydrogenase; and a 4-hydroxybutyrate lac transaminase; a 4-hydroxybutyrate dehydrogenase; a 4-hy tonase (see FIG. 1, steps J/K, L, M/N, C, AB). droxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; 0156. In a further aspect of each of the above embodi a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and ments, the exogenous nucleic acid is a heterologous nucleic E) 1,4-butanediol dehydrogenase (see FIG. 1, steps J/K, L, acid. M/N, C, O, P, Q, E); a glutamate dehydrogenase or gluta 0157. In a further aspect of each of the above embodi mate transaminase; a glutamate decarboxylase; a 4-amin ments, the conditions include Substantially anaerobic culture obutyrate dehydrogenase or a 4-aminobutyrate transami conditions. nase, a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu 0158. In a further aspect of each of the above embodi tyrylase; and a 4-hydroxybutyryl-CoA reductase (alcohol ments, the 14-BDO and GBL products are produced at a forming) (see FIG. 1, steps J/K, L, M/N, C, O, P, W); a predetermined ratio. In some aspects of the invention, the glutamate dehydrogenase or a glutamate transaminase; a predetermined ratio of 14-BDO to GBL is 1:1, or alterna glutamate decarboxylase; a 4-aminobutyrate dehydrogenase tively 1:2, or alternatively 1:3, or alternatively 1:4, or or a 4-aminobutyrate transaminase; a 4-hydroxybutyrate alternatively 1:5, or alternatively 1:6, or alternatively 1:7, or dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxy alternatively 1:8, or alternatively 1:9, or alternatively 1:10, butyryl-phosphate reductase; and a 1,4-butanediol dehydro or alternatively 1:15, or alternatively about 1:20, or alter genase; (see FIG. 1, steps J/K, L, M/N, C, O. R. E) a natively 1:25, or alternatively 1:30, or alternatively 1:35, or glutamate dehydrogenase or a glutamate transaminase; a alternatively 1:40, or alternatively 1:45, or alternatively glutamate decarboxylase; a 4-aminobutyrate dehydrogenase about 1:50, or alternatively 1:60, or alternatively 1:70, or or a 4-aminobutyrate transaminase; a 4-hydroxybutyrate alternatively 1:80, or alternatively 1:90, or alternatively dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a 1:100. In some aspects of the invention, the predetermined 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl ratio of 14-BDO to GBL is 2:1, or alternatively 3:1, or CoA synthetase; a 4-hydroxybutyryl-CoA reductase (alde alternatively 4:1 or alternatively 5:1, or alternatively 6:1, or hyde forming); and a 1,4-butanediol dehydrogenase (see alternatively 7:1, or alternatively 8:1, or alternatively 9:1, or FIG. 1, steps J/K, L, M/N, C, T/U/V. Q, E); a glutamate alternatively 10:1, or alternatively 15:1, or alternatively dehydrogenase or a glutamate transaminase; a glutamate about 20:1, or alternatively 25:1, or alternatively 30:1, or decarboxylase; a 4-aminobutyrate dehydrogenase or a alternatively 35:1, or alternatively 40:1, or alternatively US 2016/03193 13 A1 Nov. 3, 2016 22

45:1, or alternatively about 50:1, or alternatively 60:1, or trans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA reduc alternatively 70:1, or alternatively 80:1, or alternatively tase (aldehyde forming); and a 1,4-butanediol dehydroge 90:1, or alternatively 100:1. nase (see FIG. 1, steps G/H/I, A, C, O, P, Q, E). 0159. In one embodiment, the invention provides a 0162. In a further aspect of the above embodiment, the method for producing 14-BDO and GBL that includes method includes a microbial organism having a 14-BDO culturing a non-naturally occurring microbial organism pathway comprising a Succinyl-CoA transferase, a Succinyl under conditions and for a sufficient period of time to CoA hydrolase or a succinyl-CoA synthetase; a Succinyl produce 14-BDO and 4-hydroxybutyryl-phosphate followed CoA reductase (aldehyde forming); a 4-hydroxybutyrate by spontaneous lactonization of 4-hydroxybutyryl-phos dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxy phate to form GBL, wherein said non-naturally occurring butyryl-phosphate reductase; and a 1,4-butanediol dehydro microbial organism comprise a microbial organism having a genase (see FIG. 1, steps G/H/I, A, C, O. R. E). 14-BDO pathway and a 4-hydroxybutyryl-phosphate path 0163. In a further aspect of the above embodiment, the way, said 14-BDO pathway comprising at least one exog method includes a microbial organism having a 14-BDO enous nucleic acid encoding a 14-BDO pathway enzyme pathway comprising a Succinyl-CoA transferase, a Succinyl expressed in a sufficient amount to produce 14-BDO, said CoA hydrolase, or a Succinyl-CoA synthetase; a Succinyl 14-BDO pathway comprising a succinyl-CoA reductase CoA reductase (aldehyde forming); a 4-hydroxybutyrate (aldehyde forming), an alpha-ketoglutarate decarboxylase, a dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl reductase, a 1,4-butanediol dehydrogenase, a Succinate CoA synthetase; a 4-hydroxybutyryl-CoA reductase (alde reductase, a Succinyl-CoA transferase, a Succinyl-CoA hyde forming); and a 1,4-butanediol dehydrogenase (see hydrolase, a Succinyl-CoA synthetase, a glutamate dehydro FIG. 1, steps G/H/I, A, C, T/U/V, Q, E). genase, a glutamate transaminase, a glutamate decarboxy 0164. In a further aspect of the above embodiment, the lase, an 4-aminobutyrate dehydrogenase, an 4-aminobu method includes a microbial organism having a 14-BDO tyrate transaminase, a 4-hydroxybutyrate kinase, a pathway comprising a Succinyl-CoA transferase, a Succinyl phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl CoA hydrolase or Succinyl-CoA synthetase; a Succinyl-CoA CoA reductase (aldehyde forming), a 4-hydroxybutyryl reductase (aldehyde forming); a 4-hydroxybutyrate dehy phosphate reductase, a Succinyl-CoA reductase (alcohol drogenase; a 4-hydroxybutyryl-CoA transferase, a 4-hy forming), a 4-hydroxybutyryl-CoA transferase, a 4-hydroxy droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA butyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA syn synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol thetase, a 4-hydroxybutyryl-CoA reductase (alcohol form forming) (see FIG. 1, steps G/H/I, A, C, T/U/V, W). ing), an alpha-ketoglutarate reductase, a 5-hydroxy-2- 0.165. In a further aspect of the above embodiment, the oXopentanoate dehydrogenase, a 5-hydroxy-2- method includes a microbial organism having a 1,4-BDO oXopentanoate decarboxylase or a 5-hydroxy-2- pathway comprising a Succinyl-CoA transferase, a Succinyl oXopentanoate dehydrogenase (decarboxylation) (see FIG. CoA hydrolase or a succinyl-CoA synthetase; a Succinyl 1, steps A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, CoA reductase (aldehyde forming); a 4-hydroxybutyrate S, T, U, V, W, X, Y, Z, AA), said 4-hydroxybutyryl dehydrogenase; a 4-hydroxybutyrate kinase; a phospho phosphate pathway comprising at least one exogenous trans-4-hydroxybutyrylase; and a 4-hydroxybutyryl-CoA nucleic acid encoding an 4-hydroxybutyryl-phosphate path reductase (alcohol forming) (see FIG. 1, steps G/H/I, A, C, way enzyme expressed in a Sufficient amount to produce O, P, W). 4-hydroxybutyryl-phosphate, said 4-hydroxybutyryl-phos 0166 In a further aspect of the above embodiment, the phate pathway comprising a succinyl-CoA reductase (alde method includes a microbial organism having a 14-BDO hyde forming), an alpha-ketoglutarate decarboxylase, a pathway comprising a Succinyl-CoA transferase, a Succinyl 4-hydroxybutyrate dehydrogenase, a Succinate reductase, a CoA hydrolase or a succinyl-CoA synthetase; a Succinyl Succinyl-CoA transferase, a Succinyl-CoA hydrolase, a suc CoA reductase (alcohol forming); a 4-hydroxybutyrate cinyl-CoA synthetase, a glutamate dehydrogenase, a gluta reductase; and 1,4-butanediol dehydrogenase (see FIG. 1, mate transaminase, a glutamate decarboxylase, a 4-amin steps G/H/I, S, D, E). obutyrate dehydrogenase, a 4-aminobutyrate transaminase, a 0167. In a further aspect of the above embodiment, the 4-hydroxybutyrate kinase or a Succinyl-CoA reductase (al method includes a microbial organism having a 14-BDO cohol forming) (see FIG. 1, steps A, B, C, F, G, H, I, J, K, pathway comprising a Succinyl-CoA transferase, a Succinyl L., M, N, O, S). CoA hydrolase or a succinyl-CoA synthetase; a Succinyl 0160. In a further aspect of the above embodiment, the CoA reductase (alcohol forming); a 4-hydroxybutyrate method includes a microbial organism having a 14-BDO kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxy pathway comprising a Succinyl-CoA transferase, a Succinyl butyryl-CoA reductase (aldehyde forming); and a 1,4-bu CoA hydrolase or a succinyl-CoA synthetase; a Succinyl tanediol dehydrogenase (see FIG. 1, steps G/H/I, S, O, P, Q, CoA reductase (aldehyde forming); a 4-hydroxybutyrate E). dehydrogenase; a 4-hydroxybutyrate reductase; and a 1,4- 0.168. In a further aspect of the above embodiment, the butanediol dehydrogenase (see FIG. 1, steps G/H/I, A, C, D, method includes a microbial organism having a 14-BDO E). pathway comprising a Succinyl-CoA transferase, a Succinyl 0161 In a further aspect of the above embodiment, the CoA hydrolase or Succinyl-CoA synthetase; a Succinyl-CoA method includes a microbial organism having a 14-BDO reductase (alcohol forming), a 4-hydroxybutyrate kinase, a pathway comprising a Succinyl-CoA transferase, a Succinyl 4-hydroxybutyryl-phosphate reductase; and a 1,4-butanediol CoA hydrolase or a succinyl-CoA synthetase; a Succinyl dehydrogenase (see FIG. 1, steps G/H/I, S. O. R. E). CoA reductase (aldehyde forming); a 4-hydroxybutyrate 0169. In a further aspect of the above embodiment, the dehydrogenase; a 4-hydroxybutyrate kinase; a phospho method includes a microbial organism having a 14-BDO US 2016/03193 13 A1 Nov. 3, 2016

pathway comprising a Succinyl-CoA transferase, a Succinyl 0178. In a further aspect of the above embodiment, the CoA hydrolase or a succinyl-CoA synthetase; a Succinyl method includes a microbial organism having a 14-BDO CoA reductase (alcohol forming); a 4-hydroxybutyryl-CoA pathway comprising an alpha-ketoglutarate decarboxylase; a transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hy 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate droxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA reductase; and 1,4-butanediol dehydrogenase (see FIG. 1, reductase (aldehyde forming); and a 1,4-butanediol dehy steps B, C, D, E). drogenase (see FIG. 1, steps G/H/I, S, T/U/V, Q, E). 0179. In a further aspect of the above embodiment, the 0170 In a further aspect of the above embodiment, the method includes a microbial organism having a 14-BDO method includes a microbial organism having a 14-BDO pathway comprising an alpha-ketoglutarate decarboxylase; a pathway comprising a Succinyl-CoA transferase, a Succinyl 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate CoA hydrolase or a succinyl-CoA synthetase; a Succinyl kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxy CoA reductase (alcohol forming); a 4-hydroxybutyryl-CoA butyryl-CoA reductase (aldehyde forming); and a 1,4-bu transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hy tanediol dehydrogenase (see FIG. 1, steps B, C, O, P, Q, E). droxybutyryl-CoA synthetase; and a 4-hydroxybutyryl-CoA 0180. In a further aspect of the above embodiment, the reductase (alcohol forming) (see FIG. 1, steps G/H/I, S. method includes a microbial organism having a 14-BDO T/U/V, W). pathway comprising an alpha-ketoglutarate decarboxylase; a 0171 In a further aspect of the above embodiment, the 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate method includes a microbial organism having a 14-BDO kinase; a phosphotrans-4-hydroxybutyrylase and 4-hy pathway comprising a Succinyl-CoA transferase, a Succinyl droxybutyryl-CoA reductase (alcohol forming) (see FIG. 1, CoA hydrolase or a succinyl-CoA synthetase; a Succinyl steps B, C, O, P, W). CoA reductase (alcohol forming); a 4-hydroxybutyrate 0181. In a further aspect of the above embodiment, the kinase; a phosphotrans-4-hydroxybutyrylase; and a 4-hy method includes a microbial organism having a 14-BDO droxybutyryl-CoA reductase (alcohol forming) (see FIG. 1, pathway comprising an alpha-ketoglutarate decarboxylase; a steps G/H/I, S, O, P, W). 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate 0172. In a further aspect of the above embodiment, the kinase; a 4-hydroxybutyryl-phosphate reductase; and a 1,4- method includes a microbial organism having a 14-BDO butanediol dehydrogenase (see FIG. 1, steps B, C, O. R. E). pathway comprising a Succinate reductase; a 4-hydroxybu 0182. In a further aspect of the above embodiment, the tyrate dehydrogenase; a 4-hydroxybutyrate reductase; and method includes a microbial organism having a 14-BDO 1,4-butanediol dehydrogenase (see FIG. 1, steps F.C, D, E). pathway comprising an alpha-ketoglutarate decarboxylase; a 0173. In a further aspect of the above embodiment, the 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl method includes a microbial organism having a 14-BDO CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or pathway comprising a Succinate reductase; a 4-hydroxybu 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA tyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phos reductase (aldehyde forming); and a 1,4-butanediol dehy photrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA drogenase (see FIG. 1, steps B, C, T/U/V. Q, E). reductase (aldehyde forming); and a 1,4-butanediol dehy 0183 In a further aspect of the above embodiment, the drogenase (see FIG. 1, steps F. C., O, P, Q, E). method includes a microbial organism having a 14-BDO 0.174. In a further aspect of the above embodiment, the pathway comprising an alpha-ketoglutarate decarboxylase; a method includes a microbial organism having a 14-BDO 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl pathway comprising a Succinate reductase; a 4-hydroxybu CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a tyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phos 4-hydroxybutyryl-CoA synthetase; and 4-hydroxybutyryl photrans-4-hydroxybutyrylase; and a 4-hydroxybutyryl CoA reductase (alcohol forming) (see FIG. 1, steps B, C, CoA reductase (alcohol forming) (see FIG. 1, steps F. C., O. T/U/V, W). P. W). 0184. In a further aspect of the above embodiment, the 0.175. In a further aspect of the above embodiment, the method includes a microbial organism having a 14-BDO method includes a microbial organism having a 14-BDO pathway comprising a glutamate dehydrogenase or a gluta pathway comprising a Succinate reductase; a 4-hydroxybu mate transaminase; a glutamate decarboxylase; a 4-amin tyrate dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hy obutyrate dehydrogenase or 4-aminobutyrate transaminase; droxybutyryl-phosphate reductase; and 1,4-butanediol dehy a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate drogenase (see FIG. 1, steps F. C. O. R. E). reductase; and a 1,4-butanediol dehydrogenase (see FIG. 1, 0176). In a further aspect of the above embodiment, the steps J/K, L, M/N, C, D, E). method includes a microbial organism having a 14-BDO 0185. In a further aspect of the above embodiment, the pathway comprising a Succinate reductase; a 4-hydroxybu method includes a microbial organism having a 14-BDO tyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase, pathway comprising a glutamate dehydrogenase or a gluta a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl mate transaminase; a glutamate decarboxylase; a 4-amin CoA synthetase; a 4-hydroxybutyryl-CoA reductase (alde obutyrate dehydrogenase or a 4-aminobutyrate transami hyde forming); and 1,4-butanediol dehydrogenase (see FIG. nase, a 4-hydroxybutyrate dehydrogenase; a 1, steps F. C. T/U/V, Q, E). 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu 0177. In a further aspect of the above embodiment, the tyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde method includes a microbial organism having a 14-BDO forming); and E) 1,4-butanediol dehydrogenase (see FIG. 1, pathway comprising a Succinate reductase; a 4-hydroxybu steps J/K, L, M/N, C, O, P, Q, E). tyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase, 0186. In a further aspect of the above embodiment, the a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl method includes a microbial organism having a 14-BDO CoA synthetase; and a 4-hydroxybutyryl-CoA reductase pathway comprising a glutamate dehydrogenase or gluta (alcohol forming) (see FIG. 1, steps F. C. T/U/V, W). mate transaminase; a glutamate decarboxylase; a 4-amin US 2016/03193 13 A1 Nov. 3, 2016 24 obutyrate dehydrogenase or a 4-aminobutyrate transami 0194 In a further aspect of the above embodiment, the nase, a 4-hydroxybutyrate dehydrogenase; a method includes a microbial organism having a 4-hydroxy 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu butyryl-phosphate pathway comprising a Succinyl-CoA tyrylase; and a 4-hydroxybutyryl-CoA reductase (alcohol transferase, a Succinyl-CoA hydrolase or a Succinyl-CoA forming) (see FIG. 1, steps J/K, L, M/N, C, O, P, W). synthetase; a Succinyl-CoA reductase (alcohol forming); and 0187. In a further aspect of the above embodiment, the a 4-hydroxybutyrate kinase (see FIG. 1, steps G/H/I, S. O). method includes a microbial organism having a 14-BDO 0.195. In a further aspect of the above embodiment, the pathway comprising a glutamate dehydrogenase or a gluta method includes a microbial organism having a 4-hydroxy mate transaminase; a glutamate decarboxylase; a 4-amin butyryl-phosphate pathway comprising a succinate reduc obutyrate dehydrogenase or a 4-aminobutyrate transami tase; a 4-hydroxybutyrate dehydrogenase; and a 4-hydroxy nase, a 4-hydroxybutyrate dehydrogenase; a butyrate kinase (see FIG. 1, steps F. C. O). 4-hydroxybutyrate kinase; a 4-hydroxybutyryl-phosphate 0196. In a further aspect of the above embodiment, the reductase; and a 1,4-butanediol dehydrogenase (see FIG. 1, method includes a microbial organism having a 4-hydroxy steps J/K, L, M/N, C, O, R, E). butyryl-phosphate pathway comprising an alpha-ketoglut 0188 In a further aspect of the above embodiment, the arate decarboxylase; a 4-hydroxybutyrate dehydrogenase; method includes a microbial organism having a 14-BDO and a 4-hydroxybutyrate kinase (FIG. 1, steps B, C, O). pathway comprising a glutamate dehydrogenase or a gluta 0.197 In a further aspect of the above embodiment, the mate transaminase; a glutamate decarboxylase; a 4-amin method includes a microbial organism having a 4-hydroxy obutyrate dehydrogenase or a 4-aminobutyrate transami butyryl-phosphate pathway comprising a glutamate dehy nase, a 4-hydroxybutyrate dehydrogenase; a drogenase or a glutamate transaminase; a glutamate decar 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl boxylase; an 4-aminobutyrate dehydrogenase or an CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; a 4-aminobutyrate transaminase; a 4-hydroxybutyrate dehy 4-hydroxybutyryl-CoA reductase (aldehyde forming); and a drogenase; and a 4-hydroxybutyrate kinase (see FIG. 1, 1,4-butanediol dehydrogenase (see FIG. 1, steps J/K, L, steps J/K, L, MN, C, O). M/N, C, T/U/V, Q, E). 0.198. In one embodiment, the invention provides a 0189 In a further aspect of the above embodiment, the method for producing 14-BDO and GBL that includes method includes a microbial organism having a 14-BDO culturing a non-naturally occurring microbial organism pathway comprising a glutamate dehydrogenase or a gluta under conditions and for a sufficient period of time to mate transaminase; a glutamate decarboxylase; a 4-amin produce 14-BDO and 4-hydroxybutyryl-phosphate followed obutyrate dehydrogenase or a 4-aminobutyrate transami by spontaneous lactonization of 4-hydroxybutyryl-phos nase, a 4-hydroxybutyrate dehydrogenase; a phate to form GBL, wherein said non-naturally occurring 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl microbial organism comprise a microbial organism having a CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and 14-BDO pathway and a 4-hydroxybutyryl-phosphate path a 4-hydroxybutyryl-CoA reductase (alcohol forming) (see way, said 14-BDO pathway comprising a first set of exog FIG. 1, steps J/K, L, M/N, C, T/U/V, W). enous nucleic acids encoding 14-BDO pathway enzymes 0190. In a further aspect of the above embodiment, the expressed in a sufficient amount to produce 14-BDO, said method includes a microbial organism having a 14-BDO first set is selected from the group consisting of a Succinyl pathway comprising an alpha-ketoglutarate reductase; a CoA transferase, a Succinyl-CoA hydrolase or a Succinyl 5-hydroxy-2-oxopentanoate dehydrogenase; a 5-hydroxy-2- CoA synthetase; a Succinyl-CoA reductase (aldehyde form oXopentanoate dehydrogenase (decarboxylation); a 4-hy ing); a 4-hydroxybutyrate dehydrogenase; a droxybutyryl-CoA reductase (aldehyde forming); and a 1,4- 4-hydroxybutyrate reductase; and a 1,4-butanediol dehydro butanediol dehydrogenase (see FIG. 1, steps X, Y, AA, Q, genase) (see FIG. 1, steps G/H/I, A, C, D, E); a succinyl CoA transferase, a Succinyl-CoA hydrolase or a Succinyl E). CoA synthetase; a Succinyl-CoA reductase (aldehyde 0191 In a further aspect of the above embodiment, the forming); a 4-hydroxybutyrate dehydrogenase; a 4-hydroxy method includes a microbial organism having a 14-BDO pathway comprising an alpha-ketoglutarate reductase; a butyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 5-hydroxy-2-oxopentanoate dehydrogenase; a 5-hydroxy-2- 4-hydroxybutyryl-CoA reductase (aldehyde forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, steps G/H/I, A, oXopentanoate dehydrogenase (decarboxylation); and a C, O, P, Q, E); a succinyl-CoA transferase, a succinyl-CoA 4-hydroxybutyryl-CoA reductase (alcohol forming) (see hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA FIG. 1, steps X, Y, AA, and W). reductase (aldehyde forming); a 4-hydroxybutyrate dehy 0.192 In a further aspect of the above embodiment, the drogenase; a 4-hydroxybutyrate kinase; a 4-hydroxybutyryl method includes a microbial organism having a 14-BDO phosphate reductase; and a 1,4-butanediol dehydrogenase pathway comprising an alpha-ketoglutarate reductase; a (see FIG. 1, steps G/H/I, A, C, O. R. E); a succinyl-CoA 5-hydroxy-2-oxopentanoate dehydrogenase; a 5-hydroxy-2- transferase, a Succinyl-CoA hydrolase, or a Succinyl-CoA oXopentanoate decarboxylase; and a 1,4-butanediol dehy synthetase; a Succinyl-CoA reductase (aldehyde forming); a drogenase (see FIG. 1, steps X, Y, Z. E). 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl 0193 In a further aspect of the above embodiment, the CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a method includes a microbial organism having a 4-hydroxy 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA butyryl-phosphate pathway comprising a Succinyl-CoA reductase (aldehyde forming); and a 1,4-butanediol dehy transferase, a Succinyl-CoA hydrolase or a Succinyl-CoA drogenase (see FIG. 1, steps G/H/I, A, C, T/U/V. Q, E); a synthetase; a Succinyl-CoA reductase (aldehyde forming); a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or Suc 4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate cinyl-CoA synthetase; a Succinyl-CoA reductase (aldehyde kinase (see FIG. 1, steps G/H/I, A, C, O). forming); a 4-hydroxybutyrate dehydrogenase; a 4-hydroxy US 2016/03193 13 A1 Nov. 3, 2016 butyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase and a 4-hydroxybutyryl-CoA reductase (alcohol forming) or a 4-hydroxybutyryl-CoA synthetase; and a 4-hydroxybu (see FIG. 1, steps F. C. T/U/V. W); an alpha-ketoglutarate tyryl-CoA reductase (alcohol forming) (FIG. 1, steps G/H/I, decarboxylase; a 4-hydroxybutyrate dehydrogenase; a 4-hy A, C, T/U/V. W); a succinyl-CoA transferase, a succinyl droxybutyrate reductase; and 1,4-butanediol dehydroge CoA hydrolase or a succinyl-CoA synthetase; a Succinyl nase (see FIG. 1, Steps B, C, D, E); an alpha-ketoglutarate CoA reductase (aldehyde forming); a 4-hydroxybutyrate decarboxylase; a 4-hydroxybutyrate dehydrogenase; a 4-hy dehydrogenase; a 4-hydroxybutyrate kinase; a phospho droxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; trans-4-hydroxybutyrylase; and a 4-hydroxybutyryl-CoA a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and reductase (alcohol forming) (see FIG. 1, steps G/H/I, A, C, a 1,4-butanediol dehydrogenase (see FIG. 1, steps B, C, O. O. P. W); a succinyl-CoA transferase, a succinyl-CoA P. Q., E); an alpha-ketoglutarate decarboxylase; a 4-hy hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA droxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; reductase (alcohol forming); a 4-hydroxybutyrate reductase; a phosphotrans-4-hydroxybutyrylase and 4-hydroxybutyryl and 1,4-butanediol dehydrogenase (see FIG. 1, steps G/H/I, CoA reductase (alcohol forming) (see FIG. 1, steps B, C, O. S, D, E); a Succinyl-CoA transferase, a Succinyl-CoA P. W); an alpha-ketoglutarate decarboxylase; a 4-hydroxy hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a reductase (alcohol forming); a 4-hydroxybutyrate kinase; a 4-hydroxybutyryl-phosphate reductase; and a 1,4-butanediol phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl dehydrogenase (see FIG. 1, steps B, C, O. R. E); an CoA reductase (aldehyde forming); and a 1,4-butanediol alpha-ketoglutarate decarboxylase; a 4-hydroxybutyrate dehydrogenase (see FIG. 1, steps G/H/I, S, O, P, Q, E; a dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a Succinyl-CoA transferase, a Succinyl-CoA hydrolase or Suc 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA cinyl-CoA synthetase; a Succinyl-CoA reductase (alcohol synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde forming), a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, phosphate reductase; and a 1,4-butanediol dehydrogenase steps B, C, T/U/V. Q, E); an alpha-ketoglutarate decarboxy (see FIG. 1, steps G/H/I, S. O. R. E); a succinyl-CoA lase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu transferase, a Succinyl-CoA hydrolase or a Succinyl-CoA tyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase synthetase; a Succinyl-CoA reductase (alcohol forming); a or a 4-hydroxybutyryl-CoA synthetase; and 4-hydroxybu 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps B, CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; a C, T/U/V. W); a glutamate dehydrogenase or a glutamate 4-hydroxybutyryl-CoA reductase (aldehyde forming); and a transaminase; a glutamate decarboxylase; a 4-aminobutyrate 1,4-butanediol dehydrogenase (see FIG. 1, steps G/H/I, S, dehydrogenase or 4-aminobutyrate transaminase; a 4-hy T/U/V. Q, E); a succinyl-CoA transferase, a succinyl-CoA droxybutyrate dehydrogenase; a 4-hydroxybutyrate reduc hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA tase; and a 1,4-butanediol dehydrogenase (see FIG. 1, steps reductase (alcohol forming); a 4-hydroxybutyryl-CoA trans J/K, L, M/N, C, D, E); a glutamate dehydrogenase or a ferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxy glutamate transaminase; a glutamate decarboxylase; a butyryl-CoA synthetase; and a 4-hydroxybutyryl-CoA 4-aminobutyrate dehydrogenase or a 4-aminobutyrate reductase (alcohol forming) (see FIG. 1, steps G/H/I, S. transaminase; a 4-hydroxybutyrate dehydrogenase; a 4-hy T/U/V. W); a succinyl-CoA transferase, a succinyl-CoA droxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and reductase (alcohol forming); a 4-hydroxybutyrate kinase; a E) 1,4-butanediol dehydrogenase (see FIG. 1, steps J/K, L, phosphotrans-4-hydroxybutyrylase; and a 4-hydroxybu M/N, C, O, P, Q, E); a glutamate dehydrogenase or gluta tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps mate transaminase; a glutamate decarboxylase; a 4-amin G/H/I, S. O. P. W); a succinate reductase; a 4-hydroxybu obutyrate dehydrogenase or a 4-aminobutyrate transami tyrate dehydrogenase; a 4-hydroxybutyrate reductase; and nase, a 4-hydroxybutyrate dehydrogenase; a 1,4-butanediol dehydrogenase (see FIG. 1, steps F.C, D, E): 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu a Succinate reductase; a 4-hydroxybutyrate dehydrogenase; tyrylase; and a 4-hydroxybutyryl-CoA reductase (alcohol a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu forming) (see FIG. 1, steps J/K, L, M/N, C, O, P, W); a tyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde glutamate dehydrogenase or a glutamate transaminase; a forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, glutamate decarboxylase; a 4-aminobutyrate dehydrogenase steps F. C., O, P, Q, E); a Succinate reductase; a 4-hydroxy or a 4-aminobutyrate transaminase; a 4-hydroxybutyrate butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxy phosphotrans-4-hydroxybutyrylase; and a 4-hydroxybu butyryl-phosphate reductase; and a 1,4-butanediol dehydro tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps F, genase (see FIG. 1, steps J/K, L, M/N, C, O. R. E. Pathway C, O, P, W); a succinate reductase; a 4-hydroxybutyrate 26: FIG. 1, steps J/K, L, M/N, C, O. R. E); a glutamate dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxy dehydrogenase or a glutamate transaminase; a glutamate butyryl-phosphate reductase; and 1,4-butanediol dehydroge decarboxylase; a 4-aminobutyrate dehydrogenase or a nase (see FIG. 1, steps F. C. O. R. E); a succinate 4-aminobutyrate transaminase; a 4-hydroxybutyrate dehy reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hy drogenase; a 4-hydroxybutyryl-CoA transferase, a 4-hy droxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; a 4-hy synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde droxybutyryl-CoA reductase (aldehyde forming); and 1,4- forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, butanediol dehydrogenase (see FIG. 1, steps F.C, T/U/V, Q, steps J/K, L, M/N, C, T/U/V, Q, E); a glutamate dehydro E); a Succinate reductase; a 4-hydroxybutyrate dehydroge genase or a glutamate transaminase; a glutamate decarboxy nase; a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybu lase; a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate tyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase: transaminase; a 4-hydroxybutyrate dehydrogenase; a 4-hy US 2016/03193 13 A1 Nov. 3, 2016 26 droxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA 45:1, or alternatively about 50:1, or alternatively 60:1, or hydrolase or a 4-hydroxybutyryl-CoA synthetase; and a alternatively 70:1, or alternatively 80:1, or alternatively 4-hydroxybutyryl-CoA reductase (alcohol forming) (see 90:1, or alternatively 100:1. FIG. 1, steps J/K, L, M/N, C, T/U/V. W); an alpha 0202 In one embodiment, the invention provides a ketoglutarate reductase; a 5-hydroxy-2-oxopentanoate dehy method for producing 14-BDO and GBL that includes drogenase; a 5-hydroxy-2-oxopentanoate dehydrogenase culturing a non-naturally occurring microbial organism (decarboxylation); a 4-hydroxybutyryl-CoA reductase (alde under conditions and for a sufficient period of time to hyde forming); and a 1,4-butanediol dehydrogenase (see produce 14-BDO and 4-hydroxybutyryl-CoA followed by FIG. 1, steps X, Y, AA, Q, E); an alpha-ketoglutarate spontaneous lactonization of 4-hydroxybutyryl-phosphate to reductase; a 5-hydroxy-2-oxopentanoate dehydrogenase; a form GBL, wherein said non-naturally occurring microbial 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxy organism comprise a microbial organism having a 14-BDO lation); and a 4-hydroxybutyryl-CoA reductase (alcohol pathway and a 4-hydroxybutyryl-CoA pathway, said forming) (see FIG. 1, steps X, Y, AA, and W); and an 14-BDO pathway comprising at least one exogenous nucleic alpha-ketoglutarate reductase; a 5-hydroxy-2-oxopentanoate acid encoding a 14-BDO pathway enzyme expressed in a dehydrogenase; a 5-hydroxy-2-oxopentanoate decarboxy sufficient amount to produce 14-BDO, said 14-BDO path lase; and a 1,4-butanediol dehydrogenase (see FIG. 1, steps way comprising a Succinyl-CoA reductase (aldehyde form X, Y, Z, E), said 4-hydroxybutyryl-phosphate pathway com ing), an alpha-ketoglutarate decarboxylase, a 4-hydroxybu prising a second set of exogenous nucleic acids encoding tyrate dehydrogenase, a 4-hydroxybutyrate reductase, a 1,4- 4-hydroxybutyryl-phosphate pathway enzymes expressed in butanediol dehydrogenase, a Succinate reductase, a Succinyl a Sufficient amount to produce 4-hydroxybutyryl-phosphate, CoA transferase, a Succinyl-CoA hydrolase, a Succinyl-CoA said second set selected from the group consisting of a synthetase, a glutamate dehydrogenase, a glutamate Succinyl-CoA transferase, a Succinyl-CoA hydrolase or a transaminase, a glutamate decarboxylase, an 4-aminobu Succinyl-CoA synthetase; a Succinyl-CoA reductase (alde tyrate dehydrogenase, an 4-aminobutyrate transaminase, a hyde forming); a 4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybu 4-hydroxybutyrate kinase (see FIG. 1, steps G/H/I, A, C, tyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde O); a Succinyl-CoA transferase, a Succinyl-CoA hydrolase forming), a 4-hydroxybutyryl-phosphate reductase, a Succi or a Succinyl-CoA synthetase; a Succinyl-CoA reductase nyl-CoA reductase (alcohol forming), a 4-hydroxybutyryl (alcohol forming); and a 4-hydroxybutyrate kinase; (see CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a FIG. 1, steps G/H/I. S. O) a succinate reductase; a 4-hy 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA droxybutyrate dehydrogenase; and a 4-hydroxybutyrate reductase (alcohol forming), an alpha-ketoglutarate reduc kinase (see FIG. 1, steps F. C. O); an alpha-ketoglutarate tase, a 5-hydroxy-2-oxopentanoate dehydrogenase, a 5-hy decarboxylase; a 4-hydroxybutyrate dehydrogenase; and a droxy-2-oxopentanoate decarboxylase or a 5-hydroxy-2- 4-hydroxybutyrate kinase (see FIG. 1, steps B, C, O); and oXopentanoate dehydrogenase (decarboxylation), said a glutamate dehydrogenase or a glutamate transaminase; a 4-hydroxybutyryl-CoA pathway comprising at least one glutamate decarboxylase; an 4-aminobutyrate dehydroge exogenous nucleic acid encoding an 4-hydroxybutyryl-CoA nase or an 4-aminobutyrate transaminase; a 4-hydroxybu pathway enzyme expressed in a Sufficient amount to produce tyrate dehydrogenase; and a 4-hydroxybutyrate kinase (see 4-hydroxybutyryl-CoA, (see FIG. 1, steps A, B, C, D, E, F, FIG. 1, steps J/K, L, M/N, C, O). G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z. AA) said 4-hydroxybutyryl-CoA pathway comprising a Suc 0199. In a further aspect of each of the above embodi cinyl-CoA reductase (aldehyde forming), an alpha-ketoglu ments, the exogenous nucleic acid is a heterologous nucleic tarate decarboxylase, a 4-hydroxybutyrate dehydrogenase, a acid. Succinate reductase, a Succinyl-CoA transferase, a Succinyl 0200. In a further aspect of each of the above embodi CoA hydrolase, a Succinyl-CoA synthetase, a glutamate ments, the conditions include Substantially anaerobic culture dehydrogenase, a glutamate transaminase, a glutamate conditions. decarboxylase, a 4-aminobutyrate dehydrogenase, a 4-amin 0201 In a further aspect of each of the above embodi obutyrate transaminase, a 4-hydroxybutyrate kinase, a phos ments, the 14-BDO and GBL products are produced at a photrans-4-hydroxybutyrylase, a Succinyl-CoA reductase predetermined ratio. In some aspects of the invention, the (alcohol forming), a 4-hydroxybutyryl-CoA transferase, a predetermined ratio of 14-BDO to GBL is 1:1, or alterna 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA tively 1:2, or alternatively 1:3, or alternatively 1:4, or synthetase, an alpha-ketoglutarate reductase, a 5-hydroxy alternatively 1:5, or alternatively 1:6, or alternatively 1:7, or 2-oxopentanoate dehydrogenase, or a 5-hydroxy-2-oxopen alternatively 1:8, or alternatively 1:9, or alternatively 1:10, tanoate dehydrogenase (decarboxylation) (see FIG. 1, steps or alternatively 1:15, or alternatively about 1:20, or alter A, B, C, F, G, H, I, J, K, L, M, N, O, P, S, T, U, V, X, Y, AA). natively 1:25, or alternatively 1:30, or alternatively 1:35, or 0203. In a further aspect of the above embodiment, the alternatively 1:40, or alternatively 1:45, or alternatively method includes a microbial organism having a 14-BDO about 1:50, or alternatively 1:60, or alternatively 1:70, or pathway comprising a Succinyl-CoA transferase, a Succinyl alternatively 1:80, or alternatively 1:90, or alternatively CoA hydrolase or a succinyl-CoA synthetase; a Succinyl 1:100. In some aspects of the invention, the predetermined CoA reductase (aldehyde forming); a 4-hydroxybutyrate ratio of 14-BDO to GBL is 2:1, or alternatively 3:1, or dehydrogenase; a 4-hydroxybutyrate reductase; and a 1,4- alternatively 4:1 or alternatively 5:1, or alternatively 6:1, or butanediol dehydrogenase (see FIG. 1, steps G/H/I, A, C, D, alternatively 7:1, or alternatively 8:1, or alternatively 9:1, or E). alternatively 10:1, or alternatively 15:1, or alternatively 0204. In a further aspect of the above embodiment, the about 20:1, or alternatively 25:1, or alternatively 30:1, or method includes a microbial organism having a 14-BDO alternatively 35:1, or alternatively 40:1, or alternatively pathway comprising a Succinyl-CoA transferase, a Succinyl US 2016/03193 13 A1 Nov. 3, 2016 27

CoA hydrolase or a succinyl-CoA synthetase; a Succinyl 4-hydroxybutyryl-phosphate reductase; and a 1,4-butanediol CoA reductase (aldehyde forming); a 4-hydroxybutyrate dehydrogenase (see FIG. 1, steps G/H/I, S. O. R. E). dehydrogenase; a 4-hydroxybutyrate kinase; a phospho 0212. In a further aspect of the above embodiment, the trans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA reduc method includes a microbial organism having a 14-BDO tase (aldehyde forming); and a 1,4-butanediol dehydroge pathway comprising a Succinyl-CoA transferase, a Succinyl nase (see FIG. 1, steps G/H/I, A, C, O, P, Q, E). CoA hydrolase or a succinyl-CoA synthetase; a Succinyl 0205. In a further aspect of the above embodiment, the CoA reductase (alcohol forming); a 4-hydroxybutyryl-CoA method includes a microbial organism having a 14-BDO transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hy pathway comprising a Succinyl-CoA transferase, a Succinyl droxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA CoA hydrolase or a succinyl-CoA synthetase; a Succinyl reductase (aldehyde forming); and a 1,4-butanediol dehy CoA reductase (aldehyde forming); a 4-hydroxybutyrate drogenase (see FIG. 1, steps G/H/I, S, T/U/V, Q, E). dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxy 0213. In a further aspect of the above embodiment, the butyryl-phosphate reductase; and a 1,4-butanediol dehydro method includes a microbial organism having a 14-BDO genase (see FIG. 1, steps G/H/I, A, C, O. R. E). pathway comprising a Succinyl-CoA transferase, a Succinyl 0206. In a further aspect of the above embodiment, the CoA hydrolase or a succinyl-CoA synthetase; a Succinyl method includes a microbial organism having a 14-BDO CoA reductase (alcohol forming); a 4-hydroxybutyryl-CoA pathway comprising a Succinyl-CoA transferase, a Succinyl transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hy CoA hydrolase, or a Succinyl-CoA synthetase; a Succinyl droxybutyryl-CoA synthetase; and a 4-hydroxybutyryl-CoA CoA reductase (aldehyde forming); a 4-hydroxybutyrate reductase (alcohol forming) (see FIG. 1, steps G/H/I, S. dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a T/U/V, W). 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl 0214. In a further aspect of the above embodiment, the CoA synthetase; a 4-hydroxybutyryl-CoA reductase (alde method includes a microbial organism having a 14-BDO hyde forming); and a 1,4-butanediol dehydrogenase (see pathway comprising a Succinyl-CoA transferase, a Succinyl FIG. 1, steps G/H/I, A, C, T/U/V, Q, E). CoA hydrolase or a succinyl-CoA synthetase; a Succinyl 0207. In a further aspect of the above embodiment, the CoA reductase (alcohol forming); a 4-hydroxybutyrate method includes a microbial organism having a 14-BDO kinase; a phosphotrans-4-hydroxybutyrylase; and a 4-hy pathway comprising a Succinyl-CoA transferase, a Succinyl droxybutyryl-CoA reductase (alcohol forming) (see FIG. 1, CoA hydrolase or Succinyl-CoA synthetase; a Succinyl-CoA steps G/H/I, S, O, P, W). reductase (aldehyde forming); a 4-hydroxybutyrate dehy 0215. In a further aspect of the above embodiment, the drogenase; a 4-hydroxybutyryl-CoA transferase, a 4-hy method includes a microbial organism having a 14-BDO droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA pathway comprising a Succinate reductase; a 4-hydroxybu synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol tyrate dehydrogenase; a 4-hydroxybutyrate reductase; and forming) (see FIG. 1, steps G/H/I, A, C, T/U/V, W). 1,4-butanediol dehydrogenase (see FIG. 1, steps F.C, D, E). 0216. In a further aspect of the above embodiment, the 0208. In a further aspect of the above embodiment, the method includes a microbial organism having a 14-BDO method includes a microbial organism having a 1,4-BDO pathway comprising a Succinate reductase; a 4-hydroxybu pathway comprising a Succinyl-CoA transferase, a Succinyl tyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phos CoA hydrolase or a succinyl-CoA synthetase; a Succinyl photrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA CoA reductase (aldehyde forming); a 4-hydroxybutyrate reductase (aldehyde forming); and a 1,4-butanediol dehy dehydrogenase; a 4-hydroxybutyrate kinase; a phospho drogenase (see FIG. 1, steps F. C., O, P, Q, E). trans-4-hydroxybutyrylase; and a 4-hydroxybutyryl-CoA 0217. In a further aspect of the above embodiment, the reductase (alcohol forming) (see FIG. 1, steps G/H/I, A, C, method includes a microbial organism having a 14-BDO O, P, W). pathway comprising a Succinate reductase; a 4-hydroxybu 0209. In a further aspect of the above embodiment, the tyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phos method includes a microbial organism having a 14-BDO photrans-4-hydroxybutyrylase; and a 4-hydroxybutyryl pathway comprising a Succinyl-CoA transferase, a Succinyl CoA reductase (alcohol forming) (see FIG. 1, steps F. C., O. CoA hydrolase or a succinyl-CoA synthetase; a Succinyl P. W). CoA reductase (alcohol forming); a 4-hydroxybutyrate 0218. In a further aspect of the above embodiment, the reductase; and 1,4-butanediol dehydrogenase (see FIG. 1, method includes a microbial organism having a 14-BDO steps G/H/I, S, D, E). pathway comprising a Succinate reductase; a 4-hydroxybu 0210. In a further aspect of the above embodiment, the tyrate dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hy method includes a microbial organism having a 14-BDO droxybutyryl-phosphate reductase; and 1,4-butanediol dehy pathway comprising a Succinyl-CoA transferase, a Succinyl drogenase (see FIG. 1, steps F. C. O. R. E). CoA hydrolase or a succinyl-CoA synthetase; a Succinyl 0219. In a further aspect of the above embodiment, the CoA reductase (alcohol forming); a 4-hydroxybutyrate method includes a microbial organism having a 14-BDO kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxy pathway comprising a Succinate reductase; a 4-hydroxybu butyryl-CoA reductase (aldehyde forming); and a 1,4-bu tyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase, tanediol dehydrogenase (see FIG. 1, steps G/H/I, S, O, P, Q, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl E). CoA synthetase; a 4-hydroxybutyryl-CoA reductase (alde 0211. In a further aspect of the above embodiment, the hyde forming); and 1,4-butanediol dehydrogenase (see FIG. method includes a microbial organism having a 14-BDO 1, steps F, C, T/U/V, Q, E). pathway comprising a Succinyl-CoA transferase, a Succinyl 0220. In a further aspect of the above embodiment, the CoA hydrolase or Succinyl-CoA synthetase; a Succinyl-CoA method includes a microbial organism having a 14-BDO reductase (alcohol forming), a 4-hydroxybutyrate kinase, a pathway comprising a Succinate reductase; a 4-hydroxybu US 2016/03193 13 A1 Nov. 3, 2016 28 tyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase, 0229. In a further aspect of the above embodiment, the a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl method includes a microbial organism having a 14-BDO CoA synthetase; and a 4-hydroxybutyryl-CoA reductase pathway comprising a glutamate dehydrogenase or gluta (alcohol forming) (see FIG. 1, steps F. C. T/U/V, W). mate transaminase; a glutamate decarboxylase; a 4-amin 0221. In a further aspect of the above embodiment, the obutyrate dehydrogenase or a 4-aminobutyrate transami method includes a microbial organism having a 14-BDO nase, a 4-hydroxybutyrate dehydrogenase; a pathway comprising an alpha-ketoglutarate decarboxylase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate tyrylase; and a 4-hydroxybutyryl-CoA reductase (alcohol reductase; and 1,4-butanediol dehydrogenase (see FIG. 1, forming) (see FIG. 1, steps J/K, L, M/N, C, O, P, W) steps B, C, D, E). 0230. In a further aspect of the above embodiment, the 0222. In a further aspect of the above embodiment, the method includes a microbial organism having a 14-BDO method includes a microbial organism having a 14-BDO pathway comprising a glutamate dehydrogenase or a gluta pathway comprising an alpha-ketoglutarate decarboxylase; a mate transaminase; a glutamate decarboxylase; a 4-amin 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate obutyrate dehydrogenase or a 4-aminobutyrate transami kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxy nase, a 4-hydroxybutyrate dehydrogenase; a butyryl-CoA reductase (aldehyde forming); and a 1,4-bu 4-hydroxybutyrate kinase; a 4-hydroxybutyryl-phosphate tanediol dehydrogenase (see FIG. 1, steps B, C, O, P, Q, E). reductase; and a 1,4-butanediol dehydrogenase (see FIG. 1, 0223) In a further aspect of the above embodiment, the steps J/K, L, M/N, C, O, R, E). method includes a microbial organism having a 14-BDO 0231. In a further aspect of the above embodiment, the pathway comprising an alpha-ketoglutarate decarboxylase; a method includes a microbial organism having a 14-BDO 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate pathway comprising a glutamate dehydrogenase or a gluta kinase; a phosphotrans-4-hydroxybutyrylase and 4-hy mate transaminase; a glutamate decarboxylase; a 4-amin droxybutyryl-CoA reductase (alcohol forming) (see FIG. 1, obutyrate dehydrogenase or a 4-aminobutyrate transami steps B, C, O, P, W). nase, a 4-hydroxybutyrate dehydrogenase; a 0224. In a further aspect of the above embodiment, the 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl method includes a microbial organism having a 14-BDO CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; a pathway comprising an alpha-ketoglutarate decarboxylase; a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate 1,4-butanediol dehydrogenase (see FIG. 1, steps J/K, L, kinase; a 4-hydroxybutyryl-phosphate reductase; and a 1,4- M/N, C, T/U/V, Q, E). butanediol dehydrogenase (see FIG. 1, steps B, C, O. R. E). 0232. In a further aspect of the above embodiment, the 0225. In a further aspect of the above embodiment, the method includes a microbial organism having a 14-BDO method includes a microbial organism having a 14-BDO pathway comprising a glutamate dehydrogenase or a gluta pathway comprising an alpha-ketoglutarate decarboxylase; a mate transaminase; a glutamate decarboxylase; a 4-amin 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl obutyrate dehydrogenase or a 4-aminobutyrate transami CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or nase, a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl reductase (aldehyde forming); and a 1,4-butanediol dehy CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and drogenase (see FIG. 1, steps B, C, T/U/V, Q, E). a 4-hydroxybutyryl-CoA reductase (alcohol forming) (see 0226. In a further aspect of the above embodiment, the FIG. 1, steps J/K, L, M/N, C, T/U/V, W). method includes a microbial organism having a 14-BDO 0233. In a further aspect of the above embodiment, the pathway comprising an alpha-ketoglutarate decarboxylase; a method includes a microbial organism having a 14-BDO 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl pathway comprising an alpha-ketoglutarate reductase; a CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 5-hydroxy-2-oxopentanoate dehydrogenase; a 5-hydroxy-2- 4-hydroxybutyryl-CoA synthetase; and 4-hydroxybutyryl oXopentanoate dehydrogenase (decarboxylation); a 4-hy CoA reductase (alcohol forming) (see FIG. 1, steps B, C, droxybutyryl-CoA reductase (aldehyde forming); and a 1,4- T/U/V, W). butanediol dehydrogenase (see FIG. 1, steps X, Y, AA, Q, 0227. In a further aspect of the above embodiment, the E). method includes a microbial organism having a 14-BDO 0234. In a further aspect of the above embodiment, the pathway comprising a glutamate dehydrogenase or a gluta method includes a microbial organism having a 14-BDO mate transaminase; a glutamate decarboxylase; a 4-amin pathway comprising an alpha-ketoglutarate reductase; a obutyrate dehydrogenase or 4-aminobutyrate transaminase; 5-hydroxy-2-oxopentanoate dehydrogenase; a 5-hydroxy-2- a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate oXopentanoate dehydrogenase (decarboxylation); and a reductase; and a 1,4-butanediol dehydrogenase (see FIG. 1, 4-hydroxybutyryl-CoA reductase (alcohol forming) (see steps J/K, L, M/N, C, D, E. FIG. 1, steps X, Y, AA, and W). 0228. In a further aspect of the above embodiment, the 0235. In a further aspect of the above embodiment, the method includes a microbial organism having a 14-BDO method includes a microbial organism having a 14-BDO pathway comprising a glutamate dehydrogenase or a gluta pathway comprising an alpha-ketoglutarate reductase; a mate transaminase; a glutamate decarboxylase; a 4-amin 5-hydroxy-2-oxopentanoate dehydrogenase; a 5-hydroxy-2- obutyrate dehydrogenase or a 4-aminobutyrate transami oXopentanoate decarboxylase; and a 1,4-butanediol dehy nase, a 4-hydroxybutyrate dehydrogenase; a drogenase (see FIG. 1, steps X, Y, Z. E). 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu 0236. In a further aspect of the above embodiment, the tyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde method includes a microbial organism having a 14-BDO forming); and E) 1,4-butanediol dehydrogenase (see FIG. 1, pathway comprising comprises a Succinyl-CoA transferase, steps J/K, L, M/N, C, O, P, Q, E). a Succinyl-CoA hydrolase or a Succinyl-CoA synthetase; a US 2016/03193 13 A1 Nov. 3, 2016 29

Succinyl-CoA reductase (aldehyde forming); a 4-hydroxy 0245. In a further aspect of the above embodiment, the butyrate dehydrogenase; a 4-hydroxybutyrate kinase; and a method includes a microbial organism having a 4-hydroxy phosphotrans-4-hydroxybutyrylase (see FIG. 1, steps G/H/I, butyryl-CoA pathway comprising a glutamate dehydroge A, C, O, P). nase or a glutamate transaminase; a glutamate decarboxy 0237. In a further aspect of the above embodiment, the lase; an 4-aminobutyrate dehydrogenase or an method includes a microbial organism having a 4-hydroxy 4-aminobutyrate transaminase; a 4-hydroxybutyrate dehy butyryl-CoA pathway comprising a Succinyl-CoA trans drogenase; and a 4-hydroxybutyryl-CoA transferase, a 4-hy ferase, a Succinyl-CoA hydrolase or a Succinyl-CoA syn droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA thetase; a Succinyl-CoA reductase (aldehyde forming); a synthetase (see FIG. 1, steps J/K, L, M/N, C, T/U/V). 4-hydroxybutyrate dehydrogenase; and a 4-hydroxybutyryl 0246. In a further aspect of the above embodiment, the CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a method includes a microbial organism having a 4-hydroxy 4-hydroxybutyryl-CoA synthetase (see FIG. 1, steps G/H/I, butyryl-CoA pathway comprising an alpha-ketoglutarate A, C, T/U/V). reductase; a 5-hydroxy-2-oxopentanoate dehydrogenase and 0238. In a further aspect of the above embodiment, the a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxy method includes a microbial organism having a 4-hydroxy lation) (see FIG. 1, steps X, Y, AA). butyryl-CoA pathway comprising a Succinyl-CoA trans 0247. In one embodiment, the invention provides a ferase, a Succinyl-CoA hydrolase or a Succinyl-CoA syn method for producing 14-BDO and GBL that includes thetase; a Succinyl-CoA reductase (alcohol forming); a culturing a non-naturally occurring microbial organism 4-hydroxybutyrate kinase; and a phosphotrans-4-hydroxy under conditions and for a sufficient period of time to butyrylase (see FIG. 1, steps G/H/I, S, O, P). produce 14-BDO and 4-hydroxybutyryl-CoA followed by 0239. In a further aspect of the above embodiment, the spontaneous lactonization of 4-hydroxybutyryl-phosphate to method includes a microbial organism having a 4-hydroxy form GBL, wherein said non-naturally occurring microbial butyryl-CoA pathway comprising a Succinyl-CoA trans organism comprise a microbial organism having a 14-BDO ferase, a Succinyl-CoA hydrolase or a Succinyl-CoA syn pathway and a 4-hydroxybutyryl-CoA pathway, said thetase; a Succinyl-CoA reductase (alcohol forming); and a 14-BDO pathway comprising a first set of exogenous 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl nucleic acids encoding 14-BDO pathway enzymes CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase (see expressed in a sufficient amount to produce 14-BDO, said FIG. 1, steps G/H/I, S, T/U/V). first set is selected from the group consisting of a Succinyl 0240. In a further aspect of the above embodiment, the CoA transferase, a succinyl-CoA hydrolase or a succinyl method includes a microbial organism having a 4-hydroxy CoA synthetase; a Succinyl-CoA reductase (aldehyde form butyryl-CoA pathway comprising a Succinate reductase; a ing); a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate 4-hydroxybutyrate reductase; and a 1,4-butanediol dehydro kinase and a phosphotrans-4-hydroxybutyrylase (see FIG. 1, genase) (see FIG. 1, steps G/H/I, A, C, D, E); a succinyl steps F, C, O, P). CoA transferase, a Succinyl-CoA hydrolase or a Succinyl 0241. In a further aspect of the above embodiment, the CoA synthetase; a Succinyl-CoA reductase (aldehyde method includes a microbial organism having a 4-hydroxy forming); a 4-hydroxybutyrate dehydrogenase; a 4-hydroxy butyryl-CoA pathway comprising a Succinate reductase; a butyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyrate dehydrogenase; and a 4-hydroxybutyryl 4-hydroxybutyryl-CoA reductase (aldehyde forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, steps G/H/I, A, CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a C, O, P, Q, E); a succinyl-CoA transferase, a succinyl-CoA 4-hydroxybutyryl-CoA synthetase (see FIG. 1, steps F. C. hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA T/U/V). reductase (aldehyde forming); a 4-hydroxybutyrate dehy 0242. In a further aspect of the above embodiment, the drogenase; a 4-hydroxybutyrate kinase; a 4-hydroxybutyryl method includes a microbial organism having a 4-hydroxy phosphate reductase; and a 1,4-butanediol dehydrogenase butyryl-CoA pathway comprising an alpha-ketoglutarate (see FIG. 1, steps G/H/I, A, C, O. R. E); a succinyl-CoA decarboxylase; a 4-hydroxybutyrate dehydrogenase; a 4-hy transferase, a Succinyl-CoA hydrolase, or a Succinyl-CoA droxybutyrate kinase; and a phosphotrans-4-hydroxybutyry synthetase; a Succinyl-CoA reductase (aldehyde forming); a lase (see FIG. 1, steps B, C, O, P). 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl 0243 In a further aspect of the above embodiment, the CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a method includes a microbial organism having a 4-hydroxy 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA butyryl-CoA pathway comprising an alpha-ketoglutarate reductase (aldehyde forming); and a 1,4-butanediol dehy decarboxylase; a 4-hydroxybutyrate dehydrogenase; and a drogenase (see FIG. 1, steps G/H/I, A, C, T/U/V. Q, E); a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl Succinyl-CoA transferase, a Succinyl-CoA hydrolase or Suc CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase (see cinyl-CoA synthetase; a Succinyl-CoA reductase (aldehyde FIG. 1, steps B, C, T/U/V). forming); a 4-hydroxybutyrate dehydrogenase; a 4-hydroxy 0244. In a further aspect of the above embodiment, the butyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase method includes a microbial organism having a 4-hydroxy or a 4-hydroxybutyryl-CoA synthetase; and a 4-hydroxybu butyryl-CoA pathway comprising a glutamate dehydroge tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps nase or a glutamate transaminase; a glutamate decarboxy G/H/I, A, C, T/U/V. W); a succinyl-CoA transferase, a lase; an 4-aminobutyrate dehydrogenase or an Succinyl-CoA hydrolase or a Succinyl-CoA synthetase; a 4-aminobutyrate transaminase; a 4-hydroxybutyrate dehy Succinyl-CoA reductase (aldehyde forming); a 4-hydroxy drogenase; a 4-hydroxybutyrate kinase; and a phosphotrans butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxybutyrylase (see FIG. 1, steps J/K, L, M/N, C, O. phosphotrans-4-hydroxybutyrylase; and a 4-hydroxybu P). tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps US 2016/03193 13 A1 Nov. 3, 2016 30

G/H/I, A, C, O, P, W); a succinyl-CoA transferase, a P. Q., E); an alpha-ketoglutarate decarboxylase; a 4-hy Succinyl-CoA hydrolase or a Succinyl-CoA synthetase; a droxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; Succinyl-CoA reductase (alcohol forming); a 4-hydroxybu a phosphotrans-4-hydroxybutyrylase and 4-hydroxybutyryl tyrate reductase; and 1,4-butanediol dehydrogenase (see CoA reductase (alcohol forming) (see FIG. 1, steps B, C, O. FIG. 1, steps G/H/I, S, D, E); a succinyl-CoA transferase, P. W); an alpha-ketoglutarate decarboxylase; a 4-hydroxy a Succinyl-CoA hydrolase or a Succinyl-CoA synthetase; a butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a Succinyl-CoA reductase (alcohol forming); a 4-hydroxybu 4-hydroxybutyryl-phosphate reductase; and a 1,4-butanediol tyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hy dehydrogenase (see FIG. 1, steps B, C, O. R. E); an droxybutyryl-CoA reductase (aldehyde forming); and a 1,4- alpha-ketoglutarate decarboxylase; a 4-hydroxybutyrate butanediol dehydrogenase (see FIG. 1, steps G/H/I, S. O. P. dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a Q, E); a Succinyl-CoA transferase, a Succinyl-CoA hydro 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA lase or Succinyl-CoA synthetase; a Succinyl-CoA reductase synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde (alcohol forming), a 4-hydroxybutyrate kinase, a 4-hydroxy forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, butyryl-phosphate reductase; and a 1,4-butanediol dehydro steps B, C, T/U/V. Q, E; an alpha-ketoglutarate decarboxy genase (see FIG. 1, steps G/H/I, S. O. R. E); a succinyl lase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu CoA transferase, a Succinyl-CoA hydrolase or a Succinyl tyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase CoA synthetase; a Succinyl-CoA reductase (alcohol or a 4-hydroxybutyryl-CoA synthetase; and 4-hydroxybu forming); a 4-hydroxybutyryl-CoA transferase, a 4-hy tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps B, droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA C, T/U/V. W); a glutamate dehydrogenase or a glutamate synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde transaminase; a glutamate decarboxylase; a 4-aminobutyrate forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, dehydrogenase or 4-aminobutyrate transaminase; a 4-hy steps G/H/I, S, T/U/V. Q, E); a succinyl-CoA transferase, a droxybutyrate dehydrogenase; a 4-hydroxybutyrate reduc Succinyl-CoA hydrolase or a Succinyl-CoA synthetase; a tase; and a 1,4-butanediol dehydrogenase (see FIG. 1, steps Succinyl-CoA reductase (alcohol forming); a 4-hydroxybu J/K, L, M/N, C, D, E); a glutamate dehydrogenase or a tyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase glutamate transaminase; a glutamate decarboxylase; a or a 4-hydroxybutyryl-CoA synthetase; and a 4-hydroxybu 4-aminobutyrate dehydrogenase or a 4-aminobutyrate tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps transaminase; a 4-hydroxybutyrate dehydrogenase; a 4-hy G/H/I, S, T/U/V. W); a succinyl-CoA transferase, a succi droxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; nyl-CoA hydrolase or a succinyl-CoA synthetase; a succi a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and nyl-CoA reductase (alcohol forming); a 4-hydroxybutyrate E) 1,4-butanediol dehydrogenase (see FIG. 1, steps J/K, L, kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxy M/N, C, O, P, Q, E); a glutamate dehydrogenase or gluta butyryl-CoA reductase (alcohol forming) (see FIG. 1, steps mate transaminase; a glutamate decarboxylase; a 4-amin G/H/I, S. O. P. W); a succinate reductase; a 4-hydroxybu obutyrate dehydrogenase or a 4-aminobutyrate transami tyrate dehydrogenase; a 4-hydroxybutyrate reductase; and nase, a 4-hydroxybutyrate dehydrogenase; a 1,4-butanediol dehydrogenase (see FIG. 1, steps F.C, D, E): 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu a Succinate reductase; a 4-hydroxybutyrate dehydrogenase; tyrylase; and a 4-hydroxybutyryl-CoA reductase (alcohol a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybu forming) (see FIG. 1, steps J/K, L, M/N, C, O, P, W); a tyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde glutamate dehydrogenase or a glutamate transaminase; a forming); and a 1,4-butanediol dehydrogenase (see FIG. 1, glutamate decarboxylase; a 4-aminobutyrate dehydrogenase steps F. C., O, P, Q, E); a Succinate reductase; a 4-hydroxy or a 4-aminobutyrate transaminase; a 4-hydroxybutyrate butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxy phosphotrans-4-hydroxybutyrylase; and a 4-hydroxybu butyryl-phosphate reductase; and a 1,4-butanediol dehydro tyryl-CoA reductase (alcohol forming) (see FIG. 1, steps F, genase (see FIG. 1, steps J/K, L, M/N, C, O. R. E); a C, O, P, W); a succinate reductase; a 4-hydroxybutyrate glutamate dehydrogenase or a glutamate transaminase; a dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxy glutamate decarboxylase; a 4-aminobutyrate dehydrogenase butyryl-phosphate reductase; and 1,4-butanediol dehydroge or a 4-aminobutyrate transaminase; a 4-hydroxybutyrate nase (see FIG. 1, steps F. C. O. R. E); a succinate dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hy 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl droxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA CoA synthetase; a 4-hydroxybutyryl-CoA reductase (alde hydrolase or a 4-hydroxybutyryl-CoA synthetase; a 4-hy hyde forming); and a 1,4-butanediol dehydrogenase (see droxybutyryl-CoA reductase (aldehyde forming); and 1,4- FIG. 1, steps J/K, L, M/N, C, T/U/V. Q, E); a glutamate butanediol dehydrogenase (see FIG. 1, steps F.C, T/U/V, Q, dehydrogenase or a glutamate transaminase; a glutamate E); a Succinate reductase; a 4-hydroxybutyrate dehydroge decarboxylase; a 4-aminobutyrate dehydrogenase or a nase; a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybu 4-aminobutyrate transaminase; a 4-hydroxybutyrate dehy tyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase: drogenase; a 4-hydroxybutyryl-CoA transferase, a 4-hy and a 4-hydroxybutyryl-CoA reductase (alcohol forming) droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA (see FIG. 1, steps F. C. T/U/V. W); an alpha-ketoglutarate synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol decarboxylase; a 4-hydroxybutyrate dehydrogenase; a 4-hy forming) (see FIG. 1, steps J/K, L, M/N, C, T/U/V. W); an droxybutyrate reductase; and 1,4-butanediol dehydroge alpha-ketoglutarate reductase; a 5-hydroxy-2-oxopentanoate nase (see FIG. 1, Steps B, C, D, E); an alpha-ketoglutarate dehydrogenase; a 5-hydroxy-2-oxopentanoate dehydroge decarboxylase; a 4-hydroxybutyrate dehydrogenase; a 4-hy nase (decarboxylation); a 4-hydroxybutyryl-CoA reductase droxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; (aldehyde forming); and a 1,4-butanediol dehydrogenase a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and (see FIG. 1, steps X, Y, AA, Q, E); an alpha-ketoglutarate a 1,4-butanediol dehydrogenase (see FIG. 1, steps B, C, O. reductase; a 5-hydroxy-2-oxopentanoate dehydrogenase; a US 2016/03193 13 A1 Nov. 3, 2016

5-hydroxy-2-oxopentanoate dehydrogenase (decarboxy 0249. In a further aspect of each of the above embodi lation); and a 4-hydroxybutyryl-CoA reductase (alcohol ments, the conditions include Substantially anaerobic culture forming) (see FIG. 1, steps X, Y, AA, and W); and an conditions. alpha-ketoglutarate reductase; a 5-hydroxy-2-oxopentanoate 0250 In a further aspect of each of the above embodi dehydrogenase; a 5-hydroxy-2-oxopentanoate decarboxy ments, the 14-BDO and GBL products are produced at a lase; and a 1,4-butanediol dehydrogenase (see FIG. 1, steps predetermined ratio. In some aspects of the invention, the X, Y, Z, E), said 4-hydroxybutyryl-CoA pathway comprising predetermined ratio of 14-BDO to GBL is 1:1, or alterna a second set of exogenous nucleic acids encoding 4-hy tively 1:2, or alternatively 1:3, or alternatively 1:4, or droxybutyryl-CoA pathway enzymes expressed in a Sufi alternatively 1:5, or alternatively 1:6, or alternatively 1:7, or cient amount to produce 4-hydroxybutyryl-CoA, said sec alternatively 1:8, or alternatively 1:9, or alternatively 1:10, ond set selected from the group consisting of a Succinyl or alternatively 1:15, or alternatively about 1:20, or alter CoA transferase, a Succinyl-CoA hydrolase or a Succinyl natively 1:25, or alternatively 1:30, or alternatively 1:35, or CoA synthetase; a Succinyl-CoA reductase (aldehyde alternatively 1:40, or alternatively 1:45, or alternatively about 1:50, or alternatively 1:60, or alternatively 1:70, or forming); a 4-hydroxybutyrate dehydrogenase; a 4-hydroxy alternatively 1:80, or alternatively 1:90, or alternatively butyrate kinase; and a phosphotrans-4-hydroxybutyrylase 1:100. In some aspects of the invention, the predetermined (see FIG. 1, steps G/H/I, A, C, O, P): a succinyl-CoA ratio of 14-BDO to GBL is 2:1, or alternatively 3:1, or transferase, a Succinyl-CoA hydrolase or a Succinyl-CoA alternatively 4:1 or alternatively 5:1, or alternatively 6:1, or synthetase; a Succinyl-CoA reductase (aldehyde forming); a alternatively 7:1, or alternatively 8:1, or alternatively 9:1, or 4-hydroxybutyrate dehydrogenase; and a 4-hydroxybutyryl alternatively 10:1, or alternatively 15:1, or alternatively CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a about 20:1, or alternatively 25:1, or alternatively 30:1, or 4-hydroxybutyryl-CoA synthetase (see FIG. 1, steps G/H/I, alternatively 35:1, or alternatively 40:1, or alternatively A, C, T/U/V); a succinyl-CoA transferase, a succinyl-CoA 45:1, or alternatively about 50:1, or alternatively 60:1, or hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA alternatively 70:1, or alternatively 80:1, or alternatively reductase (alcohol forming); a 4-hydroxybutyrate kinase; 90:1, or alternatively 100:1. and a phosphotrans-4-hydroxybutyrylase (see FIG. 1, steps 0251. In one embodiment, the invention provides a G/H/I, S, O, P): a succinyl-CoA transferase, a succinyl-CoA method for producing 14-BDO and GBL that includes hydrolase or a Succinyl-CoA synthetase; a Succinyl-CoA culturing a non-naturally occurring microbial organism reductase (alcohol forming); and a 4-hydroxybutyryl-CoA under conditions and for a sufficient period of time to transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hy produce 14-BDO and 4-hydroxybutyryl-phosphate followed droxybutyryl-CoA synthetase (see FIG. 1, steps G/H/I, S. by spontaneous lactonization of 4-hydroxybutyryl-phos T/U/V): a succinate reductase; a 4-hydroxybutyrate dehy phate to form GBL, wherein said non-naturally occurring drogenase; a 4-hydroxybutyrate kinase and a phosphotrans microbial organism comprise a microbial organism having a 4-hydroxybutyrylase (see FIG. 1, steps F. C., O, P): a 14-BDO pathway and a 4-hydroxybutyryl-phosphate path Succinate reductase; a 4-hydroxybutyrate dehydrogenase; way, said 14-BDO pathway comprising at least one exog and a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybu enous nucleic acid encoding a 14-BDO pathway enzyme tyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase expressed in a sufficient amount to produce 14-BDO, wherein the 14-BDO pathway comprises an alpha-ketoglu (see FIG. 1, steps F. C. T/U/V); an alpha-ketoglutarate tarate decarboxylase; a 4-hydroxybutyrate dehydrogenase; a decarboxylase; a 4-hydroxybutyrate dehydrogenase; a 4-hy 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl droxybutyrate kinase; and a phosphotrans-4-hydroxybutyry CoA hydrolase or 4-hydroxybutyryl-CoA synthetase; a lase (see FIG. 1, steps B, C, O, P): an alpha-ketoglutarate 4-hydroxybutyryl-CoA reductase (aldehyde forming); and decarboxylase; a 4-hydroxybutyrate dehydrogenase; and a said 4-hydroxybutyryl-phosphate pathway comprising at 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl least one exogenous nucleic acid encoding an 4-hydroxy CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase (see butyryl-phosphate pathway enzyme expressed in a sufficient FIG. 1, steps B, C, T/U/V); a glutamate dehydrogenase or amount to produce 4-hydroxybutyryl-phosphate (see steps a glutamate transaminase; a glutamate decarboxylase; an B, C, T/U/V. Q, E), said 4-hydroxybutyryl-phosphate path 4-aminobutyrate dehydrogenase or an 4-aminobutyrate way comprising an alpha-ketoglutarate decarboxylase; a transaminase; a 4-hydroxybutyrate dehydrogenase; a 4-hy 4-hydroxybutyrate dehydrogenase; and a 4-hydroxybutyrate droxybutyrate kinase; and a phosphotrans-4-hydroxybutyry kinase see steps B, C, O). lase (see FIG. 1, steps J/K, L, M/N, C, O, P): a glutamate 0252. In a further aspect of the above embodiment, the dehydrogenase or a glutamate transaminase; a glutamate non-naturally occurring microbial organism does not com decarboxylase; an 4-aminobutyrate dehydrogenase or an prise an exogenous nucleic acid encoding a phosphotrans 4-aminobutyrate transaminase; a 4-hydroxybutyrate dehy 4-hydroxybutyrylase. In on apsect, the microbial organism drogenase; and a 4-hydroxybutyryl-CoA transferase, a 4-hy comprises a gene disruption in a gene encoding a phospho droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA trans-4-hydroxybutyrylase, wherein the gene disruption synthetase (see FIG. 1, steps J/K, L, M/N, C, T/U/V); and confers production of a predetermined ratio of 14-BDO to an alpha-ketoglutarate reductase; a 5-hydroxy-2-oxopen GBL. In some aspects of the invention, the predetermined tanoate dehydrogenase and a 5-hydroxy-2-oxopentanoate ratio is 1:1, or alternatively 1:2, or alternatively 1:3, or dehydrogenase (decarboxylation) (see FIG. 1, steps X, Y, alternatively 1:4, or alternatively 1:5, or alternatively 1:6, or AA). alternatively 1:7, or alternatively 1:8, or alternatively 1:9, or 0248. In a further aspect of each of the above embodi alternatively 1:10, or alternatively 1:15, or alternatively ments, the exogenous nucleic acid is a heterologous nucleic about 1:20, or alternatively 1:25, or alternatively 1:30, or acid. alternatively 1:35, or alternatively 1:40, or alternatively US 2016/03193 13 A1 Nov. 3, 2016 32

1:45, or alternatively about 1:50, or alternatively 1:60, or by the liquid phase hydrogenation of gamma-butyrolactone alternatively 1:70, or alternatively 1:80, or alternatively over a copper oxide-zinc oxide catalyst. Further exemplified 1:90, or alternatively 1:100. In some aspects of the inven is a continuous flow tubular reactor operating at high total tion, the predetermined ratio of 14-BDO to GBL is 2:1, or reactor pressures and high reactant and product partial alternatively 3:1, or alternatively 4:1, or alternatively 5:1, or pressures. And U.S. Pat. No. 4,652,685, which describes the alternatively 6:1, or alternatively 7:1, or alternatively 8:1, or hydrogenation of lactones to glycols. alternatively 9:1, or alternatively 10:1, or alternatively 15:1, 0257 Suitable purification and/or assays to test for the or alternatively about 20:1, or alternatively 25:1, or alter production of 14-BDO and GBL can be performed using natively 30:1, or alternatively 35:1, or alternatively 40:1, or well known methods. Suitable replicates such as triplicate alternatively 45:1, or alternatively about 50:1, or alterna cultures can be grown for each engineered strain to be tested. tively 60:1, or alternatively 70:1, or alternatively 80:1, or For example, product and byproduct formation in the engi alternatively 90:1, or alternatively 100:1. neered production host can be monitored. The final product 0253) In a further aspect of the above embodiment, the and intermediates, and other organic compounds, can be exogenous nucleic acid is a heterologous nucleic acid. analyzed by methods such as HPLC (High Performance 0254. In a further aspect of the above embodiment, the Liquid Chromatography), GC-MS (Gas Chromatography conditions include Substantially anaerobic culture condi Mass Spectroscopy) and LC-MS (Liquid Chromatography tions. Mass Spectroscopy) or other suitable analytical methods 0255 Embodiments described herein include the down using routine procedures well known in the art. The release stream compound that can be produced from the 14-BDO of product in the fermentation broth can also be tested with and GBL producing non-naturally occurring microbial the culture Supernatant. Byproducts and residual glucose can organisms of the invention, for example, 14-BDO. This be quantified by HPLC using, for example, a refractive compound can be synthesized by, for example, chemical index detector for glucose and alcohols, and a UV detector hydrogenation of GBL. Chemical hydrogenation reactions for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 are well known in the art. One exemplary procedure includes (2005)), or other suitable assay and detection methods well the chemical reduction of GBL or a mixture of GBL and known in the art. The individual enzyme or protein activities 14-BDO deriving from the culture using a heterogeneous or from the exogenous DNA sequences can also be assayed homogeneous hydrogenation catalyst together with hydro using methods well known in the art. gen, or a hydride-based reducing agent used stoichiometri (0258. The 14-BDO and GBL products can be separated cally or catalytically, to produce 14-BDO. from other components in the culture using a variety of 0256. Other procedures well known in the art are equally methods well known in the art. Such separation methods applicable for the above chemical reaction and include, for include, for example, extraction procedures as well as meth example, WO No. 82/03854 (Bradley, et al.), which ods that include continuous liquid-liquid extraction, per describes the hydrogenolysis of gamma-butyrolactone in the vaporation, membrane filtration, membrane separation, vapor phase over a copper oxide and Zinc oxide catalyst. reverse osmosis, electrodialysis, distillation, crystallization, British Pat. No. 1,230.276, which describes the hydrogena centrifugation, extractive filtration, ion exchange chroma tion of gamma-butyrolactone using a copper oxide-chro tography, size exclusion chromatography, adsorption chro mium oxide catalyst. The hydrogenation is carried out in the matography, and ultrafiltration. All of the above methods are liquid phase. Batch reactions also are exemplified having well known in the art. high total reactor pressures. Reactant and product partial (0259. The 14-BDO and GBL can be separated from other pressures in the reactors are well above the respective dew components in the culture using a variety of methods well points. British Pat. No. 1,314,126, which describes the known in the art. Such separation methods include, for hydrogenation of gamma-butyrolactone in the liquid phase example, extraction procedures as well as methods that over a nickel-cobalt-thorium oxide catalyst. Batch reactions include continuous liquid-liquid extraction, pervaporation, are exemplified as having high total pressures and compo membrane filtration, membrane separation, reverse osmosis, nent partial pressures well above respective component dew electrodialysis, distillation, crystallization, centrifugation, points. British Pat. No. 1,344,557, which describes the extractive filtration, ion exchange chromatography, size hydrogenation of gamma-butyrolactone in the liquid phase exclusion chromatography, adsorption chromatography, and over a copper oxide-chromium oxide catalyst. A vapor phase ultrafiltration. All of the above methods are well known in or vapor-containing mixed phase is indicated as Suitable in the art. Some instances. A continuous flow tubular reactor is exem 0260 Any of the non-naturally occurring microbial plified using high total reactor pressures. British Pat. No. organisms described herein can be cultured to produce 1,512,751, which describes the hydrogenation of gamma and/or secrete the biosynthetic products of the invention. For butyrolactone to 1,4-butanediol in the liquid phase over a example, the 14-BDO and GBL producers can be cultured copper oxide-chromium oxide catalyst. Batch reactions are for the biosynthetic production of 14-BDO and GBL. exemplified with high total reactor pressures and, where 0261) For the production of 14-BDO and GBL, the determinable, reactant and product partial pressures well recombinant strains are cultured in a medium with carbon above the respective dew points. U.S. Pat. No. 4.301,077. Source and other essential nutrients. It is sometimes desir which describes the hydrogenation to 1,4-butanediol of able and can be highly desirable to maintain anaerobic gamma-butyrolactone over a Ru-Ni-Co—Zn catalyst. conditions in the fermenter to reduce the cost of the overall The reaction can be conducted in the liquid or gas phase or process. Such conditions can be obtained, for example, by in a mixed liquid-gas phase. Exemplified are continuous first sparging the medium with nitrogen and then sealing the flow liquid phase reactions at high total reactor pressures flasks with a septum and crimp-cap. For strains where and relatively low reactor productivities. U.S. Pat. No. growth is not observed anaerobically, microaerobic or Sub 4,048,196, which describes the production of 1,4-butanediol stantially anaerobic conditions can be applied by perforating US 2016/03193 13 A1 Nov. 3, 2016 the septum with a small hole for limited aeration. Exemplary pounds such as acetate as long as hydrogen is present to anaerobic conditions have been described previously and are Supply the necessary reducing equivalents (see for example, well-known in the art. Exemplary aerobic and anaerobic Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New conditions are described, for example, in United State pub York, (1994)). This can be summarized by the following lication 2009/0047719, filed Aug. 10, 2007. Fermentations equation: can be performed in a batch, fed-batch or continuous man ner, as disclosed herein. 0262. If desired, the pH of the medium can be maintained 0267 Hence, non-naturally occurring microorganisms at a desired pH, in particular neutral pH, such as a pH of possessing the Wood-Ljungdahl pathway can utilize CO around 7 by addition of a base, such as NaOH or other bases, and H mixtures as well for the production of acetyl-CoA or acid, as needed to maintain the culture medium at a and other desired products. desirable pH. The growth rate can be determined by mea 0268. The Wood-Ljungdahl pathway is well known in the Suring optical density using a spectrophotometer (600 nm), art and consists of 12 reactions which can be separated into and the glucose uptake rate by monitoring carbon Source two branches: (1) methyl branch and (2) carbonyl branch. depletion over time. The methyl branch converts syngas to methyl-tetrahydrofo 0263. The growth medium can include, for example, any late (methyl-THF) whereas the carbonyl branch converts carbohydrate Source which can Supply a source of carbon to methyl-THF to acetyl-CoA. The reactions in the methyl the non-naturally occurring microorganism. Such sources branch are catalyzed in order by the following enzymes or include, for example, Sugars such as glucose, Xylose, arab proteins: ferredoxin , formate dehydroge inose, galactose, mannose, fructose, Sucrose and starch. nase, formyltetrahydrofolate synthetase, methenyltetrahy Other sources of carbohydrate include, for example, renew drofolate cyclodehydratase, methylenetetrahydrofolate able feedstocks and biomass. Exemplary types of biomasses dehydrogenase and methylenetetrahydrofolate reductase. that can be used as feedstocks in the methods of the The reactions in the carbonyl branch are catalyzed in order invention include cellulosic biomass, hemicellulosic bio by the following enzymes or proteins: methyltetrahydrofo mass and lignin feedstocks or portions of feedstocks. Such late:corrinoid protein methyltransferase (for example, biomass feedstocks contain, for example, carbohydrate Sub AcSE), corrinoid iron-sulfur protein, nickel-protein assem strates useful as carbon Sources such as glucose, Xylose, bly protein (for example, AcsF), ferredoxin, acetyl-CoA arabinose, galactose, mannose, fructose and starch. Given synthase, carbon monoxide dehydrogenase and nickel-pro the teachings and guidance provided herein, those skilled in tein assembly protein (for example, CooC). Following the the art will understand that renewable feedstocks and bio teachings and guidance provided herein for introducing a mass other than those exemplified above also can be used for Sufficient number of encoding nucleic acids to generate a culturing the microbial organisms of the invention for the 14-BDO and GBL pathway, those skilled in the art will production of 14-BDO and GBL. understand that the same engineering design also can be 0264. In addition to renewable feedstocks such as those performed with respect to introducing at least the nucleic exemplified above, the 14-BDO and GBL microbial organ acids encoding the Wood-Ljungdahl enzymes or proteins isms of the invention also can be modified for growth on absent in the host organism. Therefore, introduction of one syngas as its source of carbon. In this specific embodiment, or more encoding nucleic acids into the microbial organisms one or more proteins or enzymes are expressed in the of the invention Such that the modified organism contains the 14-BDO and GBL producing organisms to provide a meta complete Wood-Ljungdahl pathway will confer syngas ulti bolic pathway for utilization of syngas or other gaseous lization ability. carbon source. 0269. Additionally, the reductive (reverse) tricarboxylic 0265 Synthesis gas, also known as Syngas or producer acid cycle is and/or hydrogenase activities can also be used gas, is the major product of gasification of coal and of for the conversion of CO, CO2 and/or H2 to acetyl-CoA and carbonaceous materials such as biomass materials, including other products such as acetate. Organisms capable of fixing agricultural crops and residues. Syngas is a mixture primar carbon via the reductive TCA pathway can utilize one or ily of H and CO and can be obtained from the gasification more of the following enzymes: ATP citrate-, citrate of any organic feedstock, including but not limited to coal, lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglut coal oil, natural gas, biomass, and waste organic matter. arate:ferredoxin oxidoreductase, Succinyl-CoA synthetase, Gasification is generally carried out under a high fuel to Succinyl-CoA transferase, fumarate reductase, fumarase, oxygen ratio. Although largely H and CO, syngas can also malate dehydrogenase, NAD(P)H:ferredoxin oxidoreduc include CO and other gases in Smaller quantities. Thus, tase, carbon monoxide dehydrogenase, and hydrogenase. synthesis gas provides a cost effective source of gaseous Specifically, the reducing equivalents extracted from CO carbon such as CO and, additionally, CO. and/or H2 by carbon monoxide dehydrogenase and hydro 0266 The Wood-Ljungdahl pathway catalyzes the con genase are utilized to fix CO2 via the reductive TCA cycle version of CO and H to acetyl-CoA and other products such into acetyl-CoA or acetate. Acetate can be converted to as acetate. Organisms capable of utilizing CO and syngas acetyl-CoA by enzymes Such as acetyl-CoA transferase, also generally have the capability of utilizing CO and acetate kinase/phosphotransacetylase, and acetyl-CoA syn CO/H mixtures through the same basic set of enzymes and thetase. Acetyl-CoA can be converted to the 14-BDO or transformations encompassed by the Wood-Ljungdahl path GBL precursors, glyceraldehyde-3-phosphate, phospho way. H-dependent conversion of CO to acetate by micro enolpyruvate, and pyruvate, by pyruvate:ferredoxin oxi organisms was recognized long before it was revealed that doreductase and the enzymes of gluconeogenesis. Following CO also could be used by the same organisms and that the the teachings and guidance provided herein for introducing same pathways were involved. Many acetogens have been a sufficient number of encoding nucleic acids to generate a shown to grow in the presence of CO and produce com 14.BDO or GBL pathway, those skilled in the art will US 2016/03193 13 A1 Nov. 3, 2016 34 understand that the same engineering design also can be between and above each of these exemplary ranges also can performed with respect to introducing at least the nucleic be achieved from the non-naturally occurring microbial acids encoding the reductive TCA pathway enzymes or organisms of the invention. proteins absent in the host organism. Therefore, introduction 0272. In some embodiments, culture conditions include of one or more encoding nucleic acids into the microbial anaerobic or Substantially anaerobic growth or maintenance organisms of the invention Such that the modified organism conditions. Exemplary anaerobic conditions have been contains the complete reductive TCA pathway will confer described previously and are well known in the art. Exem syngas utilization ability. plary anaerobic conditions for fermentation processes are 0270. Accordingly, given the teachings and guidance described herein and are described, for example, in U.S. provided herein, those skilled in the art will understand that publication 2009/0047719, filed Aug. 10, 2007. Any of these a non-naturally occurring microbial organism can be pro conditions can be employed with the non-naturally occur duced that secretes the biosynthesized compounds of the ring microbial organisms as well as other anaerobic condi invention when grown on a carbon Source Such as a carbo tions well known in the art. Under such anaerobic or hydrate. Such compounds include, for example, 14-BDO, substantially anaerobic conditions, the 14-BDO and GBL GBL and any of the intermediate metabolites in the 14-BDO producers can synthesize 14-BDO and GBL at intracellular or GBL pathway. All that is required is to engineer in one or concentrations of 5-10 mM or more as well as all other more of the required enzyme or protein activities to achieve concentrations exemplified herein. It is understood that, biosynthesis of the desired compound or intermediate even though the above description refers to intracellular including, for example, inclusion of some or all of the concentrations, 14-BDO and GBL producing microbial 14-BDO and GBL biosynthetic pathways. Accordingly, the organisms can produce 14-BDO and GBL intracellularly invention provides a non-naturally occurring microbial and/or secrete the products into the culture medium. organism that produces and/or secretes 14-BDO and GBL 0273. In addition to the culturing and fermentation con when grown on a carbohydrate or other carbon source and ditions disclosed herein, growth condition for achieving produces and/or secretes any of the intermediate metabolites biosynthesis of 14-BDO and GBL can include the addition shown in the 14-BDO or GBL pathway when grown on a of an osmoprotectant to the culturing conditions. In certain carbohydrate or other carbon source. The 14-BDO and GBL embodiments, the non-naturally occurring microbial organ producing microbial organisms of the invention can initiate isms of the invention can be Sustained, cultured or fermented synthesis from an intermediate, for example, a Succinyl-CoA as described herein in the presence of an osmoprotectant. reductase (aldehyde forming), an alpha-ketoglutarate decar Briefly, an osmoprotectant refers to a compound that acts as boxylase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxy an osmolyte and helps a microbial organism as described butyrate reductase, a 1,4-butanediol dehydrogenase, a suc herein Survive osmotic stress. Osmoprotectants include, but cinate reductase, a Succinyl-CoA transferase, a Succinyl are not limited to, betaines, amino acids, and the Sugar CoA hydrolase, a Succinyl-CoA synthetase (or Succinyl-CoA trehalose. Non-limiting examples of Such are glycine ligase), a glutamate dehydrogenase, a glutamate transami betaine, praline betaine, dimethylthetin, dimethyl slfonio nase, a glutamate decarboxylase, a 4-aminobutyrate dehy proprionate, 3-dimethylsulfonio-2-methylproprionate, pipe drogenase, a 4-aminobutyrate transaminase, a 4-hydroxybu colic acid, dimethylsulfonioacetate, choline, L-carnitine and tyrate kinase, a phosphotrans-4-hydroxybutyrylase, a ectoine. In one aspect, the osmoprotectant is glycine betaine. 4-hydroxybutyryl-CoA reductase (aldehyde forming), a It is understood to one of ordinary skill in the art that the 4-hydroxybutyryl-phosphate reductase, a Succinyl-CoA amount and type of osmoprotectant Suitable for protecting a reductase (alcohol forming), a 4-hydroxybutyryl-CoA trans microbial organism described herein from osmotic stress ferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybu will depend on the microbial organism used. The amount of tyryl-CoA synthetase (or 4-hydroxybutyryl-CoA ligase), a osmoprotectant in the culturing conditions can be, for 4-hydroxybutyryl-CoA reductase (alcohol forming), an example, no more than about 0.1 mM, no more than about alpha-ketoglutarate reductase, a 5-hydroxy-2-oxopentanoate 0.5 mM, no more than about 1.0 mM, no more than about 1.5 dehydrogenase, a 5-hydroxy-2-oxopentanoate decarboxy mM, no more than about 2.0 mM, no more than about 2.5 lase, a 5-hydroxy-2-oxopentanoate dehydrogenase (decar mM, no more than about 3.0 mM, no more than about 5.0 boxylation) or a 4-hydroxybutyrate lactonase. mM, no more than about 7.0 mM, no more than about 10 0271 The non-naturally occurring microbial organisms mM, no more than about 50 mM, no more than about 100 of the invention are constructed using methods well known mM or no more than about 500 mM. in the art as exemplified herein to exogenously express at 0274 The culture conditions can include, for example, least one nucleic acid encoding a 14-BDO and GBL pathway liquid culture procedures as well as fermentation and other enzyme or protein in sufficient amounts to produce 14-BDO large scale culture procedures. As described herein, particu and GBL. It is understood that the microbial organisms of larly useful yields of the biosynthetic products of the inven the invention are cultured under conditions sufficient to tion can be obtained under anaerobic or substantially anaero produce 14-BDO and GBL. Following the teachings and bic culture conditions. guidance provided herein, the non-naturally occurring 0275. As described herein, one exemplary growth con microbial organisms of the invention can achieve biosyn dition for achieving biosynthesis of 14-BDO and GBL thesis of 14-BDO and GBL resulting in intracellular con includes anaerobic culture or fermentation conditions. In centrations between about 0.1-200 mM or more. Generally, certain embodiments, the non-naturally occurring microbial the intracellular concentration of 14-BDO and GBL is organisms of the invention can be sustained, cultured or between about 0.01-1000 mM, particularly 3-150 mM, more fermented under anaerobic or Substantially anaerobic con particularly between about 5-125 mM and more particularly ditions. Briefly, anaerobic conditions refers to an environ between about 8-100 mM, including about 10 mM, 20 mM, ment devoid of oxygen. Substantially anaerobic conditions 50 mM, 80 mM, or more. Intracellular concentrations include, for example, a culture, batch fermentation or con US 2016/03193 13 A1 Nov. 3, 2016

tinuous fermentation such that the dissolved oxygen con 4-hydroxybutyryl-CoA are secreated, the spontaneous lac centration in the medium remains between 0 and 10% of tonization of these products can occur extracellularly. saturation. Substantially anaerobic conditions also includes 0280. To generate better producers, metabolic modeling growing or resting cells in liquid medium or on Solid agar can be utilized to optimize growth conditions. Modeling can inside a sealed chamber maintained with an atmosphere of also be used to design gene knockouts that additionally less than 1% oxygen. The percent of oxygen can be main optimize utilization of the pathway (see, for example, U.S. tained by, for example, sparging the culture with an N2/CO patent publications US 2002/0012939, US 2003/0224363, mixture or other Suitable non-oxygen gas or gases. US 2004/0029149, US 2004/0072723, US 2003/0059792, 0276. The culture conditions described herein can be US 2002/0168654 and US 2004/000.9466, and U.S. Pat. No. scaled up and grown continuously for manufacturing of 7,127.379). Modeling analysis allows reliable predictions of 14-BDO and GBL. Exemplary growth procedures include, the effects on cell growth of shifting the metabolism towards for example, fed-batch fermentation and batch separation; more efficient production of 14-BDO and GBL. fed-batch fermentation and continuous separation, or con 0281 One computational method for identifying and tinuous fermentation and continuous separation. All of these designing metabolic alterations favoring biosynthesis of a processes are well known in the art. Fermentation proce desired product is the OptKnock computational framework dures are particularly useful for the biosynthetic production (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)). of commercial quantities of 14-BDO and GBL. Generally, OptKnock is a metabolic modeling and simulation program and as with non-continuous culture procedures, the continu that Suggests gene deletion or disruption strategies that result ous and/or near-continuous production of 14-BDO and GBL in genetically stable microorganisms which overproduce the will include culturing a non-naturally occurring 14-BDO target product. Specifically, the framework examines the and GBL producing organism of the invention in Sufficient complete metabolic and/or biochemical network of a micro nutrients and medium to Sustain and/or nearly Sustain organism in order to suggest genetic manipulations that growth in an exponential phase. Continuous culture under force the desired biochemical to become an obligatory Such conditions can be include, for example, growth for 1 byproduct of cell growth. By coupling biochemical produc day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous tion with cell growth through Strategically placed gene culture can include longer time periods of 1 week, 2, 3, 4 or deletions or other functional gene disruption, the growth 5 or more weeks and up to several months. Alternatively, selection pressures imposed on the engineered strains after organisms of the invention can be cultured for hours, if long periods of time in a bioreactor lead to improvements in suitable for a particular application. It is to be understood performance as a result of the compulsory growth-coupled that the continuous and/or near-continuous culture condi biochemical production. Lastly, when gene deletions are tions also can include all time intervals in between these constructed there is a negligible possibility of the designed exemplary periods. It is further understood that the time of strains reverting to their wild-type states because the genes culturing the microbial organism of the invention is for a selected by OptKnock are to be completely removed from sufficient period of time to produce a sufficient amount of the genome. Therefore, this computational methodology can product for a desired purpose. be used to either identify alternative pathways that lead to 0277 Fermentation procedures are well known in the art. biosynthesis of a desired product or used in connection with Briefly, fermentation for the biosynthetic production of the non-naturally occurring microbial organisms for further 14-BDO and GBL can be utilized in, for example, fed-batch optimization of biosynthesis of a desired product. fermentation and batch separation; fed-batch fermentation 0282 Briefly, OptKnock is a term used herein to refer to and continuous separation, or continuous fermentation and a computational method and system for modeling cellular continuous separation. Examples of batch and continuous metabolism. The OptKnock program relates to a framework fermentation procedures are well known in the art. of models and methods that incorporate particular con straints into flux balance analysis (FBA) models. These 0278. In addition to the above fermentation procedures constraints include, for example, qualitative kinetic infor using the 14-BDO and GBL producers of the invention for mation, qualitative regulatory information, and/or DNA continuous production of substantial quantities of 14-BDO microarray experimental data. OptKnock also computes and GBL, the 14-BDO and GBL producers also can be, for Solutions to various metabolic problems by, for example, example, simultaneously Subjected to chemical synthesis tightening the flux boundaries derived through flux balance procedures to convert the product to other compounds or the models and Subsequently probing the performance limits of product can be separated from the fermentation culture and metabolic networks in the presence of gene additions or sequentially Subjected to chemical conversion to convert the deletions. OptKnock computational framework allows the product to other compounds, if desired. construction of model formulations that allow an effective 0279. In addition to the above fermentation procedures query of the performance limits of metabolic networks and using the 14-BDO and GBL producers of the invention for provides methods for solving the resulting mixed-integer continuous production of substantial quantities of 14-BDO linear programming problems. The metabolic modeling and and GBL, 14-BDO and 4-hydroxybutyryl-phosphate pro simulation methods referred to herein as OptKnock are ducers or 14-BDO and 4-hydroxybutyryl-CoA producers described in, for example, U.S. publication 2002/0168654, also can be, for example, simultaneously subjected to spon filed Jan. 10, 2002, in International Patent No. PCT/US02/ taneous lactonization procedures to convert the products 00660, filed Jan. 10, 2002, and U.S. publication 2009/ 4-hydroxybutyryl-phosphate or 4-hydroxybutyryl-CoA to 0047719, filed Aug. 10, 2007. GBL as described herein. In some aspects, the spontaneous 0283 Another computational method for identifying and lactonization of 4-hydroxybutyryl-phosphate or 4-hydroxy designing metabolic alterations favoring biosynthetic pro butyryl-CoA to form GBL can occur intracellularly. Alter duction of a product is a metabolic modeling and simulation natively, if the products 4-hydroxybutyryl-phosphate or system termed SimPheny(R). This computational method and US 2016/03193 13 A1 Nov. 3, 2016 36 system is described in, for example, U.S. publication 2003/ deletion of each encoding gene. However, in some instances, 0233218, filed Jun. 14, 2002, and in International Patent it can be beneficial to disrupt the reaction by other genetic Application No. PCT/US03/18838, filed Jun. 13, 2003. aberrations including, for example, mutation, deletion of SimPheny(R) is a computational system that can be used to regulatory regions such as promoters or cis binding sites for produce a network model in silico and to simulate the flux regulatory factors, or by truncation of the coding sequence of mass, energy or charge through the chemical reactions of at any of a number of locations. These latter aberrations, a biological system to define a solution space that contains resulting in less than total deletion of the gene set can be any and all possible functionalities of the chemical reactions useful, for example, when rapid assessments of the coupling in the system, thereby determining a range of allowed of a product are desired or when genetic reversion is less activities for the biological system. This approach is referred likely to occur. to as constraints-based modeling because the Solution space 0288 To identify additional productive solutions to the is defined by constraints such as the known Stoichiometry of above described bilevel OptKnock problem which lead to the included reactions as well as reaction thermodynamic further sets of reactions to disrupt or metabolic modifica and capacity constraints associated with maximum fluxes tions that can result in the biosynthesis, including growth through reactions. The space defined by these constraints coupled biosynthesis of a desired product, an optimization can be interrogated to determine the phenotypic capabilities method, termed integer cuts, can be implemented. This and behavior of the biological system or of its biochemical method proceeds by iteratively solving the Optiknock prob components. lem exemplified above with the incorporation of an addi 0284. These computational approaches are consistent tional constraint referred to as an integer cut at each itera with biological realities because biological systems are tion. Integer cut constraints effectively prevent the Solution flexible and can reach the same result in many different procedure from choosing the exact same set of reactions ways. Biological systems are designed through evolutionary identified in any previous iteration that obligatorily couples mechanisms that have been restricted by fundamental con product biosynthesis to growth. For example, if a previously straints that all living systems must face. Therefore, con identified growth-coupled metabolic modification specifies straints-based modeling strategy embraces these general reactions 1, 2, and 3 for disruption, then the following realities. Further, the ability to continuously impose further constraint prevents the same reactions from being simulta restrictions on a network model via the tightening of con neously considered in Subsequent solutions. The integer cut straints results in a reduction in the size of the Solution space, method is well known in the art and can be found described thereby enhancing the precision with which physiological in, for example, Burgard et al., Biotechnol. Prog. 17:791 performance or phenotype can be predicted. 797 (2001). As with all methods described herein with 0285) Given the teachings and guidance provided herein, reference to their use in combination with the OptKnock those skilled in the art will be able to apply various com computational framework for metabolic modeling and simu putational frameworks for metabolic modeling and simula lation, the integer cut method of reducing redundancy in tion to design and implement biosynthesis of a desired iterative computational analysis also can be applied with compound in host microbial organisms. Such metabolic other computational frameworks well known in the art modeling and simulation methods include, for example, the including, for example, SimPheny(R). computational systems exemplified above as SimPheny(R) 0289. The methods exemplified herein allow the con and OptKnock. For illustration of the invention, some meth struction of cells and organisms that biosynthetically pro ods are described herein with reference to the OptKnock duce a desired product, including the obligatory coupling of computation framework for modeling and simulation. Those production of a target biochemical product to growth of the skilled in the art will know how to apply the identification, cell or organism engineered to harbor the identified genetic design and implementation of the metabolic alterations alterations. Therefore, the computational methods described using OptKnock to any of Such other metabolic modeling herein allow the identification and implementation of meta and simulation computational frameworks and methods well bolic modifications that are identified by an in silico method known in the art. selected from OptKnock or SimPheny(R). The set of meta 0286 The methods described above will provide one set bolic modifications can include, for example, addition of one of metabolic reactions to disrupt. Elimination of each reac or more biosynthetic pathway enzymes and/or functional tion within the set or metabolic modification can result in a disruption of one or more metabolic reactions including, for desired product as an obligatory product during the growth example, disruption by gene deletion. phase of the organism. Because the reactions are known, a 0290. As discussed above, the OptKnock methodology solution to the bilevel OptKnock problem also will provide was developed on the premise that mutant microbial net the associated gene or genes encoding one or more enzymes works can be evolved towards their computationally pre that catalyze each reaction within the set of reactions. dicted maximum-growth phenotypes when subjected to long Identification of a set of reactions and their corresponding periods of growth selection. In other words, the approach genes encoding the enzymes participating in each reaction is leverages an organism’s ability to self-optimize under selec generally an automated process, accomplished through cor tive pressures. The OptKnock framework allows for the relation of the reactions with a reaction database having a exhaustive enumeration of gene deletion combinations that relationship between enzymes and encoding genes. force a coupling between biochemical production and cell 0287. Once identified, the set of reactions that are to be growth based on network stoichiometry. The identification disrupted in order to achieve production of a desired product of optimal gene/reaction knockouts requires the solution of are implemented in the target cell or organism by functional a bilevel optimization problem that chooses the set of active disruption of at least one gene encoding each metabolic reactions such that an optimal growth solution for the reaction within the set. One particularly useful means to resulting network overproduces the biochemical of interest achieve functional disruption of the reaction set is by (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)). US 2016/03193 13 A1 Nov. 3, 2016 37

0291. An in silico stoichiometric model of E. coli levels, to increase protein yields and overall pathway flux: metabolism can be employed to identify essential genes for oxygen stability, for operation of air sensitive enzymes metabolic pathways as exemplified previously and described under aerobic conditions; and anaerobic activity, for opera in, for example, U.S. patent publications US 2002/0012939, tion of an aerobic enzyme in the absence of oxygen. US 2003/0224363, US 2004/0029149, US 2004/0072723, 0294. A number of exemplary methods have been devel US 2003/0059792, US 2002/0168654 and US 2004/ oped for the mutagenesis and diversification of genes to 0009466, and in U.S. Pat. No. 7,127,379. As disclosed target desired properties of specific enzymes. Such methods herein, the OptKnock mathematical framework can be are well known to those skilled in the art. Any of these can applied to pinpoint gene deletions leading to the growth be used to alter and/or optimize the activity of a 14-BDO or coupled production of a desired product. Further, the solu GBL pathway enzyme or protein. Such methods include, but tion of the bilevel Optiknock problem provides only one set are not limited to EpiPCR, which introduces random point of deletions. To enumerate all meaningful solutions, that is, mutations by reducing the fidelity of DNA polymerase in all sets of knockouts leading to growth-coupled production PCR reactions (Pritchard et al., J Theor: Biol. 234:497-509 formation, an optimization technique, termed integer cuts, (2005)); Error-prone Rolling Circle Amplification (epRCA), can be implemented. This entails iteratively solving the which is similar to epPCR except a whole circular plasmid OptKnock problem with the incorporation of an additional is used as the template and random 6-mers with exonuclease constraint referred to as an integer cut at each iteration, as resistant thiophosphate linkages on the last 2 nucleotides are discussed above. used to amplify the plasmid followed by transformation into 0292. As disclosed herein, a nucleic acid encoding a cells in which the plasmid is re-circularized at tandem desired activity of a 14-BDO or GBL pathway can be repeats (Fujii et al., Nucleic Acids Res. 32:e 145 (2004); and introduced into a host organism. In some cases, it can be Fujii et al., Nat. Protoc. 1:2493-2497 (2006)); DNA or desirable to modify an activity of a 14-BDO or GBL Family Shuffling, which typically involves digestion of two pathway enzyme or protein to increase production of or more variant genes with nucleases Such as Dnase I or 14-BDO and GBL. For example, known mutations that EndoW to generate a pool of random fragments that are increase the activity of a protein or enzyme can be intro reassembled by cycles of annealing and extension in the duced into an encoding nucleic acid molecule. Additionally, presence of DNA polymerase to create a library of chimeric optimization methods can be applied to increase the activity genes (Stemmer, Proc Natl AcadSci USA 91: 10747-10751 of an enzyme or protein and/or decrease an inhibitory (1994); and Stemmer, Nature 370:389-391 (1994)); Stag activity, for example, decrease the activity of a negative gered Extension (StEP), which entails template priming regulator. followed by repeated cycles of 2 step PCR with denaturation 0293. One such optimization method is directed evolu and very short duration of annealing/extension (as short as tion. Directed evolution is a powerful approach that involves 5 sec) (Zhao et al., Nat. Biotechnol. 16:258-261 (1998)); the introduction of mutations targeted to a specific gene in Random Priming Recombination (RPR), in which random order to improve and/or alter the properties of an enzyme. sequence primers are used to generate many short DNA Improved and/or altered enzymes can be identified through fragments complementary to different segments of the tem the development and implementation of sensitive high plate (Shao et al., Nucleic Acids Res 26:681-683 (1998)). throughput screening assays that allow the automated 0295 Additional methods include Heteroduplex Recom screening of many enzyme variants (for example, >10). bination, in which linearized plasmid DNA is used to form Iterative rounds of mutagenesis and screening typically are heteroduplexes that are repaired by mismatch repair (Volkov performed to afford an enzyme with optimized properties. et al. Nucleic Acids Res. 27:e18 (1999); and Volkov et al., Computational algorithms that can help to identify areas of Methods Enzymol. 328:456-463 (2000)); Random Chime the gene for mutagenesis also have been developed and can ragenesis on Transient Templates (RACHITT), which significantly reduce the number of enzyme variants that need employs Dnase I fragmentation and size fractionation of to be generated and screened. Numerous directed evolution single stranded DNA (ssDNA) (Coco et al., Nat. Biotechnol. technologies have been developed (for reviews, see Hibbert 19:354-359 (2001)); Recombined Extension on Truncated et al., Biomol. Eng 22:11-19 (2005); Huisman and Lalonde, templates (RETT), which entails template switching of In Biocatalysis in the pharmaceutical and biotechnology unidirectionally growing strands from primers in the pres industries pgs. 717-742 (2007), Patel (ed.), CRC Press: ence of unidirectional ssDNA fragments used as a pool of Otten and Quax. Biomol. Eng 22:1-9 (2005).; and Sen et al., templates (Lee et al., J. Molec. 26:119-129 Appl Biochem. Biotechnol 143:212-223 (2007)) to be effec (2003)); Degenerate Oligonucleotide Gene Shuffling tive at creating diverse variant libraries, and these methods (DOGS), in which degenerate primers are used to control have been successfully applied to the improvement of a wide recombination between molecules: (Bergquist and Gibbs, range of properties across many enzyme classes. Enzyme Methods Mol. Biol 352:191-204 (2007); Bergquist et al., characteristics that have been improved and/or altered by Biomol. Eng 22:63-72 (2005): Gibbs et al., Gene 271:13-20 directed evolution technologies include, for example: selec (2001)); Incremental Truncation for the Creation of Hybrid tivity/specificity, for conversion of non-natural Substrates; Enzymes (ITCHY), which creates a combinatorial library temperature stability, for robust high temperature process with 1 base pair deletions of a gene or gene fragment of ing; pH stability, for bioprocessing under lower or higher pH interest (Ostermeier et al., Proc. Natl. Acad. Sci. USA conditions; Substrate or product tolerance, so that high 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotech product titers can be achieved; binding (K), including mol. 17:1205-1209 (1999)); Thio-Incremental Truncation for broadening Substrate binding to include non-natural Sub the Creation of Hybrid Enzymes (THIO-ITCHY), which is strates; inhibition (K), to remove inhibition by products, similar to ITCHY except that phosphothioate dNTPs are Substrates, or key intermediates; activity (kcat), to increases used to generate truncations (Lutz et al., Nucleic Acids Res enzymatic reaction rates to achieve desired flux, expression 29:E16 (2001)); SCRATCHY, which combines two methods US 2016/03193 13 A1 Nov. 3, 2016 for recombining genes, ITCHY and DNA shuffling (Lutz et Automation (PDA), which is an optimization algorithm that al., Proc. Natl. Acad. Sci. USA 98:11248-11253 (2001)); anchors the structurally defined protein backbone possessing Random Drift Mutagenesis (RNDM), in which mutations a particular fold, and searches sequence space for amino acid made via epPCR are followed by screening/selection for substitutions that can stabilize the fold and overall protein those retaining usable activity (Bergquist et al., Biomol. Eng. energetics, and generally works most effectively on proteins 22:63-72 (2005)); Sequence Saturation Mutagenesis with known three-dimensional structures (Hayes et al., Proc. (SeSaM), a random mutagenesis method that generates a Natl. Acad. Sci. USA 99:15926-15931 (2002)); and Iterative pool of random length fragments using random incorpora Saturation Mutagenesis (ISM), which involves using knowl tion of a phosphothioate nucleotide and cleavage, which is edge of structure/function to choose a likely site for enzyme used as a template to extend in the presence of “universal improvement, performing Saturation mutagenesis at chosen bases such as inosine, and replication of an inosine-contain site using a mutagenesis method such as Stratagene ing complement gives random base incorporation and, con QuikChange (Stratagene; San Diego Calif.), Screening/se sequently, mutagenesis (Wong et al., Biotechnol. J. 3:74-82 lecting for desired properties, and, using improved clone(s), (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and starting over at another site and continue repeating until a Wong et al., Anal. Biochem. 341:187-189 (2005)); Synthetic desired activity is achieved (Reetz et al., Nat. Protoc. Shuffling, which uses overlapping oligonucleotides 2:891-903 (2007); and Reetz et al., Angew. Chem. Int. Ed designed to encode “all genetic diversity in targets and Engl. 45:7745-7751 (2006)). allows a very high diversity for the shuffled progeny (Ness 0298 Any of the aforementioned methods for mutagen et al., Nat. Biotechnol. 20:1251-1255 (2002)); Nucleotide esis can be used alone or in any combination. Additionally, Exchange and Excision Technology NeXT, which exploits a any one or combination of the directed evolution methods combination of dUTP incorporation followed by treatment can be used in conjunction with adaptive evolution tech with uracil DNA glycosylase and then piperidine to perform niques, as described herein. endpoint DNA fragmentation (Muller et al., Nucleic Acids 0299. It is understood that modifications which do not Res. 33:e117 (2005)). substantially affect the activity of the various embodiments 0296. Further methods include Sequence Homology-In of this invention are also provided within the definition of dependent Protein Recombination (SHIPREC), in which a the invention provided herein. Accordingly, the following linker is used to facilitate fusion between two distantly examples are intended to illustrate but not limit the present related or unrelated genes, and a range of chimeras is invention. generated between the two genes, resulting in libraries of single-crossover hybrids (Sieber et al., Nat. Biotechnol. Example I 19:456-460 (2001)); Gene Site Saturation MutagenesisTM (GSSMTM), in which the starting materials include a super Pathways for Co-Production of 14-BDO and GBL coiled double stranded DNA (dsDNA) plasmid containing from Succinate and Alpha-Ketoclutarate an insert and two primers which are degenerate at the desired (0300. The 14-BDO and GBL coproduction pathways are site of mutations (Kretz et al., Methods Enzymol. 388:3-11 exemplified in FIG.1. As shown in FIG. 1A, central metabo (2004)); Combinatorial Cassette Mutagenesis (CCM), lism intermediates can be first channeled into alpha-keto which involves the use of short oligonucleotide cassettes to glutarate or Succinate through the oxidative and/or the replace limited regions with a large number of possible reductive branches of the TCA cycle. Alpha-ketoglutarate or amino acid sequence alterations (Reidhaar-Olson et al. Succinate can then be converted to 4-hydroxybutyrate Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson (4-HB) through different routes. When starting from alpha et al. Science 241:53-57 (1988)); Combinatorial Multiple ketoglutarate, it can be converted to Succinic semialdehyde Cassette Mutagenesis (CMCM), which is essentially similar through a single transformation (Step B in FIG. 1A) or to CCM and uses epPCR at high mutation rate to identify hot multiple-step conversions (Steps J or K plus Step L plus spots and hot regions and then extension by CMCM to cover Steps M or N in FIG. 1B), and then succinic semialdehyde a defined region of protein sequence space (Reetz et al., can be converted to 4-HB through Step C. When starting Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the from Succinate, it can be converted to Succinyl-CoA (Steps Mutator Strains technique, in which conditional is mutator G or H or I) then to succinic semialdehyde (Step A) then plasmids, utilizing the mutD5 gene, which encodes a mutant further to 4-HB (Step C). Alternatively, succinate can be subunit of DNA polymerase III, to allow increases of 20 to directly converted to succinic semialdehyde (Step F) or 4000-X in random and natural mutation frequency during succinyl-CoA can be directly converted to 4-HB (Step S). selection and block accumulation of deleterious mutations 0301 4-HB is an important intermediate for both when selection is not required (Selifonova et al., Appl. 14-BDO and GBL production. For 14-BDO production, Environ. Microbiol. 67:3645-3649 (2001)); Low et al., J. 4-HB can be converted to 4-hydroxybutyrul-phosphate Mol. Biol. 260:359-3680 (1996)). (Step O) then to 4-hydroxybutyryl-CoA (Step P) then to 0297. Additional exemplary methods include Look 4-hydroxybutanal (Step Q) and finally to 14-BDO (Step E). Through Mutagenesis (LTM), which is a multidimensional There are several possible alternative routes for the conver mutagenesis method that assesses and optimizes combina sion from 4-HB to 14-BDO, such as the direct conversions torial mutations of selected amino acids (Rajpal et al., Proc. from 4-HB to 4-hydroxybutanal (Step D), or from 4-hy Natl. Acad. Sci. USA 102:8466-8471 (2005)); Gene Reas droxybutyryl-phosphate to 4-hydroxylbutanal (Step R), or sembly, which is a DNA shuffling method that can be from 4-HB to 4-hydrobutyryl-CoA (Steps T or U or V), or applied to multiple genes at one time or to create a large from 4-hydrobutyryl-CoA to 14-BDO (Step W). Addition library of chimeras (multiple mutations) of a single gene ally, alpha-ketoglutarate can be converted to 2,5-dioxopen (Tunable GeneReassembly TM (TGRTM) Technology sup tanoic acid (Step X), then to 5-hydroxy-2-oxopentanoic acid plied by Verenium Corporation), in Silico Protein Design (Step Y), then further to 14-BDO (Step Z). An alternative US 2016/03193 13 A1 Nov. 3, 2016 39 route for this pathway will transform 5-hydroxy-2-oxopen TABLE 5 tanoic acid to 4-hydroxybutyryl-CoA (Step AA) that will be further converted to 14-BDO as described herein. Classes of Enzyme Transformations Depicted in FIG. 1 0302 For GBL production, 4-HB can be directly con LABEL FUNCTION verted to GBL through Step AB, or both 4-hydroxybutyryl 1.1.1.a Oxidoreductase (Oxo to alcohol) phosphate and 4-hydrobutyryl-CoA can be converted to 1.1.1.c Oxidoreductase (2 step, acyl-CoA to alcohol) GBL by non-enzymatic transformations (Step AC and Step 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde) 1.2.1.c Oxidoreductase (2-oxoacid to acyl-CoA, AD). decarboxylation) 1.2.1.d Oxidoreductase (phosphonate reductase) 0303 Under anaerobic growth conditions, the production 1.2.1.e. Acid reductase of 14-BDO and GBL using the above describe pathways can 1.4.1.a Oxidoreductase (aminating) be redox-balanced. The coproduction strain can produce 2.3.1.a. Acyltransferase (transferring phosphate group to CoA) both 14-BDO and GBL at different ratios based on the 2.6.1.a. Aminotransferase 2.7.2.a. Phosphotransferase (carboxy acceptor) activities of pathways for 14-BDO and GBL, which can be 2.8.3.a. CoA transferase adjust using metabolic engineering techniques. Therefore, 3.1.1.a Hydroxyacylhydrolase the ratios of 14-BDO and GBL can be modified in engi 3.1.2.a. CoA hydrolase neered coproduction strains. The maximum theoretical 4.1.1.a. Carboxy-lyase yields of 14-BDO and GBL in a coproduction strain can be 6.2.1.a. CoA synthetase found based on the following equation: Glucose->1.09a 14-BDO--1.33b GBL+(1.64a--0.67b) CO2+(0.55a-2b)H2O 1.1.1.a Oxidoreductase (Oxo to Alcohol) where a is the molar percentage of glucose converted to 14-BDO, and b is the percentage of glucose converted to 0308 Aldehyde to Alcohol. GBL, and a +b=100% 0309 Three transformations described in FIG. 1 involve 0304 For example, if 90% glucose is converted to the conversion of an aldehyde to alcohol. These are 4-hy 14-BDO and 10% glucose is converted to GBL, the maxi droxybutyrate dehydrogenase (Step C), 1,4-butanediol mum theoretical yields, in a coproduction strain, will be dehydrogenase (Step E), and 5-hydroxy-2-oxopentanoate 0.981 moles 14-BDO and 0.133 moles GBL per mole dehydrogenase (Step Y). Exemplary genes encoding glucose consumed. In another example, if 70% glucose is enzymes that catalyze the conversion of an aldehyde to converted to 14-BDO and 30% glucose is converted to GBL, alcohol, that is, alcohol dehydrogenase or equivalently alde the maximum theoretical yields, in a coproduction strain, hyde reductase, include alrA encoding a medium-chain will be 0.763 moles 14-BDO and 0.4 moles GBL per mole alcohol dehydrogenase for C2-C14 (Tani et al. Appl. Envi glucose consumed. ron. Microbiol. 66:5231-5235 (2000)), ADH2 from Saccha 0305 For 14-BDO and GBL coproduction, all routes can romyces cerevisiae (Atsumi et al. Nature 451:86-89 (2008)), lead to high theoretical yields for combined 14-BDO and ychD from E. coli which has preference for molecules GBL assuming glucose as the carbon Source. Similar high longer than C3 (Sulzenbacher et al. Journal of Molecular theoretical yields can be obtained from additional substrates Biology 342:489-502 (2004)), and bdh I and bdh II from C. including Sucrose, Xylose, arabinose, synthesis gas, among acetobutyllicum which converts butyrylaldehyde into butanol many others. (Walter et al. Journal of Bacteriology 174:7149-7158 (1992)). The protein sequences for each of these exemplary gene products, if available, can be found using the following Example II GenBank accession numbers shown in Table 6.

Enzyme Classification System for Co-Production of TABLE 6 14-BDO and GBL Gene Accession No. GI No. Organism 0306 This example describes the enzyme classification alrA BAB12273.1 9967138 Acinetobacter sp. Strain M-1 system for the exemplary pathways described in Example I ADH2 NP 014032.1 6323961 Saccharomyces cerevisiae for production of 1,4-butanediol (14-BDO) and gamma ych) NP 417484.1 16130909 Escherichia coi butyrolactone (GBL). All transformations depicted in FIGS. bdh I NP 349892.1 15896543 Clostridium acetobutyllicum 1A and 1B fall into the general categories of transformations bdh II NP 349891.1 15896542 Clostridium acetobutyllicum shown in Table 5. Below is described a number of biochemi cally characterized genes in each category. Specifically 0310 Enzymes exhibiting 4-hydroxybutyrate dehydroge listed are genes that can be applied to catalyze the appro nase activity (EC 1.1.1.61) also fall into this category. Such priate transformations in FIGS. 1A and 1B when properly enzymes have been characterized in Ralstonia eutropha cloned and expressed. (Bravo et al. J. Forensic Sci. 49:379-387 (2004), 0307 Table 5 shows the enzyme types useful to convert Clostridium kluyveri (Wolfetal. Protein Expr: Purif. 6:206 common central metabolic intermediates into 14-BDO and 212 (1995)) and Arabidopsis thaliana (Breitkreuz et al. J. GBL. The first three digits of each label correspond to the Biol. Chem. 278:41552-41556 (2003)). The protein first three digits which denote sequences for each of these exemplary gene products, if the general type of transformation independent of Substrate available, can be found using the following GenBank acces specificity. sion numbers shown in Table 7. US 2016/03193 13 A1 Nov. 3, 2016 40

TABLE 7 identified (Gokam et al., U.S. Pat. No. 7,393,676; Liao et al., US Publication No. 2005/0221466). Additional gene candi Gene Accession No. GI No. Organism dates from other organisms including Rhodobacter 113867564 Ralstonia eutropha H16 spaeroides can be inferred by sequence similarity. The 146348486 Clostridium kluyveri DSM 555 protein sequences for each of these exemplary gene prod 752498.05 Arabidopsis thaliana ucts, if available, can be found using the following GenBank accession numbers shown in Table 10.

0311. The adh1 gene from Geobacillus thermoglucosi TABLE 10 dasius M10EXG (Jeon et al., J Biotechnol 135:127-133 (2008)) was shown to exhibit high activity on both 4-hy Gene Accession No. GI No. Organism droxybutanal and butanal as shown in Example VIII. Thus mmsB AAA25892.1 151363 Pseudomonas aeruginosa this enzyme exhibits 1,4-butanediol dehydrogenase activity. mmsB NP 252259.1 15598765 Pseudomonas aeruginosa PAO1 The protein sequence for the exemplary gene product, if mmsB NP 746775.1 26991350 Pseudomonas putida KT2440 available, can be found using the following GenBank acces mmsB JCT926 60729613 Pseudomonas putida E23 sion numbers shown in Table 8. orfB1 AAL26884 16588720 Rhodobacter spaeroides

TABLE 8 0314. The conversion of malonic semialdehyde to 3-HP Gene Accession No. GI No. Organism can also be accomplished by two other enzymes: NADH dependent 3-hydroxypropionate dehydrogenase and adh1 AAR91477.1 40795.502 Geobacilius NADPH-dependent malonate semialdehyde reductase. An thermoglucosidasius M1OEXG NADH-dependent 3-hydroxypropionate dehydrogenase is thought to participate in beta-alanine biosynthesis pathways 0312 Another exemplary enzyme is 3-hydroxyisobu from propionate in bacteria and plants (Rathinasabapathi, B. tyrate dehydrogenase which catalyzes the reversible oxida Journal of Plant Pathology 159:671-674 (2002); Stadtman, tion of 3-hydroxyisobutyrate to methylmalonate semialde E. R. J. Am. Chem. Soc. 77:5765-5766 (1955)). This enzyme hyde. This enzyme participates in Valine, leucine and has not been associated with a gene in any organism to date. isoleucine degradation and has been identified in bacteria, NADPH-dependent malonate semialdehyde reductase cata eukaryotes, and mammals. The enzyme encoded by P84067 from Thermus thermophilus HB8 has been structurally char lyzes the reverse reaction in autotrophic CO-fixing bacteria. acterized (Lokanath et al. J Mol Biol 352:905-17 (2005)). Although the enzyme activity has been detected in Metal The reversibility of the human 3-hydroxyisobutyrate dehy losphaera sedulla, the identity of the gene is not known drogenase was demonstrated using isotopically-labeled Sub (Alber et al. J. Bacteriol. 188:8551-8559 (2006)). strate (Manning et al. Biochem J 231:481-484 (1985)). Additional genes encoding this enzyme include 3hidh in 1.1.1.c Oxidoreductase (2 Step, Acyl-CoA to Alcohol) Homo sapiens (Hawes et al. Methods Enzymol. 324:218-228 (2000)) and Oryctolagus cuniculus (Chowdhury et al. Bio 0315 Steps S and W of FIG. 1 depict bifunctional reduc sci. Biotechnol Biochem. 60:2043-2047 (1996); Hawes et al. tase enzymes that can form 4-hydroxybutyrate and 1,4- Methods Enzymol. 324:218-228 (2000)), mmsb in butanediol from succinyl-CoA and 4-hydroxybutyryl-CoA, Pseudomonas aeruginosa, and dhat in Pseudomonas putida respectively. Exemplary 2-step oxidoreductases that convert (Aberhartet al...J Chem. Soc. (Perkin16:1404-1406 (1979); an acyl-CoA to alcohol include those that transform sub Chowdhury et al. Biosci. Biotechnol Biochem. 67:438-441 strates such as acetyl-CoA to ethanol (for example, adhE (2003); Chowdhury et al. Biosci. Biotechnol Biochem. from E. coli (Kessler et al. FEBS. Lett. 281:59-63 (1991)) 60:2043-2047 (1996)). The protein sequences for each of and butyryl-CoA to butanol (for example, adhE2 from C. these exemplary gene products, if available, can be found acetobutyllicum (Fontaine et al. J. Bacteriol. 184:821-830 using the following GenBank accession numbers shown in (2002)). The C. acetobutyllicum adhE2 gene was shown to Table 9. convert 4-hydroxybutyryl-CoA to 1,4-butanediol, as described in Example IV. In addition to reducing acetyl-CoA TABLE 9 to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxide the branched chain Gene Accession No. GI No. Organism compound isobutyraldehyde to isobutyryl-CoA (Kazahaya P84O67 P84O67 75345323 Thermus thermophilus et al. J. Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al. mmsb P288.11.1 127211 Pseudomonas aeruginosa Biotechnol Lett. 27:505-510 (2005)). The protein sequences dhat Q59477.1 2842618 Pseudomonas putida 3hidh P31937.2 12643395 Homo sapiens for each of these exemplary gene products, if available, can 3hidh P32185.1 416872 Oryctolagus cuniculus be found using the following GenBank accession numbers shown in Table 11.

0313 Several 3-hydroxyisobutyrate dehydrogenase TABLE 11 enzymes have also been shown to convert malonic semial dehyde to 3-hydroxyproprionic acid (3-HP). Three gene Gene Accession No. GI No. Organism candidates exhibiting this activity are mmsB from adhE NP 415757.1 161292.02 Escherichia coi Pseudomonas aeruginosa PAO1(62), mmsB from adhE2 AAKO9379.1 12958626 Clostridium acetobutyllicum Pseudomonas putida KT2440 (Liao et al., US Publication adhE AAV66O76.1 558.18563 Leuconostoc mesenteroides 2005/0221466) and mmsB from Pseudomonas putida E23 (Chowdhury et al., Biosci. Biotechnol. Biochem. 60:2043 2047 (1996)). An enzyme with 3-hydroxybutyrate dehydro 0316. Another exemplary enzyme can convert malonyl genase activity in Alcaligenes faecalis M3A has also been CoA to 3-HP. An NADPH-dependent enzyme with this US 2016/03193 13 A1 Nov. 3, 2016

activity has characterized in Chloroflexus aurantiacus where hyde-forming Succinyl-CoA reductase (Takahashi et al. J. it participates in the 3-hydroxypropionate cycle (Hugler et Bacteriol. 182:4704-4710 (2000)). The enzyme acylating al., J. Bacteriol. 184:2404-2410 (2002); Strauss and Fuchs, acetaldehyde dehydrogenase in Pseudomonas sp. encoded Eur: J. Biochem. 215:633-643 (1993)). This enzyme, with a by bphC, is yet another as it has been demonstrated to mass of 300 kDa, is highly substrate-specific and shows little oxidize and acylate acetaldehyde, propionaldehyde, butyr sequence similarity to other known oxidoreductases (Hugler aldehyde, isobutyraldehyde and formaldehyde (Powlowski et al., J. Bacteriol. 184:2404-2410 (2002)). No enzymes in et al. J Bacteriol. 175:377-385 (1993)). In addition to other organisms have been shown to catalyze this specific reducing acetyl-CoA to ethanol, the enzyme encoded by reaction; however there is bioinformatic evidence that other adhE in Leuconostoc mesenteroides has been shown to organisms may have similar pathways (Klatt et al., Environ. oxidize the branched chain compound isobutyraldehyde to Microbiol. 9:2067-2078 (2007)). Enzyme candidates in isobutyryl-CoA (Koo et al., Biotechnol Lett. 27:505-510 other organisms including Roseiflexus castenholzii, Eryth (2005)). Butyraldehyde dehydrogenase catalyzes a similar robacter sp. NAP1 and marine gamma proteobacterium reaction, conversion of butyryl-CoA to butyraldehyde, in HTCC2080 can be inferred by sequence similarity. The Solventogenic organisms such as Clostridium saccharoper protein sequences for each of these exemplary gene prod butylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. ucts, if available, can be found using the following GenBank 71:58-68 (2007)). The protein sequences for each of these accession numbers shown in Table 12. exemplary gene products, if available, can be found using the following GenBank accession numbers shown in Table TABLE 12 14.

Gene Accession No. GI No. Organism TABLE 1.4 C AAS204291 42561982 Chloroflexus attrantiacus Gene Accession No. GI No. Organism Ricas 2929 YP 001433009.1 156742880 Roseiflexus castenholzii acr 1 YP 047869.1 50086359 Acinetobacter Caicoaceticus NAP1 O2720 ZP 01039179.1 85708113 Erythrobacter sp. acr 1 AAC45217 1684886 Acinetobacter baylvi NAP1 acr 1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1 MGP2080 OO535 ZR O1626393.1 1195.04313 marine gamma SucD P38947.1 730847 Clostridium kluyveri proteobacterium SucD NP 904963.1 3454.0484 Porphyromonas gingivais HTCC2O8O bphO BAAO3892.1 425213 Pseudomonas sp adhE AAV66O76.1 558.18563 Leuconostoc mesenteroides blo AAP42563.1 31075383 Cliostridium 0317 Longer chain acyl-CoA molecules can be reduced Saccharoperhittylacetonictim by enzymes Such as the jojoba (Simmondsia chinensis) FAR which encodes an alcohol-forming fatty acyl-CoA reductase. 0319. An additional enzyme type that converts an acyl Its overexpression in E. coli resulted in FAR activity and the CoA to its corresponding aldehyde is malonyl-CoA reduc accumulation of fatty alcohol (Metz et al. Plant Physiology tase which transforms malonyl-CoA to malonic semialde 122:635-644) 2000)). The protein sequence for the exem hyde. Malonyl-CoA reductase is a key enzyme in plary gene product, if available, can be found using the autotrophic carbon fixation via the 3-hydroxypropionate following GenBank accession numbers shown in Table 13. cycle in thermoacidophilic archael bacteria (Berg et al. Science 318:1782-1786 (2007); Thauer, R. K. Science 3.18: TABLE 13 1732-1733 (2007)). The enzyme utilizes NADPH as a Gene Accession No. GINo. Organism cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al. J. Bacteriol. 188:8551-8559 FAR AAD38O39.1 5020215 Simmondsia chinensis (2006); Hugler et al. J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed 0709 in Metallosphaera sedula (Alber et al. J. Bacteriol. 188:8551-8559 (2006): 1.2.1.b Oxidoreductase (Acyl-CoA to Aldehyde) Berg et al. Science 3 18:1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was 0318 Step A of FIG. 1 involves the conversion of suc cloned and heterologously expressed in E. coli (Alber et al. cinyl-CoA to succinate semialdehyde by an aldehyde form J. Bacteriol. 188:8551-8559 (2006)). Although the aldehyde ing succinyl-CoA reductase. Step Q of FIG. 1 depicts the dehydrogenase functionality of these enzymes is similar to conversion of 4-hydroxybutyryl-CoA to 4-hydroxybutanal the bifunctional dehydrogenase from Chloroflexus aurantia by an aldehyde-forming 4-hydroxybutyryl-CoA reductase. cus, there is little sequence similarity. Both malonyl-CoA Several acyl-CoA dehydrogenases are capable of reducing reductase enzyme candidates have high sequence similarity an acyl-CoA to its corresponding aldehyde. Exemplary to aspartate-semialdehyde dehydrogenase, an enzyme cata genes that encode such enzymes include the Acinetobacter lyzing the reduction and concurrent dephosphorylation of calcoaceticus acr1 encoding a fatty acyl-CoA reductase aspartyl-4-phosphate to aspartate semialdehyde. Additional (Reiser and Somerville, J. Bacteriology 179:2969-2975 gene candidates can be found by sequence homology to (1997)), the Acinetobacter sp. M-1 fatty acyl-CoA reductase proteins in other organisms including Sulfolobus solfatari (Ishige et al. Appl. Environ. Microbiol. 68: 1192-1195 cus and Sulfolobus acidocaldarius. Yet another candidate for (2002)), and a CoA- and NADP-dependent succinate semi CoA-acylating aldehyde dehydrogenase is the ald gene from aldehyde dehydrogenase encoded by the SucD gene in Clostridium beijerinckii (Toth et al., Appl Environ. Micro Clostridium kluyveri (Sohling and Gottschalk J Bacteriol biol 65:4973-4980 (1999)). This enzyme has been reported 178:871-80 (1996); Sohling and Gottschalk J Bacteriol. to reduce acetyl-CoA and butyryl-CoA to their correspond 178:871-880 (1996)). SucD of P gingivalis is anotheralde ing aldehydes. This gene is very similar to eut, that encodes US 2016/03193 13 A1 Nov. 3, 2016 42 acetaldehyde dehydrogenase of Salmonella typhimurium complexes varies in different organisms, but generally and E. coli (Toth et al., Appl Environ. Microbiol 65:4973 branched-chain ketoacid dehydrogenases have the broadest 4980 (1999)). The protein sequences for each of these Substrate range. exemplary gene products, if available, can be found using 0322 Alpha-ketoglutarate dehydrogenase (AKGD) con the following GenBank accession numbers shown in Table verts alpha-ketoglutarate to Succinyl-CoA and is the primary 15. site of control of metabolic flux through the TCA cycle (Hansford, R. G. Curr. Top. Bioenerg. 10:217-278 (1980)). TABLE 1.5 Encoded by genes sucA, sucB and lpd in E. coli, AKGD Gene Accession No. GI No. Organism gene expression is downregulated under anaerobic condi tions and during growth on glucose (Park et al. Mol. Micro Msed 0709 YP 0011908.08.1 146303492 Metaliosphaera sedulla biol. 15:473-482 (1995)). Structural studies of the catalytic C NP 3781.67.1 15922498 Sulfolobus tokodai core of the E2 component pinpoint specific residues respon asd-2 NP 343563.1 15898958 Sulfolobus solfatanicus Saci. 2370 YP 256941.1 70608071 Sulfolobus sible for substrate specificity (Knapp et al. J. Mol. Biol. acidocaidarius 280:655-668 (1998)). The Bacillus subtilis AKGD, encoded Ald AAT66436 49473535 Clostridium beijerinckii by odh AB (E1 and E2) and pdhD (E3, shared domain), is eut AAA80209 687645 Salmonella typhimurium regulated at the transcriptional level and is dependent on the eut P77.445 2498.347 Escherichia coi carbon Source and growth phase of the organism (Resnekov et al. Mol. Gen. Genet. 234:285-296 (1992)). In yeast, the LPD1 gene encoding the E3 component is regulated at the 1.2.1.c Oxidoreductase (2-Oxo Acid to Acyl-CoA, transcriptional level by glucose (Roy and Dawes J. Gen. Decarboxylation). Microbiol. 133:925-933 (1987)). The E1 component, encoded by KGD1, is also regulated by glucose and acti 0320 Step AA in FIG. 1 depicts the conversion of 5-hy vated by the products of HAP2 and HAP3 (Repetto and droxy-2-oxopentanoic acid to 4-hydroxybutyryl-CoA. Can Tzagoloff Mol Cell Biol. 9:2695-2705 (1989)). The AKGD didate enzymes for this transformation include 1) branched enzyme complex, inhibited by products NADH and succi chain 2-keto-acid dehydrogenase, 2) alpha-ketoglutarate nyl-CoA, is well-studied in mammalian systems, as dehydrogenase, and 3) the pyruvate dehydrogenase multi impaired function of has been linked to several neurological enzyme complex (PDHC). These enzymes are multi-enzyme diseases (Tretter and dam-Vizi Philos. Trans. R. Soc. Lond complexes that catalyze a series of partial reactions which B Biol. Sci. 360:2335-2345 (2005)). The protein sequences result in acylating oxidative decarboxylation of 2-keto for each of these exemplary gene products, if available, can acids. Each of the 2-keto-acid dehydrogenase complexes be found using the following GenBank accession numbers occupies key positions in intermediary metabolism, and shown in Table 16. enzyme activity is typically tightly regulated (Fries et al. Biochemistry 42:6996-7002 (2003)). The enzymes share a TABLE 16 complex but common structure composed of multiple copies of three catalytic components: alpha-ketoacid decarboxylase Gene Accession No. GI No. Organism (E1), dihydrolipoamide acyltransferase (E2) and dihydroli SucA NP 415254.1 16128701 Escherichia Coi str. K12 poamide dehydrogenase (E3). The E3 component is shared SucB NP 415255.1 16128702 Escherichia Coi str. K12 among all 2-keto-acid dehydrogenase complexes in an lpd NP 414658.1 16128109 Escherichia Coi str. K12 odh A P23129.2 51704265 Bacilius Subiiis organism, while the E1 and E2 components are encoded by OdhB P16263.1 129041 Bacilius subtiis different genes. The enzyme components are present in pdhD P21880.1 118672 Bacilius subtiis numerous copies in the complex and utilize multiple cofac KGD1 NP 012141.1 6322066 Saccharomyces cerevisiae tors to catalyze a directed sequence of reactions via Substrate KGD2 NP 010432.1 6320352 Saccharomyces cerevisiae channeling. The overall size of these dehydrogenase com LPD1 NP 116635.1 14318501 Saccharomyces cerevisiae plexes is very large, with molecular masses between 4 and 10 million Da (that is, larger than a ribosome). 0323 Branched-chain 2-keto-acid dehydrogenase com 0321 Activity of enzymes in the 2-keto-acid dehydroge plex (BCKAD), also known as 2-oxoisovalerate dehydro nase family is normally low or limited under anaerobic genase, participates in branched-chain amino acid degrada conditions in E. coli. Increased production of NADH (or tion pathways, converting 2-keto acids derivatives of valine, NADPH) could lead to a redox-imbalance, and NADH itself leucine and isoleucine to their acyl-CoA derivatives and serves as an inhibitor to enzyme function. Engineering CO. The complex has been studied in many organisms efforts have increased the anaerobic activity of the E. coli including Bacillus subtilis (Wang et al. Eur: J. Biochem. pyruvate dehydrogenase complex (Kim et al. Appl. Environ. 213:1091-1099 (1993)), Rattus norvegicus (Namba et al. J. Microbiol. 73:1766-1771 (2007); Kim et al. J. Bacteriol. Biol. Chem. 244:4437-4447 (1969)) and Pseudomonas 190:3851-3858) 2008); Zhou et al. Biotechnol. Lett. 30:335 putida (Sokatch J. Bacteriol. 148:647-652 (1981)). In Bacil 342 (2008)). For example, the inhibitory effect of NADH lus subtilis the enzyme is encoded by genes pdhD (E3 can be overcome by engineering an H322Y mutation in the component), bfmBB (E2 component), bfmBAA and E3 component (Kim et al. J. Bacteriol. 190:3851-3858 bfmBAB (E1 component) (Wang et al. Eur: J. Biochem. (2008)). Structural studies of individual components and 213:1091-1099 (1993)). In mammals, the complex is regu how they work together in complex provide insight into the lated by phosphorylation by specific phosphatases and pro catalytic mechanisms and architecture of enzymes in this tein kinases. The complex has been studied in rathepatocites family (Aevarsson et al. Nat. Struct. Biol. 6:785-792 (1999); (Chicco et al. J. Biol. Chem. 269:19427-19434 (1994)) and Zhou et al. Proc. Natl. Acad. Sci. U.S.A. 98: 14802-14807 is encoded by genes Bckdha (E1 alpha), Bckdhb (E1 beta), (2001)). The substrate specificity of the dehydrogenase Dbt (E2), and Dld (E3). The E1 and E3 components of the US 2016/03193 13 A1 Nov. 3, 2016

Pseudomonas putida BCKAD complex have been crystal TABLE 1.8 lized (Aevarsson et al. Nat. Struct. Biol. 6:785-792 (1999); Mattevi Science 255:1544-1550 (1992)) and the enzyme Gene Accession No. GINo. Organism complex has been studied (Sokatch et al. J. Bacteriol. aceE NP 414656.1 161281.07 Escherichia Coi Str. K12 148:647-652 (1981)). Transcription of the Pputida BCKAD aceF NP 414657.1 161281.08 Escherichia Coi Str. K12 lpd NP 414658.1 16128109 Escherichia Coi Str. K12 genes is activated by the gene product of bkdR (Hester et al. pdh.A P21882.3 3123238 Bacilius Subiiis Eur: J. Biochem. 233:828-836 (1995)). In some organisms pdhB P21882.2 129068 Bacilius subtiis including Rattus norvegicus (Paxton et al. Biochem. J. pdhC P21883.2 129054 Bacilius subtiis pdhD P21880.1 118672 Bacilius subtiis 234:295-303 (1986)) and Saccharomyces cerevisiae (Sin aceE YP OO1333808.1 1529.68699 Klebsiella pneumonia clair et al. Biochem. Mol. Biol. Int. 31:911-922 (1993)), this MGH78578 complex has been shown to have a broad Substrate range that aceF YP OO1333809.1 152968.700 Klebsiella pneumonia includes linear oxo-acids such as 2-oxobutanoate and alpha MGH78578 lpdA YP OO13338.10.1 1529.68701 Klebsiella pneumonia ketoglutarate, in addition to the branched-chain amino acid MGH78578 precursors. The active site of the bovine BCKAD was Poha1 NP 001004072.2 124430510 Rattus norvegicus engineered to favor alternate Substrate acetyl-CoA (Meng Poha2 NP 446446.1 16758900 Rattus norvegicus and Chuang, Biochemistry 33:12879-12885 (1994)) The Dlat NP 112287.1 78365255 Rattus norvegicus protein sequences for each of these exemplary gene prod Dld NP 95.5417.1 40786469 Rattus norvegicus ucts, if available, can be found using the following GenBank accession numbers shown in Table 17. 0325 As an alternative to the large multienzyme 2-keto TABLE 17 acid dehydrogenase complexes described above, some anaerobic organisms utilize enzymes in the 2-ketoacid oxi Gene Accession No. GINo. Organism doreductase family (OFOR) to catalyze acylating oxidative bfnBB NP 390283.1 16079459 Bacilius Subiiis decarboxylation of 2-keto-acids. Unlike the dehydrogenase bfmBAA NP 390285.1 1607.9461 Bacilius Subiiis complexes, these enzymes contain iron-sulfur clusters, ulti bfmBAB NP 390284.1 1607.9460 Bacilius Subiiis pdhD P21880.1 118672 Bacilius subtiis lize different cofactors, and use ferredoxin or flavodixin as lpdV PO9063.1 118677 Pseudomonas putida electron acceptors in lieu of NAD(P)H. While most enzymes bkdB PO9062.1 129044 Pseudomonas putida in this family are specific to pyruvate as a substrate (POR) bkdA1 NP 746515.1 26991090 Pseudomonas putida Some 2-keto-acid:ferredoxin oxidoreductases have been bkdA2 NP 746516.1 26991.091 Pseudomonas putida Bckdha NP 036914.1 77736548 Rattus norvegicus shown to accept a broad range of 2-ketoacids as Substrates Bckdhb NP 062140.1 158749538 Rattus norvegicus including alpha-ketoglutarate and 2-oxobutanoate (Fukuda Dbt NP 445764.1 158749632 Rattus norvegicus and Wakagi Biochim. Biophys. Acta 1597:74-80 (2002); Dld NP 95.5417.1 40786469 Rattus norvegicus Zhang et al. J. Biochem. 120:587-599 (1996)). One such enzyme is the OFOR from the thermoacidophilic archaeon 0324. The pyruvate dehydrogenase complex, catalyzing Sulfolobus tokodai 7, which contains an alpha and beta the conversion of pyruvate to acetyl-CoA, has also been subunit encoded by gene ST2300 (Fukuda and Wakagi extensively studied. In the E. coli enzyme, specific residues Biochim. Biophys. Acta 1597:74-80 (2002); Zhang et al. J. in the E1 component are responsible for substrate specificity Biochem. 120:587-599 (1996)). A plasmid-based expression (Bisswanger, H. J. Biol Chem. 256:815-822 (1981); Bremer, system has been developed for efficiently expressing this J. Eur: J Biochem. 8:535-540 (1969); Gong et al. J Biol protein in E. coli (Fukuda et al. Eur: J. Biochem. 268:5639 Chem. 275:13.645-13653 (2000)). As mentioned previously, 5646 (2001)) and residues involved in substrate specificity enzyme engineering efforts have improved the E. coli PDH were determined (Fukuda and Wakagi Biochim. Biophys. enzyme activity under anaerobic conditions (Kim et al. Appl. Acta 1597:74-80 (2002)). Two OFORs from Aeropyrum Environ. Microbiol. 73:1766-1771 (2007); Kim J. Bacteriol. pernix str. K1 have also been recently cloned into E. coli, 190:3851-3858 (2008); Zhou et al. Biotechnol. Lett. 30:335 characterized, and found to react with a broad range of 342 (2008)). In contrast to the E. coli PDH, the B. subtilis 2-oxoacids (Nishizawa et al. FEBS Lett. 579:2319-2322 complex is active and required for growth under anaerobic (2005)). The gene sequences of these OFOR candidates are conditions (Nakano J. Bacteriol. 179:6749-6755 (1997)). available, although they do not have GenBank identifiers The Klebsiella pneumoniae PDH, characterized during assigned to date. There is bioinformatic evidence that similar growth on glycerol, is also active under anaerobic conditions enzymes are present in all archaea, some anaerobic bacteria (Menzel et al. J. Biotechnol. 56:135-142 (1997)). Crystal structures of the enzyme complex from bovine kidney (Zhou and amitochondrial eukarya (Fukuda and Wakagi Biochim. et al. Proc. Natl. Acad. Sci. U.S.A. 98:14802-14807 (2001)) Biophys. Acta 1597:74-80 (2005)). This class of enzyme is and the E2 catalytic domain from Azotobacter vinelandii are also interesting from an energetic standpoint, as reduced available (Mattevi et al. Science 255:1544-1550 (1992)). ferredoxin could be used to generate NADH by ferredoxin Some mammalian PDH enzymes complexes can react on NAD reductase (Petitclemange et al. Biochim. Biophys. Acta alternate Substrates Such as 2-oxobutanoate, although com 421:334-337 (1976)). Also, since most of the enzymes are parative kinetics of Rattus norvegicus PDH and BCKAD designed to operate under anaerobic conditions, less enzyme indicate that BCKAD has higher activity on 2-oxobutanoate engineering may be required relative to enzymes in the as a substrate (Paxton et al. Biochem. J. 234:295-303 2-keto-acid dehydrogenase complex family for activity in an (1986)). The protein sequences for each of these exemplary anaerobic environment. The protein sequence for the exem gene products, if available, can be found using the following plary gene product, if available, can be found using the GenBank accession numbers shown in Table 18. following GenBank accession numbers shown in Table 19. US 2016/03193 13 A1 Nov. 3, 2016 44

TABLE 19 Bacteriol. 156: 1249-1262 (1983)) and Campylobacter jejuni (Louie et al., 240:29-35 (1993)) were cloned and expressed Gene Accession No. GINo. Organism in E. coli. The protein sequences for each of these exemplary ST2300 NP 378302.1 15922633 Sulfolobus tokodai 7 gene products, if available, can be found using the following GenBank accession numbers shown in Table 21.

TABLE 21 1.2.1.d Oxidoreductase (Phosphonate Reductase) 0326. The conversion of 4-hydroxybutyryl-phosphate to Gene Accession No. GI No. Organism 4-hydroxybutanal (Step R) can be catalyzed by an oxi gap A POA9B2.2 7115.9358 Escherichia coi argC NP 418393.1 16131796 Escherichia coi doreductase in the EC class 1.2.1. Aspartate semialdehyde proA NP 414778.1 16128229 Escherichia coi dehydrogenase (ASD, EC 1.2.1.11) catalyzes the NADPH proA NP 4593.19.1 16763704 Salmonella typhimurium dependent reduction of 4-aspartyl phosphate to aspartate-4- proA P53OOO.2 9087222 Campylobacter jejuni semialdehyde. ASD participates in amino acid biosynthesis and recently has been studied as an antimicrobial target (Hadfield et al., 40:14475-14483 (2001)). The E. coli ASD structure has been solved (Hadfield et al., 289:991-1002 1.2.1.e Acid Reductase (1999)) and the enzyme has been shown to accept the 0328 Several steps in FIG. 1 depict the conversion of alternate substrate beta-3-methylaspartyl phosphate unactivated acids to aldehydes by an acid reductase. These (Shames et al., 259:15331-15339 (1984)). The Haemophilus include the conversion of 4-hydroxybutyrate to 4-hy influenzae enzyme has been the Subject of enzyme engineer droxybutanal (Step D). Succinate to Succinate semialdehyde ing studies to alter Substrate binding affinities at the active (Step F), and alpha-ketoglutarate to 2,5-dioxopentanoate site (Blanco et al., Acta Crystallogr. D. Biol. Crystallogr: (Step X). One notable carboxylic acid reductase can be 60:1388-1395 (2004); Blanco et al., Acta Crystallogr. D. found in Nocardia iowensis which catalyzes the magnesium, Biol. Crystallogr. 60:1808-1815 (2004)). Other ASD candi ATP and NADPH-dependent reduction of carboxylic acids dates are found in Mycobacterium tuberculosis (Shafiani et to their corresponding aldehydes (Venkitasubramanian et al., al., J Appl Microbiol 98:832-838 (2005)), Methanococcus J Biol. Chem. 282:478-485 (2007)). This enzyme is encoded jannaschii (Faehnle et al., 353:1055-1068 (2005)), and the by the car gene and was cloned and functionally expressed infectious microorganisms Vibrio cholera and Heliobacter in E. coli (Venkitasubramanian et al., J Biol. Chem. 282: pylori (Moore et al., Protein Expr: Purif 25:189-194 478-485 (2007)). Expression of the npt gene product (2002)). A related enzyme candidate is acetylglutamylphos improved activity of the enzyme via post-transcriptional phate reductase (EC 1.2.1.38), an enzyme that naturally modification. The npt gene encodes a specific phosphopan reduces acetylglutamylphosphate to acetylglutamate-5- tetheline transferase (PPTase) that converts the inactive apo semialdehyde, found in S. cerevisiae (Pauwels et al., Eur: J enzyme to the active holo-enzyme. The natural substrate of Biochem. 270:1014-1024 (2003)), B. subtilis (O'Reilly et this enzyme is vanillic acid and the enzyme exhibits broad al., 140 (Pt 5):1023-1025 (1994)) and other organisms. The acceptance of aromatic and aliphatic Substrates (Venkitasu protein sequences for each of these exemplary gene prod bramanian et al. “Biocatalytic Reduction of Carboxylic ucts, if available, can be found using the following GenBank Acids: Mechanism and Applications' Chapter 15 in Bioca accession numbers shown in Table 20. talysis in the Pharmaceutical and Biotechnology Industires, ed. R. N. Patel, CRC Press LLC, Boca Raton, Fla. (2006)). TABLE 20 The protein sequences for each of these exemplary gene products, if available, can be found using the following Gene Accession No. GI No. Organism GenBank accession numbers shown in Table 22. asd NP 417891.1 16131307 Escherichia coi asd YP 248335.1 68249223 Haemophilus influenzae TABLE 22 asd AAB4.9996 1899206 Mycobacterium tuberculosis Gene Accession No. GINo. Organism VC2O36 NP 231670 15642038 Vibrio cholera asd YP 002301787.1 210135348 Heliobacter pylori C8 AAR91681.1 4O796O3S Nocardia iowensis ARG5, 6 NP 010992.1 6320913 Saccharomyces cerevisiae (sp. NRRL 5646) argC NP 3890.01.1 16078184. Bacilius Subiiis mpt ABI83656.1 11484.8891 Nocardia iowensis (sp. NRRL 5646) 0327 Other exemplary enzymes in this class include glyceraldehyde 3-phosphate dehydrogenase which converts 0329. Additional car and npt genes can be identified glyceraldehyde-3-phosphate into D-glycerate 1,3-bisphos based on sequence homology. The protein sequences for phate (e.g., E. coli gap.A (Branlant et al., Eur: J. Biochem. each of these exemplary gene products, if available, can be 150:61-66 (1985))), N-acetyl-gamma-glutamyl-phosphate found using the following GenBank accession numbers reductase which converts N-acetyl-L-glutamate-5-semialde shown in Table 23. hyde into N-acetyl-L-glutamyl-5-phosphate (e.g., E. coli argC (Parsot et al., Gene. 68:275-283 (1988))), and gluta TABLE 23 mate-5-semialdehyde dehydrogenase which converts L-glu tamate-5-semialdehyde into L-glutamyl-5-phospate (e.g., E. Gene Accession No. GI No. Organism coli proA (Smith et al., J. Bacteriol. 157:545-551 (1984a))). fad)9 YP 978699.1 121638475 Mycobacterium bovis Genes encoding glutamate-5-semialdehyde dehydrogenase enzymes from Salmonella typhimurium (Mahan et al., J US 2016/03193 13 A1 Nov. 3, 2016

TABLE 23-continued TABLE 25 Gene Accession No. GI No. Organism Gene Accession No. GI No. Organism BCG 2812c YP 978898.1 121638,674. Mycobacterium bovis LYS2 AAA34747.1 171867 Saccharomyces cerevisiae BCG LYS5 PSO 113.1 1708896 Saccharomyces cerevisiae infa2O150 YP 118225.1 54023983 Nocardia farcinica LYS2 AACO2241.1 2853226 Candida albicans IFM 101S2 LYS5 AAO26020.1 28136195 Candida albicans infa40540 YP 120266.1 54.026024 Nocardia farcinica Lys1p P4O976.3 13124791 Schizosaccharomyces pombe IFM 101S2 Lys7p Q10474.1 1723561 Schizosaccharomyces pombe SGR 6790 YP 001828302.1 182440583 Streptomyces griseus Lys2 CAA743 OO1 3282044 Penicillium chrysogenium Subsp. grisetts NBRC 13350 SGR 665 YP 001822177.1 182434458 Streptomyces griseus Subsp. grisetts NBRC 13350 1.4.1.a Oxidoreductase (Aminating) 0332 Glutamate dehydrogenase (Step J, FIG. 1B) and 4-aminobutyrate dehydrogenase (Step M. FIG. 1B) can be 0330. An additional enzyme candidate found in Strepto catalyzed by aminating oxidoreductases. Enzymes in this myces griseus is encoded by the griC and grid genes. This EC class catalyze the oxidative deamination of alpha-amino enzyme is believed to convert 3-amino-4-hydroxybenzoic acids with NAD" or NADP as acceptor, and the reactions acid to 3-amino-4-hydroxybenzaldehyde as deletion of are typically reversible. Exemplary oxidoreductases operat either griC or griLD led to accumulation of extracellular ing on amino acids include glutamate dehydrogenase 3-acetylamino-4-hydroxybenzoic acid, a shunt product of (deaminating), encoded by gcdhA, leucine dehydrogenase 3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., (deaminating), encoded by lah, and aspartate dehydrogenase J. Antibiot. 60(6):380-387 (2007)). Co-expression of griC (deaminating), encoded by nadX. The gdh A gene product and griLD with SGR 665, an enzyme similar in sequence to from Escherichia coli (Korber et al. J. Mol. Biol. 234:1270 the Nocardia iowensis npt, may be beneficial. The protein 1273 (1993); McPherson and Wootton Nucleic. Acids Res. sequences for each of these exemplary gene products, if 11:5257-5266 (1983)), gdh from Thermotoga maritima available, can be found using the following GenBank acces (Kort et al. Extremophiles 1:52-60 (1997); Lebbink, et al. J. sion numbers shown in Table 24. Mol. Biol. 280:287-296 (1998)); Lebbink et al. J. Mol. Biol. 289:357-369 (1999)), and gdhal from Halobacterium sali TABLE 24 narum (Ingoldsby et al. Gene 349:237-244 (2005)) catalyze the reversible interconversion of glutamate to 2-oxoglutarate Gene Accession No. GINo. Organism and ammonia, while favoring NADP(H), NAD(H), or both, griC 182438O36 YP 001825755.1 Streptomyces griseus Subsp. respectively. The ldh gene of Bacillus cereus encodes the griseus NBRC 13350 Leul)H protein that has a wide of range of substrates grid 1824.38037 YP 001825756.1 Streptomyces griseus Subsp. including leucine, isoleucine, Valine, and 2-aminobutanoate griseus NBRC 13350 (Ansorge and Kula Biotechnol Bioeng. 68:557-562 (2000); Stoyan et al. 1 Biotechnol. 54:77-80 (1997)). The nadX gene from Thermotoga maritime encoding for the aspartate dehy 0331. An enzyme with similar characteristics, alpha drogenase is involved in the biosynthesis of NAD (Yang et aminoadipate reductase (AAR, EC 1.2.1.31), participates in al. J. Biol. Chem. 278:8804-8808 (2003)). The protein lysine biosynthesis pathways in Some fungal species. This sequences for each of these exemplary gene products, if enzyme naturally reduces alpha-aminoadipate to alpha available, can be found using the following GenBank acces aminoadipate semialdehyde. The carboxyl group is first sion numbers shown in Table 26. activated through the ATP-dependent formation of an adenylate that is then reduced by NAD(P)H to yield the TABLE 26 aldehyde and AMP. Like CAR, this enzyme utilizes mag nesium and requires activation by a PPTase. Enzyme can Gene Accession No. GI No. Organism didates for AAR and its corresponding PPTase are found in gdh A POO370 1185.47 Escherichia coi Saccharomyces cerevisiae (Morris et al., Gene 98:141-145 gdh P96110.4 6226595 Thermotoga maritima gdh A1 NP 27965.1.1 15789827 Haiobacterium sainarum (1991)), Candida albicans (Guo et al., Mol. Genet. Genom ldh POA393 61222614 Bacilius cereus ics 269:271-279 (2003)), and Schizosaccharomyces pombe nadX NP 229443.1 15644391 Thermotoga maritima (Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombe exhibited significant activity when expressed in E. coli (Guo et al., Yeast 21:1279-1288 (2004)). The AAR 0333 Additional glutamate dehydrogenase gene candi from Penicillium chrysogenium accepts S-carboxymethyl-L- dates are found in Bacillus subtilis (Khan et al., Biosci. cysteine as an alternate substrate, but did not react with Biotechnol Biochem. 69:1861-1870 (2005)), Nicotiana taba adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J cum (Purnell et al., Planta 222:167-180 (2005)), Oryza Biol. Chem 278:8250-8256 (2003)). The gene encoding the sativa (Abiko et al., Plant Cell Physiol 46:1724-1734 P. chrysogenium PPTase has not been identified to date and (2005)), Haloferax mediterranei (Diaz et al., Extremophiles. no high-confidence hits were identified by sequence com 10:105-115 (2006)) and Halobactreium salinarum (Hayden parison homology searching. The protein sequences for each et al., FEMS Microbiol Lett. 211:37-41 (2002)). The Nico of these exemplary gene products, if available, can be found tiana tabacum enzyme is composed of alpha and beta using the following GenBank accession numbers shown in subunits encoded by gcdh1 and gcdh2 (Purnell et al., Planta Table 25. 222:167-180 (2005)). Overexpression of the NADH-depen US 2016/03193 13 A1 Nov. 3, 2016 46 dent glutamate dehydrogenase was found to improve ethanol TABLE 29 production in engineered strains of S. cerevisiae (Roca et al., Appl Environ. Microbiol 69:4732-4736 (2003)). The protein Gene Accession No. GI No. Organism sequences for each of these exemplary gene products, if kdd AAL93966.1 19713113 Fusobacterium nucleatum available, can be found using the following GenBank acces mxan 439 ABF87267.1 108462082 Myxococcus xanthus sion numbers shown in Table 27. pg. 1069 AAQ66183.1 34397119 Porphyromonas gingivais

TABLE 27 2.3.1.a Acyltransferase (Transferring Phosphate Group to Gene Accession No. GI No. Organism CoA) rocG NP 391659.1 1608.0831 Bacilius Subiiis gdh1 AAR11534.1 38146335 Nicotiana tabacum 0336 Step P of FIG. 1 depicts the transformation of gdh2 AAR11535.1 38146337 Nicotiana tabacum 4-hydroxybutyryl-CoA to 4-hydroxybutyryl-phosphate. GDH Q852MO 75243660 Oryza sativa Exemplary phosphate transferring acyltransferases include GDH Q977U6 744.99858 Haloferax mediterranei phosphotransacetylase, encoded by pta, and phosphotrans GDH P29051 118549 Haiobactreiun sainarum butyrylase, encoded by ptb. The pta gene from E. coli GDH2 NP 010066.1 6319986 Saccharomyces cerevisiae encodes an enzyme that can convert acetyl-CoA into acetyl phosphate, and vice versa (Suzuki, T. Biochim. Biophys. Acta 191:559-569 (1969)). This enzyme can also utilize 0334. An exemplary enzyme for catalyzing the conver propionyl-CoA instead of acetyl-CoA forming propionate in sion of aldehydes to their corresponding primary amines is the process (Hesslinger et al. Mol. Microbiol 27:477-492 lysine 6-dehydrogenase (EC 1.4.1.18), encoded by the (1998)). Similarly, the ptb gene from C. acetobutyllicum lysDH genes. The lysine 6-dehydrogenase (deaminating), encodes an enzyme that can convert butyryl-CoA into encoded by lysDH gene, catalyze the oxidative deamination butyryl-phosphate (Walter et al. Gene 134(1): p. 107-11 of the e-amino group of L-lysine to form 2-aminoadipate (1993)); Huang et al. J Mol. Microbiol Biotechnol 2(1): p. 6-semialdehyde, which in turn nonenzymatically cyclizes to 33-38 (2000). Additional ptb genes can be found in butyrate form Al-piperideine-6-carboxylate (Misono and Nagasaki.J. producing bacterium L2-50 (Louis et al. J. Bacteriol. 186: Bacteriol. 150:398-401 (1982)). The lysDH gene from Geo 2099-2106 (2004)) and Bacillus megaterium (Vazquez et al. bacillus Stearothermophilus encodes a thermophilic NAD Curr. Microbiol 42:345-349 (2001)). The protein sequences dependent lysine 6-dehydrogenase (Heydari et al. Appl for each of these exemplary gene products, if available, can Environ. Microbiol 70.937-942 (2004)). The lysDH gene be found using the following GenBank accession numbers from Aeropyrum permix K1 is identified through homology shown in Table 30. from genome projects. Additional enzymes can be found in Agrobacterium tumefaciens (Hashimoto et al., J Biochem. TABLE 30 106:76-80 (1989); Misono et al., JBacteriol. 150:398-401 (1982)) and Achromobacter denitrificans (Ruldeekultham Gene Accession No. GI No. Organism rong et al., BMB. Rep. 41:790-795 (2008)). The protein pta NP 416800.1 16130232 Escherichia coi ptb NP 349676 15896327 Clostridium acetobutyllicum sequences for each of these exemplary gene products, if ptb AAR19757.1 38425288 butyrate-producing bacterium available, can be found using the following GenBank acces L2-SO sion numbers shown in Table 28. ptb CACO7932.1 10046659 Bacilius megaterium

TABLE 28 Gene Accession No. GI No. Organism 2.6.1.a Aminotransferase lysDH BAB39707 1342.9872 Geobacilius Stearothermophilus 0337 Steps K and N in FIG. 1 can be catalyzed by lysDH NP 147035.1 14602185 Aeropyrum pernix K1 aminotransferases that reversibly convert an aldehyde or lysDH NP 353966 15888.285 Agrobacterium tumefaciens ketone to an amino group. Common amino donor/acceptor lysDH AAZ94428 74026644 Achromobacter denitrificans combinations include glutamate/alpha-ketoglutarate, ala nine/pyruvate, and aspartate/oxaloacetate. Several enzymes have been shown to convert aldehydes to primary amines, 0335 An enzyme that converts 3-oxoacids to 3-amino and vice versa. Lysine-6-aminotransferase (EC 2.6.1.36) is acids is 3,5-diaminohexanoate dehydrogenase (EC 1.4.1. one exemplary enzyme capable of forming a primary amine. 11), an enzyme found in organisms that ferment lysine. The This enzyme function, converting lysine to alpha-aminoadi gene encoding this enzyme, kdd, was recently identified in pate semialdehyde, has been demonstrated in yeast and Fusobacterium nucleatum (Kreimeyer et al., 282:7191-7197 bacteria. Candidates from Candida utilis (Hammer et al., J (2007)). The enzyme has been purified and characterized in Basic Microbiol 32:21-27 (1992)). Flavobacterium lute other organisms (Baker et al., 247:7724-7734 (1972); Baker scens (Fujii et al., J Biochem. 128:391-397 (2000)) and et al., 13:292-299 (1974)) but the genes associated with Streptomyces clavuligenus (Romero et al., J Ind. Microbiol these enzymes are not known. Candidates in Myxococcus Biotechnol 18:241-246 (1997)) have been characterized. A xanthus, Porphyromonas gingivalis W83 and other recombinant lysine-6-aminotransferase from S. clavulligenus sequenced organisms can be inferred by sequence homol was functionally expressed in E. coli (Tobin et al., J Bac ogy. The protein sequences for each of these exemplary gene teriol. 173:6223-6229 (1991)). The F. lutescens enzyme is products, if available, can be found using the following specific to alpha-ketoglutarate as the amino acceptor (Soda GenBank accession numbers shown in Table 29. et al., 7:4110-4119 (1968)). Other enzymes which convert US 2016/03193 13 A1 Nov. 3, 2016 47 aldehydes to terminal amines include the dat gene product in TABLE 33 Acinetobacter baumani encoding 2,4-diaminobutanoate:2- ketoglutarate 4-transaminase (Ikai et al., J Bacteriol. 179: Gene Accession No. GI No. Organism 5118-5125 (1997)). In addition to its natural substrate, ygG NP 417544 145698310 Escherichia coi 2,4-diaminobutyrate, DAT transaminates the terminal spuC AAGO3688 9946143 Pseudomonas aeruginosa amines of lysine, 4-aminobutyrate and ornithine. The protein sequences for each of these exemplary gene products, if 0340 Enzymes that transaminate 3-oxoacids include available, can be found using the following GenBank acces GABA aminotransferase (described above), beta-alanine/ sion numbers shown in Table 31. alpha-ketoglutarate aminotransferase and 3-amino-2-meth ylpropionate aminotransferase. Beta-alanine/alpha-ketoglu TABLE 31 tarate aminotransferase (WO08027742) reacts with beta alanine to form malonic semialdehyde, a 3-oxoacid. The Gene Accession No. GI No. Organism gene product of SkPYD4 in Saccharomyces kluyveri was lat BAB13756.1 10336502 Flavobacterium initesCens shown to preferentially use beta-alanine as the amino group lat AAA26777.1 153343 Streptomyces clavulligenus donor (Andersen et al., Gene 124:105-109 (1993)). dat PS6744.1 6685373 Acinetobacter battmani SkUGA1 encodes a homologue of Saccharomyces cerevi siae GABA aminotransferase, UGA1 (Ramos et al., Eur: J. Biochem. 149:401–404 (1985)), whereas SkPYD4 encodes 0338. The conversion of an aldehyde to a terminal amine an enzyme involved in both beta-alanine and GABA can also be catalyzed by gamma-aminobutyrate transami transamination (Andersen and Hansen, Gene 124:105-109 nase (GABA transaminase or 4-aminobutyrate transami (1993)). 3-Amino-2-methylpropionate transaminase cata nase). This enzyme naturally interconverts Succinic semial lyzes the transformation from methylmalonate semialde dehyde and glutamate to 4-aminobutyrate and alpha hyde to 3-amino-2-methylpropionate. The enzyme has been ketoglutarate and is known to have a broad Substrate range characterized in Rattus norvegicus and Sus scrofa and is (Schulz et al., 56:1-6 (1990); Liu et al., 43:10896-10905 encoded by Abat (Tamaki et al., 324:376-389 (2000); (2004)). The two GABA transaminases in E. coli are Kakimoto et al., 156:374-380 (1968)). The protein encoded by gabT (Bartsch et al., J Bacteriol. 172:7035-7042 sequences for each of these exemplary gene products, if (1990)) and puuE (Kurihara et al., J. Biol. Chem. 280:4602 available, can be found using the following GenBank acces 4608 (2005)). GABA transaminases in Mus musculus, sion numbers shown in Table 34. Pseudomonas fluorescens, and Sus scrofa have been shown to react with a range of alternate Substrates including TABLE 34 6-aminocaproic acid (Cooper, 113:80-82 (1985): SCOTT et al., 234:932-936 (1959)). The protein sequences for each of Gene Accession No. GI No. Organism these exemplary gene products, if available, can be found SkyPYD4 ABFS8893.1 98.626772 Lachancea kluyveri SkUGA1 ABFS8894.1 98.626792 Lachancea kluyveri using the following GenBank accession numbers shown in UGA1 NP 011533.1 6321456 Saccharomyces cerevisiae Table 32. Abat P50554.3 122065191 Rattus norvegicus Abat P8O147.2 120968 Sus scrofa TABLE 32 Gene Accession No. GI No. Organism 0341. Several aminotransferases transaminate the amino groups of amino acids to form 2-oxoacids. Aspartate ami gabT NP 41.7148.1 16130576 Escherichia coi puuE NP 415818.1 16129263 Escherichia coi notransferase is an enzyme that naturally transfers an oxo Abat NP 766549.2 37202121 Mus musculius group from oxaloacetate to glutamate, forming alpha-keto gabT YP 257332.1 70733692 Pseudomonas fluorescens glutarate and aspartate. Aspartate is similar in structure to abat NP 999428.1 475236.00 Sus scrofa OHED and 2-AHD. Aspartate aminotransferase activity is catalyzed by, for example, the gene products of aspC from Escherichia coli (Yagi et al., 100:81-84 (1979); Yagi et al., 0339. Additional enzyme candidates for interconverting 113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae aldehydes and primary amines are putrescine transminases (Yagi et al., 92:35-43 (1982)) and ASPS from Arabidopsis or other diamine aminotransferases. The E. coli putrescine thaliana (Kwok et al., 55:595-604 (2004); de la et al., aminotransferase is encoded by the ygG gene and the 46:414-425 (2006); Wilkie et al., Protein Expr: Purif. purified enzyme also was able to transaminate cadaverine 12:381-389 (1998)). The enzyme from Rattus norvegicus and spermidine (Samsonova et al., BMC. Microbiol 3:2 has been shown to transaminate alternate Substrates such as (2003)). In addition, activity of this enzyme on 1,7-diamino 2-aminohexanedioic acid and 2,4-diaminobutyric acid (Re heptane and with amino acceptors other than 2-oxoglutarate casens et al., 19:4583-4589 (1980)). Aminotransferases that (e.g., pyruvate, 2-oxobutanoate) has been reported (Sam work on other amino-acid Substrates may also be able to sonova et al., BMC. Microbiol 3:2 (2003); KIM, 239:783 catalyze this transformation. Valine aminotransferase cata 786 (1964)). A putrescine aminotransferase with higher lyzes the conversion of Valine and pyruvate to 2-ketoisoval activity with pyruvate as the amino acceptor than alpha erate and alanine. The E. coli gene, avta, encodes one Such ketoglutarate is the spuC gene of Pseudomonas aeruginosa enzyme (Whalen et al., J. Bacteriol. 150:739-746 (1982)). (Lu et al., J Bacteriol. 184:3765-3773 (2002)). The protein This gene product also catalyzes the transamination of sequences for each of these exemplary gene products, if alpha-ketobutyrate to generate C.-aminobutyrate, although available, can be found using the following GenBank acces the amine donor in this reaction has not been identified sion numbers shown in Table 33. (Whalen et al., J. Bacteriol. 158:571-574 (1984)). The gene US 2016/03193 13 A1 Nov. 3, 2016 48 product of the E. coli serC catalyzes two reactions, phos good candidates: acetate kinase and gamma-glutamyl phoserine aminotransferase and phosphohydroxythreonine kinase. The E. coli acetate kinase, encoded by ackA aminotransferase (Lam et al., J. Bacteriol. 172:6518-6528 (Skarstedt et al., J. Biol. Chem. 251:6775-6783 (1976)), (1990)), and activity on non-phosphorylated substrates phosphorylates propionate in addition to acetate (Hesslinger could not be detected (Drewke et al., FEBS. Lett. 390:179 et al., Mol. Microbiol 27:477-492 (1998)). The E. coli 182 (1996)). The protein sequences for each of these exem gamma-glutamyl kinase, encoded by proB (Smith et al., J. plary gene products, if available, can be found using the Bacteriol. 157:545-551 (1984b)), phosphorylates the following GenBank accession numbers shown in Table 35. gamma carbonic acid group of glutamate. The protein sequences for each of these exemplary gene products, if TABLE 35 available, can be found using the following GenBank acces Gene Accession No. GI No. Organism sion numbers shown in Table 37. aspC NP 415448.1 16128895 Escherichia coi AAT2 P23S42.3 1703040 Saccharomyces cerevisiae TABLE 37 ASPS P46248.2 20532373 Arabidopsis thaliana Got2 POO507 112987 Rattus norvegicus Gene Accession No. GI No. Organism avt.A YP O26231.1 49176374 Escherichia coi buk1 NP 349675 15896326 Clostridium acetobutyllicum serC NP 415427.1 16128874 Escherichia coi buk2 Q97II1 20137415 Clostridium acetobutyllicum buk2 Q9X278.1 6685256 Thermotoga maritima lysC NP 418.448.1 16131850 Escherichia coi 0342 Another enzyme candidate is alpha-aminoadipate ackA NP 416799.1 16130231 Escherichia coi aminotransferase (EC 2.6.1.39), an enzyme that participates proB NP 414777.1 16128228 Escherichia coi in lysine biosynthesis and degradation in some organisms. This enzyme interconverts 2-aminoadipate and 2-oxoadi pate, using alpha-ketoglutarate as the amino acceptor. Gene 0344 Acetylglutamate kinase phosphorylates acetylated candidates are found in Homo sapiens (Okuno et al., Enzyme glutamate during arginine biosynthesis. This enzyme is not Protein 47:136-148 (1993)) and Thermus thermophilus (Mi known to accept alternate Substrates; however, several resi yazaki et al., 150:2327-2334 (2004)). The Thermus thermo dues of the E. coli enzyme involved in substrate binding and philus enzyme, encoded by lysN, is active with several phosphorylation have been elucidated by site-directed muta alternate Substrates including oxaloacetate, 2-oxoiso genesis (Marco-Marin et al., 334:459-476 (2003); Ramon caproate, 2-oxoisovalerate, and 2-oxo-3-methylvalerate. Maiques et al., Structure. 10:329-342 (2002)). The enzyme The protein sequences for each of these exemplary gene is encoded by argB in Bacillus subtilis and E. coli (Parsot et products, if available, can be found using the following al., Gene 68:275-283 (1988)), and ARG5.6 in S. cerevisiae GenBank accession numbers shown in Table 36. (Pauwels et al., Eur: J Biochem. 270: 1014-1024 (2003)). The ARG5.6 gene of S. cerevisiae encodes a polyprotein pre TABLE 36 cursor that is matured in the mitochondrial matrix to become Gene Accession No. GI No. Organism acetylglutamate kinase and acetylglutamylphosphate reduc lysN BACT 6939.1 31096548 Thermus thermophilus tase. The protein sequences for each of these exemplary AadAT-II Q8N5Z0.2 46395904 Homo sapiens gene products, if available, can be found using the following GenBank accession numbers shown in Table 38.

2.7.2.a Phosphotransferase (Carboxy Acceptor) TABLE 38 (0343 Step O of FIG. 1 involves the conversion of 4-hy Gene Accession No. GI No. Organism droxybutyrate to 4-hydroxybutyryl-phosphate by the phos argB NP 418394.3 145698.337 Escherichia coi photransferase enzymes in the EC class 2.7.2 that transform argB NP 3890O3.1 16078186 Bacilius Subiiis carboxylic acids to phosphonic acids with concurrent hydro ARG5, 6 NP 010992.1 6320913 Saccharomyces cerevisiae lysis of one ATP. Butyrate kinase (EC 2.7.2.7) carries out the reversible conversion of butyryl-phosphate to butyrate dur ing acidogenesis in C. acetobutylicum (Cary et al., 56:1576 1583 (1990)). This enzyme is encoded by either of the two 2.8.3.a CoA Transferase buk gene products (Huang et al., J Mol. Microbiol Biotech not 2:33-38 (2000)). Other butyrate kinase enzymes are (0345 Steps G and T in FIG. 1 involve CoA transferase found in C. butyricum and C. tetanomorphum (TWAROG et activities. The gene products of catl, cat2, and cat3 of al., J Bacteriol. 86: 112-117 (1963)). Related enzyme isobu Clostridium kluyveri have been shown to exhibit succinyl tyrate kinase from Thermotoga maritima has also been CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltrans expressed in E. coli and crystallized (Diao et al., J Bacteriol. ferase activities (Seedorf et al. Proc Natl Acad Sci U.S.A. 191:2521-2529 (2009); Diao et al., Acta Crystallogr. D. 105(6):2128-2133 (2008); Sohling and GottschalkJ Bacte Biol. Crystallogr. 59:1100-1102 (2003)). Aspartokinase riol 178(3):871-880 (1996)). Similar CoA transferase activi catalyzes the ATP-dependent phosphorylation of aspartate ties are also present in Trichomonas vaginalis (van Grinsven and participates in the synthesis of several amino acids. The et al., J. Biol. Chem. 283:141 1-1418 (2008)) and Trypano aspartokinase III enzyme in E. coli, encoded by lysC, has a soma bruceii (Riviere et al., J. Biol. Chem. 279:45337-45346 broad Substrate range and the catalytic residues involved in (2004)). The protein sequences for each of these exemplary Substrate specificity have been elucidated (Keng et al., gene products, if available, can be found using the following 335:73-81 (1996)). Two additional kinases in E. coli are also GenBank accession numbers shown in Table 39. US 2016/03193 13 A1 Nov. 3, 2016 49

TABLE 39 TABLE 41 Gene Accession No. GI No. Organism Gene Accession No. GI No. Organism getA CAAS71991 559392 Acidaminococcus fermenians gctB CAAS72001 catl P38946.1 729048 Clostridium kluyveri 559393 Acidaminococcus fermenians cat2 P38942.2 1705614 Clostridium kluyveri cats EDK35586.1 146349050 Clostridium kluyveri 3.1.1.a Hydroxyacylhydrolase TVAG 395550 XP OO1330176 123975034 Trichomonas vaginalis 0348 Step AB in FIG. 1 is the transformation of 4-hy droxybutyrate to GBL. This step can be catalyzed by Tb11.02.0290 XP 828352 71754.875 Trypanosoma brucei enzymes in the 3.1.1 family that act on carboxylic ester bonds molecules for the interconversion between cyclic lactones and the open chain hydroxycarboxylic acids. The 0346. An additionally useful enzyme for this type of 1.4-lacton hydroxyacylhydrolase (EC 3.1.1.25), also known transformation is acyl-CoA:acetate-CoA transferase, also as 1.4-lactonase or gamma-lactonase, is specific for 1,4- known as acetate-CoA transferase (EC 2.8.3.8), which has lactones with 4-8 carbon atoms. It does not hydrolyze simple been shown to transfer the CoA moiety to acetate from a aliphatic esters, acetylcholine, or Sugar lactones. The gamma variety of branched and linear acyl-CoA substrates, includ lactonase in human blood and rat liver microsomes was ing isobutyrate (Matthies and Schink Appl Environ Micro purified (Fishbeinet al., J Biol Chem 241:4835-4841 (1966)) biol 58:1435-1439 (1992)), Valerate (Vanderwinkel et al. and the lactonase activity was activated and stabilized by Biochem. Biophys. Res Commun. 33:902-908 (1968)) and calcium ions (Fishbein et al., J Biol Chem 241:4842-4847 butanoate (Vanderwinkel, supra (1968)). This enzyme is (1966)). The optimal lactonase activities were observed at encoded by atoA (alpha subunit) and ato D (beta subunit) in pH 6.0, whereas high pH resulted in hydrolytic activities E. coli sp. K12 (Korolev et al. Acta Crystallogr. D Biol (Fishbein and Bessman, J Biol Chem 241:4842-4847 Crystallogr. 58:21 16-2121 (2002); Vanderwinkel, supra (1966)). The following genes have been annotated as 1,4- (1968)). Similar enzymes exist in Corynebacterium gluta lactonase and can be utilized to catalyze the transformation micum ATCC 13032 (Duncan et al., 68:5186-5190 (2002)), of 4-hydroxybutyrate to GBL, including a lactonase from Clostridium saccharoperbutylacetonicum (Kosaka et al., Fusarium oxysporum (Zhang et al., Appl Microbiol Biotech Biosci. Biotechnol Biochem. 71:58-68 (2007)), and nol 75:1087-1094 (2007)). The protein sequences for each of Clostridium acetobutyllicum (Cary et al., 56:1576-1583 these exemplary gene products, if available, can be found (1990); Wiesenborn et al., 55:323-329 (1989)). The protein using the following GenBank accession numbers shown in sequences for each of these exemplary gene products, if Table 42. available, can be found using the following GenBank acces TABLE 42 sion numbers shown in Table 40. Gene Accession No. GI No. Organism TABLE 40 Xccb100 2516 YP 001903921.1 188991911 Xanthomonas campestris Gene Accession No. GI No. Organism An16g06620 CAK46996.1 134083519 Aspergillus niger atoA P76459.1 2492994 Escherichia Coi K12 BAA34062 BAA34062.1 3810873 Fusarium oxysporum ato) P764.58.1 2492990 Escherichia Coi K12 actA YP 2268.09.1 62391407 Corynebacterium glutamicum cg0592 YP 224801.1 62389399 Corynebacterium glutamicum 0349 Additionally, it has been reported that lipases such ctfA NP 149326.1 15004.866 Clostridium acetobutyllicum as Candida antarcticalipase B can catalyze the lactonization ctfB NP 1493.27.1 15004867 Clostridium acetobutyllicum of 4-hydroxybutyrate to GBL (Efeet al., Biotechnol Bioeng ctfA AAP42564.1 31075384 Cliostridium Saccharoperbait-lacetonictim 99:1392-1406 (2008)). Therefore, the following genes cod ctfB AAP42565.1 31075385 Cliostridium ing for lipases can also be utilized for Step AB in FIG.1. The Saccharoperbait-lacetonictim protein sequences for each of these exemplary gene prod ucts, if available, can be found using the following GenBank accession numbers shown in Table 43. 0347 The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium Acidaminococcus fer TABLE 43 mentans reacts with diacid glutaconyl-CoA and 3-butenoyl CoA (Mack and Buckel FEBS Lett. 405:209-212 (1997)). Gene Accession No. GI No. Organism The genes encoding this enzyme are gctA and gctB. This calB P41365.1 1170790 Candida antarctica enzyme has reduced but detectable activity with other CoA lip B P41773.1 1170792 Pseudomonas fittorescens derivatives including glutaryl-CoA, 2-hydroxyglutaryl estA P37957.1 7676155 Bacilius subtilis CoA, adipyl-CoA and acrylyl-CoA (Buckel et al. Eur: J. Biochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mac et al. Eur: J. Biochem. 3.1.2.a CoA Hydrolase 226:41-51 (1994)). The protein sequences for each of these 0350 Steps H and U in FIG. 1 involve enzymes in the exemplary gene products, if available, can be found using 3.1.2 family that hydrolyze acyl-CoA molecules to their the following GenBank accession numbers shown in Table corresponding acids. Nevertheless, perhaps such enzymes 41. can be modified to embark CoA-ligase or synthetase func US 2016/03193 13 A1 Nov. 3, 2016 50 tionality if coupled to an energy source Such as a proton for each of these exemplary gene products, if available, can pump or direct ATP hydrolysis. Several eukaryotic acetyl be found using the following GenBank accession numbers CoA (EC 3.1.2.1) have broad substrate specific shown in Table 46. ity. For example, the enzyme from Rattus norvegicus brain (Robinson, Jr. et al., 71:959-965 (1976)) can react with TABLE 46 butyryl-CoA, hexanoyl-CoA and malonyl-CoA. Though its sequence has not been reported, the enzyme from the Gene Accession No. GI No. Organism mitochondrion of the pea leaf also has a broad substrate getA CAAS71991 559392 Acidaminococcus fermenians specificity, with demonstrated activity on acetyl-CoA, pro gctB CAAS72001 559393 Acidaminococcus fermenians pionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, and crotonyl-CoA (Zeiher et al., Plant. 0353. Additional hydrolase enzymes include 3-hydroxy Physiol. 94:20-27 (1990)). The acetyl-CoA hydrolase, isobutyryl-CoA hydrolase which has been described to effi ACH1, from S. cerevisiae represents another candidate ciently catalyze the conversion of 3-hydroxyisobutyryl-CoA hydrolase (Buu et al., J. Biol. Chem. 278:17203-17209 to 3-hydroxyisobutyrate during valine degradation (Shimo (2003)). The protein sequences for each of these exemplary mura et al., J Biol Chem. 269: 14248-14253 (1994)). Genes gene products, if available, can be found using the following encoding this enzyme include hibch of Rattus norvegicus GenBank accession numbers shown in Table 44. (Shimomura et al., Supra; Shimomura et al., Methods Enzy mol. 324:229-240 (2000)) and Homo sapiens (Shimomura et TABLE 44 al., Supra). Candidate genes by sequence homology include hibch of Saccharomyces cerevisiae and BC 2292 of Bacillus Gene Accession No. GI No. Organism cereus. The protein sequences for each of these exemplary acot12 NP 570103.1 18543355 Rattus norvegicus gene products, if available, can be found using the following ACH1 NP O09538 6319456 Saccharomyces cerevisiae GenBank accession numbers shown in Table 47.

TABLE 47 0351. Another candidate hydrolase is the human dicar boxylic acid thioesterase, acot:8, which exhibits activity on Gene Accession No. GI No. Organism glutaryl-CoA, adipyl-CoA, Suberyl-CoA, sebacyl-CoA, and hibch Q5XIE6.2 146324906 Rattus norvegicus dodecanedioyl-CoA (Westin et al., 280:38125-38132 hibch Q6NVY1.2 146324905 Homo sapiens (2005)) and the closest E. coli homolog, tesB, which can hibch P28817.2 2506374 Saccharomyces cerevisiae also hydrolyze a broad range of CoA thioesters (Naggert et BC 2292 APO92S6 29895.975 Bacilius ceretts al., 266:11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana, Biochem. Int. 26:767-773 (1992)). Other potential E. coli thioester hydro 4.1.1.a Carboxy-Lyase lases include the gene products of tesA (Bonner et al., 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS 0354 Alpha-ketoglutarate decarboxylase (Step B), glu Microbiol Rev 29:263-279 (2005); Zhuang et al., 516:161 tamate decarboxylase (Step L), and 5-hydroxy-2-oxopen 163 (2002)), paal (Song et al., 281:11028-11038 (2006)), tanoic acid decarboxylase (Step Z) in FIG. 1 all involve the andybdB (Leduc et al., J Bacteriol. 189:7112-7126 (2007)). decarboxylation of an alpha-ketoacid. The decarboxylation The protein sequences for each of these exemplary gene of keto-acids is catalyzed by a variety of enzymes with products, if available, can be found using the following varied Substrate specificities, including pyruvate decarboxy GenBank accession numbers shown in Table 45. lase (EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1. 7), alpha-ketoglutarate decarboxylase and branched-chain TABLE 45 alpha-ketoacid decarboxylase. 0355 Pyruvate decarboxylase (PDC), also termed keto Gene Accession No. GI No. Organism acid decarboxylase, is a key enzyme in alcoholic fermenta acot& CAA1SSO2 3.19.1970 Homo sapiens tion, catalyzing the decarboxylation of pyruvate to acetal tesB NP 41.4986 16128437 Escherichia coi dehyde. The enzyme from Saccharomyces cerevisiae has a acot& NP 570112 51036669 Ratti is norvegicus broad Substrate range for aliphatic 2-keto acids including tes.A NP 415027 16128478 Escherichia coi ybgC NP 415264 16128711 Escherichia coi 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvate and paa NP 415914 16129357 Escherichia coi 2-phenylpyruvate (22). This enzyme has been extensively ybdB NP 415129 16128580 Escherichia coi studied, engineered for altered activity, and functionally expressed in E. coli (Killenberg-Jabs et al., 268: 1698-1704 (2001): Li et al., Biochemistry. 38:10004-10012 (1999); ter 0352 Yet another candidate hydrolase is the glutaconate Schure et al., Appl. Environ. Microbiol. 64:1303-1307 CoA-transferase from Acidaminococcus fermentans. This (1998)). The PDC from Zymomonas mobilus, encoded by enzyme was transformed by site-directed mutagenesis into pdc, also has a broad Substrate range and has been a subject an acyl-CoA hydrolase with activity on glutaryl-CoA, of directed engineering studies to alter the affinity for acetyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS. Lett. different substrates (Siegert et al., 18:345-357 (2005)). The 405:209-212 (1997)). This suggests that the enzymes encod crystal structure of this enzyme is available (Killenberg-Jabs ing Succinyl-CoA:3-ketoacid-CoA transferases and et al., 268: 1698-1704 (2001)). Other well-characterized acetoacetyl-CoA:acetyl-CoA transferases may also serve as PDC candidates include the enzymes from Acetobacter candidates for this reaction step but would require certain pasteurians (Chandra et al., 176:443-451 (2001)) and mutations to change their function. The protein sequences Kluyveromyces lactis (Krieger et al., 269:3256-3263 US 2016/03193 13 A1 Nov. 3, 2016

(2002)). The protein sequences for each of these exemplary identified by testing candidate genes containing this N-ter gene products, if available, can be found using the following minal sequence for KDC activity. The protein sequences for GenBank accession numbers shown in Table 48. each of these exemplary gene products, if available, can be found using the following GenBank accession numbers TABLE 48 shown in Table 50. Gene Accession No. GI No. Organism TABLE 50 pdc PO6672.1 118391 Zymomonas mobilus pdc1 PO6169 30923172 Saccharomyces cerevisiae Gene Accession No. GI No. Organism pdc AM21208 20385191 Acetobacter pasteurians pdc1 Q12629 52788279 Kluyveromyces lactis kgd OSO463.4 160395583 Mycobacterium tuberculosis kgd NP 767092.1 27375.563 Bradyrhizobium japonicum kgd NP 105204.1 13473636 Mesorhizobium ioti 0356. Like PDC, benzoylformate decarboxylase (EC 4.1. 1.7) has a broad Substrate range and has been the target of enzyme engineering studies. The enzyme from Pseudomo 0358. A fourth candidate enzyme for catalyzing this nas putida has been extensively studied and crystal struc reaction is branched chain alpha-ketoacid decarboxylase tures of this enzyme are available (Polovnikova et al., (BCKA). This class of enzyme has been shown to act on a 42:1820-1830 (2003); Hasson et al., 37:9918-9930 (1998)). variety of compounds varying in chain length from 3 to 6 Site-directed mutagenesis of two residues in the active site carbons (Oku et al., 263:18386-18396 (1988); Smit et al., of the Pseudomonas putida enzyme altered the affinity (Kim) 71:303-311 (2005)). The enzyme in Lactococcus lactis has of naturally and non-naturally occurring Substrates (Siegert been characterized on a variety of branched and linear et al., 18:345-357 (2005)). The properties of this enzyme Substrates including 2-oxobutanoate, 2-oxohexanoate, have been further modified by directed engineering (Lingen 2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2- et al., Chembiochem. 4:721-726 (2003); Lingen et al., Pro oxobutanoate and isocaproate (Smit et al., 71:303-311 tein Eng 15:585-593 (2002)). The enzyme from Pseudomo (2005)). The enzyme has been structurally characterized nas aeruginosa, encoded by mdlC, has also been character (Berg et al., 318:1782-1786 (2007)). Sequence alignments ized experimentally (Barrowman et al., 34:57-60 (1986)). between the Lactococcus lactis enzyme and the pyruvate Additional gene candidates from Pseudomonas Stutzeri, decarboxylase of Zymomonas mobilus indicate that the Pseudomonas fluorescens and other organisms can be catalytic and substrate recognition residues are nearly iden inferred by sequence homology or identified using a growth tical (Siegert et al., 18:345-357 (2005)), so this enzyme selection system developed in Pseudomonas putida (Hen would be a promising candidate for directed engineering. ning et al., Appl. Environ. Microbiol. 72:7510-7517 (2006)). Decarboxylation of alpha-ketoglutarate by a BCKA was The protein sequences for each of these exemplary gene detected in Bacillus subtilis; however, this activity was low products, if available, can be found using the following (5%) relative to activity on other branched-chain substrates GenBank accession numbers shown in Table 49. (Oku and Kaneda, 263:18386-18396 (1988)) and the gene encoding this enzyme has not been identified to date. Addi TABLE 49 tional BCKA gene candidates can be identified by homology to the Lactococcus lactis protein sequence. Many of the Gene Accession No. GINo. Organism high-scoring BLASTp hits to this enzyme are annotated as mdC P20906.2 3915757 Pseudomonas putida indolepyruvate decarboxylases (EC 4.1.1.74). Indolepyru mdC Q9HUR2.1 81539678 Pseudomonas aeruginosa vate decarboxylase (IPDA) is an enzyme that catalyzes the dpg|B ABN804231 1262021.87 Pseudomonas Stutzeri decarboxylation of indolepyruvate to indoleacetaldehyde in ilwB-1 YP 260581.1 70730840 Pseudomonas fluorescens plants and plant bacteria. The protein sequence for the exemplary gene product, if available, can be found using the 0357. A third enzyme capable of decarboxylating 2-ox following GenBank accession numbers shown in Table 51. oacids is alpha-ketoglutarate decarboxylase (KGD). The Substrate range of this class of enzymes has not been studied TABLE 51 to date. The KDC from Mycobacterium tuberculosis (Tianet al., 102:10670-10675 (2005)) has been cloned and function Gene Accession No. GINo. Organism ally expressed in other internal projects at Genomatica. kdcA AAS49166.1 44921617 Lactococci is lactis However, it is not an ideal candidate for strain engineering because it is large (~130 kDa) and GC-rich. KDC enzyme activity has been detected in several species of rhizobia 0359 Recombinant branched chain alpha-keto acid including Bradyrhizobium japonicum and Mesorhizobium decarboxylase enzymes derived from the E1 subunits of the loti (Green et al., 182:2838-2844 (2000)). Although the mitochondrial branched-chain keto acid dehydrogenase KDC-encoding gene(s) have not been isolated in these complex from Homo sapiens and Bos taurus have been organisms, the genome sequences are available and several cloned and functionally expressed in E. coli (Davie et al., genes in each genome are annotated as putative KDCs. A 267:16601-16606 (1992); Wynn et al., 267: 12400-12403 KDC from Euglena gracilis has also been characterized but (1992); Wynn et al., 267: 1881-1887 (1992)). In these stud the gene associated with this activity has not been identified ies, the authors found that co-expression of chaperonins to date (Shigeoka et al., 288:22-28 (1991)). The first twenty GroEL and GroES enhanced the specific activity of the amino acids starting from the N-terminus were sequenced decarboxylase by 500-fold (Wynn et al., 267:12400-12403 MTYKAPVKDVKFLLDKVFKV (SEQ ID NO: 3) (Shi (1992)). These enzymes are composed of two alpha and two geoka and Nakano, 288:22-28 (1991)). The gene could be beta subunits. The protein sequences for each of these US 2016/03193 13 A1 Nov. 3, 2016 52 exemplary gene products, if available, can be found using TABLE 54-continued the following GenBank accession numbers shown in Table 52. Gene Accession No. GI No. Organism bioW NP 390902.2 50812281 Bacilius Subiiis TABLE 52 AACS NP 084486.1 21313520 Mus musculius AACS NP O76417.2 31982927 Homo sapiens Gene Accession No. GI No. Organism Msed 1422 YP 001191504 146304188 Metaliosphaera sedulla BCKDHB NP 898.871.1 34101272 Homo sapiens BCKDHA NP OOO700.1 11386135 Homo sapiens 0362 ADP-forming acetyl-CoA synthetase (ACD, EC BCKDHB P21839 1155.02434 Bos tattrits 6.2.1.13) is another candidate enzyme that couples the BCKDHA P111.78 129030 Bos tattrits conversion of acyl-CoA esters to their corresponding acids with the concurrent synthesis of ATP. Several enzymes with broad substrate specificities have been described in the 6.2.1.a CoA Synthetase literature. ACDI from Archaeoglobus fulgidus, encoded by 0360 Step I of FIG. 1 involves CoA synthetase or ligase AF1211, was shown to operate on a variety of linear and reactions for Succinate or 4-hydroxybutyrate as the Sub branched-chain Substrates including acetyl-CoA, propionyl strates. Exemplary genes encoding enzymes likely to carry CoA, butyryl-CoA, acetate, propionate, butyrate, isobu out these transformations include the sucCD genes of E. coli tyrate, isovalerate. Succinate, fumarate, phenylacetate, which naturally form a Succinyl-CoA synthetase complex. indoleacetate (Musfeldt et al., J Bacteriol 184:636-644 This enzyme complex naturally catalyzes the formation of (2002)). The enzyme from Haloarcula marismortui (anno Succinyl-CoA from Succinate with the concaminant con tated as a Succinyl-CoA synthetase) accepts propionate, sumption of one ATP, a reaction which is reversible in vivo butyrate, and branched-chain acids (isovalerate and isobu (Buck et al., Biochem. 24.6245-6252 (1985)). The protein tyrate) as Substrates, and was shown to operate in the sequences for each of these exemplary gene products, if forward and reverse directions (Brasen et al., Arch Microbiol available, can be found using the following GenBank acces 182:277-287 (2004)). The ACD encoded by PAE3250 from sion numbers shown in Table 53. hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized TABLE 53 ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred Substrate) and phenylacetyl-CoA (Brasen et al., Supra). The Gene Accession No. GI No. Organism enzymes from A. fulgidus, H. marismortui and P. aerophi SucC NP 415256.1 16128.703 Escherichia coi lum have all been cloned, functionally expressed, and char SucD AAC738231 1786949 Escherichia coi acterized in E. coli (Musfeldt et al., Supra; Brasen et al., Supra). The protein sequences for each of these exemplary 0361 Additional exemplary CoA-ligases include the rat gene products, if available, can be found using the following dicarboxylate-CoA ligase for which the sequence is yet GenBank accession numbers shown in Table 55. uncharacterized (Vamecq et al., Biochemical Journal 230: 683-693 (1985)), either of the two characterized phenylac TABLE 55 etate-CoA ligases from P. chrysogenium (Lamas-Maceiras et Gene Accession No. GI No. Organism al., Biochem. J. 395:147-155 (2005); Wang et al., Biochem AF1211 NP 070039.1 11498.810 Archaeoglobus fulgidus Biophy Res Commun 360(2):453-458 (2007)), the pheny DSM 4304 lacetate-CoA ligase from Pseudomonas putida (Martinez SCS YP 135572.1 55377722 Haioarctia marismortii Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)), and the ATCC 43O49 6-carboxyhexanoate-CoA ligase from Bacillus subtilis PAE32SO NP 560604.1 18313937 Pyrobaculum aerophilum (Boweret al., J. Bacteriol. 178(14):4122-4130 (1996)). Str. IM2 Additional candidate enzymes are acetoacetyl-CoA syn thetases from Mus musculus (Hasegawa et al., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens Example III (Ohgami et al., Biochem Pharmacol 65:989-994 (2003)) which naturally catalyze the ATP-dependant conversion of Biosynthesis of 4-Hydroxybutanoic Acid (4-HB) acetoacetate into acetoacetyl-CoA. 4-hydroxybutyryl-CoA synthetase activity has been demonstrated in Metallospha 0363 This example describes exemplary biochemical era sedula (Berget al., Science 318:1782-1786 (2007)). This pathways for 4-HB production. 4-HB is the common inter function has been tentatively assigned to the Msed 1422 mediate for both 14-BDO and GBL productions. gene. The protein sequences for each of these exemplary 0364 Previous reports of 4-HB synthesis in microbes gene products, if available, can be found using the following have focused on this compound as an intermediate in GenBank accession numbers shown in Table 54. production of the biodegradable plastic poly-hydroxyal kanoate (PHA) (U.S. Pat. No. 6,117,658). The use of 4-HB/ TABLE 54 3-hydroxybutyrate (3-EIB) copolymers over poly-3-hy droxybutyrate polymer (PHB) can result in plastic that is Gene Accession No. GI No. Organism less brittle (Saito and Doi, Intl. J. Biol. Macromol. 16:99 phl CA15517.1 77019264 Penicilium chrysogenium 104 (1994)). The production of monomeric 4-HB described phlB ABS19624.1 152002983 Penicilium chrysogenium herein is a fundamentally distinct process for several rea paaF AAC24333.2 22711873 Pseudomonas putida sons: (1) the product is secreted, as opposed to PHA which is produced intracellularly and remains in the cell; (2) for US 2016/03193 13 A1 Nov. 3, 2016 organisms that produce hydroxybutanoate polymers, free 0367 The microbial production capabilities of 4-HB 4-HB is not produced, but rather the Coenzyme A derivative were explored in two microbes, E. coli and S. cerevisiae, is used by the polyhydroxyalkanoate synthase; (3) in the using in silico metabolic models of each organism. Potential case of the polymer, formation of the granular product pathways to 4-HB proceed via a succinate. Succinyl-CoA, or changes thermodynamics; and (4) extracellular pH is not an alpha-ketoglutarate intermediate as shown in FIG. 1. issue for production of the polymer, whereas it will affect 0368 A first step in the 4-HB production pathway from whether 4-HB is present in the free acid or conjugate base Succinate involves the conversion of Succinate to Succinic state, and also the equilibrium between 4-HB and GBL. semialdehyde via an NADH- or NADPH-dependant suc 0365 4-HB can be produced in two enzymatic reduction cinic semialdehyde dehydrogenase. In E. coli, gab) is an steps from succinate, a central metabolite of the TCA cycle, NADP-dependant succinic semialdehyde dehydrogenase with succinic semialdehyde as the intermediate (FIG. 1). and is part of a gene cluster involved in 4-aminobutyrate The first of these enzymes, succinic semialdehyde dehydro uptake and degradation (Schneider et al., J. Bacteriol. 184: genase, is native to many organisms including E. coli, in 6976-6986 (2002); Niegemann et al., Arch. Microbiol 160: which both NADH- and NADPH-dependent enzymes have 454-460 (1993)). Sad is believed to encode the enzyme for been found (Donnelly and Cooper, Eur: J. Biochem. 113: NAD-dependant Succinic semialdehyde dehydrogenase 555-561 (1981); Donnelly and Cooper, J. Bacteriol. 145: activity (Marek et al., J Bacteriol. 170:991-994 (1988)). S. 1425-1427 (1981); Marek and Henson, J. Bacteriol. 170: cerevisiae contains only the NADPH-dependant succinic 991-994 (1988)). There is also evidence supporting succinic semialdehyde dehydrogenase, putatively assigned to UGA2, semialdehyde dehydrogenase activity in S. cerevisiae (Ra which localizes to the cytosol (Huh et al., 425:686-691 mos et al., Eur: J. Biochem. 149:401–404 (1985)), and a (2003)). The maximum yield calculations assuming the putative gene has been identified by sequence homology. succinate pathway to 4-HB in both E. coli and S. cerevisiae However, most reports indicate that this enzyme proceeds in require only the assumption that a non-native 4-HB dehy the direction of Succinate synthesis (Donnelly and Cooper, drogenase has been added to their metabolic networks. supra; Lutke-Eversloh and Steinbuchel, FEMS Microbiol. 0369. The pathway from succinyl-CoA to 4-hydroxybu Lett. 181:63-71 (1999)), participating in the degradation tyrate was described in U.S. Pat. No. 6,117,658 as part of a pathway of 4-HB and gamma-aminobutyrate. Succinic process for making polyhydroxyalkanoates comprising semialdehyde also is natively produced by certain microbial 4-hydroxybutyrate monomer units. Clostridium kluyveri is organisms such as E. coli through the TCA cycle interme one example organism known to possess CoA-dependant diate O-ketogluterate via the action of two enzymes: gluta Succinic semialdehyde dehydrogenase activity (Sohling et mate:Succinic semialdehyde transaminase and glutamate al., Eur: J Biochem. 212:121-127 (1993); Sohling and decarboxylase. An alternative pathway, used by the obligate Gottschalk, J Bacteriol. 178:871-880 (1996)). In this study, anaerobe Clostridium kluyveri to degrade Succinate, acti it is assumed that this enzyme, from C. kluyveri or another Vates Succinate to Succinyl-CoA, then converts Succinyl organism, is expressed in E. coli or S. cerevisiae along with CoA to Succinic semialdehyde using an alternative Succinic a non-native or heterologous 4-HB dehydrogenase to com semialdehyde dehydrogenase which is known to function in plete the pathway from succinyl-CoA to 4-HB. The pathway this direction (Sohling and Gottschalk, Eur: Biochem. 212: from alpha-ketoglutarate to 4-HB was demonstrated in E. 121-127 (1993)). However, this route has the energetic cost coli resulting in the accumulation of poly(4-hydroxybutyric of ATP required to convert succinate to succinyl-CoA. acid) to 30% of dry cell weight (Song et al., Wei Sheng Wu Xue. Bao. 45:382-386 (2005)). As E. coli and S. cerevisiae 0366. The second enzyme of the pathway, 4-hydroxybu natively or endogenously possess both glutamate:Succinic tanoate dehydrogenase, is not native to E. coli or yeast but semialdehyde transaminase and glutamate decarboxylase is found in various bacteria Such as C. kluyveri and Ralstonia (Coleman et al., J Biol Chem. 276:244-250 (2001)), the eutropha (Lutke-Eversloh et al., FEMS Microbiol Lett. 181: pathway from alpha-ketoglutarate to 4-HB can be completed 63-71 (1999); Sohling et al., J Bacteriol. 178:871-880 in both organisms by assuming only that a non-native 4-HB (1996); Wolff et al., Protein Expr: Purif. 6:206-212 (1995); dehydrogenase is present. Valentin et al., Eur: J Biochem. 227:43-60 (1995)). These enzymes are known to be NADH-dependent, though NADPH-dependent forms also exist. An additional pathway Example IV to 4-HB from alpha-ketoglutarate was demonstrated in E. Biosynthesis of 1,4-Butanediol from Succinate and coli resulting in the accumulation of poly(4-HB) (Song et Alpha-Ketoglutarate al., Wei Sheng Wu Xue. Bao. 45:382-386 (2005)). The recombinant Strain required the overexpression of three 0370. This example illustrates the construction and bio heterologous genes, PHA synthase (R. eutropha), 4-hy synthetic production of 4-HB and 14-BDO from microbial droxybutyrate dehydrogenase (R. eutropha) and 4-hydroxy organisms. Pathways for 4-HB and 14-BDO are disclosed butyrate:CoA transferase (C. kluyveri), along with two herein. native E. coli genes: glutamate:Succinic semialdehyde 0371. There are several alternative enzymes that can be transaminase and glutamate decarboxylase. Alpha-ketoglu utilized in the pathway described above. The native or tarate can be directly converted to succinic semialdehyde by endogenous enzyme for conversion of Succinate to Succinyl an alpha-ketoglutarate decarboxylase Such as the one iden CoA can be replaced by a CoA transferase such as that tified in Euglena gracilis (Shigeoka et al., Arch. Biochem. encoded by the cat1 gene C. kluyveri (Sohling and Biophys. 288:22-28 (1991); Shigeoka et al., Biochem. J. 282 Gottschalk, Eur: J Biochem. 212:121-127 (1993)), which (Pt 2):319-323 (1992); Shigeoka et al., Biochem. J. 292 (Pt functions in a similar manner to the conversion of 4-HB to 2):463-467 (1993)). However, this enzyme has not previ 4-hydroxybutyryl-CoA. However, the production of acetate ously been applied to impact the production of 4-HB or by this enzyme may not be optimal, as it might be secreted related polymers in any organism. rather than being converted back to acetyl-CoA. In this US 2016/03193 13 A1 Nov. 3, 2016 54 respect, it also can be beneficial to eliminate acetate forma alternative is to use an alpha-ketoglutarate decarboxylase tion. As one alternative to this CoA transferase, a mechanism that can perform this conversion in one step (Tian et al., Proc can be employed in which the 4-HB is first phosphorylated Natl AcadSci USA 102:10670-10675 (2005)). by ATP and then converted to the CoA derivative, similar to 0373 For the construction of different strains of the acetate kinase/phosphotransacetylase pathway in E. coli 14-BDO-producing microbial organisms, a list of applicable genes was assembled for corroboration. Briefly, one or more for the conversion of acetate to acetyl-CoA. The net cost of genes within the 4-HB and/or 14-BDO biosynthetic path this route is one ATP, which is the same as is required to ways were identified for each step of the complete 14-BDO regenerate acetyl-CoA from acetate. The enzymes phospho producing pathway shown in FIG. 1, using available litera transbutyrylase (ptb) and butyrate kinase (bk) are known to ture resources, the NCBI genetic database, and homology carry out these steps on the non-hydroxylated molecules for searches. The genes cloned and assessed in this study are butyrate production in C. acetobutyllicum (Cary et al., Appl presented below in Table 56, along with the appropriate Environ Microbiol 56:1576-1583 (1990); Valentine, R. C. references and gene accession and GI numbers to the poly and R. S. Wolfe, J Biol Chem. 235:1948-1952 (1960)). peptide sequence. As discussed further below, Some genes These enzymes are reversible, allowing synthesis to proceed were synthesized for codon optimization while others were in the direction of 4-HB. cloned via PCR from the genomic DNA of the native or 0372 14-BDO also can be produced via alpha-ketoglut wild-type organism. For Some genes both approaches were arate in addition to or instead of through Succinate. As used, and in this case the native genes are indicated by an described herein, one pathway to accomplish product bio “n” suffix to the gene identification number when used in an synthesis is with the production of Succinic semialdehyde experiment. Note that only the DNA sequences differ; the via alpha-ketoglutarate using the endogenous enzymes. An proteins are identical. TABLE 56 Genes expressed in host 14-BDO-producting microbial organisms. Gene ID Reaction Gene Source number (FIG. 1) name organism Enzyme name GI number Reference OOO1 T Cat? Cliostridium 4-hydroxybutyrate 1228100 1 kluyveri coenzyme A DSM 555 transferase O002 Q/E adhE Cliostridium Aldehydefalcohol 1SOO4739 2 acetobutylictim dehydrogenase ATCC 824 O003 Q/E adhE2 Cliostridium Aldehydefalcohol 1SOO486S 2 acetobutylictim dehydrogenase ATCC 824 OOO4 G Cat1 Cliostridium Succinate coenzyme 1228100 1 kluyveri A transferase DSM 555 OOO8 A SucD Cliostridium Succinic 1228100 1 kluyveri semialdehyde DSM 555 dehydrogenase (CoA dependent) OOO9 C 4-HBd Ralstonia 4-hydroxybutyrate 113867S64 2 eutropha H16 dehydrogenase (NAD-dependent) OO10 C 4-HBd Ciostridium 4-hydroxybutyrate 1228100 1 kluyveri dehydrogenase DSM 555 (NAD-dependent) O011 Q/E adhE E. coi Aldehydefalcohol 161292O2 dehydrogenase O012 Q/E yghD E. coli Aldehydefalcohol 16130909 dehydrogenase OO13 E bdhB Cliostridium Butanol 15896542 2 acetobutylictim dehydrogenase II ATCC 824 OO2O P ptb Cliostridium Phospho- 15896327 2 acetobutylictim transbutyrylase ATCC 824 OO21 O buk1 Cliostridium Butyrate kinase I 20137334 2 acetobutylictim ATCC 824 OO22 O buk2 Cliostridium Butyrate kinase II 20137415 2 acetobutylictim ATCC 824 OO23 E adhEm isolated from Alcohol 8 metalibrary dehydrogenase of anaerobic sewage digester microbial consortia US 2016/03193 13 A1 Nov. 3, 2016 55

TABLE 56-continued Genes expressed in host 14-BDO-producting microbial organisms.

Gene ID Reaction Gene Source number (FIG. 1) name organism Enzyme name GI number Reference

OO24 E adhE Cliostridium Alcohol 125972944 thermocelium enydrogenase OO25 E al Cliostridium Coenzyme A- 4903.6681 9 beijerinckii acylating aldehyde enydrogenase OO26 E bdha Cliostridium Butanol 15896.543 2 acetobiitvictim enydrogenase ATCC 824 0027 Q b Cliostridium Butyraldehyde 31075383 4 Saccharoperbativiacetonictim enydrogenase OO28 E bdh Cliostridium Butanol 124221917 4 Saccharoperbativiacetonictim enydrogenase 0029 Q/E adhE Cliostridium Aldehydefalcohol 28211045 tetani enydrogenase 0030 Q/E adhE Cliostridium Aldehydefalcohol 18311513 perfingens enydrogenase 0031 Q/E adhE Cliostridium Aldehydefalcohol 1267OOS86 difficile enydrogenase OO32 B SucA Mycobacterium alpha-ketoglutarate 121637177 5 bovis ecarboxylase BCG, Pasteur OO33 T cat2 Cliostridium 4-hydroxybutyrate 6249316 aminobutyricum coenzyme A transferase OO34 T cat2 Porphyromonas 4-hydroxybutyrate 34541558 gingivais coenzyme A W83 transferase OO3S A SucD Porphyromonas Succinic 3454O484 gingivais semialdehyde W83 dehydrogenase (CoA dependent) OO36 C 4-HBd Porphyromonas NAD-dependent 4- 3454O485 gingivais hydroxybutyrate W83 dehydrogenase OO37 C gbd Uncultured 4-hydroxybutyrate 591.6168 6 bacterium dehydrogenase OO38 G sucCD E. coi Succinyl-CoA 16128703 and synthetase 16128704

'Sohling and Gottschalk, Eur, J. Biochem. 212: 121-127 (1993); Sohling and Gottschalk, J. Bacteriol. 178: 871-880 (1996) 'Nolling et al., J. J. Bacteriol. 183: 4823-4838 (2001) Pohlmann et al., Nat. Biotechnol. 24; 1257-1262 (2006) Kosaka et al., Biosci. Biotechnoi. Biochem. 71:58-68 (2007) Brosch et al., Proc. Nati. Acad. Sci. U.S.A. 104:5596-5601 (2007) Henne et al., Appi. Environ. Microbioi. 65: 390.1-3907 (1999) (Fontaine et al., J Bacteriol. 184: 821-830 (2002)) (Wexler et al., Environ Microbiol 7: 1917-1926 (2005)) (Toth et al., Appi Environ Microbioi 65: 4973-4980 (1999))

0374 Expression Vector Construction for 14-BDO Path way. lacZalpha-RI 0375 Vector backbones and some strains were obtained (SEQ ID NO : 4) from Dr. Rolf Lutz of Expressys. The vectors and strains are 5 GACGAATTCGCTAGCAAGAGGAGAAGTCGACATGTCCAATTCAC based on the pZ Expression System developed by Dr. Rolf TGGCCGTCGTTTTAC3" Lutz and Prof. Hermann Bujard (Lutz, R. and H. Bujard, lacZalpha 3 'BB Nucleic Acids Res 25:1203-1210 (1997)). Vectors obtained (SEO ID NO; 5) were pZE131uc, pZA33luc, pZS*13luc and pZE22luc and 5 - GACCCTAGGAAGCTTTCTAGAGTCGACCTATGCGGCATCAGAG contained the luciferase gene as a stuffer fragment. To CAGA-3 '' . replace the luciferase stuffer fragment with a lacZ-alpha 0376. This generated a fragment with a 5' end of EcoRI fragment flanked by appropriate restriction enzyme sites, the site, Nhe site, a Ribosomal , a SalI site and the luciferase stuffer fragment was first removed from each start codon. On the 3' end of the fragment contained the stop vector by digestion with EcoRI and Xbal. The lacZ-alpha codon, Xbal, HindIII, and Avril sites. The PCR product was fragment was PCR amplified from puC19 with the follow digested with EcoRI and Avril and ligated into the base ing primers: vectors digested with EcoRI and Xbal (Xbal and Avril have US 2016/03193 13 A1 Nov. 3, 2016 56 compatible ends and generate a non-site). Because Nhe and genes. Cells were grown aerobically in LB media (Difico) Xbal restriction enzyme sites generate compatible ends that containing the appropriate antibiotics for each construct, and can be ligated together (but generate a Nhe/Xbal non-site induced by addition of IPTG at 1 mM when the optical that is not digested by either enzyme), the genes cloned into density (OD600) reached approximately 0.5. Cells were the vectors could be “Biobricked” together. Briefly, this harvested after 6 hours, and enzyme assays conducted as method allows joining an unlimited number of genes into the discussed below. vector using the same 2 restriction sites (as long as the sites 0383. In Vitro Enzyme Assays. do not appear internal to the genes), because the sites 0384. To obtain crude extracts for activity assays, cells between the genes are destroyed after each addition. were harvested by centrifugation at 4,500 rpm (Beckman 0377 All vectors have the pZ designation followed by Coulter, Allegera X-15R) for 10 min. The pellets were letters and numbers indication the origin of replication, resuspended in 0.3 mL BugBuster (Novagen) reagent with antibiotic resistance marker and promoter/regulatory unit. benzonase and lysozyme, and lysis proceeded for 15 min The origin of replication is the second letter and is denoted utes at room temperature with gentle shaking. Cell-free by E for ColE1. A for p 15A and S for pSC101-based origins. lysate was obtained by centrifugation at 14,000 rpm (Eppen The first number represents the antibiotic resistance marker dorf centrifuge 5402) for 30 min at 4°C. Cell protein in the (1 for Ampicillin, 2 for Kanamycin, 3 for Chloramphenicol, sample was determined using the method of Bradford et al., 4 for Spectinomycin and 5 for Tetracycline). The final Anal. Biochem. 72:248-254 (1976), and specific enzyme number defines the promoter that regulated the gene of assays conducted as described below. Activities are reported interest (1 for Piero-1, 2 for Pino-1, 3 for Patto-1, and 4 in Units/mg protein, where a unit of activity is defined as the for P). The MCS and the gene of interest follows amount of enzyme required to convert 1 umol of Substrate in immediately after. For the work discussed here we employed 1 min. at room temperature. In general, reported values are two base vectors, pZA33 and pZE13, modified for the averages of at least 3 replicate assays. biobricks insertions as discussed above. Once the gene(s) of 0385 Succinyl-CoA transferase (Cat1) activity was interest have been cloned into them, resulting plasmids are determined by monitoring the formation of acetyl-CoA from indicated using the four digit gene codes given in Table 56; Succinyl-CoA and acetate, following a previously described e.g., p7A33-XXXX-YYYY-. procedure Sohling and Gottschalk, J. Bacteriol. 178:871 0378 Host Strain Construction. 880 (1996). Succinyl-CoA synthetase (SucCD) activity was 0379 The parent strain in all studies described here is E. determined by following the formation of succinyl-CoA Coli K-12 strain MG 1655. Markerless deletion strains in from succinate and CoA in the presence of ATP. The adhE, gab), and aldA were constructed under service con experiment followed a procedure described by Cha and tract by a third party using the redET method (Datsenko, K. Parks, J. Biol. Chem. 239:1961-1967 (1964). CoA-depen A. and B. L. Wanner, Proc Natl Acad Sci U. S.A 97:6640 dent Succinate semialdehyde dehydrogenase (SucD) activity 6645 (2000)). Subsequent strains were constructed via bac was determined by following the conversion of NAD to teriophage P1 mediated transduction (Miller, J. Experiments NADH at 340 nm in the presence of succinate semialdehyde in Molecular Genetics, Cold Spring Harbor Laboratories, and CoA (Sohling and Gottschalk, Eur: J. Biochem. 212: New York (1973)). Strain C600Z1 (lacii, PN25-tetR, Sp', 121-127 (1993)). 4-HB dehydrogenase (4-HBd) enzyme lacY1, leuB6, mcrB+, supE44, thi-1, thr-1, tonA21) was activity was determined by monitoring the oxidation of obtained from Expressys and was used as a Source of a lacrl NADH to NAD at 340 nm in the presence of succinate allele for P1 transduction. Bacteriophage P1 vir was grown semialdehyde. The experiment followed a published proce on the C600Z1 E. coli strain, which has the spectinomycin dure Gerhardt et al. Arch. Microbiol. 174: 189-199 (2000). resistance gene linked to the lacI. The P1 lysate grown on 4-HB CoA transferase (Cat2) activity was determined using C600Z1 was used to infect MG 1655 with selection for a modified procedure from Scherf and Buckel, Appl. Envi spectinomycin resistance. The spectinomycin resistant colo ron. Microbiol. 57:2699-2702 (1991). The formation of nies were then screened for the linked lacI by determining 4-HB-CoA or butyryl-CoA formation from acetyl-CoA and the ability of the transductants to repress expression of a 4-HB or butyrate was determined using HPLC. gene linked to a Pro-1 promoter. The resulting strain was 0386 Alcohol (ADH) and aldehyde (ALD) dehydroge designated MG1655 lacI. A similar procedure was used to nase was assayed in the reductive direction using a proce introduce lacI into the deletion strains. dure adapted from several literature sources (Durre et al., 0380 Production of 4-HB from Succinate. FEMS Microbiol. Rev 17:251-262 (1995); Palosaari and 0381 For construction of a 4-HB producer from succi Rogers, J. Bacteriol. 170:2971-2976 (1988) and Welch et nate, genes encoding steps from Succinate to 4-HB and al., Arch. Biochem. Biophy's. 273:309-318 (1989)). The 4-HB-CoA (Steps G, A, C, O in FIG. 1) were assembled onto oxidation of NADH is followed by reading absorbance at the pZA33 and pZE 13 vectors as described below. Various 340 nM every four seconds for a total of 240 seconds at combinations of genes were assessed, as well as different room temperature. The reductive assays were performed in constructs bearing incomplete pathways as controls (Tables 100 mM MOPS (adjusted to pH 7.5 with KOH), 0.4 mM 2 and 3). The plasmids were then transformed into host NADH, and from 1 to 50 ul of cell extract. The reaction is strains containing lacI, which allow inducible expression started by adding the following reagents: 100 ul of 100 mM by addition of isopropyl B-D-1-thiogalactopyranoside acetaldehyde or butyraldehyde for ADH, or 100 ul of 1 mM (IPTG). Both wild-type and hosts with deletions in genes acetyl-CoA or butyryl-CoA for ALD. The Spectrophotom encoding the native Succinic semialdehyde dehydrogenase eter is quickly blanked and then the kinetic read is started. were tested. The resulting slope of the reduction in absorbance at 340 nM 0382 Activity of the heterologous enzymes were first per minute, along with the molar extinction coefficient of tested in in vitro assays, using strain MG1655 lacI as the NAD(P)H at 340 nM (6000) and the protein concentration of host for the plasmid constructs containing the pathway the extract, can be used to determine the specific activity. US 2016/03193 13 A1 Nov. 3, 2016 57

(0387. The enzyme activity of PTB is measured in the 0390 Chemical and sample preparation was performed direction of butyryl-CoA to butyryl-phosphate as described as follows. Briefly, CoA, AcCoA, BuCoA and all other in Cary et al. J. Bacteriol. 170:4613-4618 (1988). It provides chemicals, were obtained from Sigma-Aldrich. The sol inorganic phosphate for the conversion, and follows the vents, methanol and acetonitrile, were of HPLC grade. increase in free CoA with the reagent 5,5'-dithiobis-(2- Standard calibration curves exhibited excellent linearity in nitrobenzoic acid), or DTNB. DTNB rapidly reacts with the 0.01-1 mg/mL concentration range. Enzymatic reaction thiol groups such as free CoA to release the yellow-colored mixtures contained 100 mM Tris HCl buffer (pH 7), aliquots 2-nitro-5-mercaptobenzoic acid (TNB), which absorbs at were taken at different time points, quenched with formic 412 nm with a molar extinction coefficient of 14,140 M cm. The assay buffer contained 150 mM potassium phos acid (0.04% final concentration) and directly analyzed by phate at pH 7.4, 0.1 mM DTNB, and 0.2 mM butyryl-CoA, HPLC. and the reaction was started by addition of 2 to 50 uL cell 0391 HPLC analysis was performed using an Agilent extract. The enzyme activity of BK is measured in the 1100 HPLC system equipped with a binary pump, degasser, direction of butyrate to butyryl-phosphate formation at the thermostated autosampler and column compartment, and expense of ATP. The procedure is similar to the assay for diode array detector (DAD), was used for the analysis. A acetate kinase previously described Rose et al., J. Biol. reversed phase column, Kromasil 100 Sum C18, 4.6x150 Chem. 211:737-756 (1954). However we have found mm (Peeke Scientific), was employed. 25 mM potassium another acetate kinase enzyme assay protocol provided by phosphate (pH 7) and methanol or acetonitrile, were used as Sigma to be more useful and sensitive. This assay links aqueous and organic solvents at 1 mL/min flow rate. Two conversion of ATP to ADP by acetate kinase to the linked conversion of ADP and phosphoenolpyruvate (PEP) to ATP methods were developed: a short one with a faster gradient and pyruvate by pyruvate kinase, followed by the conver for the analysis of well-resolved CoA, AcCoA and BuCoA, sion of pyruvate and NADH to lactate and NAD" by lactate and a longer method for distinguishing between closely dehydrogenase. Substituting butyrate for acetate is the only eluting AcCoA and 4-HBCoA. Short method employed major modification to allow the assay to follow BK enzyme acetonitrile gradient (O min—5%, 6 min 30%. 6.5 min activity. The assay mixture contained 80 mM trietha 5%, 10 min 5%) and resulted in the retention times 2.7, 4.1 nolamine buffer at pH 7.6, 200 mM sodium butyrate, 10 mM and 5.5 min for CoA, AcCoA and BuCoA, respectively. In MgCl, 0.1 mM NADH, 6.6 mM ATP 1.8 mM phospho the long method methanol was used with the following enolpyruvate. Pyruvate kinase, lactate dehydrogenase, and linear gradient: 0 min—5%, 20 min 35%, 20.5 min 5%, myokinase were added according to the manufacturers 25 min -5%. The retention times for CoA, AcCoA, 4-HB instructions. The reaction was started by adding 2 to 50 cell CoA and BuCoA were 5.8, 8.4, 9.2 and 16.0 min, respec extract, and the reaction was monitored based on the tively. The injection volume was 5 LL, column temperature decrease in absorbance at 340 nm indicating NADH oxida 30° C., and UV absorbance was monitored at 260 nm. tion. 0392 The results demonstrated activity of each of the 0388 Analysis of CoA Derivatives by HPLC. four pathway steps (Table 57), though activity is clearly 0389. An HPLC based assay was developed to monitor dependent on the gene source, position of the gene in the enzymatic reactions involving coenzyme A (CoA) transfer. vector, and the context of other genes with which it is The developed method allowed enzyme activity character expressed. For example, gene 0035 encodes a Succinic ization by quantitative determination of CoA, acetyl CoA semialdehyde dehydrogenase that is more active than that (AcCoA), butyryl CoA (BuCoA) and 4-hydroxybutyrate encoded by 0008, and 0.036 and 0010n are more active 4-HB CoA (4-HBCoA) present in in-vitro reaction mixtures. Sen dehydrogenase genes than 0009. There also seems to be sitivity down to low uM was achieved, as well as excellent better 4-HB dehydrogenase activity when there is another resolution of all the CoA derivatives of interest. gene preceding it on the same operon. TABLE 57 In vitro enzyme activities in cell extracts from MG 1655 lacI containing the plasmids expressing genes in the 4-HB-CoA pathway. Activities are reported in Units/mg protein, where a unit of activity is defined as the amount of enzyme required to convert 1 Imol of Substrate in 1 min. at room temperature. Sample # pZE13 (a) pZA33 (b) OD600 Cell Prot (c) Cat1 SucD 4HBd Cat2 1 cat1 (0.004) 2.71 6.43 1.232 OOO 2 cat1 (0.004)-sucD (0035) 2.03 S.OO 0.761 2.57 3 cat1 (0.004)-sucD (O008) 1.04 3.01 O.783 0.01 4 sucD (0035) 2.31 6.94 2.32 5 sucD (0008) 1.10 4.16 O.OS 6 4hbd (0.009) 2.81 7.94 O.OO3 O.25 7 4hbd (0036) 2.63 7.84 3.31 8 4hbd (001On) 2.OO 5.08 2.57 9 cat1 (0.004)-sucD (0035) 4hbd (0.009) 2.07 S.04 O.6OO 1.85 0.01 10 cat1 (0.004)-sucD (0035) 4hbd (0036) 2.08 S.40 O.694 1.73 O.41 11 cat1 (0.004)-sucD (0035) 4hbd (001On) 2.44 4.73 0.679 2.28 O.37 12 cat1 (0.004)-sucD (O008) 4hbd (0.009) 1.08 3.99 O.S72 -0.01 O.O2 13 cat1 (0.004)-sucD (O008) 4hbd (0036) O.77 2.60 O898 -0.01 O.04 14 cat1 (0.004)-sucD (O008) 4hbd (001On) O.63 2.47 O.776 O.OO O.OO 15 cat2 (0034) 2.56 7.86 1.283 US 2016/03193 13 A1 Nov. 3, 2016

TABLE 57-continued In vitro enzyme activities in cell extracts from MG 1655 lacI containing the plasmids expressing genes in the 4-HB-CoA pathway. Activities are reported in Units/mg protein, where a unit of activity is defined as the amount of enzyme required to convert 1 Imol of Substrate in 1 min. at room temperature. Sample # pZE13 (a) pZA33 (b) OD600 Cell Prot (c) Catl SucD 4HBd Cat? 16 cat2(0034)-4-hbd(0036) 3.13 8.04 24.86 0.993 17 cat2(0034)-4-hbd(001On) 2.38 7.03 7.45 0.675 18 4hbd(0036)-cat2(0034) 2.69 8.26 2.15 7.490 19 4hbd(001On)-cat2(0034) 2.44 6.59 O59 4.101 (a) Genes expressed from Plac on p7E13, a high-copy plasmid with colB1 origin and amplicillin resistance, Gene identification numbers are as given in Table 56. (b) Genes expressed from Plac on p7A33, a medium-copy plasmid with pACYC origin and chloramphenicol resistance, (c) Cell protein given as mg protein per mL extract,

0393 Recombinant strains containing genes in the 4-HB experiments was aimed to determine whether endogenous pathway were then evaluated for the ability to produce 4-HB Succinate produced by the cells during growth on glucose in vivo from central metabolic intermediates. Cells were could fuel the 4-HB pathway. Cells were grown anaerobi grown anaerobically in LB medium to OD600 of approxi cally in M9 minimal medium (6.78 g/L NaHPO, 3.0 g/L mately 0.4, then induced with 1 mM IPTG. One hour later, KHPO, 0.5 g/L NaCl, 1.0 g/L NHCl, 1 mM MgSO, 0.1 Sodium Succinate was added to 10 mM, and samples taken mM CaCl) supplemented with 20 g/L glucose, 100 mM for analysis following an additional 24 and 48 hours. 4-HB 3-(N-morpholino)propanesulfonic acid (MOPS) to improve in the culture broth was analyzed by GC-MS as described the buffering capacity, 10 g/mL thiamine, and the appro below. The results indicate that the recombinant strain can priate antibiotics. 0.25 mM IPTG was added when OD600 produce over 2 mM 4-HB after 24 hours, compared to reached approximately 0.2, and samples taken for 4-HB essentially zero in the control strain (Table 58). analysis every 24 hours following induction. In all cases TABLE 58 Production of 4-HB from Succinate in E. coli strains harboring plasmids expressing Various combinations of 4-HB pathway genes. Sample 24 Hours 48 Hours i Host Strain pZE13 pZA33 OD600 4HB, 1M 4HB norm. (a) OD600 4HB, 1M 4HB norm. (a) 1 MG1655 laco cat1 (0.004)-sucD (0035) 4hbd (0.009) O.47 487 1036 1.04 1780 1711 2 MG1655 laco cat1 (0.004)-sucD (0035) 4hbd (0027) 0.41 111 270 O.99 214 217 3 MG 1655 laco cat1 (0.004)-sucD (0035) 4hbd (0036) O.47 863 1835 O.48 215.2 4484 4 MG1655 laco cat1 (0.004)-sucD (0035) 4hbd (001On) O46 956 2O78 O.49 2221 4533 5 MG.1655 laco cat1 (0.004)-sucD (0.008) 4hbd (0.009) O38 493 1296 0.37 1338 3616 6 MG1655 laco cat1 (0.004)-sucD (0.008) 4hbd (0027) O.32 26 81 0.27 87 323 7 MG1655 laco cat1 (0.004)-sucD (0.008) 4hbd (0036) O.24 SO6 2108 O.31 1448 4672 8 MG1655 laco cat1 (0.004)-sucD (0.008) 4hbd (001On) O.24 78 324 O.S6 233 416 9 MG 1655 laclagabD cat1 (0.004)-sucD (0035) 4hbd (0.009) OS3 656 1237 1.03 1643 1595 10 MG 1655 laclagabD cat1 (0.004)-sucD (0035) 4hbd (0027) 0.44 92 209 O.98 214 218 11 MG 1655 laclagabD cat1 (0.004)-sucD (0035) 4hbd (0036) OS1 1072 2102 0.97 2358 2431 12 MG 1655 laclagabD cat1 (0.004)-sucD (0035) 4hbd (001On) OS1 981 1924 0.97 2121 2186 13 MG 1655 laclagabD cat1 (0.004)-sucD (0.008) 4hbd (O009) O3S 407 1162 0.77 1178 1530 14 MG 1655 laclagabD cat1 (0.004)-sucD (0.008) 4hbd (0027) OS1 19 36 1.07 50 47 15 MG.1655 laclagabD cat1 (0.004)-sucD (0.008) 4hbd (0036) O3S S84 1669 O.78 1350 1731 16 MG 1655 laclagabD cat1 (0.004)-sucD (0.008) 4hbd (001On) O.32 74 232 O.82 232 283 17 MG1655 laco vector only vector only O.8 1 2 1.44 3 2 18 MG1655 laclagabD vector only vector only O89 1 2 1.41 7 5 (a) Normalized 4-HB concentration, uMOD600 units

0394 An alternate to using a CoA transferase (catl) to 4-HB plateaued after 24 hours, with a maximum of about 1 produce Succinyl-CoA from Succinate is to use the native E. mM in the best strains (FIG. 2a), while the succinate coli sucCD genes, encoding Succinyl-CoA synthetase. This concentration continued to rise (FIG.2b). This indicates that gene cluster was cloned onto pZE13 along with candidate the Supply of Succinate to the pathway is likely not limiting, genes for the remaining steps to 4-HB to create pZE13 and that the bottleneck may be in the activity of the enzymes OO38-OO35-OO36. themselves or in NADH availability. Gens 0035 and 0.036 0395 Production of 4-HB from Glucose. are clearly the best candidates for CoA-dependent succinic 0396 Although the above experiments demonstrate a semialdehyde dehydrogenase and 4-HB dehydrogenase, functional pathway to 4-HB from a central metabolic inter respectively. The elimination of one or both of the genes mediate (Succinate), an industrial process would require the encoding known (gab)) or putative (aldA) native Succinic production of chemicals from low-cost carbohydrate feed semialdehyde dehydrogenases had little effect on perfor stocks Such as glucose or Sucrose. Thus, the next set of mance. Finally, it should be noted that the cells grew to a US 2016/03193 13 A1 Nov. 3, 2016 59 much lower OD in the 4-HB-producing strains than in the taining each gene candidate expressed on pZA33 were controls (FIG. 2C). grown in the presence of 0.25 mM IPTG for four hours at 0397. An alternate pathway for the production of 4-HB 37° C. to fully induce expression of the enzyme. Four hours from glucose is via alpha-ketoglutarate. We explored the use after induction, cells were harvested and assayed for ADH of an alpha-ketoglutarate decarboxylase from Mycobacte and ALD activity as described above. Since 4-HB-CoA and rium tuberculosis (Tian et al., Proc. Natl. Acad. Sci. USA 4-hydroxybutyraldehyde are not available commercially, 102:10670-10675 (2005)) to produce succinic semialdehyde assays were performed using the non-hydroxylated Sub directly from alpha-ketoglutarate (Step B in FIG. 1). To strates (Table 59). The ratio in activity between 4-carbon and demonstrate that this gene (0032) was functional in vivo, we 2-carbon substrates for C. acetobutyllicum adhE2 (0002) and expressed it on pZE13 in the same host as 4-HB dehydro E. coli adhE (0011) were similar to those previously reported genase (gene 0036) on pZA33. This strain was capable of in the literature (Atsumi et al., Biochim. Biophys. Acta. producing over 1.0 mM 4-HB within 24 hours following 1207: 1-11 (1994)). induction with 1 mM IPTG (FIG. 3). Since this strain does not express a CoA-dependent Succinic semialdehyde dehy TABLE 59 drogenase, the possibility of Succinic semialdehyde produc In vitro enzyme activities in cell extracts from MG 1655 tion via Succinyl-CoA is eliminated. It is also possible that lacI containing pZA33 expressing gene candidates for the native genes responsible for producing Succinic semial aldehyde and alcohol dehydrogenases. Activities are expressed in dehyde could function in this pathway; however, the amount Imol mining cell protein'. of 4-HB produced when the pZE13-0032 plasmid was left out of the host is the negligible. Aldehyde dehydrogenase Alcohol dehydrogenase 0398. Production of 14-BDO from 4-HB. Butyryl- Acetyl- Butyr- Acet 0399. The production of 14- 14-BDO from 4-HB Gene Substrate CoA CoA aldehyde aldehyde required two reduction steps, catalyzed by dehydrogenases. Alcohol and aldehyde dehydrogenases (ADH and ALD, respectively) are NAD+/H and/or NADP+/H-dependent enzymes that together can reduce a carboxylic acid group on a molecule to an alcohol group, or in reverse, can perform the oxidation of an alcohol to a carboxylic acid. This biotransformation has been demonstrated in wild-type Clostridium acetobutyllicum (Jewell et al., Current Microbi N.D., not determined. ology, 13:215-19 (1986)), but neither the enzymes respon sible nor the genes responsible were identified. In addition, 04.01 For the 14-BDO production experiments, cat? from it is not known whether activation to 4-HB-CoA is first Porphyromonas gingivalis W83 (gene 0034) was included required (Step T in FIG. 1), or if the aldehyde dehydroge on pZA33 for the conversion of 4-HB to 4-HB-CoA, while nase (Step Q) can act directly on 4-HB. We developed a list the candidate dehydrogenase genes were expressed on of candidate enzymes from C. acetobutylicum and related pZE13. The host strain was MG 1655 lacI. Along with the organisms based on known activity with the non-hydroxy alcohol and aldehyde dehydrogenase candidates, we also lated analogues to 4-HB and pathway intermediates, or by tested the ability of CoA-dependent succinic semialdehyde similarity to these characterized genes (Table 56). Since dehydrogenases (sucD) to function in this step, due to the Some of the candidates are multifunctional dehydrogenases, similarity of the substrates. Cells were grown to an OD of they could potentially catalyze both the NAD(P)H-depen about 0.5 in LB medium supplemented with 10 mM 4-HB, dent reduction of the acid (or CoA-derivative) to the alde induced with 1 mM IPTG, and culture broth samples taken hyde, and of the aldehyde to the alcohol. Before beginning after 24 hours and analyzed for 14-BDO as described below. work with these genes in E. coli, we first validated the result The best 14-BDO production occurred using adhE2 from C. referenced above using C. acetobutylicum ATCC 824. Cells acetobutyllicum, sucD from C. kluyveri, or sucD from P were grown in Schaedler broth (Accumedia, Lansing, gingivalis (FIG. 4). Interestingly, the absolute amount of Mich.) supplemented with 10 mM 4-HB, in an anaerobic 14-BDO produced was higher under aerobic conditions: atmosphere of 10% CO, 10% H and 80% N, at 30° C. however, this is primarily due to the lower cell density Periodic culture samples were taken, centrifuged, and the achieved in anaerobic cultures. When normalized to cell broth analyzed for 14-BDO by GC-MS as described below. OD, the 14-BDO production per unit biomass is higher in 14-BDO concentrations of 0.1 mM, 0.9 mM, and 1.5 mM anaerobic conditions (Table 60). were detected after 1 day, 2 days, and 7 days incubation, respectively. No 14-BDO was detected in culture grown TABLE 60 without 4-HB addition. To demonstrate that the 14-BDO Absolute and normalized 14-BDO concentrations from cultures of cells produced was derived from glucose, we grew the best expressing adhE2 from C. acetobutyllicum, sucD from C. kluyveri, 14-BDO producing strain MG 1655 lacI pZE13-0004 or sucD from Egingivalis, as well as the negative control. 0035-0002 pZA33-0034-0036 in M9 minimal medium supplemented with 4 g/L uniformly labeled C-glucose. Gene 14-BDO OD 14 Cells were induced at OD of 0.67 with 1 mM IPTG, and a expressed Conditions (IM) (600 nm) BDOOD sample taken after 24 hours. Analysis of the culture Super Ole Aerobic O 13.4 O natant was performed by mass spectrometry. Ole Microaerobic O.S 6.7 O.09 Ole Anaerobic 2.2 1.26 1.75 04.00 Gene candidates for the 4-HB to 14-BDO conver OOO2 Aerobic 138.3 9.12 15.2 sion pathway were next tested for activity when expressed in OOO2 Microaerobic 48.2 5.52 8.73 the E. coli host MG1655 lacI. Recombinant strains con US 2016/03193 13 A1 Nov. 3, 2016 60

TABLE 60-continued TABLE 61

Absolute and normalized 14-BDO concentrations from cultures of cells Absolute and normalized 14-BDO concentrations from cultures expressing adhE2 from C. acetobutyllicum, sucD from C. kluyveri, of cells expressing adhE2 from C. acetobutyllicum in pZE13 or sucD from R gingivalis, as well as the negative control. along with either cat2 from P gingivais (0034) or the PTB/BK genes from C. acetobutyllicum on pZA33. Host Gene 14-BDO OD 14 strains were either MG 1655 lacIC or MG 1655 AadhE lacI9. expressed Conditions (IM) (600 nm) BDOOD 14-BDO OD 14 OOO2 Anaerobic 54.7 1.35 40.5 Genes Host Strain (IM) (600 nm) BDO/OD O008n Aerobic 255.8 5.37 47.6 OO34 MG1655 lacI9 O.827 19.9 O.042 O008n Microaerobic 127.9 3.05 41.9 O020 + 0021 MG1655 lacI9 O.007 9.8 O.OOO7 O008n Anaerobic 60.8 O.62 98.1 OO34 MG1655 AadhE 2.084 12.5 O.166 OO35 Aerobic 21.3 14.0 1.52 lacI9 OO35 Microaerobic 13.1 4.14 3.16 0020 + 0021 MG1655 AadhE 0.975 18.8 O.OS2 OO35 Anaerobic 21.3 1.06 20.1 lacI9

0403. Production of 14-BDO from Glucose. The final 0402. As discussed above, it may be advantageous to use step of pathway corroboration is to express both the 4-HB a route for converting 4-HB to 4-HB-CoA that does not and 14-BDO segments of the pathway in E. coli and generate acetate as a byproduct. To this aim, we tested the demonstrate production of 14-BDO in glucose minimal use of phosphotransbutyrylase (ptb) and butyrate kinase (bk) medium. New plasmids were constructed so that all the from C. acetobutylicum to carry out this conversion via required genes fit on two plamids. In general, catl, adhE, Steps 0 and P in FIG. 1. The native ptb/bk operon from C. and SucD genes were expressed from pzE13, and cat2 and acetobutyllicum (genes 0020 and 0021) was cloned and 4-HBd were expressed from pZA33. Various combinations of gene source and gene order were tested in the MG 1655 expressed in pZA33. Extracts from cells containing the lacI background. Cells were grown anaerobically in M9 resulting construct were taken and assayed for the two minimal medium (6.78 g/L NaHPO, 3.0 g/L KHPO, 0.5 enzyme activities as described herein. The specific activity g/L NaCl, 1.0 g/L NHCl, 1 mM MgSO, 0.1 mM CaCl) of BK was approximately 65 U/mg, while the specific supplemented with 20 g/L glucose, 100 mM 3-(N-mor activity of PTB was approximately 5 U/mg. One unit (U) of pholino)propanesulfonic acid (MOPS) to improve the buff activity is defined as conversion of 1 M substrate in 1 ering capacity, 10 ug/mL thiamine, and the appropriate minute at room temperature. Finally, the construct was antibiotics. 0.25 mM IPTG was added approximately 15 tested for participation in the conversion of 4-HB to hours following inoculation, and culture Supernatant 14-BDO. Host strains were transformed with the pZA33 samples taken for 14-BDO, 4-HB, and succinate analysis 24 and 48 hours following induction. The production of 0020-0021 construct described and pZE13-0002, and com 14-BDO appeared to show a dependency on gene order pared to use of cat2 in 14-BDO production using the aerobic (Table 62). The highest 14-BDO production, over 0.5 mM, procedure used above in FIG. 4. The BK/PTB strain pro was obtained with cat? expressed first, followed by 4-HBd duced 1 mM 14-BDO, compared to 2 mM when using cat? on pZA33, and cat1 followed by P gingivalis sucD on (Table 61). Interestingly, the results were dependent on pZE13. The addition of C. acetobutyllicum adhE2 in the last whether the host strain contained a deletion in the native position on pZE13 resulted in slight improvement. 4-HB and adhE gene. Succinate were also produced at higher concentrations. TABLE 62 Production of 14-BDO, 4-HB, and succinate in recombinant E. coii strains expressing combinations of 14-BDO pathway genes, grown in minimal medium Supplemented with 20 g/L glucose. Concentrations are given in InM.

Induction 24 Hours 48 Hours

Sample pZE13 pZA33 OD OD600 nm Su 4HB BDO OD600 nm Su 4HB BDO 1 cat1 (0.004)-sucD(0035) 4hbd (0036)-cat2(0034) O.92 1.29 5.44 1.37 0.240 1.24 6.42 1.49 0.28O 2 cat1 (0.004)-sucD(O008N) 4hbd (0036)-cat2(0034) O.36 1.11 6.90 124 OO11 1.06 7.63 1.33 O.O11 3 adhE(0002)-cat1 (0.004)- 4hbd (0036)-cat2(0034) O.20 0.44 O34 1.84 O.OSO O.6O 1.93 2.67 0.119 sucD(0035) 4 cat1 (0.004)-sucD(0035)- 4hbd (0036)-cat2(0034) 1.31 1.90 9.02 0.73 O.O73 1.95 9.73 0.82 0.077 adhE(OOO2) 5 adhE(0002)-cat1 (0.004)- 4hbd (0036)-cat2(0034) O.17 O.45 1.04 104 OOO8 O.94 7.13 1.02 0.017

6 cat1 (0.004)-sucD(O008N)- 4hbd (0036)-cat2(0034) 1.30 1.77 10.47 0.25 O.OO4 18O 1149 0.28 O.OO3 adhE(OOO2) 7 cat1 (0.004)-sucD(0035) cat2(0034)-4-hbd(0036) 1.09 1.29 S.63 2.15 0.461 1.38 6.66 2.30 O.S2O 8 cat1 (0.004)-sucD(O008N) cat2(0034)-4-hbd(0036) 1.81 2.01 11.28 O.O2 OOOO 2.24 1113 O.O2 O.OOO 9 adhE(0002)-cat1 (0.004)- cat2(0034)-4-hbd(0036) O.24 1.99 2.02 2.32 0.106 O.89 4.85 2.41 0.186 sucD(0035) 10 cat1 (0.004)-sucD(0035)- cat2(0034)-4-hbd(0036) O.98 1.17 S.30 2.08 O.S69 1.33 6.15 2.14 0.640 adhE(OOO2) 11 adhE(0002)-cat1 (0.004)- cat2(0034)-4-hbd(0036) O.20 O.S3 1.38 2.30 O.O19 O.91 8.1O 149 0.034 US 2016/03193 13 A1 Nov. 3, 2016 61

TABLE 62-continued Production of 14-BDO, 4-HB, and succinate in recombinant E. coii strains expressing combinations of 14-BDO pathway genes, grown in minimal medium Supplemented with 20 g/L glucose. Concentrations are given in mM. Induction 24 Hours 48 Hours

Sample pZE13 pZA33 OD OD600 nm Su 4HB BDO OD600 nm Su 4HB BDO 12 cat1(O004)-sucD(O008N)- cat2(0034)-4hbd(0036) 2.14 2.73 12.07 O.16 O.OOO 3.10 11.79 0.17 O.OO2 adhE(0002) 13 vector only vector only 2.11 2.62 9.O3 O.O1 O.OOO 3.00 12.0S O.O1 O.OOO

0404 Analysis of 14-BDO, 4-HB and Succinate by were constructed using analyte solutions in the correspond GCMS. ing cell culture or fermentation medium to match sample 04.05 14-BDO, 4-HB and succinate in fermentation and matrix as close as possible. GCMS data were processed cell culture samples were derivatized by silylation and using Environmental Data Analysis ChemStation software quantitatively analyzed by GCMS using methods adapted (Agilent Technologies). from literature reports (Song et al., Wei Sheng Wu Xue. Bao. 0408. The results indicated that most of the 4-HB and 45:382-386 (2005)). The developed method demonstrated 14-BDO produced were labeled with 'C (FIG. 6, right-hand good sensitivity down to 1 uM, linearity up to at least 25 sides). Mass spectra from a parallel culture grown in unla mM, as well as excellent selectivity and reproducibility. beled glucose are shown for comparison (FIG. 6, left-hand 0406 Sample preparation was performed as follows: sides). Note that the peaks seen are for fragments of the 1004, filtered (0.2 um or 0.45 um syringe filters) samples, derivatized molecule containing different numbers of carbon e.g. fermentation broth, cell culture or standard Solutions, atoms from the metabolite. The derivatization reagent also were dried down in a Speed Vac Concentrator (Savant contributes some carbon and silicon atoms that naturally SVC-100H) for approximately 1 hour at ambient tempera occurring label distribution, so the results are not strictly ture, followed by the addition of 20 uL 10 mM cyclohexanol quantitative. solution, as an internal standard, in dimethylformamide. The 04.09 Production of 14-BDO from 4-HB Using Alternate mixtures were vortexed and sonicated in a water bath Pathways. (Branson 3510) for 15 minto ensure homogeneity. 100 uL 0410 The various alternate pathways were also tested for silylation derivatization reagent, N.O-bis(trimethylsilyl)tri 14-BDO production. This includes use of the native E. coli flouro-acetimide (BSTFA) with 1% trimethylchlorosilane, SucCD enzyme to convert succinate to succinyl-CoA (Table was added, and the mixture was incubated at 70° C. for 30 63, rows 2-3), use of alpha-ketoglutarate decarboxylase in min. The derivatized samples were centrifuged for 5 min, the alpha-ketoglutarate pathway (Table 63, row 4), and use and the clear solutions were directly injected into GCMS. of PTB/BK as an alternate means to generate the CoA All the chemicals and reagents were from Sigma-Aldrich, derivative of 4-HB (Table 63, row 1). Strains were con with the exception of 14-BDO which was purchased from J. structed containing plasmids expressing the genes indicated T. Baker. in Table 63, which encompass these variants. The results 04.07 GCMS was performed on an Agilent gas chromato show that in all cases, production of 4-HB and 14-BDO graph 6890N, interfaced to a mass-selective detector (MSD) occurred (Table 63). 5973N operated in electron impact ionization (EI) mode has been used for the analysis. A DB-5MS capillary column (J&W Scientific, Agilent Technologies), 30mx0.25 mm i.d.x TABLE 63 0.25 um film thickness, was used. The GC was operated in Production of 14-BDO, 4-HB, and succinate in recombinant a split injection mode introducing 1 uL of sample at 20:1 E. coli strains genes for different 14-BDO pathway variants, grown anaerobically in minimal medium Supplemented with split ratio. The injection port temperature was 250° C. 20 g/L glucose, and harvested 24 hours after induction with Helium was used as a carrier gas, and the flow rate was 0.1 mM IPTG. Concentrations are given in mM. maintained at 1.0 mL/min. A temperature gradient program was optimized to ensure good resolution of the analytes of Genes on Genes on interest and minimum matrix interference. The oven was pZE13 pZA33 Succinate 4-HB 14-BDO initially held at 80° C. for 1 min, then ramped to 120° C. at OOO2 - OOO4 - OO2On-OO21m- O.336 2.91 O.230 2° C./min, followed by fast ramping to 320° C. at 100° OO35 OO36 C./min and final hold for 6 min at 320°C. The MS interface OO38 - OO3S OO34-OO36 O.814 2.81 O.126 OO38 - OO3S OO36-OO34 O.741 2.57 O.114 transfer line was maintained at 280° C. The data were OO35 - OO32 OO34-OO36 S.O1 O.S38 O.154 acquired using lowmass MS tune settings and 30-400 m/z. mass-range scan. The total analysis time was 29 min includ ing 3 min Solvent delay. The retention times corresponded to 5.2, 10.5, 14.0 and 18.2 min for BSTFA-derivatized cyclo Example V hexanol, 14-BDO, 4-HB and succinate, respectively. For quantitative analysis, the following specific mass fragments Biosynthesis of GBL were selected (extracted ion chromatograms): m/z, 157 for internal standard cyclohexanol, 116 for 14-BDO, and 147 0411. This example describes exemplary biochemical for both 4-HB and succinate. Standard calibration curves pathways for GBL production. US 2016/03193 13 A1 Nov. 3, 2016 62

0412. As shown in FIG. 1, 4-HB can be biosynthesized 0415. The transformation from 4-hydroxybutyryl-CoA to from Succinate or alpha-ketoglutarate. 4-HB, 4-hydroxybu GBL through a non-enzymatic lactonization reaction (Step tyryl-phoaphate, and 4-hydroxybutyryl-CoA can all be con AD in FIG. 1) is demonstrated by the following results. The verted into GBL (Steps AB, AC, and AD in FIG. 1). host strain used in the experiments was strain 432 (AadhE 0413 Previous studies have demonstrated the transfor Aldh AApflB AlpdA::K.p.lpdA322 Amdh AarcAgltAR163L mation from 4-HB to GBL. The conversion from 4-HB to fimD: E. coli sucCD, P gingivalis sucD, P gingivalis 4hbd GBL can be catalyzed by either the 1.4-lacton hydroxyacyl fimD:: M. bovis sucA, C. kluyveri 4hbd). This strain con hydrolase (EC 3.1.1.25) or the lipases (Step AB in FIG. 1). tained the components of the 14-BDO pathway leading to In vitro activities of 1.4-lacton hydroxyacylhydrolase, or 4HB integrated into the chromosome of E. coli at the fiml) gamma lactonase on 4-HB has been described (Fishbein and locus. It also contained additional genetic modifications to direct metabolic flux into the 4-HB pathway. Cells were Bessman, J Biol Chem 241:4835-4841 (1966)), (Fishbein grown in bottles with M9 minimal medium (6.78 g/L and Bessman, J Biol Chem 241:4842-4847 (1966)). Addi NaHPO, 3.0 g/L KHPO, 0.5 g/L NaCl, 1.0 g/L NHCl, tionally, lipases such as Candida antarctica lipase B has 1 mM MgSO, 0.1 mM CaCl) supplemented with glucose been tested to catalyze the lactonization of 4-hydroxybu under batch culture conditions. Gene expression was tyrate to GBL (Efe et al., Biotechnol Bioeng 99:1392-1406 induced by 0.25 mM IPTG and culture supernatant samples (2008)). were taken for 14-BDO, GBL 4-HB, and other products 0414. The transformation from 4-hydroxybutyryl analysis. Cultured with glucose, strain 432 produced 4-HB phoaphate to GBL can be a non-enzymatic step (Step AC in but produced neither GBL nor 14-BDO (Table 65, row 1). FIG. 1). This is demonstrated by the following results. The When genes coding for the 4-hydroxybutyryl-CoA trans hose strain used was E. coli strain MG 1655. Cells were ferase (034) or the combined butyrate kinase/phosphotrans grown in bottles with M9 minimal medium (6.78 g/L butyrylase (020/021) were expressed, the resulting enzymes NaHPO, 3.0 g/L KHPO, 0.5 g/L NaCl, 1.0 g/L NHCl, allowed the engineered strain to convert 4-HB to 4-hydroxy 1 mM MgSO, 0.1 mM CaCl) supplemented with glucose butyryl-CoA. The activated 4-hydroxybutyryl-CoA resulted under batch culture conditions. Gene expression was in the production of significant amount of GBL through the induced by 0.25 mM IPTG and culture supernatant samples non-enzymatic lactonization reaction (Table 65, rows 2-3). were taken for 14-BDO, GBL, 4-HB, and other products In the case described by Table 65, row 3, there were no analysis. When the 4-HB pathway was expressed in the host exogenous genes expressed to enable the production of the strain, the cells produced 4-HB from glucose. However, intermediate, 4-hydoxybutyryl-phosphate. 4-Hydroxybu 4-HB was not converted to GBL or 14-BDO when no tyryl-CoA transferase enabled the production of 4-hydroxy downstream gene presents (Table 64, row 1). When the butyryl-CoA from 4-hydroxybutyrate. Thus the experiment 4-HB kinase (021) was expressed from the pZA335 plamid, summarized in Table 65, row 3, demonstrated that 4-hy the cells were able to convert 4-HB to GBL through 4-hy droxybutyryl-CoA is capable of undergoing non-enzymatic droxybutyryl-phospate, whereas 14-BDO was not produced lactonization to form GBL. The high amount of GBL (Table 64, row 2). Thus, activated 4-hydroxybutyryl-phos detected in this experiment was unexpected because, to our phate can be converted to GBL through the non-enzymatic knowledge, the lactonization of 4-hydroxybutyryl-CoA had lactonization reaction (Step AC in FIG. 1). The high amount never before been demonstrated. The conversion of 4-hy of GBL detected in this experiment was unexpected because, droxybutyrate to GBL is thermodynamically favored at a to our knowledge, the lactonization of 4-hydroxybutyryl non-basic pH (Efe, et al., Biotechnol Bioeng 99:1392-1406 phosphate had never before been demonstrated. The con (2008)), though it requires a sufficient driving force to version of 4-hydroxybutyrate to GBL is thermodynamically overcome the energy of activation (Perez-Prior, et al., Jour favored at a non-basic pH (Efe, et al., Biotechnol Bioeng nal of Organic Chemistry, 70, 420-426 (2005)). It was not 99:1392-1406 (2008)), though it requires a sufficient driving obvious beforehand that the CoA group attached to 4-hy force to overcome the energy of activation (Perez-Prior, et droxybutyrate would provide sufficient activation to enable al., Journal of Organic Chemistry, 70, 420-426 (2005)). It GBL formation at such a high level. Without downstream was not obvious beforehand that the phosphate group enzymes for 14-BDO biosynthesis, none or very little attached to 4-hydroxybutyrate would provide sufficient acti 14-BDO was produced. The little amount of 14-BDO that vation to enable GBL formation at such a high level. These was produced was likely due to some residual endogenous results demonstrate the conversion of 4-hydroxybutyryl aldehyde dehydrogenase activities. These results demon phosphate to GBL through the non-enzymatic lactonization strate the conversion of 4-hydroxybutyryl-CoA to GBL reaction. through the non-enzymatic lactonization reaction. TABLE 64 Transformation of 4-hydroxybutyryl-phosphate to GBL in recombinant E. coli strain.

Accession Time OD 4-HB GBL 14-BDO Host Strain Genes Enzyme Name Source organism Number (h) 600 (mM) (mM) (mM) MG16SS - None 48 1.OO 13.25 O O 4-HB pathway MG16SS - pZA33-021 butyrate kinase Clostridium acetobutyllicum NP 349675 48 1.80 48.8O 2.77 O US 2016/03193 13 A1 Nov. 3, 2016 63

TABLE 65 Transformation of 4-hydroxybutyryl-CoA to GBL in recombinant E. coli strain. Host Accession Time OD 4-HB GBL 14-BDO Strain Genes Enzyme Name Source organism Number (h) 600 (mM) (mM) (mM) 432. None 48 1.06 4.41 O O 432 pZs*- butyrate kinase & Cliostridium NP 349675 & 48 1.13 3.60 1.35 O 2021C phosphotransbutyrylase acetobutyllicum NP 349676 432 F-034 4-hydroxybutyryl-CoA Porphyromonas YP OO1928841 48 0.99 2.94 O.85 O.11 transferase gingivalis ATCC 33277

Example VI lase and other enzymes leading to 14-BDO production can be utililized to adjust 14BDO to GBL ratio in coproduction. Control of the 14-BDO and GBL ratio during co-production 0418. As described above, the activities of the acyl-CoA reductase (aldehyde forming) can be utilized to adjust the 0416. One important aspect of coproduction of 14-BDO ratio of 14-BDO to GBL in coproduction. Different acyl and GBL is to control the ratio between the two coproducts CoA reductases (aldehyde forming, also known as aldehyde to match the market needs. Depending on different region, dehydrogenase) were tested for their aldehyde dehydroge the local market needs for 14-BDO and GBL can be differ nase activities by in vivo transformation measurements. The ent, thus it is important to coproduce 14-BDO and GBL with host strain used in the experiments was strain 761 (AadhE tailored ratio to satisfy the market needs. In this example, we Aldh A ApflB Asad Agab) AlpdA::K.p.lpdA322 Amdh demonstrate a method to coproduce 14-BDO and GBL at AarcAgltAR163L fiml): E. coli sucCD, P gingivalis sucD, different ratios. P. gingivalis 4hbd fiml): M. bovis sucA, C. kluyveri 4hbd). 0417. To adjust the ratio of 14-BDO and GBL, several This strain contained the components of the BDO pathway different methods can be employed. First, 4-hydroxybutyryl leading to 4HB integrated into the chromosome of E. coli at CoA can be converted to GBL by the non-enzymatic lac the fim) locus. It also contained additional genetic modi tonization reaction (Step AD in FIG. 1) or to BDO through fications to direct metabolic flux into the 4-HB pathway. The 4-hydroxybutanal by an acyl-CoA reductase (aldehyde 4-hydroxybutyryl-CoA transferase (034) was expressed forming) plus 14-BDL dehydrogenase (Steps Q and E in from the F" plamid and allowed the engineered strains to FIG. 1). An acyl-CoA reductase (aldehyde forming) with convert 4-HB to 4-hydroxybutyryl-CoA. Genes encoding high activity will convert more 4-hydroxybutyryl-CoA to different aldehyde dehydrogenase were expressed from 4-hydroxybutanal, which is Subsequently converted to pZs* plasmid. Cells were grown in fermentor with M9 14-BDO, therefore reducing the amount of GBL formed minimal medium (6.78 g/L NaHPO, 3.0 g/L KHPO, 0.5 from 4-hydroxybutyryl-CoA. This will result in a higher g/L NaCl, 1.0 g/L NHCl, 1 mM MgSO, 0.1 mM CaCl) 14-BDO to GBL ratio. Similarly, low activity of acyl-CoA Supplemented with glucose under batch fermentation con reductase (aldehyde forming) may result in a lower 14-BDO ditions. Gene expression of aldehyde dehydrogenases was to GBL ratio. Additionally, 4-hydroxybutyryl-CoA can be induced by 0.25 mM IPTG and culture supernatant samples directly converted 14-BDO by an acyl-CoA reductase (alco were taken for 14-BDO, GBL, and other products analysis. hol forming) (Step W in FIG. 1), thus 14-BDO to GBL ratio As shown in Table 66, aldehyde dehydrogenases from can be adjusted by the acyl-CoA reductase (alcohol forming) different organism results in different 14-BDO and GBL activities. Third, 4-hydroxybutyryl-phosphate can be con titers in the fermentation broth and different ratios between verted to GBL by the non-enzymatic lactonization reaction the 14-BDO and GBL coproducts. Among the aldehyde (Step AC in FIG. 1) or indirectly to 14-BDO through a dehydrogenase tested, gene 704 encoding the Salmonella phosphate transferring acyltransferases (Step P in FIG. 1) or enterica Tiphimurium aldehyde dehydrogenase resulted in an acyl-phosphate reductase (Step R in FIG. 1). Therefore, the lowest 14-BDO to GBL ratio at 0.5, allowing two moles the activities of phosphate transferring acyltransferases or of GBL produced for every mole of 14-BDO produced. On acyl-phosphate reductase in the 14-BDO production path the other hand, gene 025B encoding the Clostridium beijer way can be utilized to adjust 14-BDO to GBL ratio. Finally, inckii NCIMB 8052 aldehyde dehydrogenase resulted in the 4-HB can be directly converted to GBL by a hydroxyacyl highest 14-BDO to GBL ratio at 7.2. Several aldehyde hydrolase (Step AB in FIG. 1) competing against reactions dehydrogenases from different source organisms produced leading to 14-BDO including 4-HB kinase (Step O in FIG. 14-BDO and GBL at ratios between 0.5 and 7.2 (Table 66). 1), 4-HB reductase (Step D in FIG. 1), and/or 4-hydroxy These genes can be utilized to generate coproduction strains butyryl-CoA transferase, hydrolase, or synthetase (Steps T. with different 14-BDO to GBL ratio to meet the needs of U, V, in FIG. 1). Therefore, activities of hydroxyacylhydro different regional markets. TABLE 66

Production of 14-BDO and GBL in recombinant E. coli strains with genes for different aldehyde dehydrogenase.

Host Accession Time OD 14-BDO GBL 14-BDO: Strain Genes Enzyme Name Source organism Number (h) 600 (mM) (mM) GBL

Aldehyde Saimoneia enterica NP 460996 12O 9.4 106 197 O.S dehydrogenase Tiphimurium US 2016/03193 13 A1 Nov. 3, 2016 64

TABLE 66-continued Production of 14-BDO and GBL in recombinant E. coli strains with genes for different aldehyde dehydrogenase.

Host Accession Time OD 14-BDO GBL 14-BDO: Strain Genes Enzyme Name Source organism Number (h) 600 (mM) (mM) GBL 761 pZs-744, Aldehyde Cliostridium ZP 03705305 120 8.6 166 131 1.3 F'-O34 dehydrogenase methylpentosum DSM 5476 761 pZs*-707, Aldehyde Lactobacilius brevis ATCC YP 795 711 12O 11.4 132 50 2.6 F'-O34 dehydrogenase 367 761 pZs*-778, Aldehyde Bacilius Seieniiireducens ZP 02169447 120 10.8 298 67 4.4 F'-O34 dehydrogenase MLS10 761 pZs-025B, Aldehyde Clostridium beijerinckii YP 001310903 144 10.3 384 53 7.2 F'-O34 dehydrogenase NCIMB 8052

Example VII organic solvent and to provide GBL (boiling point 204°C.) and 14-BDO (boiling point 230°C.) which are distilled and Biosynthesis Coproduction of 1.4 BDO and GBL isolated as purified liquids. 0419. This Example describes the biosynthetic coproduc 0423 Fermentation Protocol to Coproduce 14-BDO/ tion of 14-BDO and GBLusing fermentation and other GBL (Fully Continuous): bioprocesses. 0424 The production organism is first grown up in batch mode using the apparatus and medium composition 0420 Methods for the integration of the 4-HB fermen described above, except that the initial glucose concentra tation step into a complete process for the coproduction of tion is 30-50 g/L. When glucose is exhausted, feed medium purified 14-BDO and GBL are described below. Since 4-HB of the same composition is Supplied continuously at a rate and GBL are in equilibrium, the fermentation broth will between 0.5 L/hr and 1 L/hr, and liquid is withdrawn at the contain both compounds. At low pH this equilibrium is same rate. The total concentration of 14-BDO, GBL, and shifted to favor GBL. Therefore, the fermentation can oper 4-HB in the bioreactor remains constant at 30-40 g/L, with ate at pH 7.5 or less, generally pH 5.5 or less. After removal a preferred ratio among the three coproducts, and the cell of biomass, the product stream enters into a separation step density remains constant between 3-5 g/L. Temperature is in which 14-BDO and GBL are removed and the remaining maintained at 30 degrees C., and the pH is maintained at 4.5 stream enriched in 4-HB is recycled. Finally, 14-BDO and using concentrated NaOH and HCl, as required. The biore GBL are distilled to remove any impurities. The process actor is operated continuously for one month, with Samples operates in one of three ways: 1) fed-batch fermentation and taken every day to assure consistency of coproducts con batch separation; 2) fed-batch fermentation and continuous centrations. In continuous mode, fermenter contents are separation; 3) continuous fermentation and continuous sepa constantly removed as new feed medium is Supplied. The ration. exit stream, containing cells, medium, and coproducts 0421 Fermentation Protocol to Coproduce 14-BDO and 14-BDO, GBL, and 4-HB, is then subjected to a continuous GBL (Batch): product separations procedure, with or without removing 0422 The production organism is grown in a 10 L cells and cell debris, and is done by standard continuous bioreactor sparged with an N/CO mixture, using 5 L broth separations methods employed in the art to separate organic containing 5 g/L potassium phosphate, 2.5 g/L ammonium products from dilute aqueous solutions, such as continuous chloride, 0.5 g/L magnesium sulfate, and 30 g/L corn steep liquid-liquid extraction using a water immiscible organic liquor, and an initial glucose concentration of 20 g/L. As the Solvent (e.g., toluene) to provide an organic Solution of cells grow and utilize the glucose, additional 70% glucose is 14-BDO and GBL. The resulting solution is subsequently fed into the bioreactor at a rate approximately balancing Subjected to standard continuous distillation methods to glucose consumption. The temperature of the bioreactor is remove and recycle the organic solvent and to provide GBL maintained at 30 degrees C. Growth continues for approxi (boiling point 204°C.) and 14-BDO (boiling point 230° C.) mately 24 hours, until the total concentration of 14-BDO, which are distilled and isolated as purified liquids. GBL, and 4-HB reaches a concentration of between 20-200 g/L with a preferred ratio among the three coproducts, with Example VIII the cell density being between 5 and 10 g/L. The pH is not controlled, and will typically decrease to pH 3-6 by the end Expression of Heterologous Genes Encoding BDO of the run. Upon completion of the cultivation period, the Pathway Enzymes fermenter contents are passed through a cell separation unit (e.g., centrifuge) to remove cells and cell debris, and the 0425 This example describes the expression of various fermentation broth is transferred to a product separations non-native pathway enzymes to provide improved produc unit. Isolation of 14-BDO and GBL is done by standard tion of 14-BDO. separations procedures employed in the art to separate 0426 4-Hydroxybutanal Reductase. organic products from dilute aqueous solutions, such as 0427 4-hydroxybutanal reductase activity of adh1 from liquid-liquid extraction using a water immiscible organic Geobacillus thermoglucosidasius (M10EXG) was utilized. Solvent (e.g., toluene) to provide an organic Solution of This led to improved 14-BDO production by increasing 14-BDO and GBL. The resulting solution is then subjected 4-hydroxybutanal reductase activity over endogenous lev to standard distillation methods to remove and recycle the els. US 2016/03193 13 A1 Nov. 3, 2016

0428 Multiple alcohol dehydrogenases were screened gltAR163L fiml): E. coli sucCD. P. gingivalis sucD, P for their ability to catalyze the reduction of 4-hydroxybuta gingivalis 4hbd fiml):: M. bovis sucA. C. kluyveri 4hbd nal to 14-BDO. Most alcohol dehydrogenases with high fimD::C. acetobutyllicum buk1, C. acetobutylicum ptb). activity on butyraldehyde exhibited far lower activity on which contains the remainder of the 14-BDO pathway on the 4-hydroxybutyraldehyde. One notable exception is the adh1 chromosome. The 084 ADH expressed in conjunction with gene from Geobacillus thermoglucosidasius M10EXG 025B showed the highest amount of 14-BDO (right arrow in (Jeon et al., J. Biotechnol. 135:127-133 (2008)) FIG.9) when compared with 025B only (left arrow in FIG. (GNM0084), which exhibits high activity on both 4-hy 9) and in conjunction with endogenous ADH functions. It droxybutanal and butanal. also produced more 14-BDO than did other ADH enzymes when paired with 025B, indicated as follows: 026A-C, 0429. The native gene sequence and encoded protein codon-optimized variants of Clostridium acetobutylicum sequence if the adh1 gene from Geobacillus thermoglucosi butanol dehydrogenase: 050, Zymomonas mobilis alcohol dasius are shown in FIG. 7. The G. thermoglucosidasius dehydrogenase I: 052, Citrobacter freundii 1.3-propanediol ald1 gene was expressed in E. coli. dehydrogenase: 053, Lactobacillus brevis 1,3-propanediol 0430. The Adh1 enzyme (084) expressed very well from dehydrogenase: 057, Bacteroides fragilis lactaldehyde its native gene in E. coli (see FIG. 8A). In ADH enzyme reductase: 058, E. coli 1,3-propanediol dehydrogenase; 071, assays, the E. coli expressed enzyme showed very high Bacillus subtilis 168 alpha-ketoglutarate semialdehyde reductive activity when butyraldehyde or 4HB-aldehyde dehydrogenase. The constructs labeled “PT5lacO” are those were used as the substrates (see FIG. 8B). The Km values in which the genes are driven by the PT5lacO promoter. In determined for these substrates were 1.2 mM and 4.0 mM, all other cases, the PA1 lacO-1 promoter was used. This respectively. These activity values showed that the Adh1 shows that inclusion of the 084 ADH in the 14-BDO enzyme was the most active on reduction of 4HB-aldehyde pathway increased 14-BDO production. of all the candidates tested. 0432. Throughout this application various publications 0431. The 084 enzyme was tested for its ability to boost have been referenced. The disclosures of these publications 14-BDO production when coupled with the C. beijerinckii in their entireties, including GenBank and GI number pub ald. The 084 gene was inserted behind the C. beijerinckii ald lications, are hereby incorporated by reference in this appli variant 025B gene to create a synthetic operon that results in cation in order to more fully describe the state of the art to coupled expression of both genes. Similar constructs linked which this invention pertains. Although the invention has 025B with other ADH candidate genes, and the effect of been described with reference to the examples provided including each ADH with 025B on 14-BDO production was above, it should be understood that various modifications tested. The host strain used was ECKh-459 (AadhE laha can be made without departing from the spirit of the inven ApflB AlpdA::fnr-pflB6-K.p.lpdA322 Amdh AarcA tion.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 5

<21 Os SEQ ID NO 1 &211s LENGTH: 1023 &212s. TYPE: DNA <213> ORGANISM: Geobacillus thermoglucosidasius 22 Os. FEATURE: <223> OTHER INFORMATION: nucleotide sequence of adh1 gene <4 OOs SEQUENCE: 1 atgaaagctg cagtag taga gcaatttaag galaccattaa aaattaaaga agtggaaaag 60

ccatc tattt catatggcga agtattagtic cqcattaaag catgcggtgt atgc catacg 12O gacttgcacg cc.gct catgg cqattggcca gtaaaac caa aact tcc titt aatc cctdgc 18O

catgaaggag toggaattgt taagaagtic ggtc.cggggg taac cc attt aaaagtggga 24 O

gaccgcgttg gaatticcittg gttat attct gcgtgcggcc attgcgaata ttgtttaa.gc 3 OO

gga caagaag cattatgtga acat Caacaa aacgc.cggct acticagtcga cqggggittat 360

gcagaatatt gcagagctgc gccagattat gtggtgaaaa titcc tacaa cittatcgttt 42O

gaagaagctg ct colt attitt ctdcgc.cgga gttact actt at aaag.cgtt aaaagt caca 48O

ggtacaaaac C9ggagaatggg tagcgat C tatggcatcg gcc.gc.cttgg acatgttgcc 54 O

gtc.cagtatg cgaaag.cgat ggggott cat gttgttgcag tigatatcgg catgagaaa 6 OO

Ctggaacttig caaaagagct tdgcgc.cgat Cttgttgtaa at CCtgcaaa agaaaatgcg 660

gcc caattta taaagagaa agtcggcgga gtacacgcgg Ctgttgttgac agctgt at ct 72O US 2016/03193 13 A1 Nov. 3, 2016 66

- Continued aaacct gctt ttcaatctgc gtaca attct atcc.gcagag gcggcacgtg cqtgcttgtc 78O ggattaccgc cqgaagaaat gcc tatt coa atc.tttgata cqg tattaaa cqgaattaaa 84 O attatcggitt C cattgtcgg cacgcggaaa gacttgcaag aag.cgct tca gttcgctgca 9 OO gaaggtaaag taaaaaccat tattgaagtg caacct cittgaaaaaattaa cqaagtattt 96.O gacagaatgc taaaaggaga aattaacgga C9ggttgttt taacgttaga aaataataat 1 O2O taa 1023

<210s, SEQ ID NO 2 &211s LENGTH: 34 O 212. TYPE: PRT <213> ORGANISM: Geobacillus thermoglucosidasius 22 Os. FEATURE: <223> OTHER INFORMATION: amino acid sequence of adh1 gene <4 OOs, SEQUENCE: 2 Met Lys Ala Ala Val Val Glu Glin Phe Lys Glu Pro Lieu Lys Ile Llys 1. 5 1O 15 Glu Val Glu Lys Pro Ser Ile Ser Tyr Gly Glu Val Lieu Val Arg Ile 2O 25 3O Lys Ala Cys Gly Val Cys His Thr Asp Lieu. His Ala Ala His Gly Asp 35 4 O 45 Trp Pro Val Llys Pro Llys Lieu Pro Leu. Ile Pro Gly His Glu Gly Val 50 55 60 Gly Ile Val Glu Glu Val Gly Pro Gly Val Thr His Leu Lys Val Gly 65 70 7s 8O Asp Arg Val Gly Ile Pro Trp Lieu. Tyr Ser Ala Cys Gly His Cys Glu 85 90 95 Tyr Cys Lieu. Ser Gly Glin Glu Ala Lieu. Cys Glu. His Glin Glin Asn Ala 1OO 105 11 O Gly Tyr Ser Val Asp Gly Gly Tyr Ala Glu Tyr Cys Arg Ala Ala Pro 115 12 O 125 Asp Tyr Val Val Lys Ile Pro Asp Asn Lieu. Ser Phe Glu Glu Ala Ala 13 O 135 14 O Pro Ile Phe Cys Ala Gly Val Thir Thr Tyr Lys Ala Leu Lys Val Thr 145 150 155 160 Gly Thr Llys Pro Gly Glu Trp Val Ala Ile Tyr Gly Ile Gly Gly Lieu. 1.65 17O 17s Gly His Val Ala Val Glin Tyr Ala Lys Ala Met Gly Lieu. His Val Val 18O 185 19 O Ala Val Asp Ile Gly Asp Glu Lys Lieu. Glu Lieu Ala Lys Glu Lieu. Gly 195 2OO 2O5

Ala Asp Lieu Val Val Asn Pro Ala Lys Glu Asn Ala Ala Glin Phe Met 21 O 215 22O

Lys Glu Lys Val Gly Gly Val His Ala Ala Val Val Thr Ala Val Ser 225 23 O 235 24 O

Llys Pro Ala Phe Glin Ser Ala Tyr Asn. Ser Ile Arg Arg Gly Gly Thr 245 250 255

Cys Val Lieu Val Gly Lieu Pro Pro Glu Glu Met Pro Ile Pro Ile Phe 26 O 265 27 O

Asp Thr Val Lieu. Asn Gly Ile Lys Ile Ile Gly Ser Ile Val Gly Thr 27s 28O 285 US 2016/03193 13 A1 Nov. 3, 2016 67

- Continued

Arg Lys Asp Lieu. Glin Glu Ala Lieu. Glin Phe Ala Ala Glu Gly Llys Val 29 O 295 3 OO Lys. Thir Ile Ile Glu Val Glin Pro Lieu. Glu Lys Ile Asn. Glu Val Phe 3. OS 310 315 32O Asp Arg Met Lieu Lys Gly Glu Ile Asin Gly Arg Val Val Lieu. Thir Lieu 3.25 330 335

Glu Asn. Asn. Asn 34 O

<210s, SEQ ID NO 3 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Euglena gracilis 22 Os. FEATURE: <223> OTHER INFORMATION: first 20 amino acids sequence from the N-terminus of KDC gene <4 OOs, SEQUENCE: 3 Met Thr Tyr Lys Ala Pro Wall Lys Asp Wall Lys Phe Lieu. Lieu. Asp Llys 1. 5 1O Val Phe Llys Val 2O

<210s, SEQ ID NO 4 &211s LENGTH: 59 & 212 TYPE DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: one of the primer pair used to PCR amplify lacz-alpha fragment from pUC19 vector

<4 OOs, SEQUENCE: 4 gacgaatt.cg Ctagdalagag gagaagttctga catgtc.caat it cactggcc.g. tcgttttac 59

<210s, SEQ ID NO 5 &211s LENGTH: 47 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: one of the primer pair used to PCR amplify lacz-alpha fragment from pUC19 vector

<4 OOs, SEQUENCE: 5 gaccCtagga agctitt Ctag agt caccita toggcatca gag caga 47

1. A method for producing 1,4-butanediol (14-BDO) and a 4-hydroxybutyryl-CoA reductase (alcohol forming), a gamma-butyrolactone (GBL), comprising culturing a non Succinyl-CoA reductase (aldehyde forming), an alpha naturally occurring microbial organism for a Sufficient ketoglutarate decarboxylase, a 4-hydroxybutyrate period of time to produce 14-BDO and 4-hydroxybutyryl dehydrogenase, a 4-hydroxybutyrate reductase, a 14 CoA followed by spontaneous lactonization of 4-hydroxy butanediol dehydrogenase, a Succinate reductase, a butyryl-CoA to form GBL, wherein said 14-BDO is pro Succinyl-CoA transferase, a Succinyl-CoA hydrolase, a duced at an intracellular concentration of at least 1 M, Succinyl-CoA synthetase, a glutamate dehydrogenase, a wherein said GBL is produced at an intracellular concen glutamate transaminase, a glutamate decarboxylase, an tration of between 0.001 mM and 10 mM, wherein said 4-aminobutyrate dehydrogenase, an 4-aminobutyrate non-naturally occurring microbial organism comprises a transaminase, a 4-hydroxybutyrate kinase, a phospho 14-BDO pathway and a 4-hydroxybutyryl-CoA pathway, trans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde forming), a 4-hydroxybutyryl said 14-BDO pathway comprising at least one exogenous phosphate reductase, a Succinyl-CoA reductase (alco nucleic acid encoding a 14-BDO pathway enzyme hol forming), a 4-hydroxybutyryl-CoA transferase, a expressed in a sufficient amount to produce 14-BDO, 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl said 14-BDO pathway comprising: CoA synthetase, an alpha-ketoglutarate reductase, a US 2016/03193 13 A1 Nov. 3, 2016 68

5-hydroxy-2-oxopentanoate dehydrogenase, a 5-hy hydroxybutyrylase; and a 4-hydroxybutyryl-CoA droxy-2-oxopentanoate decarboxylase or a 5-hydroxy reductase (alcohol forming); 2-oxopentanoate dehydrogenase (decarboxylation), (vii) a Succinyl-CoA transferase, a Succinyl-CoA hydro said 4-hydroxybutyryl-CoA pathway comprising at least lase or a Succinyl-CoA synthetase; a Succinyl-CoA one exogenous nucleic acid encoding an 4-hydroxybu reductase (alcohol forming); a 4-hydroxybutyrate tyryl-CoA pathway enzyme expressed in a Sufficient reductase; and 1,4-butanediol dehydrogenase; amount to produce 4-hydroxybutyryl-CoA, said 4-hy (viii) a Succinyl-CoA transferase, a Succinyl-CoA hydro droxybutyryl-CoA pathway comprising: lase or a Succinyl-CoA synthetase; a Succinyl-CoA a 4-hydroxybutyryl-CoA transferase, a Succinyl-CoA reductase (alcohol forming); a 4-hydroxybutyrate reductase (aldehyde forming), an alpha-ketoglutarate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hy decarboxylase, a 4-hydroxybutyrate dehydrogenase, a droxybutyryl-CoA reductase (aldehyde forming); and a Succinate reductase, a Succinyl-CoA transferase, a suc cinyl-CoA hydrolase, a Succinyl-CoA synthetase, a 1,4-butanediol dehydrogenase; glutamate dehydrogenase, a glutamate transaminase, a (ix) Succinyl-CoA transferase, a Succinyl-CoA hydrolase glutamate decarboxylase, a 4-aminobutyrate dehydro or Succinyl-CoA synthetase; a Succinyl-CoA reductase genase, a 4-aminobutyrate transaminase, a 4-hydroxy (alcohol forming), a 4-hydroxybutyrate kinase, a 4-hy butyrate kinase, a phosphotrans-4-hydroxybutyrylase, droxybutyryl-phosphate reductase; and a 1,4-butane a Succinyl-CoA reductase (alcohol forming), a 4-hy diol dehydrogenase; droxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA (X) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase synthetase, an alpha-ketoglutarate reductase, a 5-hy or a succinyl-CoA synthetase; a Succinyl-CoA reduc droxy-2-oxopentanoate dehydrogenase, or a 5-hy tase (alcohol forming); a 4-hydroxybutyryl-CoA trans droxy-2-oxopentanoate dehydrogenase (decarboxy ferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hy lation) and droxybutyryl-CoA synthetase; a 4-hydroxybutyryl wherein said non-naturally occurring microbial organism CoA reductase (aldehyde forming); and a 14 is a bacteria, yeast or fungus. butanediol dehydrogenase; 2. The method of claim 1, wherein the 14-BDO pathway (xi) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase comprises a pathway selected from the group consisting of or a succinyl-CoA synthetase; a Succinyl-CoA reduc (i) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase tase (alcohol forming); a 4-hydroxybutyryl-CoA trans or a succinyl-CoA synthetase; a succinyl-CoA reduc ferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hy tase (aldehyde forming); a 4-hydroxybutyrate dehydro droxybutyryl-CoA synthetase; and a 4-hydroxybutyryl genase; a 4-hydroxybutyrate reductase; and a 1,4-bu CoA reductase (alcohol forming); tanediol dehydrogenase, (xii) a Succinyl-CoA transferase, a Succinyl-CoA hydro (ii) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase lase or a Succinyl-CoA synthetase; a Succinyl-CoA or a succinyl-CoA synthetase; a Succinyl-CoA reduc reductase (alcohol forming); a 4-hydroxybutyrate tase (aldehyde forming); a 4-hydroxybutyrate dehydro kinase; a phosphotrans-4-hydroxybutyrylase; and a genase; a 4-hydroxybutyrate kinase; a phosphotrans-4- 4-hydroxybutyryl-CoA reductase (alcohol forming); hydroxybutyrylase; a 4-hydroxybutyryl-CoA reductase (xiii) a Succinate reductase; a 4-hydroxybutyrate dehy (aldehyde forming); and a 1,4-butanediol dehydroge drogenase; a 4-hydroxybutyrate reductase; and 1,4- nase, butanediol dehydrogenase; (iii) a Succinyl-CoA transferase, a Succinyl-CoA hydro (xiv) a Succinate reductase; a 4-hydroxybutyrate dehy lase or a Succinyl-CoA synthetase; a Succinyl-CoA drogenase; a 4-hydroxybutyrate kinase; a phospho reductase (aldehyde forming); a 4-hydroxybutyrate trans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hy reductase (aldehyde forming); and a 1,4-butanediol droxybutyryl-phosphate reductase; and a 1,4-butane dehydrogenase; diol dehydrogenase; (XV) a Succinate reductase; a 4-hydroxybutyrate dehydro (iv) a Succinyl-CoA transferase, a Succinyl-CoA hydro genase; a 4-hydroxybutyrate kinase; a phosphotrans-4- lase, or a succinyl-CoA synthetase; a Succinyl-CoA hydroxybutyrylase; and a 4-hydroxybutyryl-CoA reductase (aldehyde forming); a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a reductase (alcohol forming); 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybu (Xvi) a Succinate reductase; a 4-hydroxybutyrate dehy tyryl-CoA synthetase; a 4-hydroxybutyryl-CoA reduc drogenase; a 4-hydroxybutyrate kinase; a 4-hydroxy tase (aldehyde forming); and a 1,4-butanediol dehydro butyryl-phosphate reductase; and 1,4-butanediol dehy genase, drogenase; (v) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase (Xvii) a Succinate reductase; a 4-hydroxybutyrate dehy or Succinyl-CoA synthetase; a Succinyl-CoA reductase drogenase; a 4-hydroxybutyryl-CoA transferase, a (aldehyde forming); a 4-hydroxybutyrate dehydroge 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybu nase; a 4-hydroxybutyryl-CoA transferase, a 4-hy tyryl-CoA synthetase; a 4-hydroxybutyryl-CoA reduc droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl tase (aldehyde forming); and 1,4-butanediol dehydro CoA synthetase; and a 4-hydroxybutyryl-CoA genase, reductase (alcohol forming); (Xviii) a Succinate reductase; a 4-hydroxybutyrate dehy (vi) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase drogenase; a 4-hydroxybutyryl-CoA transferase, a or a succinyl-CoA synthetase; a Succinyl-CoA reduc 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybu tase (aldehyde forming); a 4-hydroxybutyrate dehydro tyryl-CoA synthetase; and genase; a 4-hydroxybutyrate kinase; a phosphotrans-4- a 4-hydroxybutyryl-CoA reductase (alcohol forming); US 2016/03193 13 A1 Nov. 3, 2016 69

(xix) an alpha-ketoglutarate decarboxylase; a 4-hydroxy 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu butyrate dehydrogenase; a 4-hydroxybutyrate reduc tyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydro tase; and 1,4-butanediol dehydrogenase; lase or a 4-hydroxybutyryl-CoA synthetase; and a 4-hy (XX) an alpha-ketoglutarate decarboxylase; a 4-hydroxy droxybutyryl-CoA reductase (alcohol forming); butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a (XXXi) an alpha-ketoglutarate reductase; a 5-hydroxy-2- phosphotrans-4-hydroxybutyrylase; a 4-hydroxybu OXopentanoate dehydrogenase; a 5-hydroxy-2-oxopen tyryl-CoA reductase (aldehyde forming); and a 1,4- tanoate dehydrogenase (decarboxylation); a 4-hy butanediol dehydrogenase; droxybutyryl-CoA reductase (aldehyde forming); and a (XXi) an alpha-ketoglutarate decarboxylase; a 4-hydroxy 1,4-butanediol dehydrogenase; butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a (XXXii) an alpha-ketoglutarate reductase; a 5-hydroxy-2- phosphotrans-4-hydroxybutyrylase and 4-hydroxybu OXopentanoate dehydrogenase; a 5-hydroxy-2-oxopen tyryl-CoA reductase (alcohol forming); tanoate dehydrogenase (decarboxylation); and a 4-hy (XXii) an alpha-ketoglutarate decarboxylase; a 4-hydroxy droxybutyryl-CoA reductase (alcohol forming); butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a (XXXiii) an alpha-ketoglutarate reductase; a 5-hydroxy-2- 4-hydroxybutyryl-phosphate reductase; and a 1,4-bu OXopentanoate dehydrogenase; a 5-hydroxy-2-oxopen tanediol dehydrogenase; tanoate decarboxylase; and a 1,4-butanediol dehydro (XXiii) an alpha-ketoglutarate decarboxylase; a 4-hy genase droxybutyrate dehydrogenase; a 4-hydroxybutyryl (XXXiv) a Succinyl-CoA reductase (aldehyde forming); a CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybu tyrate reductase; and 1,4-butanediol dehydrogenase; tyryl-CoA reductase (aldehyde forming); and a 1,4- (XXXV) a Succinyl-CoA reductase (aldehyde forming); a butanediol dehydrogenase; 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu (XXiv) an alpha-ketoglutarate decarboxylase; a 4-hy tyrate kinase; a phosphotrans-4-hydroxybutyrylase; a droxybutyrate dehydrogenase; a 4-hydroxybutyryl 4-hydroxybutyryl-CoA reductase (aldehyde forming); CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or and a 1,4-butanediol dehydrogenase; a 4-hydroxybutyryl-CoA synthetase; and 4-hydroxybu (XXXVi) a Succinyl-CoA reductase (aldehyde forming); a tyryl-CoA reductase (alcohol forming); 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu (XXV) a glutamate dehydrogenase or a glutamate transami tyrate kinase; a phosphotrans-4-hydroxybutyrylase and nase; a glutamate decarboxylase; a 4-aminobutyrate 4-hydroxybutyryl-CoA reductase (alcohol forming); dehydrogenase or 4-aminobutyrate transaminase; a (XXXVii) a Succinyl-CoA reductase (aldehyde forming); a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu tyrate reductase; and a 1,4-butanediol dehydrogenase; tyrate kinase; a 4-hydroxybutyryl-phosphate reductase; (XXVi) a glutamate dehydrogenase or a glutamate and a 1,4-butanediol dehydrogenase; transaminase; a glutamate decarboxylase; a 4-aminobu (XXXViii) a Succinyl-CoA reductase (aldehyde forming); a tyrate dehydrogenase or a 4-aminobutyrate transami 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu nase; a 4-hydroxybutyrate dehydrogenase; a 4-hy tyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydro droxybutyrate kinase; a phosphotrans-4- lase or 4-hydroxybutyryl-CoA synthetase; a 4-hydroxy hydroxybutyrylase; a 4-hydroxybutyryl-CoA reductase butyryl-CoA reductase (aldehyde forming); and a 1,4- (aldehyde forming); and a 1,4-butanediol dehydroge butanediol dehydrogenase; and nase, (XXVii) a glutamate dehydrogenase or glutamate transami (XXXix) a Succinyl-CoA reductase (aldehyde forming); a nase; a glutamate decarboxylase; a 4-aminobutyrate 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu dehydrogenase or a 4-aminobutyrate transaminase; a tyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydro 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu lase or a 4-hydroxybutyryl-CoA synthetase; and 4-hy tyrate kinase; a phosphotrans-4-hydroxybutyrylase; droxybutyryl-CoA reductase (alcohol forming). and a 4-hydroxybutyryl-CoA reductase (alcohol form 3-34. (canceled) ing); 35. The method of claim 1, wherein the 4-hydroxybu (XXXiii) a glutamate dehydrogenase or a glutamate tyryl-CoA pathway comprises a pathway selected from the transaminase; a glutamate decarboxylase; a 4-aminobu group consisting of tyrate dehydrogenase or a 4-aminobutyrate transami (i) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase nase; a 4-hydroxybutyrate dehydrogenase; a 4-hy or a succinyl-CoA synthetase; a Succinyl-CoA reduc droxybutyrate kinase; a 4-hydroxybutyryl-phosphate tase (aldehyde forming); a 4-hydroxybutyrate dehydro reductase; and a 1,4-butanediol dehydrogenase; genase; a 4-hydroxybutyrate kinase; and a phospho (XXiX) a glutamate dehydrogenase or a glutamate trans-4-hydroxybutyrylase; transaminase; a glutamate decarboxylase; a 4-aminobu (ii) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase tyrate dehydrogenase or a 4-aminobutyrate transami or a succinyl-CoA synthetase; a Succinyl-CoA reduc nase; a 4-hydroxybutyrate dehydrogenase; a 4-hy tase (aldehyde forming); a 4-hydroxybutyrate dehydro droxybutyryl-CoA transferase, a 4-hydroxybutyryl genase; and a 4-hydroxybutyryl-CoA transferase, a CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase: 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybu a 4-hydroxybutyryl-CoA reductase (aldehyde form tyryl-CoA synthetase: ing); and a 1,4-butanediol dehydrogenase; (iii) a Succinyl-CoA transferase, a Succinyl-CoA hydro (XXX) a glutamate dehydrogenase or a glutamate transami lase or a Succinyl-CoA synthetase; a Succinyl-CoA nase; a glutamate decarboxylase; a 4-aminobutyrate reductase (alcohol forming); a 4-hydroxybutyrate dehydrogenase or a 4-aminobutyrate transaminase; a kinase; and a phosphotrans-4-hydroxybutyrylase; US 2016/03193 13 A1 Nov. 3, 2016 70

(iv) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase ketoglutarate decarboxylase, a 4-hydroxybutyrate or a succinyl-CoA synthetase; a Succinyl-CoA reduc dehydrogenase, a 4-hydroxybutyrate reductase, a 14 tase (alcohol forming); and a 4-hydroxybutyryl-CoA butanediol dehydrogenase, a Succinate reductase, a transferase, a 4-hydroxybutyryl-CoA hydrolase or a Succinyl-CoA transferase, a Succinyl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase; Succinyl-CoA synthetase, a glutamate dehydrogenase, a (v) a Succinate reductase; a 4-hydroxybutyrate dehydro glutamate transaminase, a glutamate decarboxylase, an genase; a 4-hydroxybutyrate kinase and a phospho 4-aminobutyrate dehydrogenase, an 4-aminobutyrate trans-4-hydroxybutyrylase; transaminase, a 4-hydroxybutyrate kinase, a phospho (vi) a Succinate reductase; a 4-hydroxybutyrate dehydro trans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA genase; and a 4-hydroxybutyryl-CoA transferase, a reductase (aldehyde forming), a 4-hydroxybutyryl 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybu phosphate reductase, a Succinyl-CoA reductase (alco tyryl-CoA synthetase: hol forming), a 4-hydroxybutyryl-CoA transferase, a (vii) an alpha-ketoglutarate decarboxylase; a 4-hydroxy 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl butyrate dehydrogenase; a 4-hydroxybutyrate kinase; CoA synthetase, an alpha-ketoglutarate reductase, a and a phosphotrans-4-hydroxybutyrylase; 5-hydroxy-2-oxopentanoate dehydrogenase, a 5-hy (viii) an alpha-ketoglutarate decarboxylase; a 4-hydroxy droxy-2-oxopentanoate decarboxylase or a 5-hydroxy butyrate dehydrogenase; and a 4-hydroxybutyryl-CoA 2-oxopentanoate dehydrogenase (decarboxylation), transferase, a 4-hydroxybutyryl-CoA hydrolase or a said 4-hydroxybutyryl-phosphate pathway comprising at 4-hydroxybutyryl-CoA synthetase; least one exogenous nucleic acid encoding an 4-hy (ix) a glutamate dehydrogenase or a glutamate transami droxybutyryl-phosphate pathway enzyme expressed in nase; a glutamate decarboxylase; an 4-aminobutyrate a sufficient amount to produce 4-hydroxybutyryl-phos dehydrogenase or an 4-aminobutyrate transaminase; a phate, said 4-hydroxybutyryl-phosphate pathway com 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu prising: tyrate kinase; and a phosphotrans-4-hydroxybutyry a 4-hydroxybutyrate kinase, a Succinyl-CoA reductase lase; (aldehyde forming), an alpha-ketoglutarate decarboxy (X) a glutamate dehydrogenase or a glutamate transami lase, a 4-hydroxybutyrate dehydrogenase, a Succinate nase; a glutamate decarboxylase; an 4-aminobutyrate reductase, a Succinyl-CoA transferase, a Succinyl-CoA dehydrogenase or an 4-aminobutyrate transaminase; a hydrolase, a Succinyl-CoA synthetase, a glutamate 4-hydroxybutyrate dehydrogenase; and a 4-hydroxybu dehydrogenase, a glutamate transaminase, a glutamate tyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydro decarboxylase, a 4-aminobutyrate dehydrogenase, a lase or a 4-hydroxybutyryl-CoA synthetase: 4-aminobutyrate transaminase or a Succinyl-CoA (xi) an alpha-ketoglutarate reductase; a 5-hydroxy-2-oxo reductase (alcohol forming). pentanoate dehydrogenase and a 5-hydroxy-2-oxopen 60. The method of claim 59, wherein the 14-BDO path tanoate dehydrogenase (decarboxylation) way comprises a pathway selected from the group consisting (xii) a Succinyl-CoA reductase (aldehyde forming); a of: 4-hydroxybutyrate dehydrogenase; and a phospho (i) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase trans-4-hydroxybutyrylase; and or a succinyl-CoA synthetase; a Succinyl-CoA reduc (xiii) a Succinyl-CoA reductase (aldehyde forming); a tase (aldehyde forming); a 4-hydroxybutyrate dehydro 4-hydroxybutyrate dehydrogenase; and a 4-hydroxybu genase; a 4-hydroxybutyrate reductase; and a 1,4-bu tyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydro tanediol dehydrogenase; lase or 4-hydroxybutyryl-CoA synthetase. (ii) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase 36-46. (canceled) or a succinyl-CoA synthetase; a Succinyl-CoA reduc 47. The method of claim 1, wherein said culturing is tase (aldehyde forming); a 4-hydroxybutyrate dehydro performed under Substantially anaerobic culture conditions. genase; a 4-hydroxybutyrate kinase; a phosphotrans-4- 48. The method of claim 1, wherein said exogenous hydroxybutyrylase; a 4-hydroxybutyryl-CoA reductase nucleic acid is a heterologous nucleic acid. (aldehyde forming); and a 1,4-butanediol dehydroge 49-58. (canceled) nase, 59. A method for producing 1,4-butanediol (14-BDO) and (iii) a Succinyl-CoA transferase, a Succinyl-CoA hydro gamma-butyrolactone (GBL), comprising culturing a non lase or a Succinyl-CoA synthetase; a Succinyl-CoA naturally occurring microbial organism for a Sufficient reductase (aldehyde forming); a 4-hydroxybutyrate period of time to produce 14-BDO and 4-hydroxybutyryl dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hy phosphate followed by spontaneous lactonization of 4-hy droxybutyryl-phosphate reductase; and a 1,4-butane droxybutyryl-phosphate to form GBL, wherein said diol dehydrogenase; 14-BDO is produced at an intracellular concentration of at (iv) a Succinyl-CoA transferase, a Succinyl-CoA hydro least 1 M, wherein said GBL is produced at an intracellular lase, or a Succinyl-CoA synthetase; a Succinyl-CoA concentration of between 0.001 mM and 10 mM, wherein reductase (aldehyde forming); a 4-hydroxybutyrate said non-naturally occurring microbial organism comprise a dehydrogenase; a 4-hydroxybutyryl-CoA transferase, a microbial organism having a 14-BDO pathway and a 4-hy 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybu droxybutyryl-phosphate pathway, said 14-BDO pathway tyryl-CoA synthetase; a 4-hydroxybutyryl-CoA reduc comprising at least one exogenous nucleic acid encoding a tase (aldehyde forming); and a 1,4-butanediol dehydro 14-BDO pathway enzyme expressed in a sufficient amount genase, to produce 14-BDO, said 14-BDO pathway comprising: (v) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase a 4-hydroxybutyryl-CoA reductase (alcohol forming), a or Succinyl-CoA synthetase; a Succinyl-CoA reductase Succinyl-CoA reductase (aldehyde forming), an alpha (aldehyde forming); a 4-hydroxybutyrate dehydroge US 2016/03193 13 A1 Nov. 3, 2016

nase; a 4-hydroxybutyryl-CoA transferase, a 4-hy tyryl-CoA synthetase; a 4-hydroxybutyryl-CoA reduc droxybutyryl-CoA hydrolase or a 4-hydroxybutyryl tase (aldehyde forming); and 1,4-butanediol dehydro CoA synthetase; and a 4-hydroxybutyryl-CoA genase, reductase (alcohol forming); (Xviii) a Succinate reductase; a 4-hydroxybutyrate dehy (vi) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase drogenase; a 4-hydroxybutyryl-CoA transferase, a or a succinyl-CoA synthetase; a Succinyl-CoA reduc 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybu tase (aldehyde forming); a 4-hydroxybutyrate dehydro tyryl-CoA synthetase; and a 4-hydroxybutyryl-CoA genase; a 4-hydroxybutyrate kinase; a phosphotrans-4- reductase (alcohol forming); hydroxybutyrylase; and a 4-hydroxybutyryl-CoA (Xix) an alpha-ketoglutarate decarboxylase; a 4-hydroxy reductase (alcohol forming); butyrate dehydrogenase; a 4-hydroxybutyrate reduc (vii) a Succinyl-CoA transferase, a Succinyl-CoA hydro tase; and 1,4-butanediol dehydrogenase; lase or a Succinyl-CoA synthetase; a Succinyl-CoA (XX) an alpha-ketoglutarate decarboxylase; a 4-hydroxy reductase (alcohol forming); a 4-hydroxybutyrate butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a reductase; and 1,4-butanediol dehydrogenase; phosphotrans-4-hydroxybutyrylase; a 4-hydroxybu (viii) a Succinyl-CoA transferase, a Succinyl-CoA hydro tyryl-CoA reductase (aldehyde forming); and a 1,4- lase or a Succinyl-CoA synthetase; a Succinyl-CoA butanediol dehydrogenase; reductase (alcohol forming); a 4-hydroxybutyrate (XXi) an alpha-ketoglutarate decarboxylase; a 4-hydroxy kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hy butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a droxybutyryl-CoA reductase (aldehyde forming); and a phosphotrans-4-hydroxybutyrylase and 4-hydroxybu tyryl-CoA reductase (alcohol forming); 1,4-butanediol dehydrogenase; (XXii) an alpha-ketoglutarate decarboxylase; a 4-hydroxy (ix) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase butyrate dehydrogenase; a 4-hydroxybutyrate kinase; a or Succinyl-CoA synthetase; a Succinyl-CoA reductase 4-hydroxybutyryl-phosphate reductase; and a 1,4-bu (alcohol forming), a 4-hydroxybutyrate kinase, a 4-hy tanediol dehydrogenase; droxybutyryl-phosphate reductase; and a 1,4-butane (XXiii) an alpha-ketoglutarate decarboxylase; a 4-hy diol dehydrogenase; droxybutyrate dehydrogenase; a 4-hydroxybutyryl (X) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or or a succinyl-CoA synthetase; a Succinyl-CoA reduc 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybu tase (alcohol forming); a 4-hydroxybutyryl-CoA trans tyryl-CoA reductase (aldehyde forming); and a 1,4- ferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hy butanediol dehydrogenase; droxybutyryl-CoA synthetase; a 4-hydroxybutyryl (XXiv) an alpha-ketoglutarate decarboxylase; a 4-hy CoA reductase (aldehyde forming); and a 14 droxybutyrate dehydrogenase; a 4-hydroxybutyryl butanediol dehydrogenase; CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or (xi) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase a 4-hydroxybutyryl-CoA synthetase; and 4-hydroxybu or a succinyl-CoA synthetase; a Succinyl-CoA reduc tyryl-CoA reductase (alcohol forming); tase (alcohol forming); a 4-hydroxybutyryl-CoA trans (XXV) a glutamate dehydrogenase or a glutamate transami ferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hy nase; a glutamate decarboxylase; a 4-aminobutyrate droxybutyryl-CoA synthetase; and a 4-hydroxybutyryl dehydrogenase or 4-aminobutyrate transaminase; a CoA reductase (alcohol forming); 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu (xii) a Succinyl-CoA transferase, a Succinyl-CoA hydro tyrate reductase; and a 1,4-butanediol dehydrogenase; lase or a Succinyl-CoA synthetase; a Succinyl-CoA (XXvi) a glutamate dehydrogenase or a glutamate reductase (alcohol forming); a 4-hydroxybutyrate transaminase; a glutamate decarboxylase; a 4-aminobu kinase; a phosphotrans-4-hydroxybutyrylase; and a tyrate dehydrogenase or a 4-aminobutyrate transami 4-hydroxybutyryl-CoA reductase (alcohol forming); nase; a 4-hydroxybutyrate dehydrogenase; a 4-hy (xiii) a Succinate reductase; a 4-hydroxybutyrate dehy droxybutyrate kinase; a phosphotrans-4- drogenase; a 4-hydroxybutyrate reductase; and 1,4- hydroxybutyrylase; a 4-hydroxybutyryl-CoA reductase butanediol dehydrogenase; (aldehyde forming); and a 1,4-butanediol dehydroge (xiv) a Succinate reductase; a 4-hydroxybutyrate dehy nase, drogenase; a 4-hydroxybutyrate kinase; a phospho (XXvii) a glutamate dehydrogenase or glutamate transami trans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA nase; a glutamate decarboxylase; a 4-aminobutyrate reductase (aldehyde forming); and a 1,4-butanediol dehydrogenase or a 4-aminobutyrate transaminase; a dehydrogenase; 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu tyrate kinase; a phosphotrans-4-hydroxybutyrylase; (XV) a Succinate reductase; a 4-hydroxybutyrate dehydro and a 4-hydroxybutyryl-CoA reductase (alcohol form genase; a 4-hydroxybutyrate kinase; a phosphotrans-4- ing); hydroxybutyrylase; and a 4-hydroxybutyryl-CoA (XXviii) a glutamate dehydrogenase or a glutamate reductase (alcohol forming); transaminase; a glutamate decarboxylase; a 4-aminobu (Xvi) a Succinate reductase; a 4-hydroxybutyrate dehy tyrate dehydrogenase or a 4-aminobutyrate transami drogenase; a 4-hydroxybutyrate kinase; a 4-hydroxy nase; a 4-hydroxybutyrate dehydrogenase; a 4-hy butyryl-phosphate reductase; and 1,4-butanediol dehy droxybutyrate kinase; a 4-hydroxybutyryl-phosphate drogenase; reductase; and a 1,4-butanediol dehydrogenase; (Xvii) a Succinate reductase; a 4-hydroxybutyrate dehy (XXiX) a glutamate dehydrogenase or a glutamate drogenase; a 4-hydroxybutyryl-CoA transferase, a transaminase; a glutamate decarboxylase; a 4-aminobu 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybu tyrate dehydrogenase or a 4-aminobutyrate transami US 2016/03193 13 A1 Nov. 3, 2016 72

nase; a 4-hydroxybutyrate dehydrogenase; a 4-hy 100. The method of claim 59, wherein said exogenous droxybutyryl-CoA transferase, a 4-hydroxybutyryl nucleic acid is a heterologous nucleic acid. CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase: 101-157. (canceled) a 4-hydroxybutyryl-CoA reductase (aldehyde form 158. The method of claim 1, wherein said microbial ing); and a 1,4-butanediol dehydrogenase; organism comprises one, two, three, four, five, six, seven or (XXX) a glutamate dehydrogenase or a glutamate transami eight exogenous nucleic acids each encoding a 14-BDO or nase; a glutamate decarboxylase; a 4-aminobutyrate 4-hydroxybutyryl-CoA pathway enzyme. dehydrogenase or a 4-aminobutyrate transaminase; a 159. The method of claim 1, wherein said method further 4-hydroxybutyrate dehydrogenase; a 4-hydroxybu comprises separating the 14-BDO from other components in tyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydro the culture. lase or a 4-hydroxybutyryl-CoA synthetase; and a 4-hy 160. The method of claim 159, wherein the separating droxybutyryl-CoA reductase (alcohol forming); comprises extraction, continuous liquid-liquid extraction, (XXXi) an alpha-ketoglutarate reductase; a 5-hydroxy-2- pervaporation, membrane filtration, membrane separation, oXopentanoate dehydrogenase; a 5-hydroxy-2-oxopen reverse osmosis, electrodialysis, distillation, crystallization, tanoate dehydrogenase (decarboxylation); a 4-hy centrifugation, extractive filtration, ion exchange chroma droxybutyryl-CoA reductase (aldehyde forming); and a tography, absorption chromatography, or ultrafiltration. 1,4-butanediol dehydrogenase; 161. The method of claim 59, wherein said microbial (XXXii) an alpha-ketoglutarate reductase; a 5-hydroxy-2- organism comprises one, two, three, four, five, six, seven or oXopentanoate dehydrogenase; a 5-hydroxy-2-oxopen eight exogenous nucleic acids each encoding a 14-BDO or tanoate dehydrogenase (decarboxylation); and a 4-hy 4-hydroxybutyryl-phosphate pathway enzyme. droxybutyryl-CoA reductase (alcohol forming); and 162. The method of claim 59, wherein said method further (XXXiii) an alpha-ketoglutarate reductase; a 5-hydroxy-2- comprises separating the 14-BDO from other components in oXopentanoate dehydrogenase; a 5-hydroxy-2-oxopen the culture. tanoate decarboxylase; and a 1,4-butanediol dehydro 163. The method of claim 162, wherein the separating genase. comprises extraction, continuous liquid-liquid extraction, 61-92. (canceled) pervaporation, membrane filtration, membrane separation, 93. The method of claim 59, wherein the 4-hydroxybu reverse osmosis, electrodialysis, distillation, crystallization, tyryl-phosphate pathway comprises a pathway selected from centrifugation, extractive filtration, ion exchange chroma the group consisting of: tography, absorption chromatography, or ultrafiltration. (i) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase 164. The method of claim 1, wherein said bacteria is or a succinyl-CoA synthetase; a Succinyl-CoA reduc selected from the group consisting of Escherichia coli, tase (aldehyde forming); a 4-hydroxybutyrate dehydro Klebsiella Oxytoca, Anaerobiospirillum succiniciproducens, genase; and 4-hydroxybutyrate kinase; Actinobacillus succinogenes, Mannheimia succiniciprod (ii) a Succinyl-CoA transferase, a Succinyl-CoA hydrolase ucens, Rhizobium etli, Bacillus subtilis, Corynebacterium or a succinyl-CoA synthetase; a Succinyl-CoA reduc glutamicum, Gluconobacter Oxydans, Zymomonas mobilis, tase (alcohol forming); and a 4-hydroxybutyrate Lactococcus lactis, Lactobacillus plantarum, Streptomyces kinase; (iii) a Succinate reductase; a 4-hydroxybutyrate dehydro coelicolor, Clostridium acetobutyllicum, Pseudomonas fluo genase; and a 4-hydroxybutyrate kinase; rescens, and Pseudomonas putida. (iv) an alpha-ketoglutarate decarboxylase; a 4-hydroxy 165. The method of claim 1, wherein said bacteria is butyrate dehydrogenase; and a 4-hydroxybutyrate Escherichia coli. kinase; and 166. The method of claim 1, wherein said yeast or fungus (v) a glutamate dehydrogenase or a glutamate transami is selected from the group consisting of Saccharomyces nase; a glutamate decarboxylase; an 4-aminobutyrate cerevisiae, Schizosaccharomyces pombe, Kluyveromyces dehydrogenase or an 4-aminobutyrate transaminase; a lactis, Kluyveromyces marxianus, Aspergillus terreus, 4-hydroxybutyrate dehydrogenase; and a 4-hydroxybu Aspergillus niger; Pichia pastoris, Rhizopus arrhizus, and tyrate kinase. Rhizopus Oryzae. 94-98. (canceled) 167. The method of claim 1, wherein said yeast is 99. The method of claim 59, wherein said culturing is Saccharomyces cerevisiae. performed under Substantially anaerobic culture conditions. k k k k k