US 201701 01638A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0101638 A1 Liao et al. (43) Pub. Date: Apr. 13, 2017

(54) ACETYL-COA CARBOXYLASES (60) Provisional application No. 61/852.387, filed on Mar. 15, 2013. (71) Applicant: Cargill, Incorporated, Wayzata, MN (US) (72) Inventors: Hans Liao, Superior, CO (US); Publication Classification Christopher Patrick Mercogliano, Minneapolis, MN (US); Travis Robert (51) Int. Cl. Wolter, Denver, CO (US); Michael Tai CI2N 9/00 (2006.01) Man Louie, Broomfield, CO (US); (52) U.S. Cl. Wendy Kathleen Ribble, Arvada, CO CPC ...... CI2N 9/93 (2013.01); C12Y 604/01002 (US); Tanya E. W. Lipscomb, Boulder, (2013.01) CO (US); Eileen Colie Spindler, Lafayette, CO (US); Michael D. Lynch, Durham, NC (US) (57) ABSTRACT (21) Appl. No.: 15/269,382 (22) Filed: Sep. 19, 2016 The present invention provides various combinations of genetic modifications to a transformed host cell that provide Related U.S. Application Data increase conversion of carbon to a chemical product. The (63) Continuation of application No. 14/215,379, filed on present invention also provides methods of fermentation and Mar. 17, 2014, now Pat. No. 9,447,438. methods of making various chemical products. Patent Application Publication US 2017/0101638A1

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ACETYL-COA CARBOXYLASES NADPH-dependent, (b) primarily NADH-dependent, (c) primarily flavin-dependent, (d) less susceptible to 3-HP CLAIM OF PRIORITY inhibition at high concentration, and/or (e) catalyzes a 0001. This application is a Continuation of and claims the reaction pathway to 3-HP that is substantially irreversible: benefit of priority to U.S. patent application Ser. No. 14/215. (6) a monofunctional malonyl-CoA reductase enzyme fused 379, Filed on Mar. 17, 2014, which claims the benefit of to one or more malonate semialdehyde dehydrogenase priority to U.S. Provisional Patent Application No. 61/852. enzymes; (7) a malonyl-CoA reductase gene that is mutated 387, filed on Mar. 15, 2013, the benefit of priority of each of to enhance its activity at lower temperatures; (8) salt-tolerant which is claimed hereby, and each of which are incorporated enzymes; (9) a gene that facilitates the exportation of a by reference herein in its entirety. chemical product of interest or the export of an inhibitory chemical from within the cell to the extracellular media; BACKGROUND OF THE INVENTION and/or (10) a gene that facilitates the importation from the extracellular media to within the cell of a reactant, precursor, 0002 There is a need for alternative production methods and/or metabolite used in the organism’s production path of industrial chemicals used for various consumer products way for producing a chemical product of interest. and fuels that are currently made from petroleum. One 0007. The present invention further relates to methods of alternative method is the use of engineered microorganisms producing a chemical product using the genetically modified to produce industrial chemicals. Currently, in the field of organisms of the invention. The present invention further bioproduced chemicals there is a need to improve microbial includes products made from these methods. In accordance enzyme performance, enhanced production rate in order to with certain embodiments that product is acetyl-CoA, malo reach the goal of becoming an at-cost replacement basis for nyl-CoA, malonate semialdehyde, 3-hydroxypropionic acid petro-based chemicals. (3-HP), acrylic acid, 1.3 propanediol, malonic acid, ethyl 0003. A common challenge faced in field of bio-produced 3-HP, propiolactone, acrylonitrile, acrylamide, methyl acry chemicals in microorganisms is that any one modification to late, a polymer including Super absorbent polymers and a host cell may require coordination with other modifica polyacrylic acid, or a consumer product. tions in or r to Successfully enhance chemical bioproduc 0008. The present invention further relates to a method of tion. producing a chemical product from a renewable carbon 0004. The current invention provides methods, systems Source through a bioproduction process that comprises a of fermentation, genetically modified microorganisms, controlled multi-phase production process wherein the ini modified enhanced enzymes for chemical production, all of tiation and/or completion of one or more phases of the which may be used in various combinations to increase production process is controlled by genetic modifications to chemical production of a desired chemical product. the organism producing the chemical product and/or is controlled by changes made to the cell environment. In INCORPORATION BY REFERENCE accordance with this aspect of the invention, the bioproduc 0005 All publications, patents, and patent applications tion process may include two or more of the following mentioned in this specification are herein incorporated by phases: (1) growth phase; (2) induction phase; and (3) reference to the same extent as if each individual publica production phase. The present invention further includes tion, patent, or patent application was specifically and indi products made from these methods. vidually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS SUMMARY OF THE INVENTION 0009. The novel features of the invention are set forth 0006. The present invention relates to genetically modi with particularity in the appended claims. A better under fied organisms capable of producing an industrial chemical standing of the features and advantages of the present product of interest, wherein the genetic modification invention will be obtained by reference to the following includes introduction of nucleic acid sequences coding for detailed description that sets forth illustrative embodiments, polynucleotides encoding one or more of the following: (1) in which the principles of the invention are utilized, and the an acetyl-CoA carboxylase gene with one or more of its accompanying drawings of which: Subunits fused together in the genetic structure of the 0010 FIG. 1 Depicts some embodiments of the metabolic organism; (2) an acetyl-CoA carboxylase gene having a pathways to produce 3-hydroxypropionic acid. predefined stoichiometric ratio of each of the four ACCase 0011 FIG. 2 Depicts some embodiments of the of various Subunits relative to one another; (3) a monofunctional malo nyl-CoA reductase gene capable of catalyzing the conver equilibrium states in the malonate semialdehyde to 3-HP sion of malonyl-CoA to malonate semialdehyde and one or reaction in a cell environment. more genes encoding one or more of the following enzymes: 0012 FIG. 3 Depicts some embodiments of the reaction ydfG, mmsB, NDSD, ruthE, and nemA; (4) a monofunctional catalyzed by acetyl-CoA carboxylase (ACCase) malonyl-CoA reductase gene capable of catalyzing the con (0013 FIG. 4 Shows the inhibition of ACCase enzyme version of malonyl-CoA to malonate semialdehyde and one activity by high salt concentration. or more genes encoding one or more enzymes capable of 0014 FIG. 5 Depicts some embodiments of the fusion converting malonate semialdehyde keto form to 3-HP, and ACCase subunit gene constructs overexpressed in E. coli. one or more genes encoding one or more enzymes capable CAT-chloramphenicol resistance marker, p15a of converting either the malonate semialdehyde enol form or rep=replication origin; red arrow promoter. the malonate semialdehyde hydrated form to 3-HP; (5) a (0015 FIG. 6 Show improved production of 3-HP in monofunctional malonyl-CoA reductase enzyme fused to a fermentors by genetically modified organism with DA dehydrogenase enzyme that is either: (a) primarily not fusion ACCase. US 2017/01 01638 A1 Apr. 13, 2017

0016 FIG. 7 Shows improved production of 3-HP in that this concept is well-understood in the art. Further, it is fermentors by genetically modified organism with overex appreciated that nucleic acid sequences may be varied and pression of rhtA exporter. still provide a functional enzyme, and Such variations are 0017 FIG. 8 Shows various embodiments of the genetic within the scope of the present invention. The term “enzyme modules used for optimizing expression in host cells. homolog' can also mean a functional variant. 0018 FIG. 9 Shows various chemical products that can 0027. The term "Functional homolog” means a polypep made from various embodiments of the invention. tide that is determined to possess an enzymatic activity and 0019 Table 1 Lists the accession numbers for genes specificity of an enzyme of interest but which has an amino encoding ACCase Subunits from Halomonas elongate. acid sequence different from Such enzyme of interest. A 0020 Table 2 Depicts some embodiments of the RBS corresponding "homolog nucleic acid sequence' may be sequences used to enhance expression of H. elongate constructed that is determined to encode such an identified ACCase subunits. enzymatic functional variant. 0021 Table 3 Shows the improvement in 3-HP produc 0028. The term “3-HP means 3-hydroxypropionic acid. tion by RBS-optimized expression of H. elongata ACCase (0029. The term “heterologous DNA,” “heterologous Subunits. nucleic acid sequence,” and the like as used herein refers to 0022 Table 4 Shows some embodiments of ACCase a nucleic acid sequence wherein at least one of the following Subunit fusions that increase and ACCase enzyme complex is true: (a) the sequence of nucleic acids is foreign to (i.e., activity. not naturally found in) a given host microorganism; (b) the 0023 Table 5 Shows some of the genetic modifications of sequence may be naturally found in a given host microor a host cell for increase chemical production. ganism, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids comprises two DETAILED DESCRIPTION OF THE or more Subsequences that are not found in the same INVENTION relationship to each other in nature. For example, regarding 0024 Definitions instance (c), a heterologous nucleic acid sequence that is 0025. The term “homology” refers to the optimal align recombinantly produced will have two or more sequences ment of sequences (either nucleotides or amino acids), from unrelated genes arranged to make a new functional which may be conducted by computerized implementations nucleic acid. Embodiments of the present invention may of algorithms. “Homology', with regard to polynucleotides, result from introduction of an expression vector into a host for example, may be determined by analysis with BLASTN microorganism, wherein the expression Vector contains a version 2.0 using the default parameters. “Homology', with nucleic acid sequence coding for an enzyme that is, or is not, respect to polypeptides (i.e., amino acids), may be deter normally found in a host microorganism. With reference to mined using a program, Such as BLASTP version 2.2.2 with the host microorganism’s genome prior to the introduction the default parameters, which aligns the polypeptide or of the heterologous nucleic acid sequence, then, the nucleic fragments (and can also align nucleotide fragments) being acid sequence that codes for the enzyme is heterologous compared and determines the extent of amino acid identity (whether or not the heterologous nucleic acid sequence is or similarity between them. It will be appreciated that amino introduced into that genome). The term "heterologous is acid “homology includes conservative Substitutions, i.e. intended to include the term “exogenous” as the latter term those that Substitute a given amino acid in a polypeptide by is generally used in the art as well as "endogenous'. another amino acid of similar characteristics. Typically seen 0030. As used in the specification and the appended as conservative Substitutions are the following replace claims, the singular forms “a,” “an,” and “the include plural ments: replacements of an aliphatic amino acid Such as Ala, referents unless the context clearly dictates otherwise. Thus, Val, Leu and He with another aliphatic amino acid; replace for example, reference to an “expression vector” includes a ment of a Ser with a Thr or vice versa; replacement of an single expression vector as well as a plurality of expression acidic residue such as Asp or GIu with another acidic vectors, either the same (e.g., the same operon) or different; residue; replacement of a residue bearing an amide group, reference to “microorganism’ includes a single microorgan Such as ASn or GIn, with another residue bearing an amide ism as well as a plurality of microorganisms; and the like. group; exchange of a basic residue such as Lys or Arg with 0031. I. Introduction another basic residue; and replacement of an aromatic 0032. The present invention relates to various genetically residue such as Phe or Tyr with another aromatic residue. modified microorganisms, methods for making the same, For example, homologs can have at least 99%, 98%, 97%, and use of the same in making industrial products. Any and 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%. 88%, 87%, all of the microorganisms herein may include a combination 86%, 85%, 84%, 83%. 82%, 81% or 80% overall amino acid of genetic alterations as described herein. The present inven or nucleotide identity to the gene or proteins of the inven tion contemplates, for example, a genetically modified tion; or can have 99%. 98%, 97%, 96%, 95%, 94%, 93%, microorganism having one or more of the following genetic 92%, 91%, 90%.89%, 88%, 87%. 86%, 85%, 84%, 83%, modifications (i) an alteration that affects the stoichiometric 82%, 81% or 80% amino acid or nucleotide to the essential ratio, expression or production of one or more ACCase protein functional domains of the gene or proteins of the enzyme genes (ii) a recombinant ACCase gene having at invention; or at least 99%, 98%, 97%, 96%, 95%, 94%, least 80% sequence homology to an ACCase gene from a 93%, 92%, 91%, 90%, 89%, 88%, 87%. 86%, 85%, 84%, salt tolerant organism (iii) a genetic alteration in one or more 83%, 82%, 81% or 80% overall amino acid or nucleotide to non-ACCase genes (iv) one or more genetic alterations that the essential binding amino acids within an essential func encodes for one or more exporters capable of exporting tional domain of the gene or proteins of the invention. 3-HP out of a cell (v) new hybrid molecules or co-expressed 0026. The above descriptions and methods for sequence of a mono-functional malonyl-CoA reductase enzyme with homology are intended to be exemplary and it is recognized various 3-HP dehydrogenase proteins that: (a) exhibit less US 2017/01 01638 A1 Apr. 13, 2017

inhibition by high 3-HP concentrations (b) that is less 0040. The conversion of acetyl-CoA to malonyl-CoA is reversible or irreversible (c) enzymes that utilized NADH an important step in the bioconversion of a renewable (d) enzymes that utilized flavin (vi) one or more genetic carbon Source (such as, for example, Sugar or natural gas) to alterations that can be used to switch the carbon in the a useful industrial chemical (Such as, for example, 3-hy standard metabolic pathways of the cells to a pathway droxypropionic acid (3-HP)). In certain organisms, such as engineered to produce a chemical. More details about each E. coli or yeast, the native ACCase expression from the of the above modifications and how the modification are chromosome alone is insufficient to enable the organism to used together to increase chemical production in a host cell produce chemicals such as 3-HP at a rate to support a is described below. commercial scale operation. Overexpression of the ACCase 0033. The present invention also relates to methods of complex has been shown to provide Some advantage U.S. fermentation. The genetically modified microorganisms are Ser. No. 12/891,760 U.S. Ser. No. 12/891,790 U.S. Ser. No. cultured under conditions that optimized a host cell for 13/055,138. increase chemical production. The bio-production process 0041 Applicants have discovered that the introduction of may include two or more of the following phases of fer an acetyl-CoA carboxylase gene with one or more of its mentation: (1) growth phase where the culture organism subunits fused is beneficial to the production of a chemical replicates itself and the carbon intermediate product is built product in a host cell. In certain aspects of the invention, up; (2) the induction phase, where the expression of key fusion is the two gene products produced from a single enzymes critical to the chemical production is induced and polypeptide controlled by a single promoter, will further the enzymes accumulate within the organism to carry out the enhance an organism's bioproduction of an industrial chemi engineered pathway reactions required to further produce cal. In certain aspects of the invention, fusion is the two gene the chemical product (3) production phase is where the products produced by at least one promoter, will further organism expresses proteins that provide for continuously enhance an organism's bioproduction of an industrial chemi production the desired chemical product. The above phases cal. In certain aspects of the invention, fusion is the two gene are further controlled by (1) addition and amount of the products produced from a single polypeptide controlled by initiating reactant added to the reaction vessel (2) key at least one inducible promoter, will further enhance an enzymes engineered into the organism using promoters that organism's bioproduction of an industrial chemical. Keep are sensitive to (e.g., activated by) the depletion of the ing components of the ACCase complex fused together in initiating reactant. Addition details about the fermentation the genetic structure of an organism can be advantageous process of the invention are disclosed below. because it enhances the stability of the non-native ACCase 0034) II. Acetyl-CoA Carboxylase genetic modification and it facilitates equimolar expression 0035. Malonyl-CoA Flux of the fused acc subunits. 0036. One of the steps in the biosynthesis of 3-HP 0042. In particular, the subunit-fused ACCase may be an involves the reaction catalyzed by acetyl-CoA carboxylase accA-accB, accA-accC, accA-accD, accB-accC, accB-accD, (ACCase) enzyme. ACCase is a primary control point in the accC-accD, accA-accB-accC, accA-accB-accD, accA-accC 3-HP pathway shown in FIG. 1 (previously described in) for accD, accB-accC-accD or accA-accB-accC-accD fused Sub the converting acetyl-CoA to malonyl-CoA and hence to unit that have having at least 80% sequence homology to E. malonate semialdehyde and 3-HP. The present invention coli accA, accB, accC and accD or is a functional homolog contemplates the use of genetic modifications that increase thereof. In addition, the organism may include any combi activity of ACCase complex enzymes to thereby increase nation of these fused subunits, or any combination of these 3-HP production in a host cell. fused subunits together with one or more of the four non 0037. Fused Subunits fused subunits. When such combinations are used, the 0038. The acetyl-CoA carboxylase complex (ACCase) is Subunits (fused and non-fused) may be expressed on the a multi-subunit protein. Prokaryotes and plants have multi same plasmid or on different plasmids or on the chromosome subunit ACCs composed of several polypeptides encoded by of the organism. distinct genes. However, humans and most other eukaryotes, 0043. In accordance with a preferred embodiment, an such as yeast, have evolved an ACC with CT and BC accA-accD fused subunit is introduced into an organism catalytic domains and biotin carboxyl carrier domains on a either alone or in combination with the accB-accC fused single polypeptide. The biotin carboxylase (BC) activity, Subunit, the accB gene, and/or the accC gene. In accordance biotin carboxyl carrier protein (BCCP), and carboxyl trans with a preferred embodiment, the organism is a bacteria, and ferase (CT) activity are each contained on a different sub preferably E. coli or Cupriavidus necator. unit. In E. coli the ACCase complex is derived from multi 0044 Composition Stoichiometry polypeptide transcribed by distinct, separable protein com 0045 Composition stoichiometry is the quantitative rela ponents known as accA, accB, accC, and accD. tionships among elements that comprise a compound. A 0039 Acetyl-CoA carboxylase is a biotin-dependent Stoichiometric ratio of a reagent is the optimum amount or enzyme that catalyzes the irreversible carboxylation of ratio where, assuming that the reaction proceeds to comple acetyl-CoA to produce malonyl-CoA through its two cata tion. Although stoichiometric terms are traditionally lytic activities, biotin carboxylase (BC) and carboxyltrans reserved for chemical compounds, theses theoretical con ferase (CT). The first reaction is carried out by BC and sideration of Stoichiometry are relevant when considering involves the ATP-dependent carboxylation of biotin with the optimal function of heterologous multi-subunit protein bicarbonate. The carboxyl group is transferred from biotin to in a host cell. acetyl-CoA to form malonyl-CoA in the second reaction, 0046. In accordance with another aspect of the invention, which is catalyzed by CT. The main function of ACCase the stoichiometric ratio of each of the four ACCase subunits complex in the cell is to provide the malonyl-CoA substrate relative to one another is important, and each Such ratio can for the biosynthesis of fatty acids. be between 0 and about 10, and preferably between about US 2017/01 01638 A1 Apr. 13, 2017

0.5 to about 2 or about 7 to about 9. In accordance with a a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, preferred embodiment the ratios for the protein subunits are 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, accA:accB:accC:accD are 1:2:1:1. In accordance with a 3:7, 3:8, 4:1, 4:3, 4:44:5, 4:6, 4:7,4:8, 5:1, 5:3, 5:4, 5:5, 5:6, preferred embodiment, an organism is genetically modified 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, to include an accA-accD fused subunit, an accB non-fused 7:7, 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium, Subunit, and an accC non-fused subunit, with the molar high or inducible expression. ratios of the accDA fusion:accB:accC being about 1:2:1, 0052. In certain aspects the invention provides for the which is close to the optimum for enzymatic activity. stoichiometry of the accD-A subunits in a 1:1, 1:2, 1:3, 1:4, 0047. In certain embodiments where an organism is engi 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, neered to make 3-HP, order to get optimal function in a host 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, cell of a heterologous ACCase enzyme complex it is impor 4:5, 4:6, 4:7,4:8, 5:1, 5:3, 5:4, 5:5,5:6, 5:7, 5:8, 6:1, 6:3, 6:4, tant to engineer the Stoichiometry of these subunits in Such 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5,7:6,7:7, 7:8, 8:1, 8:3, 8:4, a way that provides maximal production of 3-HP such that 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducible the subunit can make a more stable enzyme complex when expression. In certain aspects the invention provides for the overexpressed in the cell. stoichiometry of the accC-B subunits in a 1:1, 1:2, 1:3, 1:4, 0048. In certain aspects the invention provides for the 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, controlled expression of the natural accA, accB, accC, and 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, accD subunits of E. coli or having at least 80% sequence 4:5, 4:6, 4:7,4:8, 5:1, 5:3, 5:4, 5:5,5:6, 5:7, 5:8, 6:1, 6:3, 6:4, homology to E.coli accA, accB, accC and accD. In certain 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5,7:6,7:7, 7:8, 8:1, 8:3, 8:4, aspects the invention provides for the inducible expression 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducible of the natural accA, accB, accC, and accDSubunits of E. coli expression. In certain aspects the invention provides for the or having at least 80% sequence homology to E. coli accA, stoichiometry of the accC-A subunits in a 1:1. 1:2, 1:3, 1:4, accB, accC, and accD. In certain aspects the invention 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, provides for the low, medium, high and/or inducible expres 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, sion of the natural accA, accB, accC, and accD subunits of 4:5, 4:6, 4:7,4:8, 5:1, 5:3, 5:4, 5:5,5:6, 5:7, 5:8, 6:1, 6:3, 6:4, E. coli or having at least 80% sequence homology to E. coli 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5,7:6,7:7, 7:8, 8:1, 8:3, 8:4, accA, accB, accC and accD. 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducible 0049. In certain aspects the invention provides for the expression. In certain aspects the invention provides for the expression of the natural accC and accD subunits of E. coli stoichiometry of the accC-B subunits in a 1:1, 1:2, 1:3, 1:4, or having at least 80% sequence homology to E. coli accA, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, accB, accC and accD in low, medium, high or inducible 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, expression. In certain aspects the invention provides for the 4:5, 4:6, 4:7,4:8, 5:1, 5:3, 5:4, 5:5,5:6, 5:7, 5:8, 6:1, 6:3, 6:4, expression of the natural accB and accA subunits of E. coli 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5,7:6,7:7, 7:8, 8:1, 8:3, 8:4, or having at least 80% sequence homology to E. coli accA, 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducible accB, accC, and accD in low, medium, high or inducible expression. expression. In certain aspects the invention provides for the 0053 III. Conversion of Malonyl-CoA to Malonate expression of the natural accC and accD subunits with the Semialdehyde accA subunit of E. coli or having at least 80% sequence 0054) One of the steps in the biosynthesis of 3-HP homology to E. coli accA, accB, accC, and accD in low, involves the conversion of malonyl-CoA (MCA) to medium, high or inducible expression. In certain aspects the malonate semialdehyde (MSA) and the conversion of invention provides for the expression of the natural accCand malonate semialdehyde (MSA) to 3-HP (WO2011/038364). accD subunits with the accB subunit of E. coli or having at In accordance with another aspect of the present invention, least 80% sequence homology to E. coli accA, accB, accC. the present invention contemplates the use of novel enzymes and accD in low, medium, high or inducible expression. and/or combinations of enzymes to catalyze the reaction in 0050. In certain aspects the invention provides for the a microorganism from MCA to MSA, which results in expression of a fusion of two, three, or all of the four enhanced cellular bioproduction of 3-HP in the host cell. ACCase subunits in one polypeptide in low, medium, high 0055. In certain aspects the invention provides novel or inducible expression. Such fusion may include any of the enzyme compositions or co-expression of a combinations of following combinations of the ACCase subunits: accA enzyme compositions to catalyze the conversion of malonyl accB, accA-accC, accA-accD, accB-accC, accB-accD, CoA to 3-HP. A general overview of the enzymes and the accC-accD, accA-accB-accC, accA-accB-accD, accA-accC relevant reaction pathways methods are shown in FIG. 1. accD, accB-accC-accD, and accA-accB-accC-accD have 0056. In accordance with this aspect of the invention, having at least 80% sequence homology to E. coli accA, malonyl-CoA is converted to malonate semialdehyde by a accB, accC and accD or is a functional homolog thereof. malonyl-CoA reductase and malonate semialdehyde is con 0051. In certain aspects the invention provides for ACC verted to 3-HP through either or both of two alternative complex in the stoichiometry of these subunits of the accCB pathways. and accDA in a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 0057. In accordance with one aspect of the invention, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, malonyl-CoA is converted to malonate semialdehyde by a 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, 4:5, 4:6, 4:7,4:8, 5:1, 5:3, monofunctional malonyl-CoA reductase that catalyzes the 5:4, 5:5, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, malonyl-CoA conversion, but does not catalyze the 7:4, 7:5, 7:6, 7:7, 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in malonate semialdehyde conversion. low, medium, high or inducible expression. In certain 0058. In one embodiment, the microorganism herein aspects the invention provides for ACC complex in the comprise a genetic modification that include the monofunc stoichiometry of these subunits of the accDA and accCB in tional malonyl-CoA reductase may be derived from Sulfolo US 2017/01 01638 A1 Apr. 13, 2017

bus tokodaii (stMCR) (SEQID NO. 15 nucleic acid, SEQID make 3-HP, wherein the genetic modification includes a NO. 16 protein sequence) or a functional homolog of stMCR polynucleotide encoding: (1) a monofunctional malonyl or a homolog with at least 80% identity. CoA reductase gene capable of catalyzing the conversion of 0059. In some embodiments, the microorganism herein malonyl-CoA to malonate semialdehyde; (2) one or more comprise a genetic modification that include the bi-func genes encoding one or more enzymes capable of converting tional malonyl-CoA reductase comprised of two protein malonate semialdehyde keto form to 3-HP; and (3) one or fragments with one fragment having malonyl-CoA reductase more genes encoding one or more enzymes capable of activity and the other fragment having malonate semialde converting eithere malonate semialdehyde enol form or the hyde dehydrogenase activity may be derived from Chloro malonate semialdehyde hydrated form to 3-HP. flexus aurantiacus (caMCR). 0066. In certain aspects the invention provides mono functional malonyl-CoA reductase enzyme fused to a dehy MCR-Dehydrogenase Enzymes for Conversion of 3-HP drogenase enzyme that is either: (1) primarily not NADPH Ions dependent; (2) primarily NADH-dependent; (3) primarily 0060. Following the conversion of the malonyl-CoA to flavin-dependent; (4) less susceptible to 3-HP inhibition at malonate semialdehyde, the malonate semialdehyde is con high concentration; and/or (5) catalyzes a reaction pathway verted to 3-HP through either or both of two alternative to 3-HP that is substantially irreversible. pathways. Malonate semialdehyde may exist in at least three 0067. In certain aspects the invention also provides states; the keto form, the enol form, and hydrate form, as monofunctional malonyl-CoA reductase enzyme fused to a shown in FIG. 2. Malonate semialdehyde in the enol form, dehydrogenase enzyme that is NADPH-dependent. which will stabilize this form when compared to other 0068 Suitable 3-HP dehydrogenase enzymes that are aldehydes where the enol form is highly unfavored in the largely NADH-dependent that can be used with the claimed equilibrium among the three forms. invention include, but are not limited to, mmsB or NDSD. 0061 The malonate semialdehyde keto form is converted Suitable malonate reductase enzymes that are flavin-depen to 3-HP utilizing a 3-hydroxy acid dehydrogenase enzyme dent include, but are not limited to, rutE and nemA. Suitable (ydfG SEQ ID NO. 21 nucleic acid, SEQ ID NO. 22 3-HP dehydrogenase enzymes that are less susceptible 3-HP protein), a 3-hydroxyisobutyrate dehydrogenase enzyme inhibition at high concentration that can be used with the (Pseudomonas aeruginosa mmsB, SEQ ID No. 23 nucleic claimed invention include, but are not limited to, yafG and acid, SEQ ID NO. 24 protein), and/or NAD+-dependent NDSD. Suitable 3-HP dehydrogenase or malonate semial serine dehydrogenase (Pseudomonas NDSD, SEQ ID NO. dehyde dehydrogenase enzymes that catalyze a reaction 25 nucleic acid, SEQID NO. 26 protein). In accordance with pathway to 3-HP that is substantially irreversible are ruthE a preferred embodiment, Pseudomonas mmSB, Pseudomo and nemA. nas NDSD, and E. coli ydfG are used. The gene, ydfG from 0069. In certain aspects the invention provides mono E. coli is largely NADPH dependent, whereas mmsB and functional malonyl-CoA reductase enzyme fused to one or NDSD from Pseudomonas can utilize either NADPH or more dehydrogenase enzymes. Malonate semialdehyde, NADH. which is the intermediate product in the conversion of 0062. The malonate semialdehyde enol form is converted malonyl-CoA to 3-HP can be very reactive. Therefore, it is to 3-HP utilizing an N-ethylmaleimide reductase (nemA, advantageous to have a reaction pathway wherein the resi SEQ ID NO. 17 nucleic acid, SEQ ID NO. 18 protein), dence time of malonate semialdehyde within the cell is and/or a malonic semialdehyde reductase (ruth, SEQ ID minimized, and its conversion to 3-HP occurs quickly. By NO. 19 nucleic acid, SEQ ID NO. 20 protein) from E. coli. fusing the malonyl-CoA reductase with the malonate semi These enzymes does not directly utilize NADPH or NADH. aldehyde dehydrogenase to create a multi-domain protein Instead, these enzymes utilize a flavin mononucleotide that (e.g., two domain protein) and having the MCR and dehy is cycled between oxidized and reduced states by NADPH or drogenase domains adjacent in the sequence, when the NADH. The enol pathway also has advantages over the keto themalonate semialdehyde is quickly is quickly converted to pathway in that equilibrium between the malonate semial 3-HP. dehyde enol form and 3-HP significantly favors 3-HP, mak 0070. In certain aspects the invention provides first ing the reaction much less reversible, and essentially irre monofunctional malonyl-CoA reductase enzyme fused to a versible. first dehydrogenase enzyme of one type and second mono 0063. The malonate semialdehyde hydrated form may functional malonyl-CoA reductase enzyme fused to a dehy also be converted to 3-HP by either the 3-HP dehydrogenase drogenase enzyme of a different type than the first dehydro or malonate semialdehyde reductase enzymes, although the genase enzyme. Suitable different dehydrogenase enzymes hydrated form is more likely to be converted to the enol form include, but are not limited to, enzymes that function on the as the equilibrium continuously readjusts. different forms of malonate semialdehyde. 0064. In one embodiment, the microorganism herein 0071. In certain aspects the invention provides for micro comprise a genetic modification that include (i.e., microor organisms comprising a genetic modification that include ganism) includes a polynucleotide encoding: (1) a mono but are not limited to the malonyl-CoA reductase from S. functional malonyl-CoA reductase gene capable of catalyZ tokadaii is fused to ydfG, mmsB, NDSD, ruthE, or nemA (or ing the conversion of malonyl-CoA to malonate Some combination thereof). The fused enzyme may include semialdehyde; and (2) one or more genes encoding one or any of the following configurations: mcr-ydfG. mcr-mmsB. more of the following enzymes: ydfG, mmsB, NDSD, ruthE, mcr-NDSD, mcr-rut, mcr-nemA, mcr-ydfG-mmsB. mcr and nemA or a functional homolog or a homolog with at ydfG-NDSD, mcr-ydfG-rut, mcr-ydfG-nemA, mcr-mmsB least 80% identity. ydfG. mcr-mmsB-NDSD, mcr-mmsB-rut, mcr-mmsB 0065. In accordance with another aspect of the invention, nemA, mcr-NDSD-ydfG, mcr-NDSD-mmsB, mcr-NDSD there is provided an organism that is genetically modified to rut, mcr-NDSD-nemA, mcr-rut-ydfG. mcr-rute-mmsB, US 2017/01 01638 A1 Apr. 13, 2017 mcr-rute-NDSD, mcr-rut-nemA, mcr-nemA-ydfG. mcr NDSD, mcr-ydfG-rut, mcr-ydfG-nemA, mcr-mmsB-ydfG, nemA-mmsB, mcr-nemA-NDSD, or mcr-nemA-rutE or mcr-mmsB-NDSD, mcr-mmsB-rutE, mcr-mmsB-nemA, functional homolog or homolog with 80% sequence identity mcr-NDSD-ydfG, mcr-NDSD-mmsB, mcr-NDSD-rute, thereof. mcr-NDSD-nemA, mcr-rut-ydfG. mcr-rute-mmsB. mcr 0072. In certain aspects the invention provides for micro rute-NDSD, mcr-rut-nemA, mcr-nemA-ydfG. mcr-nemA organisms comprising a genetic modification that include mmsB, mcr-nemA-NDSD, or mcr-nemA-rutE or functional but are not limited to the malonyl-CoA reductase from C. homolog or homolog with 80% sequence identity thereof. aggregains is fused to yafG, mmsB, NDSD, rut, or nemA (0079 IV. Salt-Tolerant Enzymes (or Some combination thereof). The fused enzyme may 0080. The growth of engineered microorganism for include any of the following configurations: mcr-ydfG, enhanced production of a chemical product, Such as E. coli mcr-mmsB. mcr-NDSD, mcr-rut, mcr-nemA, mcr-ydfG is severely inhibited by high salt concentrations accumulated mmsB. mcr-ydfG-NDSD, mcr-ydfG-rut, mcr-ydfG-nemA, when the chemical product is produced at high rate within mcr-mmsB-ydfG. mcr-mmsB-NDSD, mcr-mmsB-rut, the organism. mcr-mmsB-nemA, mcr-NDSD-ydfG. mcr-NDSD-mmsB, I0081 Dose-dependent studies with increasing amounts mcr-NDSD-rut, mcr-NDSD-nemA, mcr-rut-ydfG. mcr of NaCl and Na-3-HP show that salt has inhibitory effects on rutE-mmsB, mcr-rutE-NDSD, mcr-rutE-nemA, mcr-nemA ACCase activity which is essential to fatty acid biosynthesis ydfG. mcr-nemA-mmsB, mer-nemA-NDSD, or mcr-nemA of membranes required for growth and propagation and for rut, or functional homolog or homolog with 80% sequence the production of 3-HP (see EXAMPLE 1). Thus, the use of identity thereof. salt-tolerant enzymes in 3-HP production should increase 0073. In certain aspects the invention provides for micro 3-HP production in a host cell. organisms comprising a genetic modification that include A. Enzymes from Halophilic Organisms but are not limited to the malonyl-CoA reductase from O. I0082 Halophiles are characterized as organisms with a trichoides is fused toydRi, mmsB, NDSD, rut, or nemA (or great affinity for salt. In some instances a halophilic organ Some combination thereof). The fused enzyme may include ism is one that requires at least 0.05M, 0.1M, 0.2M, 0.3M or but are not limited to any of the following configurations: 0.4M concentrations of salt (NaCl) for growth. Halophiles mcr-ydfG. mcr-mmsB. mcr-NDSD, mcr-rut, mcr-nemA, live in hypersaline environments that are generally defined mcr-ydfG-mmsB. mcr-ydfG-NDSD, mcr-ydfG-rut, mcr occurring to their salt concentration of their habitats. Halo ydfG-nemA, mcr-mmsB-ydfG. mcr-mmsB-NDSD, mcr philic organisms that are defined as “Slight salt affinity” mmsB-rut, mcr-mmsB-nemA, mcr-NDSD-ydfG. mcr have optimal growth at 2-5% NaCl, moderate halophiles NDSD-mmsB, mcr-NDSD-rutE, mcr-NDSD-nemA, mcr have optimal growth at 5-20% NaCl and extreme halophiles rut-ydfG. mcr-rute-mmsB. mcr-rutE-NDSD, mcr-rut have optimal growth at 20-30% NaCl. nemA, mcr-nemA-ydfG. mcr-nemA-mmsB. mcr-nemA I0083. Depending on the conditions of that the genetically NDSD, or mcr-nemA-rutE or functional homolog or engineered microorganism is under one might use homolo homolog with 80% sequence identity thereof. gous enzymes of the invention specifically, for example, 0074 Enhanced Mutated Monofunctional MCR for Bio from a moderate halophiles or an extreme halophiles production depending on the engineered cell's environment. 0075. In certain aspects the invention provides for micro I0084. In certain aspects the invention provides for micro organisms comprising a genetic modification that include organisms comprising a genetic modification that includes mutated form of stMCR that has enhanced activity at about enzymes of the invention provided herein from slight halo 20° C. to about 44°C., about 30° C. to about 37°C., or about philes organisms. In certain aspects the invention provides 32° C. to about 38°C. Such mutate forms may be designed for microorganisms comprising a genetic modification that based on the crystal structure now available for stMCR includes enzymes of the invention provided herein from Demmer et al., J. Biol. Chem. 288: 6363-6370, 2013. moderate halophiles organisms. In certain aspects the inven 0076. It is also contemplated the carboxylase domains of tion provides for microorganisms comprising a genetic the malonyl-CoA reductase derived from Chloroflexus modification that includes homologous enzymes of the aggregans, Oscillochloris trichoides can be enhanced by invention provided herein from extreme halophiles organ mutations in the carboxylase binding domain to provide 1SS. increased 3-HP production over the natural occurring I0085 Homology with genes provided by the invention enzyme. may be determined by analysis with BLASTN version 2.0 0077. The carboxylase activity of the malonyl-CoA provided through the NCBI website. Homology with pro reductase derived from Chloroflexus aurantiacus can be teins provided by the invention may be determined by enhanced activity. In certain aspects the invention provides analysis with BLASTP version 2.2.2 provided through the for mutated form of it carboxylase domain to provide NCBI website. This program with aligns the disclosed increased 3-HP production over the natural occurring fragments being compared and determines the extent of enzyme. identity or similarity between them. 0078. In certain aspects the invention provides for micro I0086 To date there are many sequenced halophilic organ organisms comprising a genetic modification that include isms which can be used with the claimed invention. carboxylase domains of the malonyl-CoA reductase derived Examples of Some sequenced halophilic organisms include from C. aggregains is fused to yafG, mmsB, NDSD, ruthE, or but are not limited to Chromohalobacter salexigens, Flex nemA (or Some combination thereof). It is contemplated that istipes sinusarabici Strain (MASLOT). Halobacterium sp. the any of the enhanced MCR by mutation, as provide NRC-1, Haloarcula marismortui, Natronomonas pharaonis, above, may be fused in any of the following configurations Halloquadratum wallsbyi, Haloferax volcanii, Halorubrum including but not limited to mcr-ydfG. mcr-mmsB. mcr lacusprofindi, Halobacterium sp. R-1, Halomicrobium NDSD, mcr-rut, mcr-nemA, mcr-ydfG-mmsB. mcr-ydfG mukohataei, Halorhabdus utahensis, Halogeometricum US 2017/01 01638 A1 Apr. 13, 2017

borinquense, Haloterrigena turkmenica, Natronobacterium (fbaB from E. coli), fructose bisphosphate aldolase mono gregoryi SP2, Halalkalicoccus jeotgali, Natrialba magadii, mer (fbaA from E. coli), triose phosphate isomerase mono Haloarcula hispanica, Halopiger xanaduensis, Halophilic mer (tpiA), glyceraldehyde 3-phosphate dehydrogenase-A archaeon DL31, Haloferax mediterranei, Halo vivax ruber, monomer (gap.A from E. coli), phosphoglycerate kinase Natronococcus gregoryi, and Natronococcus occultus. (pgk), 2,3-bisphosphoglycerate-independent phosphoglyc 0087. Examples of suitable moderate halophilic organ erate mutase (gpmM from E. coli), 2,3-bisphosphoglycerate isms in which homologous enzymes of the invention can be dependent or tdcE (from E. coli), phosphoglycerate mutase derived from include but are not limited to eukaryotes such (gpmA), enolase (eno from E. coli), phosphoenolpyruvate as crustaceans (e.g. Artemia salina), insects (e.g. Ephydra carboxylase (ppc from E. coli), malate dehydrogenase hians), certain plants from the genera Salicornia spp., algae (mdh), fumarase A (fum from E. coli), fumarase B (fumB), (e.g. Dunaliella viridis), fungi, and protozoa (e.g. Fabrea fumarase C (fumC from E. coli), phosphoenolpyruvate salina), phototrophic organisms. Such as planktonic and synthetase (ppSA from E. coli), pyruvate kinase I monomer microbial mat-formers cyanobacteria as well as other (pykF from E. coli), pyruvate kinase II monomer (pykA anaerobic red and green Sulphur bacteria from the genera from E. coli), fumarate reductase (fra ABCD from E. coli), Ectothiorhodospira spp.) and non-Sulphur bacteria from the lipoamide dehydrogenase (lpd from E. coli), pyruvate dehy genera Chromatium spp.; gram-negative anaerobic bacteria, drogenase (aceE from E. coli), pyruvate dehydrogenase for example from the genera Halo anaerobacter spp. Some of (aceF from E. coli), pyruvate formate-lyase (pflB from E. which are methanogenic, for example from the genera coli), acetyl-CoA carboxylase (accABCD from E. coli), Methanohalophilus spp. and either aerobic or facultative malonyl CoA reductase (mcr), 3HP dehydrogenase (mmsB, Such as species from the genera Halomonas, Chromoha NDSD, or ydfG), malonate semialdehyde reductase (nemA, lobacter, Salinovibrio, Pseudomonas, for example (e.g. rut from E. coli), or a combination thereof. Halomonase elongate); gram-positive bacteria from genera Such as Halobacillus, Bacillus, Marinococcus, etc. as well as 0092 Suitable salt-tolerant enzyme homologs that can be Some actinomycetes, for example, Actinopolyspora halo used with the claimed invention can have at least 99%, 98%, phila. 97%, 96%, 95%, 94%, 93%, 92%, 91%.90%, 89%, 88%, 87%. 86%, 85%, 84%, 83%, 82%, 81% or 80%, overall Genomic and Proteomic Hallmarks of Halophilic Organisms amino acid or nucleotide identity to the above enzymes. Suitable salt-tolerant enzyme homologs that can be used 0088 Comparative genomic and proteomic studies of with the claimed invention can have at least 99%, 98%, halophiles and non-halophiles reveal some common trends 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, in the genomes and proteomes of halophiles. At the protein 87%. 86%, 85%, 84%, 83%, 82%, 81% or 80%, amino acid level, halophilic organisms are characterized by low hydro or nucleotide to the essential protein function domains of the phobicity, over-representation of acidic residues, especially enzymes above. Suitable salt-tolerant enzyme homologs that Asp, under-representation of Cys, lower propensities for can be used with the claimed invention can have at least helix formation and higher propensities for coil structure. 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 0089. At the DNA level, halophilic organisms are char 89%, 88%, 87%. 86%, 85%, 84%, 83%, 82%, 81% or 80% acterized by the dinucleotide abundance profiles of halo overall amino acid or nucleotide to the essential binding philic genomes bear some common characteristics, which amino acids within an essential protein function domain of are quite distinct from those of non-halophiles, and hence the enzymes above. may be regarded as specific genomic signatures for salt adaptation. The synonymous codon usage in halophiles also 0093. In accordance with a preferred embodiment of the exhibits similar patterns regardless of their long-term evo invention, Suitable salt-tolerant enzyme homologs are lutionary history. enzymes from one of the following organisms: Halomonas 0090. In certain aspects the invention provides for micro elongata, Salinibacter rubur, or Halobacterium species (Ar organisms comprising a genetic modification that the pro chaea). teins provided by the invention that are modified for salt 0094. In accordance with a preferred embodiment of the tolerance such that they has low hydrophobicity, over present invention, there is provided a non-salt-tolerant representation of acidic residues, especially Asp, under organism that is genetically modified to make 3-HP, wherein representation of Cys, lower propensities for helix formation the genetic modification includes a polynucleotide encoding and higher propensities for coil structure. an acetyl-CoA carboxylase from a salt-tolerant organism. In 0091 Suitable salt-tolerant enzymes can include accordance with a preferred embodiment, the acetyl-CoA enzymes from salt-tolerant organisms. Salt-tolerant organ carboxylase Subunits accA, accB, accC and accD is from isms (such as, for example, halophiles) include any living Halomonas elongata. organism that are adapted to living in conditions of high salinity. Suitable salt-tolerant enzymes can include enzymes 0.095 V. Chemical Transporter from Salt-tolerant organism that are homologs of the fol 0096. In accordance with another aspect of the present lowing enzymes: Sucrose-6-phosphate hydrolase (cScA invention, any of the microorganisms herein may be geneti from E. coli), glucose-6-phosphate isomerase (pgi from E. cally modified to introduce a nucleic acid sequence coding coli), fructokinase (cscK from E. coli), fructose-1,6-bispho for a polypeptide that: (1) facilitates the exportation of the sphatase (yggF from E. coli), fructose 1,6-bisphosphatase chemical of interest or the export of an inhibitory chemical (ybh A from E. coli), fructose 1,6-bisphosphatase II (glpX from within the cell to the extracellular media; and/or (2) from E. coli), fructose-1,6-bisphosphatase monomer (fbp facilitates the importation from the extracellular media to from E. coli), 6-phosphofructokinase-1 monomer (pfkA within the cell of a reactant, precursor, and/or metabolite from E. coli), 6-phosphofructokinase-2 monomer (pfkB used in the organism's production pathway for producing from E. coli), fructose bisphosphate aldolase monomer the chemical of interest. US 2017/01 01638 A1 Apr. 13, 2017

0097. 3-HP Exporter cell to increase the chemical production of 3-HP in a host 0098. In accordance with a preferred embodiment, this cell. In certain aspects the invention provides for at least of invention relates to the bioproduction of 3-HP using a the exporters provided herein to be expressed in a host cell genetically modified E. coli organism. Thus, the present and with a genetic modification of tig to increase the invention contemplates of a host cell genetically modified to chemical production of 3-HP in a host-cell. express or increase expression of an exporter that can function to transfer 3HP from the cellular environment 0105. In certain aspects the invention provides for one extracellularly. exporter to be further modified by on one more genetic 0099 Bacterial cells, such as E. coli, have at least five modulates so that the expression level and timing of expres different types of exporters: the major facilitator superfamily sion of the exporter can be controlled in the host cell. In MFS); the ATP-binding cassette superfamily (ABC); the certain aspects the invention provides for one exporter to be small multidrug resistance family (SMR); the resistance further modified by an inducible promoter, RBS, high, nodulation-cell division superfamily (RND); and the multi mutlicopy plasmid or combination thereof, as provide antimicrobial extrusion protein family (MATE). In addition, herein, in order to control its expression in the host cell. amino acid exporters, which are common to almost all host 0106. In certain aspects the invention provides exporters cells, are likely to export 3-HP. Additionally, solvent toler provide herein to be expressed in a host cell in equal ratio. ance transporters, for example bromoacetate, butanol, isobu In certain aspects the invention provides exporters provide tanol and the alike may be used to export 3-HP. herein to be expressed in a host cell in equal 1:2 ratio. In 0100. In certain aspects the invention provides a host cell certain aspects the invention. provides exporters provide with a recombinant exporter wherein the exporter is an MFS herein to be expressed in a host cell in equal 1:3 ratio. In exporter, ABC exporter, SMR exporter, RND exporter, certain aspects the invention provides exporters provide MATE exporter, amino acid exporter, solvent tolerance herein to be expressed in a host cell in equal 1:4 ratio. In transporter or a combination thereof. certain aspects the invention provides exporters provide 0101 Suitable exporters that can be used with the s herein to be expressed in a host cell in equal 2:3 ratio. herein invention include but are not limited to acrD, bcr, 0107. In certain aspects the invention provides for the cuSA, dedA, eamA, eamB, eamH. emaA, emaB, emrB, exporter to maintain the host cell at pH 7.0–7.4 during the emrl D. emrKY, emry, garP. gudP, hsrA, leuE, mdlB, mdtD, continuous production phase. In certain aspects the inven mdtG, mdtL, mdtM, mhpT, rhtA, rht3, rhtC, thtB, yahN. tion provides for the exporter and the means for importing yajR, ybbP, ybiF, ybjJ, ycaP. ydcC. yddG, ydel D, ydgE, a base inside the cell in order to maintain the host cell at pH yddG, ydhC, ydhP ydiN, ydiM, ydE, ydI, yd K. yeaS, 7.0–7.4 during the continuous production phase. In certain yedA, yeeO, yegh, yggA, yfc.J., yfiK, yhE, yidE, yigk, yig J. aspects the invention provides for the exporter maintain the yiE, yi, yji.J., ypiD, ytff ytifL or functional homolog or host cell at pH 3.0 to pH 4.0, pH 4.0 to pH 5.0, pH 5.0 to homolog with 80% sequence identity thereof. Other poten pH 6.0, pH 6.0 to pH 7.0, pH 7.0 to pH 8.0, pH 8.0 to pH tial transporter proteins may be identified using topology 9.0, or pH 9.0 to pH 10.0 pH 7.0-7.3 during the continuous analysis as illustrated in Daley et al., Science 308: 1321 production phase. In certain aspects the invention provides 1323, 2005). for the exporter and the means for importing abase inside the 0102. In certain aspects the invention provides the cell in order to maintain the host cell at pH 3.0 to pH 4.0, pH exporter to be improved for binding to 3-HP. In certain 4.0 to pH 5.0, pH 5.0 to pH 6.0, pH 6.0 to pH 7.0, pH 7.0 aspects the invention provides the exporters named to be to pH 8.0, pH 8.0 to pH 9.0, or pH 9.0 to pH 10.0 pH 7.0–7.3 further enhance by genetic modification of the predicted during the continuous production phase. cytoplasmic domain to increase 3-HP binding. In certain 0108. In accordance with this aspect of the present inven aspects the invention provides the exporter to be improved tion, addition modifications to the host cell may be made to for binding to 3-HP. In certain aspects the invention provides further enhance the transporter's function. In particular, the exporters named to be further enhance by genetic deletion of the tig gene from the genome of the host cell may modification of the predicted transmembrane binding enhance expression and total activity of integral membrane domain to increase 3-HP binding or incorporation into the proteins such as exporters and importers. host cell membrane. 0103 Suitable exporter homologs that can be used with 0109 Bicarbonate Importer the claimed invention can have at least 99%, 98%, 97%, 0110. One of the key steps in the conversion of biomass 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, to 3-HP is the conversion of acetyl-CoA to malonyl-CoA, 86%, 85%, 84%, 83%. 82%, 81% or 80% overall amino acid which is illustrated in FIG. 3. or nucleotide identity to the above exporters. Suitable 0111. As shown in FIG. 3, this reaction is catalyzed by the exporter homologs that can be used with the claimed inven acetyl-CoA carboxylase, and bicarbonate is a reactant tion can have 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, needed to drive the reaction. One of the primary sources of 91%, 90% 89%, 88%, 87%. 86%, 85%, 84%, 83%, 82%, bicarbonate to drive this reaction is carbon dioxide within 81% or 80% amino acid or nucleotide to the essential protein the cell. Carbon dioxide is readily diffusible through a cells function domains of the exporters above. Suitable exporter membrane, and a natural equilibrium will be reached homologs that can be used with the claimed invention can between the intracellular and extracellular carbon dioxide. have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, As a cell produces carbon dioxide it migrates through the 91%, 90%, 89%, 88%, 87%. 86%, 85%, 84%, 83%, 82%, cell, and since it is not very soluble in the media, it will 81% or 80% overall amino acid or nucleotide to the essential bubble out of the system and more intracellular carbon binding amino acids within an essential exporter domain of dioxide will migrate out of the cell to maintain the equilib the enzymes above. rium. This process impedes the production of 3-HP since 0104. In certain aspects the invention provides for at least bicarbonate (which is in equilibrium with the dissolved of the exporters provided herein to be expressed in a host carbon dioxide in the form of carbonic acid) is needed to US 2017/01 01638 A1 Apr. 13, 2017

drive the acetyl-CoA->malonyl-CoA reaction, and the intra can impact cell growth, and even the increased concentration cellular carbon dioxide is the primary source for intracellular of the chemical product as it is produced can impede cell bicarbonate. replication. 0112. In accordance with one aspect of the present inven 0119 Table. 5 tion, an organism is provided that includes a heterologous 0120 VI. Multi-Phase Fermentation gene encoded therein that acts as a carbon dioxide importer I0121. In accordance with another aspect of the present (i.e., it enhances the importation of carbon dioxide into the invention, there is provided a method of producing a chemi cell or inhibits the exportation of carbon dioxide from the cal product from a carbon Source through a bioproduction cell), which results in increased intracellular carbon dioxide. process that comprises a controlled multi-phase production Use of CO2an importer mitigates against the natural outflow process. The multi-phase production process includes an of carbon dioxide. initiation and/or completion. of one or more phases of the 0113. In accordance with this aspect of the invention, production process is controlled by genetic modifications to there is provided an organism that is genetically modified, the organism producing the chemical product and/or is wherein the genetic modification includes a polynucleotide controlled by changes made to the cell environment. encoding a gene capable of importing extracellular carbon I0122. In accordance with this aspect of the invention, the dioxide from the media to within the cell membrane or bioproduction process may include two or more of the inhibiting the exportation of intracellular carbon dioxide following phases: (1) growth phase; (2) induction phase; and from within the cell membrane to the media. In accordance (3) production phase. During the growth phase, the organism with a preferred embodiment of the present invention, a replicates itself and the biocatalyst needed to produce the microorganism is genetically modified to encode one or chemical product is built up. During the induction phase, more of the following heterologous genes: bicA from Syn expression of key enzymes critical to the production of the echococcus species, yehM gene product of E. coli, yidE chemical is induced and the enzymes accumulate within the gene product of E. coli, any of the bicarbonate transporters biocatalyst to carry out the reactions required to produce the as described in Pelee and Saier, J. Mol. Microbiol. Bio product. During the production phase organism produces the technol. 8: 169-176, 2004 or any amino acid sequences desired chemical product. homologous thereof (e.g., at least 80%, 85%, 90%. 95%, or I0123. The initiation and/or completion of the growth, 99% homologous to the amino acids sequences of the CO2 induction and/or production phases are controlled. In accor importer/exporters described herein. dance with the present invention, the growth phase is 0114 Bioproduction Methods dependent on the presence of a critical external reactant that will initiate growth. The initiation and completion of the 0115. In some applications of the invention the host cell growth phase is controlled by the addition and amount of the is genetically modified for increased malonyl-CoA flux by at initiating reactant added to the reaction vessel. least one heterologous ACCase complex, Such as Table 4 to 0.124. In accordance with certain preferred embodiments further increase chemical bio-production in host cell. In of the present invention, the chemical product is 3-HP and Some applications of the invention the host cell is genetically the production organism is E. coli or yeast. The critical modified with heterologous salt tolerant enzymes, such as external reactant may be phosphate, which is needed for Table 5 to increase chemical bio-production in a host cell. In replication of E. coli cells. In accordance with a preferred Some applications of the invention the host cell is genetically embodiment, the growth phase is initiated by the addition of modified with heterologous 3-HP exporters to further phosphate to a reaction vessel (together with a carbon Source increase chemical bio-production in a host cell. Such as Sugar and the E. coli cells), and the duration of the 0116. In some applications of the invention the host cell growth phase is controlled by the amount of phosphate is genetically modified by at least one heterologous gene added to the system. and/or salt tolerant heterologous gene of FIG. 1 or Table 5 0.125. The induction phase is controlled by genetic modi and at least one 3-HP exporter provided herein to further fications to the producing organism. The key enzymes increase chemical bioproduction in a host cell. triggered during this phase are engineered into the organism 0117. In some applications of the invention the host cell using promoters that are sensitive to (e.g., activated by) the is genetically modified with a heterologous gene for depletion of the initiating reactant. As a result, once the increased malonyl-CoA flux, 3-HP export, at least one initiating reactant is depleted, the growth phase ends, e key heterologous and/or salt tolerant heterologous gene, as pro enzymes are activated and the induction phase begins. vided herein, to increase chemical bio-production in a host I0126. In accordance with a preferred embodiment, the cell. In some applications of the invention the host cell is induction phase is controlled by key genes that encode for genetically modified for increased malonyl-CoA flux, 3-HP enzymes in the biosynthetic pathway for the product into the export, at least one heterologous gene and/or salt tolerant production organism using promoters that are activated by heterologous gene and the host cell is genetically modified phosphate depletion. In one embodiment where the chemical by at least one gene, as provided herein to increase chemical product is 3-HP and the production organism is E. coli, the bioproduction in a host cell. key genetic modifications may include one or more of the 0118 When utilizing certain organisms to create certain following: mcr, mmsB, ydfG, rut, nemA and NDSD; genes products, it may be advantageous to control each phase that encode individual or fused subunits of ACCase, such as discretely. For example, depending on the pathway accA, accB, accC, accD, accDA fusion, and accCB fusion, involved, reactions, reactants, intermediates and byproducts and the promoters may include one or more of the promoters created during cell growth can inhibit enzyme induction that direct expression of the following E. coli genes: amn, and/or the organisms ability to produce the desired chemi tktE, xas A, yibD, ytfK, pstS, phoH, phnC, or other phos cal product. Similarly, reactions, reactants, intermediates phate-regulated genes as described in Back and Lee, FEMS and byproducts created as part of the production pathway Microbiol Lett 264: 104-109, 2006. In accordance with this US 2017/01 01638 A1 Apr. 13, 2017

embodiment, once the phosphate is depleted, expression of Verting said carbon Source to said chemical product and the key enzymes is activated by their promoters and the is not required for the genetically modified organism to induction phase begins. replicate; and (b) a gene encoding a temperature 0127. The production phase may also be controlled by sensitive enzyme; genetic modifications. For example, the organism can be 0.133 (2) forming a culture system comprising said engineered to included mutated forms of enzymes critical to carbon Source in an aqueous medium and said geneti the initiation of production of the chemical product. These cally modified microorganism; initiation enzymes may facilitate. initiation of production 0.134 (3) maintaining the culture system under condi either by: (1) becoming active and serving a key function in tions that allow the genetically modified microorgan the production pathway; and/or (2) becoming inactive and ism to replicate comprising maintaining a Sufficient thereby turning off a branch pathway or other competitive level of inorganic phosphate within said culture system; pathway that prevents or limits the production pathway 0.135 (4) allowing the inorganic phosphate to deplete leading to the chemical product. In accordance with a thereby triggering the expression of the gene regulated preferred erribodiment, initiation enzymes are mutated to by a promoter sensitive to inorganic phosphate levels; form temperature sensitive variants of the enzymes that are and either activated by or deactivated at certain temperatures. As 0.136 (5) changing the temperature of the culture sys a result, the production phase is initiated by changing the tem thereby activating or deactivating said tempera changing the temperature within the reaction vessel. ture-sensitive enzyme and initiating the production of 0128. In one embodiment, the production phase is con said chemical product. trolled by genetically modifying the microorganism with a 0.137 In accordance with the present invention, there is heterologous nucleotide sequence encoding i one or more of provided a method of producing 3-hydropropionic acid the following temperature sensitive enzymes: fabl (SEQID (3-HP) from a renewable carbon source, comprising: NO, 27), fabB' (SEQ NO.28) and fablD' (SEQ NO. 29). 0.138 (1) constructing a genetically modified organism These enzymes are deactivated or shut-off at the desired capable of converting said renewable carbon Source to temperature for production of the chemical product. These 3-HP, wherein said genetically modified organism enzymes play a key role shuttling carbon atoms into the fatty requires inorganic phosphate for growth and com acid synthesis pathway. Although fatty acid synthesis path prises: (a) at least one heterologous gene whose expres way is critical during the growth phase, it inhibits production sion is regulated by a promoter sensitive to inorganic of the chemical product. Once the reaction vessel tempera phosphate levels within a culture system, wherein said ture is changed, the temperature sensitive enzymes are gene is selected from the group consisting of mcr, deactivated and the fatty acid synthesis pathway shuts down mmsB, ydfG, rut, nemA, NDSD, accA, accB, accC. thereby allowing the production pathway of the chemical accD, accDA fusion, and accCB fusion; and (b) a gene product to ramp up. encoding a temperature-sensitive enzyme selected from 0129. In accordance with the present invention, the the group consisting of fahl, fabB and fab); growth phase can last be between 10 to 40 hours, or about 0.139 (2) forming a culture system comprising said 15 to about 35 hours, or about 20 to about 30 hours. The carbon Source in an aqueous medium, phosphate and induction phase may be for about 1 to about 6 hours, about said genetically modified microorganism, and thereby 1 to about hours, or about 2 to about 4 hours. The production initiating a growth phase during which the genetically phase may be greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, modified microorganism replicates; 50, 55, 60, 65,70, 75, 80, 85,90, 95 or 100 hours depending 0140 (3) maintaining a sufficient level of inorganic on the amount of chemical product that is desired. phosphate within said culture system until the desired 0130. In accordance with the present invention, the level of cell growth is achieved; growth phase and induction phase are conducted at a tem 0141 (4) allowing the inorganic phosphate to deplete perature of about 25° C. to about 35° C., about 28°C. to thereby initiating an induction phase which begins the about 32° C., or about 30° C. The production phase is expression of said gene regulated by a promoter sen conducted at a temperature of about 35° C. to about 45° C. sitive to inorganic phosphate levels; and about 3520 C. to about 40°C., or about 36° C. to about 38° 0.142 (5) changing the temperature of the culture sys C. Preferably, the production phase temperature is higher tem thereby activating or deactivating said tempera than the induction phase temperature, and the increase in ture-sensitive enzyme and initiating a growth phase temperature that initiates the production phase occurs over a during which said genetically modified microorganism period of about 1 to about 5 hours, about 1 to about 3 hours, produces 3-HP. about 2 hours, or about 1 hour. 0131. In accordance with the present invention, there is Fermentation Conditions provided a method of producing a chemical product from a 0.143 Depending on the host cell fermentation may be renewable carbon Source through a bioproduction process performed under aerobic, microaerobic, or anaerobic con comprising: ditions, with or without agitation. The operation of culture 0132 (1) constructing a genetically modified organism systems to achieve aerobic, microaerobic and anaerobic capable of converting said renewable carbon Source to conditions are well known to those of ordinary skill in the said chemical product, wherein said genetically modi art. fied organism requires inorganic phosphate for growth 0144. Suitable pH ranges for fermentation depend on the and comprises: (a) at least one heterologous gene multiple factors such as the host cell. In some applications whose expression is regulated by a promoter sensitive of the invention fermentation can occur between various pH to inorganic phosphate levels within a culture system, ranges for example, pH 3.0 to pH 4.0, pH 4.0 to pH 5.0, pH wherein said gene provides a critical function in con 5.0 to pH 6.0, pH 6.0 to pH 7.0, pH 7.0 to pH 8.0 pH 8.0 to US 2017/01 01638 A1 Apr. 13, 2017

pH 9.0, or pH 9.0 to pH 10.0. However, the actual pH coli), Polypeptide: predicted transcriptional regulator (yielP conditions for a particular application are not meant to be from E. coli), Blastocyin resistance gene (BSD from Schi limited by these ranges and can be between the expressed pH zosaccharomyces pombe), Enzyme: pyridine nucleotide ranges if it provides more optimal production of the fer transhydrogenase (udha from E. coli), Protein: Cra DNA mentation process, such as increased 3-HP production. binding transcriptional dual regulator (fruR from E. coli), (SCB from E. coli), enzyme: aldehyde dehydrogenase B VII. Genes and Proteins for the Bioproduction of Chemicals (aldB from E. coli), Enzyme: carbonic anhydrase (cynT 0145 An overview of the engineered pathways provided from E. coli), Enzyme: cyanase (cynS from E. coli), DNA by the invention in a host cell is shown in FIG. 1. Various gyrase toxin-antitoxin system (ccdAB from E. coli), combinations of the pathways shown can be carried out by Enzyme: phosphoglycerate mutase (pgi from E. coli), ArcA various combinations of genetic modifications to key transcriptional dual regulator or Aerobic respiration control enzymes either in the intrinsic pathways or Supplied through (arcA from E. coli), Enzyme: 6-phosphofructokinase (pfk the transformation of a heterologous gene. from E. coli), Enzyme: glyceraldehyde:3-phosphate dehy 0146 In some applications of the genetically modified drogenase-A complex (gap.A from E. coli), aldehyde dehy microorganism of the invention may comprise a single drogenase A (alda from E. coli), Enzyme: glutamate dehy genetic modification, or one or more genetic modifications. drogenase (gdh Afrom E. coli), Enzyme: NADH-dependent Various types of genetic modifications that can be used with serine dehydrogenase (NDSD from Pseudomonas aerugi the invention are disclosed herein. nosa), Protein: threonine/homoserine efflux transporter 0147 In some embodiments the genetic modified organ (rhtA from E. coli), Enzyme: glyceraldehyde 3-phosphate ism of the invention can comprise a genetic modification to dehydrogenase (gapN from E. coli), Phosphotransferase the following gene/proteins or a homolog with at least 80% system (pts from E. coli), Enzyme: 6-phosphofructokinase II identity to or a functional homolog of bifunctional malonyl (pfkB from E. coli), Enzyme: methylmalonate-semialde CoA reductase (MCR from Chloroflexus aurantiacus), hyde dehydrogenase (mms.A from Pseudomonas aerugi monofunctional malonyl-CoA reductase (caMCR from nosa), Oxaloacetate:beta-alanine aminotransferase (OAT-1 Chloroflexus aurantiacus), malonyl-CoA reductase (stMCR from Bacillus cereus), Enzyme: aspartate 1-decarboxylase from Sulfolobus tokodaii), Enzyme: malonyl-CoA reductase (panD from E. coli), Gene that confers resistance to valine (cgMCR from Chloroflexus aggregans), Enzyme: malonyl (ValR from E. coli), Enzyme: glucokinase (glk from E. coli), CoA reductase (otMCR from Oscillochloris trichoides), Polypeptide: 30S ribosomal Sununit protein S12 (rpsL from Polypeptide: host restriction; endonuclease R (hsdR from E. E. coli), Polypeptide: CynR DNA-binding transcriptional coli), lactose metabolism (lac from E. coli), L-rhamnulose repressor (cynR from E. coli), Transporter: galactose:H+ kinase (rhab from E. coli), rhamnulose-1-phosphate aldo Symporter (galP from E. coli), aspartate aminotransferase lase (rhal) from E. coli), Enzyme: B-galactosidase (lac7. (aspC from E. coli), Enzyme: alpha-ketoglutarate reductase from E. coli), L-ribulose 5-phosphate 4-epimerase (ara) (serA from E. coli), Enzyme: 6-phosphofructokinase I (pfkA from E. coli), L-ribulokinase (araB from E. coli), Enzyme: from E. coli), Enzyme: phosphoenolpyruvate carboxylase D-lactate dehydrogenase-fermentative (Idha from E. coli), (ppc from E. coli), Enzyme: Succinate-semialdehyde dehy enzyme: pyruvate formate-lyase (pflB from E. coli), drogenase (NADP+) (gab) from E. coli), Enzyme: pyruvate Enzyme: phosphate acetyltransferase/phosphate propionyl kinase (pyk from E. coli), Enzyme: Oxaloacetate 4-decar transferase (pta from E. coli), Enzyme: pyruvate oxidase boxylase (OAD from Leuconostoc mesenteroides), Enzyme: (poxB from E. coli), Enzyme: methylglyoxal synthase trigger factor, a molecular chaperone involved in cell divi (mgSA from E. coli), enzyme: Acetate kinase (ackA from E. sion (tig from E. coli), Transcription Unit (ptsHIcrr from E. coli), enzymes: phosphotransacetylase-acetate kinase (pta coli), Enzyme: acetyl-CoA acetaldehyde dehydrogenase/ ack from E. coli), Enzyme: enoyl-acyl-carrier-protein alcohol dehydrogenase (adhE from E. coli), Enzyme: fattya reductase (fabl from E. coli), Protein: Zeocin binding protein cyl thioesterase I (tes.A from E. coli), Enzyme: guanosine (ZeoR from Streptoalloteichus Hindustanus), Enzyme: car 3'-diphosphate 5'-triphosphate 3'-diphosphatase (spoT from boxytransferase moiety of acetyl-CoA carboxylase (accAD E. coli), combination of genes encoding accABCD Subunits from E. coli), Enzyme: triose phosphate isomerase (tpiA (from E. coli and Halomonas elongata), pol (from E. coli), from E. coli), Enzyme: biotoin carboxylase moiety of acetyl Enzyme: GDP pyrophosphokinase/GTP pyrophosphokinase CoA carboxylase (accBC from E. coli), Enzyme: transhy (relA from E. coli), Enzyme Name (me from E. coli), drogenase (pntAB from E. coli), Polypeptide: Lael DNA Enzyme: citrate synthase (gltA from E. coli), Polypeptide: binding transcriptional repressor (lacl from E. coli), DNA gyrase, subunit A (gyra from E. coli). En/you: multi Enzyme: B-ketoacyl-ACP synthases I (fabB from E. coli), functional 2-keto-3-deoxygluconate 6-phosphate aldolase Enzyme: B-ketoacyl-ACP synthases II (fabF from E. coli), and 2-keto-4-hydroxyglu (arate aldolase and oxaloacetate Enzyme: malonyl-CoA-ACP transacylase (fab) from E. decarboxylase (eda from E. coli), thiamin biosynthesis (thi coli), Enzyme: pantothenate kinase (coaA from E. coli), from E. coli), Polypeptide: acetolactate synthase II (ilvG Enzyme: pyruvate dehydrogenase complex (aceEF from E. from E. coli), acetyl CoA carboxylase (accDACB from E. coli), Enzyme: 3-hydroxyisobutyrate/3-HP dehydrogenase coli), Citrate synthase (ArCS from Arthrobacter aurescens), (mmsB from Pseudomonas aeruginosa), Enzyme: lipoam Acetyl-CoA carboxylase from Corynebacter glutamicum ide dehydrogenase (lpd from E. coli), Enzyme: Y-glutamyl (CgACC from Corynebacter glutatnicum), Polypeptide: fer y-aminobutyraldehyde dehydrogenase (puuC from E. coli), richrome/phage/antibiotic outer membrane porin FhuA Enzyme: malate synthase A (aceB from E. coli), Enzyme: (fhuA from E. coli), Transporter: phosphate:H+ symporter isocitrate lyase (aceA from E. coli), Enzyme: isocitrate PitA (pitA from E. coli), Transporter: uracil:H+ symporter dehydrogenase phosphatase/kinase (aceK from E. coli), (uraA from E. coli), Enzyne: uracil phosphoribosyltrans Enzyme: 3-hydroxy acid dehydrogenase (ydfG from E. ferase (upp from E. coli), Enzyme: acylphosphatase (yccX coli), Enzyme: acetyl CoA carboxylase (accADBC from E. from E. coli), acetyl-CoA synthetase acSA from E. coli), US 2017/01 01638 A1 Apr. 13, 2017

Polypeptide: restriction of methylated adenine (mirr from E. Enzyme: B-ketoacyl-ACP synthases II (fabF front E. coli), coli), Protein: TrpR transcriptional repressor (trpR from E. Enzyme: malonyl-CoA-ACP transacylase (fab) from E. coli), Enzymes: glutamate 5-semialdehyde dehydrogenase/ coli), Enzyme: pantothenate kinase (coaA from E. coli), gamma-glutamyl kinase (proAB from E. coli), methylcyto Enzyme: pyruvate dehydrogenase complex (aceEF from E. sine restriction system (mcrBC from E. coli), Protein: citrate coli), Enzyme: 3-hydroxyisobutyrate/3-HP dehydrogenase lyase, citrate-ACP transferase component (citf from E. coli), (mmsB from Pseudomonas aeruginosa), Enzyme: lipoam Enzyme: thioesterase II (tesB from E. coli), Enzyme: DNA ide dehydrogenase (Ipd from E. coli), Enzyme: Y-glutamyl specific endonuclease I (endA from E. coli), Enzyme: phos y-aminobutyraldehyde dehydrogenase (puuC from E. coli), phate acetyltransferase (cutD from E. coli), Enzyme: propi Enzyme: malate synthase A (aceB from E. coli), Enzyme: onate kinase (tdcD from E. coli), tRNA: tRNA glnV (supE isocitrate lyase (aceA from E. coli), Enzyme: isocitrate from E. coli), Enzyme: DNA-binding, ATP-dependent pro dehydrogenase phosphatase/kinase (aceK from E. coli), tease La (Ion from E. coli), Polypeptide: DNA strand Enzyme: 3-hydroxy acid dehydrogenase (ydfG from E. exchange and recombination protein with protease and coli), Enzyme: acetyl CoA carboxylase (accADBC from E. nuclease activity (recA from E. coli), Transcription Unit: coli), Polypeptide: predicted transcriptional regulator (yielP restriction endonulease component of EcolKI restriction from E. coli). Blastocyin resistance gene (BSD from Schi modification system (hsdRMS from E. coli), Enzyme: zosaccharomyces pombe), Enzyme: pyridine nucleotide restriction of DNA at 5-methylcytosine residues (mcra from transhydrogenase (udha from E. coli), Protein: Cra DNA E. coli) arald (from E. coli), araB (from E. coli), rhaD (from binding transcriptional dual regulator (fruR from E. coli), E. coli), rhaE (from E. coli), ack (from E. coli), fruR (from (SCB from E. coli), enzyme: aldehyde dehydrogenase B E. coli), gap.A (from E. coli), lad (from E. coli), lacz (from (aldB from E. coli), Enzyme: carbonic anhydrase (cynT E. coli), ldha (from E. coli), mgSA (from E. coli), pfkA from E. coli), Enzyme: cyanase (cynS from E. coli), DNA (from E. coli), pflB (from E. coli), pgi (from E. coli), poxB gyrase toxin-antitoxin system (ccdAB from E. coli), (from E. coli), pta-ack (from E. coli), ptsH (from E. coli), Enzyme: phosphoglycerate mutase (pgi from E. coli), ArcA glut 1 (from E. coli) and/or ack (from E. coli) or any transcriptional dual regulator or Aerobic respiration control combination thereof. (arcA from E. coli), Enzyme: 6-phosphofructokinase (pfk 0148. The use of genetic modifications in genetic ele from E. coli), Enzyme: glyceraldehyde 3-phosphate dehy ments, genes, proteins or the use of compounds, such as drogenase-A complex (gap.A from E. coli), aldehyde dehy siRNA technology, anti-sense technology, and Small mol drogenase A (alda from E. coli), Enxyune: glutamate dehy ecule inhibitors supplied to the host cell that modulate the drogenase (gdh Afrom E. coli), Enzyme: NADH-dependent expression of gene and proteins provided b the present serine dehydrogenase (NDSD from Pseudomonas aerugi invention are also contemplated. nosa), Protein: threonine/homoserine efflux transporter 0149. In some embodiments the genetic modified organ (rhtA from E. coli), Enzyme: glyceraldehyde 3-phosphate ism of the invention uses genetic elements such as siRNA dehydrogenase (gapN from E. coli), Phosphotransferase ect, genes, proteins or compounds Supplied to the host cell system (pts from E. coli), Enzyme: 6-phosphofructokinase II to modulate one or more of the following: bifunctional (pfkB from E. coli), Enzyme: methylmalonate-semialde rnalonyl-CoA reductase (MCR from Chloroflexus aurantia hyde dehydrogenase (mms.A from Pseudomonas aerugi cus), monofunctional malonyl-CoA reductase (caMCR from nosa), Oxaloacetate:beta-alanine aminotransferase (OAT-1 Chloroflexus aurantiacus), malonyl-CoA reductase (stMCR from Bacillus cereus), Enzyme: aspartate 1-decarboxylase from Sulfolobus tokodaii), Enzyme: malonyl-CoA reductase (panD from E. coli), Gene that confers resistance to valine (cgMCR from Chloroflexus aggregans), Enzyme: malonyl (ValR from E. coli), Enzyme: glucokinase (glk from E. coli), CoA reductase (otMCR from Oscillochloris trichoides), Polypeptide: 30S ribosomal Sununit protein S12 (rpsL from Polypeptide: host restriction; endonuclease R (hsdR from E. E. coli), Polypeptide: CynR DNA-binding transcriptional coli), lactose metabolism (lac from E. coli), L-rhamnulose repressor (cynR, from E. coli), Transporter: galactose:H+ kinase (rhab from E. coli), rhamnulose-1-phosphate aldo Symporter (galP from E. coli), aspartate aminotransferase lase (rhal) from E. coli), Enzyme: B-galactosidase (lac7. (aspC from E. coli), Enzyme: alpha-ketoglutarate reductase from E. coli), L-ribulose 5-phosphate 4-epimerase (ara) (serA from E. coli), Enzyme: 6-phosphofructokinase I (pfkA from E. coli), L-ribulokinase (araB from E. coli), Enzyme: from E. coli), Enzyme: phosphoenolpyruvate carboxylase D-lactate dehydrogenase-fermentative (Idha from E. coli), (ppc from E. coli), Enzyme: Succinate-semialdehyde dehy enzyme: pyruvate formate-lyase (pflB from E. coli), drogenase (NADP+) (gab) from E. coli), Enzyme: pyruvate Enzyme: phosphate acetyltransferase/phosphate propionyl kinase (pyk from E. coli), Enzyme: Oxaloacetate 4-decar transferase (pta from E. coli), Enzyme: pyruvate oxidase boxylase (OAD from Leuconostoc mesenteroides), Enzyme: (poxB from E. coli), Enzyme: methylglyoxal synthase trigger factor, a molecular chaperone involved in cell divi (mgSA from E. coli). enzyme: Acetate kinase (ackA from E. sion (tig from E. coli), Transcription Unit (ptsHIcrr from E. coli), enzymes: phosphotransacetylase-acetate kinase (pta coli), Enzyme: acetyl-CoA acetaldehyde dehydrogenase/ ack from E. coli), Enzyme: enoyl-acyl-carrier-protein alcohol dehydrogenase (adhE from E. coli), Enzyme: fattya reductase (fabl from E. coli), Protein: Zeocin binding protein cyl thioesterase I (tes.A from E. coli), Enzyme: guanosine (ZeoR from Streptoalloteichus Hindustanus), Enzyme: car 3'-diphosphate 5'-triphosphate 3'-diphosphatase (spoT from boxytransferase moiety of acetyl-CoA carboxylase (accAD E. coli), combination of genes encoding aceABCD Subunits from E. coli), Enzyme: triose phosphate isomerase (tpiA (from E. coli and Halomonas elongata), pol (from E. coli), from E. coli), Enzyme: biotoin carboxylase moiety of acetyl Enzyme: GDP pyrophosphokinase/GTP pyrophosphokinase CoA carboxylase (accBC from E. coli), Enzyme: transhy (relA from E. coli), Enzyme Name (me from E. coli), drogenase (pntAB front E. coli), Polypeptide: LacI DNA Enxyme: citrate synthase (gitA from E. coli), Polypeptide: binding transcriptional repressor (lacI from E. coli), DNA gyrase, Subunit A (gyrA from E. coli), Enzyme: Enzyme: B-ketoacyl-ACP synthases I (fabB from E. coli), multifunctional 2-keto-3-deoxygluconate 6-phosphate aldo US 2017/01 01638 A1 Apr. 13, 2017

lase and 2-keto-4-hydroxygiutarate aldolase and Oxaloac Genetic Modifications etate decarboxylase (eda from E. coli), thiamin biosynthesis (thi front E. coli), Polypeptide: acetolactate synthase II (ilvG 0155 Various methods to achieve such genetic modifi from E. coli), acetyl CoA carboxylase (accDACB from E. cation in a host strain are well known to one skilled in the coli), Citrate synthase (ArCS from Arthrobacter aurescens), art. Example of genetic modification that can be used by the Acetyl-CoA carboxylase from Corynebacter glutamicum claimed invention include, but are not limited to, increasing (CgACC from Corynebacter glutamicum), Polypeptide: fer expression of an endogenous genetic element, increasing richrome/phage/antibiotic outer membrane porin FhuA expression of an exogenous genetic element; decreasing (fhuA from E. coli), Transporter: phosphate:H+ symporter functionality of a repressor gene; increasing functionality of PitA (pitA from E. coli), Transporter: uracil:H+ symporter a repressor gene; increasing functionality of a activator (uraA from E. coli), Enzyme: uracil phosphoribosyltrans gene; decreasing functionality of a activator gene; introduc ferase (upp from E. coli), Enzyme: acylphosphatase (yccX ing a genetic change or element integrated in the host from E. coli), acetyl-CoA synthetase (acSA from E. coli:). genome, introducing a heterologous genetic element perma Polypeptide: restriction of methylated adenine (mirr from E. nently, by integration into the genome or transiently by coli.), Protein: TrpR transcriptional repressor (trpR from E. transformation with plasmid, increasing copy number of a coli), Enzymes: glutamate 5-semialdehyde dehydrogenase/ nucleic acid sequence encoding a polypeptide catalyzing an gamma-glutamyl kinase (proAB from E. coli), methylcyto enzymatic conversion step; mutating a genetic element to sine restriction system (mcrBC from E. coli), Protein: citrate provide a mutated protein to increase specific enzymatic lyase, citrate-ACP transferase component (citF from E. coli), activity; mutating a genetic element to provide a mutated Enzyme: thioesterase II (tesB from E. coli), Enzyme: DNA protein to decrease specific enzymatic activity; over-ex specific endonuclease I (endA from E. coli), Enzyme: phos pressing of gene; reduced the expression of a gene; knocking phate acetyltransferase (eutD from E. coli), Enzyme: propi out or deleting a gene; altering or modifying feedback onate kinase (tdcD from E. coli), tRNA: tRNA ginV (supE inhibition; providing an enzyme variant comprising one or from E. coli), Enzyme: DNA-binding, ATP-dependent pro more of an impaired binding sites or active sites; increasing tease La (lon from E. coli), Polypeptide: DNA strand functionality of a siRNA, decreasing functionality of a exchange and recombination protein with protease and siRNA, increasing functionality of a antisense molecule, nuclease activity (recA from E. coli), Transcription Unit: decreasing functionality of a antisense molecule, addition of restriction endonulease component of EcoKI restriction genetic modules such as RBS, 3 UTR elements to increase modification system (hsdRMS from E. coli), Enzyme: mRNA stability or translation; deletion of genetic modules restriction of DNA at 5-methylcytosine residues (mcra from such as RBS, 3 UTR elements to decrease mRNA stability E. coli). In some embodiment the genetic modifications or translation; addition or modification of genetic modules listed above are modified further with the genetic modules such as 5 UTR elements to increase transcription; deletion provided herein. or modification of genetic modules such as '5 uTR and elements to increase transcription. In addition other genetic 0150 in some embodiment the genetic modification of modules, provide herein, such a multicopy plasmids and the genes, proteins and enzymes of the invention can be for various promoters can be used to further modify of the the method of bioproduction of various chemicals which can genetic modifications provide herein. Additionally, as be used to make various consumer products described known to those of ordinarily skill in the art compounds such herein. as siRNA technology, anti-sense technology, and Small 0151. In some embodiment the genetic modification of molecule in inhibitors can be used to alter gene expression the genes, proteins and enzymes of the invention can be for in the same manner as a genetic modification. the bioproduction of 1,4-butanediol (1,4-BDO) (U.S. Pub. No. 20110190513). In some embodiment the genetic modi 0156 Screening methods, such as SCALE in combina fication of the genes, proteins and enzymes of the invention tion with random mutagenesis may be practiced to provide can be for the bioproduction of butanol (U.S. app. Ser. No. genetic modifications that provide a benefit to increased 13/057,359). In some embodiment the genetic modification production of 3-HP in a host cell. Examples of random of the genes, proteins and enzymes of the invention can be mutagenesis can include insertions, deletions and Substitu for the bioproduction of isobutanol (U.S. app. Ser. No. tions of one or more nucleic acids in a nucleic acid of interest. In various embodiments a genetic modification 13/057,359) results in improved enzymatic specific activity and/or turn 0152 in some embodiment the genetic modification of over number of an enzyme. Without being limited, changes the genes, proteins and enzymes of the invention can be for may be measured by one or more of the following: KM: the bioproduction of 3-HP such and its aldehyde metabolites Kcat, Kavidity, gene expression level, protein expression (U.S. app. Ser. No. 13/062,917). level, level of a product known to be produced by the 0153. In some embodiment the genetic modification of enzyme, 3-HP tolerance, or by 3-HP production or by other the genes, proteins and enzymes of the invention can be for CaS. the bioproduction of polyketide chemical products (U.S. app. Ser. No. 13/575,581). Host Cells 0154) In some embodiment the genetic modification of the genes, proteins and enzymes of the invention can be for 0157. In some applications of the invention the host cell the bioproduction of fatty acid methyl esters (U.S. Pub. No. can be a gram-negative bacterium. In some applications of 2011 0124063). In some embodiment the genetic modifica the invention the host cell can be from the genera Zymomo tion of the genes, proteins and enzymes of the invention can nas, Escherichia, Pseudomonas, Alcaligenes or Klebsiella. be for the bioproduction of C4-C18 fatty acids (U.S. app Ser. In some applications of the invention the host cell can be No. 61/682,127). Escherichia coli, Cupriavidus necator, Oligotropha carboxi US 2017/01 01638 A1 Apr. 13, 2017 dovorans, or Pseudomonas putida. In some applications of as the deprotonated form, 3-hydroxypropionate; or (iii) as the invention the host cell is one or more an E. coli strains. mixtures of the protonated and deprotonated forms. Gener 0158. In some applications of the invention the host cell ally, the fraction of 3-HP present as the acid versus the salt can be a gram-positive bacterium. In some applications of will depend on the pH, the presence of other ionic species in the invention the host cell can be from the genera Solution, temperature (which changes the equilibrium con Clostridium, Salmonella, Rhodococcus, Bacillus, Lactoba stant relating the acid and salt forms), and, to Some extent, cillus, Enterococcus, Paenibacillus, Arthrobacter, Coryne pressure. Many chemical conversions may be carried out bacterium, or Brevibacterium, in some applications of the from either of the 3-HP forms, and overall process econom invention the host cell is Bacillus licheniformis, Paeniba ics will typically dictate the form of 3-HP for downstream cillus macerans, Rhodococcus erythropolis, Lactobacillus conversion. plantarum, Enterococcus faecium, Enterococcus galli (0166 Acrylic acid obtained from 3-HP purified by the narium, Enterococcus aecalis, or Bacillus subtilis, In some methods described in this disclosure may be further con applications of the invention the host cell is 1B. subtilis verted to various polymers. For example, the free-radical strain. polymerization of acrylic acid takes place by polymerization 0159. In some applications of the invention the host cell methods known to the skilled worker and can be carried out, is yeast. In some applications of the invention the host cell for example, in an emulsion or Suspension in aqueous can be from the genera Pichia, Candida, Hansenula or Solution or another solvent. Initiators, such as but not limited Sacchammyces. In some applications of the invention the to organic peroxides, are often added to aid in the polym host cell is Saccharomyces cerevisiae. In some applications erization. Among the classes of organic peroxides that may of the invention the host cell is Saccharomyces pombe. be used as initiators are diacyls, peroxydicarbonates, 0160. In some applications of the invention the host cell monoperoxycarbonates, peroxyketals, peroxyesters, is an alga. In some applications of the invention the host cell dialkyls, and hydroperoxides. Another class of initiators is is a halophile. In some applications of the invention the host aZo initiators, which may be used for acrylate polymeriza cell is an alga, in some applications of the invention the host tion as well as co-polymerization with other monomers. U.S. cell is a chemolithotrophic bacterium. Pat. Nos. 5,470,928: 5,510,307; 6,709,919; and 7,678,869 0161 In some applications of the invention the host cell teach various approaches to polymerization using a number is comprised of multiple host cell types. In some applica of initiators, including organic peroxides, azo compounds, tions of the invention the host cell is comprised of one host and other chemical types, and are incorporated by reference cell type. In some applications of the invention the host cell for such teachings as applicable to the polymers described is comprised of one more species or strain of a host cell type. herein. 0.167 Accordingly, it is further possible for co-mono Downstream Consumer Products Chemicals mers, such as crosslinkers, to be present during the polym 0162 3-HP purified according to the methods provided in erization. The free-radical polymerization of the acrylic acid this disclosure may be converted to various other products obtained from dehydration of 3-HP, as produced herein, in at having industrial uses including, but not limited to, acry least partly neutralized form and in the presence of cross larinide, acrylic acid, esters of acrylic acid, 1,3-propanediol. linkers is practiced in certain embodiments. This polymer and other chemicals, collectively referred to as “downstream ization may result in hydrogels which can then be commu chemical products' or “downstream products.” In some nicated, ground and, where appropriate, Surface-modified, instances the conversion is associated with the separation by known techniques. and/or purification steps. These downstream chemical prod 0168 An important commercial use of polyacrylic acid is ucts are useful for producing a variety of consumer products for superabsorbent polymers. This specification hereby which are described in more detail below. The methods of incorporates by reference Modern Superabsorbent Polymer the present invention include steps to produce downstream Technology, Buchholz and Graham (Editors), Wiley-VCH, products of 3-HP. 1997, in its entirety for its teachings regarding Superabsor 0163 As a C3 building block, 3-HP offers much potential bent polymers components, manufacture, properties and in a variety of chemical conversions o commercially impor uses. Superabsorbent polymers are primarily used as absor tant intermediates, industrial end products, and consumer bents for water and aqueous Solutions for diapers, adult products. For example, 3-HP may be converted to acrylic incontinence products, feminine hygiene products, and simi acid, acrylates (e.g., acrylic acid salts and es(ers), 1.3- lar consumer products. In Such consumer products, Super propanediol, malonic acid, ethyl-3-hydroxypropionate, ethyl absorbent materials can replace traditional absorbent mate ethoxy propionate, propiolactone, acrylamide, or acryloni rials such as cloth, cotton, paper wadding, and cellulose trile. fiber. Superabsorbent polymers absorb, and retain under a 0164. Additionally, 3-HP may be oligomerized or polym slight mechanical pressure, up to 25 times or more their erized to form poly(3-hydroxypropionate) homopolymers, weight in liquid. The Swollengel holds the liquid in a solid, or co-polymerized with one or more other monomers to rubbery state and prevents the liquid from leaking. Super form various co-polymers. Because 3-HP has a single Ste absorbent polymer particles can be surface-modified to reoisomer, polymerization of 3-HP is not complicated by the produce a shell structure with the shell being more highly Stereo-specificity of monomers during chain growth. This is cross-linked than the rest of the particle. This technique in contrast to (S)-2-hydroxypropanoic acid (also known as improves the balance of absorption, absorption under load, lactic acid), which has two (D, L) stereoisomers that should and resistance to gel-blocking. It is recognized that Super be considered during its polymerizations, absorbent polymers have uses in fields other than consumer 0.165. As will be further described, 3-HP can be con products, including agriculture, horticulture, and medicine. verted into derivatives starting (i) Substantially as the pro 0169 Superabsorbent polymers are prepared from acrylic tonated form of 3-hydroxypropionic acid; (ii) substantially acid (such as acrylic acid derived from 3-HP provided US 2017/01 01638 A1 Apr. 13, 2017 herein) and a crosslinker, by solution or Suspension polym linked or lightly cross-linked, depending on the specific erization. Exemplary methods include those provided in application. The molecular weights are typically from about U.S. Pat. Nos. 5,145,906; 5,350,799; 5,342,899; 4,857,610; 200 to about 1,000,000 g/mol. Preparation of these low 4,985,518; 4,708, 997; 5,180,798; 4,666,983; 4,734,478: molecular weight polyacrylic acid polymers is described in and 5.331,059, each incorporated by reference for their U.S. Pat. Nos. 3,904,685; 4,301.266: 2,798,053; and 5,093, teachings relating to Superabsorbent polymers. 472, each of which is incorporated by reference for its 0170 Among consumer products, a diaper, a feminine teachings relating to methods to produce these polymers. hygiene product, and an adult incontinence product are 0.175 Acrylic acid may be co-polymerized with one or made with superabsorbent polymer that itself is made sub more other monomers selected from acrylamide, 2-acry stantially from acrylic acid converted from 3-HP made in lamido-2-methylpropanesulfonic acid, N,N-dimethylacryl accordance with the present invention. amide, N-isopropylacrylamide, methacrylic acid, and meth 0171 Diapers and other personal hygiene products may acrylamide, to name a few. The relative reactivities of the be produced that incorporate Superabsorbent polymers made monomers affect the microstructure and thus the physical from acrylic acid made from 3-HP which is produced and properties of the polymer. Co-monomers may be derived purified by the teachings of the present application. The from 3-HP, or otherwise provided, to produce co-polymers. following provides general guidance for making a diaper Ullmann's Encyclopedia of Industrial Chemistry, Polyacry that incorporates Such Superabsorbent polymer. The Super lamides and Poly(Acrylic Acids), Wiley VCH Verlag absorbent polymer first is molded into an absorbent pad that GmbH, Wienham (2005), is incorporated by reference may be vacuum formed, and in which other materials. Such herein for its teachings of polymer and co-polymer process as a fibrous material (e.g., wood pulp) are added. The 1ng. absorbent pad then is assembled with sheet(s) of fabric, generally a nonwoven fabric (e.g., made from one or more 0176 Acrylic acid can in principle be copolymerized of nylon, polyester, polyethylene, and polypropylene plas with almost any free-radically polymerizable monomers tics) to form diapers. including styrene, butadiene, acrylonitrile, acrylic esters, 0172 More particularly, in one non-limiting process, maleic acid, maleic anhydride, vinyl chloride, acrylamide, multiple pressurized nozzles, located above a conveyer belt, itaconic acid, and so on. End-use applications typically spray Superabsorbent polymer particles (e.g., about 400 dictate the co-polymer composition, which influences prop micron size or larger), fibrous material, and/or a combina erties. Acrylic acid also may have a number of optional tion of these onto the conveyer belt at designated spaces/ Substitutions and, after such substitutions, may be used as a intervals. The conveyor belt is perforated and under vacuum monomer for polymerization, or co-polymerization reac from below, no that the sprayed on materials are pulled tions. As a general rule, acrylic acid (or one of its co toward the belt surface to form a flat pad. In various polymerization monomers) may be substituted by any Sub embodiments, fibrous material is applied first on the belt, stituent that does not interfere with the polymerization followed by a mixture of fibrous material and the superab process, such as alkyl, alkoxy, aryl, heteroaryl, benzyl, vinyl, sorbent polymer particles, followed by fibrous material, so allyl, hydroxy, epoxy, amide, ethers, esters, ketones, that the superabsorbent polymer is concentrated in the maleimides, Succinimides, Sulfoxides, glycidyl and silyl (see middle of the pad. A leveling roller may be used toward the e.g., U.S. Pat. No. 7,678,869, incorporated by reference end of the belt path to yield pads of uniform thickness. Each above, for further discussion). The following paragraphs pad thereafter may be further processed, such as to cut it to provide a few non-limiting examples of copolymerization a proper shape for the diaper, or the pad may be in the form applications. of a long roll sufficient for multiple diapers. Thereafter, the 0177 Paints that comprise polymers and copolymers of pad is sandwiched between a top sheet and a bottom sheet acrylic acid and its esters are in wide use as industrial and of fabric (one generally being liquid pervious, the other consumer products. Aspects of the technology for making liquid impervious), for example on a conveyor belt, and such paints can be found in e.g., U.S. Pat. Nos. 3,687,885 these are attached together, for example by gluing, heating and 3,891.591, incorporated by reference for their teachings or ultrasonic welding, and cut into diaper-sized units (if not of Such paint manufacture. Generally, acrylic acid and its previously so cut). Additional features may be provided, esters may form homopolymers or copolymers among them Such as elastic components, strips of tape, etc., for fit and selves or with other monomers, such as amides, methacry ease of wearing by a person. lates, acrylonitrile, vinyl, styrene and butadiene. A desired 0173 The ratio of the fibrous material to polymer par mixture of homopolymers and/or copolymers, referred to in ticles is known to affect performance characteristics. In the paint industry as “vehicle' (or “binder') are added to an some cases, this ratio is between 75:25 and 90:10 (see e.g., aqueous solution and agitated Sufficiently to form an aque U.S. Pat. No. 4,685,915, incorporated by reference for its ous dispersion that includes Sub-micrometer sized polymer teachings of diaper manufacture). Other disposable absor particles. The paint cures by coalescence of these vehicle bent articles may be constructed in a similar fashion, such as particles as the water and any other solvent evaporate. Other absorbent articles for adult incontinence, feminine hygiene additives to the aqueous dispersion may include pigment, (sanitary napkins), tampons, etc. (see, for example, U.S. Pat. filler (e.g., calcium carbonate, aluminum silicate), solvent Nos. 5,009,653: 5,558,656; and 5,827,255 incorporated by (e.g., acetone, benzol, alcohols, etc., although these are not reference for their teachings of Sanitary napkin manufac found in certain no VOC paints), thickener, and additional ture). additives depending on the conditions, applications, 0.174 Low molecular weight polyacrylic acid has uses for intended Surfaces, etc. In many paints, the weight percent of water treatment, and as a flocculant and thickener for various the vehicle portion may range from about nine to about 26 applications including cosmetics and pharmaceutical prepa percent, but for other paints the weight percent may vary rations. For these applications, the polymer may be uncross beyond this range. US 2017/01 01638 A1 Apr. 13, 2017

0.178 Acrylic-based polymers are used for many coatings with use of Suitable stabilizing agents or inhibiting agents in addition to paints. For example, for paper coating latexes, reducing the likelihood of polymer formation. See, for acrylic acid is used from 0.1-5.0%, along with styrene and example, U.S. Publication No. 2007/0219390, incorporated butadiene, to enhance binding to the paper and modify by reference in its entirety. Stabilizing agents and/or inhib rheology, freeze-thaw stability and shear stability. In this iting agents include, but are not limited to, e.g., phenolic context, U.S. Pat. Nos. 3,875,101 and 3,872,037 are incor compounds (e.g., dimethoxyphenol (DMP) or alkylated phe porated by reference for their teachings regarding Such nolic compounds such as di-tert-butyl phenol), quinones latexes. Acrylate-based polymers also are used in many inks, (e.g., t-butyl hydroquinone or the monomethyl ether of particularly UV curable printing inks. For water treatment, hydroquinone (MEHQ)), and/or metallic copper or copper acrylamide and/or hydroxy ethyl acrylate are commonly salts (e.g., copper Sulfate, copper chloride, or copper co-polymerized with acrylic acid to produce low molecular acetate). Inhibitors and/or stabilizers can be used individu weight linear polymers. In this context, U.S. Pat. Nos. ally or in combinations as will be known by those of skill in 4,431,547 and 4,029,577 are incorporated by reference for the art. their teachings of Such polymers. Co-polymers of acrylic 0182. In some cases, the one or more downstream com acid with maleic acid or itaconic acid are also produced for pounds are recovered at a molar yield of up to about 100 water-treatment applications, as described in U.S. Pat. No. percent, or a molar yield in the range from about 70 percent 5,135,677, incorporated by reference for that teaching. to about 90 percent, or a molar yield in the range from about Sodium acrylate (the Sodium salt of glacial acrylic acid) can 80 percent to about 100 percent, or a molar yield in the range be co-polymerized with acrylamide (which may be derived from about 90 percent to about 100 percent. Such yields may from acrylic acid via amidation chemistry) to make an be the result of single-pass (batch or continuous) or iterative anionic co-polymer that is used as a flocculant in water separation and purification steps in a particular process. treatment. 0183. The methods described in this disclosure can also 0179 For thickening agents, a variety of co-monomers be used to produce downstream compounds derived from can be used, such as those described in U.S. Pat. Nos. 3-HP, such as but not limited to, polymerized-3-HP (poly 4.268,641 and 3.915,921, incorporated by reference for their 3-HP), acrylic acid, polyacrylic acid (polymerized acrylic description of these co-monomers. U.S. Pat. No. 5,135,677 acid, in various forms), copolymers of acrylic acid and. describes a number of co-monomers that can be used with acrylic esters, acrylamide, acrylonitrile, propiolactone, ethyl acrylic acid to produce water-soluble polymers, and is 3-HP, malonic acid, and 1,3-propanediol. Also, among esters incorporated by reference for such description. that are formed are methyl acrylate, ethyl acrylate, n-butyl 0180. In some cases, conversion to downstream products acrylate, hydroxypropyl acrylate, hydroxyethyl acrylate, may be made enzymatically. For example, 3-HP may be isobutyl acrylate, and 2-ethylhexyl acrylate. These and/or converted to 3-HP-CoA, which then may be converted into other acrylic acid and/or other acrylate esters may be com polymerized 3-HP with an enzyme having polyhydroxy acid bined, including with other compounds, to form various synthase activity (EC 2.3.1.-). Also, 1,3-propanediol can be known acrylic acid-based polymers. Numerous approaches made using polypeptides having oxidoreductase activity or may be employed for Such downstream conversions, gen reductase activity (e.g., enzymes in the EC 1.1.1.—class of erally falling into enzymatic, catalytic (chemical conversion enzymes). Alternatively, when creating 1,3-propanediol process using a catalyst), thermal, and combinations thereof from 3-HP, a combination of (1) a polypeptide having (including some wherein a desired pressure is applied to aldehyde dehydrogenase activity (e.g., an enzyme from the accelerate a reaction). For example, without being limiting, 1.1.1.34 class) and (2) a polypeptide having alcohol dehy acrylic acid may be made from 3-HP via a dehydration drogenase activity (e.g., an enzyme front the 1.1.1.32 class) reaction, methyl acrylate may be made from 3-HP via can be used. Polypeptides having lipase activity may be used dehydration and esterification, the latter to add a methyl to form esters. Enzymatic reactions such as these may be group (such as using methanol), acrylamide may be made conducted in vitro. Such as using cell-free extracts, or in from 3-HP via dehydration and amidation reactions, acry V1VO. lonitrile may be made via a dehydration reaction and form 0181. Thus, various embodiments described in this dis ing a nitrile moiety, propiolactone may be made from 3-HP closure, such as methods of making a chemical, include via a ring-forming internal esterification reaction, ethyl-3- conversion steps to any downstream products of microbially HP may be made from 3-HP via esterification with ethanol, produced 3-HP, including but not limited to those chemicals malonic acid may be made from 3-HP via an oxidation described herein, in the incorporated references, and known reaction, and 1,3-propanediol may be made from 3-HP via in the art. For example, in some cases, 3-HP is produced and a reduction reaction. Additionally, it is appreciated that converted to polymerized-3-HP (poly-3-HP) or acrylic acid. various derivatives of the derivatives of 3-HP and acrylic In some cases, 3-HP or acrylic acid can be used to produce acid may be made. Such as the various known polymers of polyacrylic acid (polymerized acrylic acid, in various acrylic acid and its derivatives. Production of such polymers forms), methyl acrylate, acrylamide, acrylonitrile, propio is considered within the scope of the present invention. lactone, ethyl 3-HP, malonic acid, 1,3-propanediol, ethyl Copolymers containing acrylic acid and/or esters have been acrylate, n-butyl acrylate, hydroxypropyl acrylate, hydroxy widely used in the pharmaceutical formulation to achieve ethyl acrylate, isobutyl acrylate, 2-ethylhexyl acrylate, and extended or Sustained release of active ingredients, for acrylic acid or an acrylic acid ester to which an alkyl or aryl example as coating material. Downstream compounds may addition may be made, and/or to which halogens, aromatic also be converted to consumer products such as diapers, amines or amides, and aromatic hydrocarbons may be carpet, paint, and adhesives. added. 0.184 Another important product, acrylamide, has been a) Reactions that form downstream compounds such as used in a number of industrial applications. Acrylamide may acrylates or acrylamides can be conducted in conjunction be produced from 3-HP for example, without being limiting, US 2017/01 01638 A1 Apr. 13, 2017 via an esterification-amidation-dehydration sequence. choosing a transcription or translation vector is the source of Refluxing an alcohol solution of 3-HP in the presence of an the DNA to be expressed. Prokaryotic genes usually have a acid or Lewis acid catalyst described herein would lead to a ribosome-binding site that is compatible with the host E. coli 3-HP ester. Treatment of the 3-HP ester with either an translation machinery, whereas eukaryotic genes do not. ammonia gas or an ammonium ion could yield 3-HP amide. Normal prokaryotic gene expression may be enhanced by Finally, dehydration of the 3-HP amide with dehydration use of an engineered promoter and ribosome-binding site. reagents described elsewhere in this disclosure could pro 0193 Promoters duce acrylamide. The steps mentioned herein may be rear 0.194. A promoter is a region of DNA that initiates ranged to produce the same final product acrylamide. transcription of a particular gene. In bacteria, transcription is Polymerization of acrylamide can be achieved, for example, initiated by the promoter being recognized by RNA poly and without being limiting, by radical polymerization. Poly merase and an associated Sigma factor, which are often acrylamide polymers have been widely used as additives for brought to the promoter site by an activator protein's bind treating municipal drinking water and waste water. In addi ing to its own DNA binding site located by the promoter. tion, they have found applications in gel electrophoresis, 0.195 Promoter selection is an important factor when oil-drilling, papermaking, ore processing, and the manufac designing an expression plasmid system. A promoter is ture of permanent press fabrics. located upstream of the ribosome-binding site. Owing to the fact that many heterologous protein products are toxic to the VIII. Expression Systems General Concepts cell, the promoter can be regulated so that the heterologous 0185. The following general concepts are applicable to protein is expressed at the appropriate amount and time to embodiments of the invention described above. reduced the burden on the cell host. 0186 Multicopy Plasmids 0196. Historically, the most commonly used promoters 0187. The researcher is faced with a myriad of genetic have been the lactose (lac) and tryptophan (trp) promoters. module options when designing a plasmid for expression of These two promoters were combined to create the hybrid a heterologous protein in a host cell. How to optimize an promoters lac and trc that are also commonly used. Other expression plasmid system often depends on the down common promoters are the phage lambda promoters, the stream use of the expressed protein. phage T7 promoter (T7), and the alkaline phosphatase 0188 Different cloning vectors or plasmids are main promoter (phoA). tained at different copy numbers, dependent on the replicon 0.197 Promoters can be constitutive and inducible. Con of the plasmid. Most general cloning plasmids can carry a stitutive promoter is active in all circumstances in the cell, DNA insert up to around 15 kb in size. while regulated or inducible promoter become active in 0189 Multicopy plasmids can be used for the expression response to specific stimuli. In addition the strength of the of recombinant genes in Escherichia coli. Examples of promoter can also differ. A strong promoter has a high include multicopy plasmids include high-copy, medium frequency of transcription and generates the heterologous copy and low-copy plasmids (see FIG. 8). The high copy protein as 10-30% of the total cellular protein production number is generally desired for maximum gene expression. (for examples see FIG. 8). Chemically-inducible promoters However, the metabolic burden effects can result from that can be used in various aspects of the invention include multiple plasmid copies could prove to be detrimental for but are not limited to promoters whose transcriptional activ maximum productivity in certain metabolic engineering ity is regulated by the presence or absence of alcohol, applications by adding significant metabolic burden to the tetracycline, steroids, metal and other compounds. Physi system. cally-inducible promoters that can be used in various aspects 0190. The low-copy plasmids for example, pBR322 is of the invention include but are not limited to including based on the original ColE1 replicon and thus has a copy promoters whose transcriptional activity is regulated by the number of 15-20. The paCYC series of plasmids are based presence or absence of light and low or high temperatures. on the p15A replicon, which has a copy number of 18-22, 0.198. In order to be an inducible promoter, the promoter whereas pSC101 has even a lower copy number around 5. should be initially be completely repressed to transcription and BACs are maintained at one copy per cell. Such low and then transcription induced with the addition of an copy plasmids may be useful in metabolic engineering inducer to allow expression at the time that expression is applications, particularly when one or more of the Substrates desired in the host cell. Alternatively, an inducible promoter used in the recombinant pathway are required for normal may be responsive to the lack of a Substance, such as cellular metabolism and can be toxic to the cell at high inorganic phosphate. Such that the absence of inorganic levels. phosphate will allow expression at the time that expression 0191) However, the used of high-copy plasmids may be is desired in the host cell (for examples see FIG. 8). useful in enhanced cellular metabolism contexts. The mutant (0199 Ribosome Binding Sites ColE1 replicon, as found in the puC series of plasmids (0200. A Ribosome Binding Sites (RBS) is an RNA produces a copy number of 500-700 as a result of a point sequence upstream of the start codon that affects the rate at mutation within the RNAII regulatory molecule. which a particular gene or open reading frame (ORF) is 0.192 There are transcription and translation vectors. translated. One can tailor an RBS site to a particular gene. Transcription vectors are utilized when the DNA to be Ribosome Binding Sites (RBSs) are typically short cloned has an ATG start codon and a prokaryotic ribosome sequences, often less than 20 base pairs. Various aspects of -binding site. Translation vectors contain an efficient ribo RBS design are known to affect the rate at which the gene some-binding site and, therefore, it is not necessary for the is translated in the cell. The RBS module can influences the target DNA to contain one. This is particularly useful in translation rate of a gene largely by two known mechanisms. cases where the initial portion of the gene may be cleaved in First, the rate at which ribosomes are recruited to the mRNA an effort to improve solubility. Another consideration when and initiate translation is dependent on the sequence of the US 2017/01 01638 A1 Apr. 13, 2017

RBS. Secondly, the sequence of the RBS can also affect the EXAMPLES stability of the mRNA in the cell, which in turn affects the number of proteins. Through the use of genetic expression Example 1 modules the expression of desired genes. Such as genes encoding enzymes in the biosynthetic pathway for 3-HP can Salt Inhibition Studies in E. COLI be tailored activity either at the transcriptional and transla 0205 The activity of ACCase complex, a critical enzyme tional level. in the conversion of acetyl-CoA to malonyl-CoA, the imme 0201 One can access the registry RBS collection as a diate precursor for 3-HP, is severely inhibited by salt. starting point for designing an RBS 2. degree of secondary structure near the RBS can affect the 5-3M. It is hypothesized that enzymes derived from any translation initiation rate. This fact can be used to produce salt-tolerant species should be more resistant to enzyme regulated translation initiation rates. inhibition by salts, such as 3-HP. Further, these enzymes that 0202 The Shine-Dalgarno portion of the RBS is critical have greater salt tolerance should in turn have extended to the strength of the RBS. The Shine-Dalgarno is found at production under high salt conditions than enzymes with the end of the 16S rRNA and is the portion that binds with lower salt tolerance. the mRNA and includes the sequence 5'ACCUCC-3'. RBSs 0207 Accordingly, the genes encoding the accA, accB. will commonly include a portion of the Shine-Dalgarno accC, accD of H. elongata described in Table 1 were sequence. One of the ribosomal proteins, S1, is known to synthesized for expression in E. coli using codons optimized bind to adenine bases upstream from the Shine-Dalgarno for this organism and supplied individually on pUC57 sequence. As a result, the RBS can be made stronger by plasmids without promoters. Synthetic operons comprising adding more adenines in the sequence upstream of the RBS. the subunits were assembled using the Gibson assembly 0203 When considering the design of the spacing method between the RBS and the start codon, it is important to think of the aligned spacing rather than just the absolute spacing. TABLE 1. While the Shine-Dalgarno portion of the RBS is critical to the strength of the RBS, the sequence upstream of the Accession numbers for genes encoding ACCase Shine-Dalgarno sequence is also important. Note that the subunits from Halomonas elongata promoter may add some bases onto the start of the mRNA Gene Accession number SEQ ID NO. that may affect the strength of the RBS by affecting S1 accA YP OO3898857.1 SEQ ID NO. 1, 2 binding. Computer programs that design RBS sequence to accB YP OO389.7250.1 SEQ ID NO. 3, 4 match protein coding sequences, desired upstream accC YP OO3897249.1 SEQ ID NO. 5, 6 sequences including regulatory mRNA sequences, and accD YP OO38973.09.1 SEQ ID NO. 7, 8 account of secondary structure are known Salis, Mirsky, and Voight, Nature Biotechnology 27: 946-950, 2009 and Each gene was amplified by PCRs with Pfu Ultra II HS were used to optimize RBSs for the ACCase subunit genes using the manufacturers instructions, and the PCR products as described in(see EXAMPLE 3). were purified using the Zymo PCR Cleanup kit. Concentra 0204 While preferred embodiments of the present inven tions of products were measured using the Nanodrop spec tion have been shown and described herein, it will be tophotometer. The Gibson Assembly kit (NEB) was used to obvious to those skilled in the art that such embodiments are construct plasmids expressing the ACCase Subunit genes as provided by way of example only. Numerous variations, directed by the manufacturer. The effect of NH3-HP and changes, and Substitutions will now occur to those skilled in NH4C1 on H. elongata ACCase was tested and compared to the art without departing from the invention. It should be the E. coli ACCase. As shown in FIG. 4, whereas the E. coli understood that various alternatives to the embodiments of ACCase is significantly inhibited by the salts, the ACCase the invention described herein may be employed in practic from the halophile is less affected by either NH3-HP or by ing the invention. It is intended that the following claims NHC1. This result indicated that use of the H. elongata define the scope of the invention and that methods and ACCase in 3-HP production strains would in beneficial in structures within the scope of these claims and their equiva relieving 3-HP inhibition of the conversion of acetyl-CoA to lents be covered thereby. malonyl-CoA, a critical step in the pathway. US 2017/01 01638 A1 Apr. 13, 2017 19

Example 3 Sites (RBS) are 15 nucleotide segments which are known to control the level of protein expression in microorganisms. RBS-Optimized Genes To enhance H. elongata ACCase expression various cus tomized RBS were constructed and optimized for E. coli 0208 Enzyme expression is regulated at transcriptional translation expression. Table 2 shows the RBS sequences and translational levels in prokaryotes. Ribosome Binding used to increase expression of the individual subunits. TABL E 2 RBS sequences used to enhance expression of H. elongate ACCase subunits. H. elongata ACC expression Modified RBS SeClelCSS preceeding ATG (underlined) plasmid He accD He accA He accC He accB

Parent 2-4 5'- 5'- 5'- 5'- GCGTAGTAAAGGA CAATTTATTTAAGGA GAAATTTCATACC GGAAGAACAAGGG GGTAACATATG GGACTCTTAAGATG ACAGGCGAAGGAG. GTGTACATG GAAAAACCATG

B2 Same as 2-4 Same as 2-4 Same as 2-4 5'- ggaagaattalagg ggga Caaggggga ataATG

13A 5'- Same as 2-4 Same as 2-4 gcgtag tagc.cggg tgataaggagcc.gt aacATG

14C 5'- Same as 2-4 Same as 2-4 Same as 2-4 gcgtag tagctgat ataaaaggaggtaa cggATG

15C Same as 2-4 5'- Same as 2-4 Same as 2-4 caatttattitttgtt cacccaaggagtatt gctaATG

17C Same as 2-4 5'- Same as 2-4 Same as 2-4 caatttatttac.cga aataaaaggagggat gcca ATG

5'- 5'- Same as 2-4 Same as 2-4 gcgtag tagc.cggg caattt attitttgtt tgataaggagcc.gt cacccaaggagtatt aacATG gctaATG

5'- 5'- Same as 2-4 Same as 2-4 gcgtag tagc.cggg caattt attt accga tgataaggagcc.gt aataaaaggagggat aacATG gcca ATG

36 C-8 5'- 5'- Same as 2-4 5'- gcgtag tagc.cggg caattt attt accga ggaagaattalagg tgataaggagcc.gt aataaaaggagggat ggga Caaggggga aacATG gcca ATG ataATG

5'- 5'- 5'- 5'- gcgtag tagc.cggg caatttatttaccga TCTTCCCACAACA GAAATTTCATACC tgataaggagcc.gt aataaaaggagggat CTGGCGGACTCCA ACAGGCGAAGGAG aacATG gcca ATG TCATG GAAAAACCATG

105F 5'- 5'- 5'- 5'- gcgtag tagc.cggg caatttattitttgtt TCTTCCCACAACA GAAATTTCATACC tgataaggagcc.gt cacccaaggagtatt CTGGCGGACTCCA ACAGGCGAAGGAG aacATG gctaATG TCATG GAAAAACCATG US 2017/01 01638 A1 Apr. 13, 2017 20

0209. The expression performance of the RBS-optimized tokadaii and E. coli ydfG providing a 3-HP dehydrogenase H. elongata ACCases was evaluated by 3-HP production in to complete the metabolic pathway from malonyl-CoA to a 96-well format, each in triplicate wells, and the averaged 3HP. These results show that the strain with the fused accDA results shown in Table 3. Specific 3HP production is shown genes had higher average specific productivity of 3-HP as g/L per ODoo. As may be seen in Table 3, enhancing the compared to the parental strain in which the overexpressed efficiency of the RBS in strains B2, 35C, and 72 B clearly ACCase is not fused. FIG. 6 shows that the benefit of the resulted in increased malonyl-CoA production leading to accDA fusion were also manifested in 3-HP production in increased 3-HP production. It is evident from these results fermentors with environmental controls of nutrient feed, pH, that combinations of enhanced RBS's before each of the aeration, and temperature. individual genes accA, accB, accC, and accD may result in strains with even higher ACCase expression and activity. Table 4: 0214 TABLE 3 TABLE 4 Improvement in 3-HP production by RBS-optimized expression of H. elongata ACCase subunits. ACC Fusions and ACCase activity H. elongata ACCase 3HP AVg specific Avg specific ACCase expression plasmid (g/l OD) Strain prodin rate prodin rate specific activity desig- (ggDCW h) (ggDCW hr) at TS - 6 Parent 2-4 O.O6 nation Plasmid at TS - 6 at TS - 20 (Umg) B2 O.81 13A O.O1 BX3 783 Parent O-160 O.146 0.057 14C O.S4 (unfused 15C O.14 ACCase) 17C O.08 BX3 829 No ACC O.069 O.062 O.OOO 35C O.68 BX3 837 ECACC O.209 O.2O1 O.OS4 36C O.31 DA fusion 36C-8 O.31 72B 0.57 105F O.19 Example 5 3-HP Exporter EXAMPLE 4 0215 Growth inhibition has been demonstrated for E. coli strains grown in the presence of 3-HP at levels as low Coordinated Expression by Subunit Fusions as 20 g/L. To produce high titers of 3-1-IP the production 0210. In nature ACCase subunit genes from prokaryotes host is required to balance production with overcoming Such as E. coli and H. elongata have been shown to have a inhibition. A known chemical exporter from E. coli that has quaternary structure: accAaccDaccE3accC. However, been previously characterized for homoserine transport, the intrinsic levels of the ACCase subunit genes are too low rhtA, was evaluated for increased production of 3-HP. A for optimal production. Therefore, for optimal production it mutant version of the exporter, rhtA(P2S) (SEQ ID NO. 30 is ideal to have overexpression to be coordinated in a similar nucleic acid, SEQ ID NO. 31 protein) was synthesized a. behind the PtpiA promoter and inserted into the pTRC 0211 Expression of the genes encoding each ACCase PyibD-MCR plasmid behind a terminator using the Gibson Subunit is regulated at transcriptional and translational lev Assemply kit (NEB) according to manufacturer's instruc els. Coordinated overexpression of the AC:Case subunit tions. The effects of overexpression of rhtA were evaluated genes, accA, accB, accC, accD should give better ACCase in IL fermentation compared to the control plasmid without activity. Examples of fusions of accC-B proteins exist in rhtA. As shown in FIG. 7, overexpression of rhtA resulted in bacteria. It is hypothesized that coordinated overexpression a significant improvement in 3HP titer compared to the may be achieved by fusion of Subunit genes should ensures control production strain. Construction of plasmids express equimolar expression of the Subunit genes at the optimal ing another putative transporter, ydcC) (SEQ ID NO. 32 time. nucleic acid, SEQ ID NO. 33 protein) is carried out in the 0212. The following ACCase subunit gene fusion were Sale a. constructed and the constructs overexpressed in E. coli: (A) Example 6 Control ABCD, (B) fusion of accC-B (SEQ ID NOS 9, 10) Subunit genes as seen in bacteria, (C) fusion of accD-A Bicarbonate Importer Prophetic Subunit genes using a flexible 15-amino acid linker (Linker 0216) Increased import of bicarbonate to increase avail sequence LSGGGGSGGGGSGGGGSGGGGSAAA; SEQ ability of bicarbonate for the ACCase reaction will increase ID NOs 11, 12) as depicted in FIG. 5. production of inalonyl-CoA and hence products derived 0213. The performance of the ACC fusions were tested metabolically from malonyl-CoA, such as 3-HP. The gene for their ACCase activity and for various 3-HP production encoding the bicA bicarbonate transporter (SEQ lD NO. 13) metrics in Table 4. ACCase activity was determined in cell of Synechococcus sp. was synthesized using codons opti lysates using an assay for malonyl-CoA production as mized for expression in E. coli (SEQ ID NO. 14) and described in Kroeger, Zarzycki, and Fuchs, Analytical expressed using the E. colital promoter in a strain cotrans Biochem. 411: 100-105, 2011. Production of 3-HP was formed with plasmids encoding ACCase and MCR func determined in cells co-transformed with a plasmid bearing tions. Production of 3-HP by this strain is compared to that the genes encoding the malonyl-CoA reductase from S. achieved by a control strain without overexpressed bicA.

US 2017/01 01638 A1 Apr. 13, 2017 37

- Continued

SEQUENCE LISTING

ASP LEU GLU WAL MET ASP GLY SER ASP PRO ALA. ALA. WAL ARG ALA GLY 65 70 7s 8O

WAL. ALA. ALA ILE ILE GLY ARG HIS GLY HIS ILE ASP ILE LEU WAL ASN 85 90 95

ASN ALA GLY SER THR GLY ALA GLN ARG ARG LEU ALA GLU ILE PRO LEU 1OO 105 11O

ASN GLU THR ASP ARG ASP LEU ASP ASP GLU GLU ALA LEU SER THR SER 115 12O 125

WAL. ALA. ASN LEU LEU GLY MET ALIA TRP HIS LEU MET ARG ILE LEU SER 13 O 135 14 O

PRO HIS MET PRO PRO GLY SER ALA ILE ILE ASN ILE SER THR ILE PHE 145 15 O 155 16 O

SER ARG ALA GLU TYR TYR GLY ARG ILE PRO TYR WAL WAL PRO LYS ALA 1.65 17 O 17s

ALA LEU ASN THR LEU THR GLN ILE ALA. ALA. ARG GLU LEU GLY ILE 18O 185 190

GLY ILE ARG WAL ASN THR ILE PHE PRO GLY PRO ILE GLU SER GLU 195 2OO 2O5

ILE GLIN THR WAL PHE GLN ARG MET ASP GLN LEU LYS GLY ARG PRO GLU 21 O 215 22 O

GLY ASP THR ALA SER GLN PHE LEU ALA. THR MET ARG LEU TYR ALA 225 23 O 235 24 O

ASN ASP GLN GLY GLN LEU GLU ARG ARG PHE PRO THR ILE CYS ASP WAL 245 250 255

ALA. ASP ALA. ALA, WAL PHE LEU ALA. SER ASP GLU ALA. ALA. ALA LEU THR 26 O 265 27 O

GLY GLU THR ILE GLU WAL THR HIS GLY MET GLU LEU PRO THR SER SER 27s 28O 285

GLU THR SER LEU LEU ALA. ARG THR ASP LEU ARG THR ILE ASP ALA ASN 29 O 295 3OO

GLY ARG THR THR LEU ILE CYS ALA GLY ASP GLN ILE GLU GLU WAL MET 3. OS 310 315 32O

ALA LEU THR GLY MET LEU ARG THR CYS GLY SER GLU WAL ILE ILE GLY 3.25 330 335

PHE ARG SER GLU ALA. ALA LEU ALA GLN PHE GLU GLN ALA ILE GLY GLU 34 O 345 350

SER ARG ARG LEU ALA GLY GLU SER PHE ILE PRO PRO ILE ALA LEU PRO 355 360 365

ILE ASP LEU ARG ASN PRO SER THR ILE ASP ALA LEU PHE ASP TRP ALA 37 O 375 38O

GLY GLU ASN THR GLY GLY ILE HIS ALA. ALA. WAL, ILE LEU PRO ALA SER 385 390 395 4 OO

GLY ARG GLU PRO ALA. THR GLN WAL, ILE ASP ILE ASP ASP ALA HIS WAL 405 41 O 415

GLN ALA PHE LEU ASN ASP GLU ILE WAL, GLY SER ILE ILE ILE ALA SER 42O 425 43 O

ARG LEU ALA. ARG TYR TRP GLN ALA GLN ARG ILE ALA PRO GLY ALA 435 44 O 445

ALA. ARG GLU PRO ARG WAL, ILE PHE LEU SER ASN GLY ALA. SER THR ALA 450 45.5 460 US 2017/01 01638 A1 Apr. 13, 2017 38

- Continued

SEQUENCE LISTING

GLY ASN PRO TYR GLY ARG ILE GLN SER ALA. ALA ILE GLU LEU ILE 465 470

ARG WAL TRP ARG HIS GLU ALA. ALA, LEU ASP TYR GLU ARG THR ALA 485 490 495

ALA GLY GLU ARG WAL LEU PRO ALA WAL TRP ALA SER GLN WAL 5 OO 5 OS 510

PHE ALA. ASN ARG SER LEU GLU GLY LEU GLU PHE CYS TRP THR 515 52O 525

ALA GLN LEU LEU HIS SER GLN ARG ARG ILE ASN ILE LEU THR 53 O 535 54 O

ILE PRO ALA ASP ILE SER ALA THR THR GLY ALA SER SER WAL 550 555 560

GLY TRP ALA GLU SER LEU ILE GLY LEU HIS LEU LYS WAL ALA LEU 565 st O sfs

ILE THR GLY GLY SER ALA GLY ILE GLY GLY GLY LEU LEU 58 O 585 590

ALA LEU SER GLY ALA. ARG WAL MET LEU ALA ASP PRO HIS LYS 595 6OO 605

LEU GLU GLN ILE GLN ALA THR ILE ARG ALA LEU ALA GLU WAL GLY 610 615 62O

TYR THR ASP WAL, GLU GLU ARG WAL, GLN ILE PRO GLY CYS ASP WAL 625 630 635 64 O

SER SER GLU GLU GLN LEU WAL ASP LEU WAL ARG THR LEU ALA ALA 645 650 655

PHE GLY THR WAL ASP TYR LEU ILE ASN ASN GLY ILE ALA GLY WAL 660 665 670

GLU GLU MET WAL, ILE ASP MET PRO WAL GLU TRP ARG ASN THR LEU 675 68O 685

TYR ALA. ASN LEU ILE SER ASN TYR SER LEU MET ARG LYS LEU ALA PRO 69 O. 695 7 OO

LEU MET LYS LYS GLN GLY SER GLY TYR WAL LEU ASN WAL SER SER TYR 7 Os 71O 71s 72O

PHE GLY GLY GLU LYS ASP ALA. ALA ILE PRO TYR PRO ASN ALA ASP 72 73 O 73

TYR ALA. WAL SER LYS ALA GLY GLN ARG ALA MET ALA GLU WAL PHE ALA 74 O 74. 7 O

ARG. PHE LEU GLY PRO GLU ILE GLN ILE ASN ALA ILE ALA PRO GLY PRO 7ss 760 765

WAL, GLU GLY ASP ARG LEU ARG GLY THR GLY GLU ARG PRO GLY LEU PHE 770 77 78O

ALA. ARG ARG ALA. ARG LEU ILE LEU GLU ASN LYS ARG LEU ASN GLU LEU 78s 79 O 79. 8 OO

HIS ALA. ALA LEU ILE THR ALA. ALA. ARG THR ASP ASN ARG PRO MET 805 810 815

GLU LEU WAL GLU LEU LEU LEU PRO ASN ASP WAL ALA ALA LEU ALA GLN 82O 825 830

HIS PRO ALA ALA PRO ASP WAL LEU ARG THR LEU ALA LYS PHE GLN 835 84 O 845

SER GLU GLY ASP PRO ALA ALA SER SER SER SER PHE LEU LEU ASN 850 855 860 US 2017/01 01638 A1 Apr. 13, 2017 39

- Continued

SEQUENCE LISTING

SER ILE ALA. ALA LYS LEU LEU ALA. ARG LEU ILE ASN GLY GLY TYR ASP 865 87O 87s 88O

LEU PRO ALA. ASP ILE PHE ALA. ASN LEU ALA. WAL PRO PRO ASP PRO PHE 885 890 895

PHE THR ARG ALA GLN ILE ASP ARG GLU ALA. ARG LYS WAL ARG ASP GLY 9 OO 9 OS 910

ILE MET GLY MET LEU TYR LEU GLN ARG MET PRO THR GLU PHE ASP WAL 915 92 O 925

ALA MET ALA. THR WAL. TYR TYR LEU ALA. ASP ARG ASN WAL SER GLY GW 93 O 935 94 O

THR PHE HIS PRO SER GLY GLY LEU ARG. TYR GW ARG THR PRO THR GLY 945 950 955 96.O

GLY GLU LEU PHE GLY LEU PRO ALA PRO GLU ARG LEU ALA GLU LEU WAL 965 97O 97.

GLY SER THR WAL. TYR LEU ILE GLY GLU HIS LEU THR GLU HIS LEU ASN 98O 985 990

LEU LEU ALA. ARG ALA TYR LEU GLU ARG TYR GLY ALA. ARG GLN WAL WAL 995 1 OOO 1005

MET ILE WAL, GLU THR GLU ALA GLY ALA GLU LYS MET ARG HIS LEU O1O 1015 102O

LEU HIS ASP HIS WAL GLU ALA GLY ARG LEU PRO ILE ILE WAL. ALA O25 1O3O O35

GLY ASP GLN ILE GLU ALA. ALA ILE ASP GLN ALA ILE ALA. ASN TYR O4 O O45 OSO

GLY ARG PRO GLY PRO WAL WAL CYS THR PRO PHE ARG PRO LEU PRO

SER ALA PRO LEU WAL GLY ARG LYS ASP SER ASP TRP SER THR WAL

LEU SER GLU ALA GLU PHE ALA GLU LEU CYS GLU HIS GLN LEU THR

HIS HIS PHE ARG WAL. ALA. ARG LYS ILE ALA LEU SER ASP GLY ALA

SER LEU ALA LEU WAL THR PRO GLU THR THR ALA. THR SER SER THR 15 2O 25

GLU GLN PHE ALA LEU ALA. ASN PHE WAL LYS THR THR LEU HIS ALA 3O 35 4 O

PHE THR ALA. THR ILE GLY WAL, GLU SER GLU ARG THR ALA GLN ARG 45 SO 55

ILE LEU ILE ASN GLN WAL ASP LEU THR ARG ARG ALA. ARG ALA GLU 60 65 70

GLU PRO ARG ASP PRO ARG GLU ARG GLN GLN GLU LEU GLU ARG PHE 7s 8O 85

ILE GLU ALA. WAL LEU LEU WAL THR ALA PRO LEU PRO PRO GLU ALA 90 95 2OO

ASP THR ARG TYR ALA GLY ARG ILE HIS ARG GLY ARG ALA ILE THR WAL 2O5 21 O 215

SEO ID NO. 108 : MCR OSCILLOCHLORIS TRICHOIDES (OTMCR) MFMTRLNDKIALITGGAGTIGEWITRRYLEEGATWWMAGRNRDKLDRYRERLITEFHALP ERVMVVRMDGSSNAEWRMGIAAV VAHFGRIDILVNNAGSAGARORLPAIPLLRSELOADE TETLADSIGNLIGITWNLIRAAAPFMPAGSSWINISTIFARTDYYGRIPYWWPKAALHAL

US 2017/01 01638 A1 Apr. 13, 2017 41

- Continued

Ala Ile Val Gly Gly Val Ala Arg Lieu. Asp Asp Llys Pro Val Met Val 1OO 105 11 O Ile Gly His Gln Lys Gly Arg Asp Wal His Glu Lys Val Arg Arg Asn 115 12 O 125 Phe Gly Met Pro Arg Pro Glu Gly Tyr Arg Lys Ala Cys Arg Lieu Met 13 O 135 14 O Glu Met Ala Glu Arg Phe His Met Pro Val Lieu. Thr Phe Ile Asp Thr 145 150 155 160 Pro Gly Ala Tyr Pro Gly Ile Asp Ala Glu Glu Arg Gly Glin Ser Glu 1.65 17O 17s Ala Ile Ala Tyr Asn Lieu. Gly Val Met Ser Arg Lieu Lys Thr Pro Ile 18O 185 19 O Ile Ser Thr Val Val Gly Glu Gly Gly Ser Gly Gly Ala Lieu Ala Ile 195 2OO 2O5

Gly Val Cys Asp Glu Lieu. A a. Met Leu Gln Tyr Ser Thr Tyr Ser Val 21 O 2 5 22O Ile Ser Pro Glu Gly Cys Ala Ser Ile Lieu. Trp Llys Ser Ala Asp Llys 225 23 O 235 24 O Ala Ser Glu Ala Ala Glin Ala Met Gly Ile Thr Ala Glu Arg Lieu Lys 245 250 255 Glu Lieu. Gly Phe Val Asp Thir Lieu. Ile Pro Glu Pro Lieu. Gly Gly Ala 26 O 265 27 O His Arg Glin Pro Ser Ala Thr Ala Glu Arg Ile Llys Thr Ala Lieu. Lieu. 27s 28O 285 Glu Ser Lieu. Asp Arg Lieu. Glu Thir Met Glu Thir Asp Ala Lieu. Lieu. Glu 29 O 295 3 OO Arg Arg Tyr Glu Arg Lieu Met Ser Tyr Gly Ala Pro Wall 3. OS 310 315

<210s, SEQ ID NO 2 &211s LENGTH: 954 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide

<4 OOs, SEQUENCE: 2 atgaatccga act atctgga ctittgaacaa ccgatcqctgaactgcaa.gc caaaatcgaa 6 O gaactg.cgta tigtgggcaa cact cacag gtgaacctgt Ctgatgaaat tdoctotg 12 O gaagaaaaaa gtc.gcaaact gaccgaatcc atctittaaag acctgtcagc gtggcaagtt 18O agccaactgt citcgtcatcc gcaacgc.ccg tataccctgg attacctgga acatgtctitt 24 O acggatttic acgaactgca C9gtgaccgt. c9ctittgcag atgacgcggc cattgttggc 3OO ggtgtc.gctic gtctggatga caaac C9gtc atggtgatcg gcc at Cagaa aggtogtgat 360 gtgcacgaaa aagttctgtcg caact tcggc atgcc.gc.gcc cqgaaggitta t cqtaaag.cg 42O tgcc.gc.ctga tiggaaatggc cgaacgctitt Cacatgc.cgg totgacctt cattgatacg 48O ccgggcgcat atc.cgggitat cacgctgaa gaacgtggcc aaag.cgaagc gattgcctac 54 O aatctgggtg titatgtc.gcg cctgaaaacc ccgattatca gcacggtggit tdgcgaaggc 6OO ggttctggcg gtgcactggc tat cqgtgtc. tcc.gatgaac togcgatgct gcaat at agt 660

US 2017/01 01638 A1 Apr. 13, 2017 43

- Continued

<210s, SEQ ID NO 5 &211s LENGTH: 446 212. TYPE: PRT <213> ORGANISM: Halomonas elongata

<4 OOs, SEQUENCE: 5 Met Lieu. Asp Llys Val Lieu. Ile Ala Asn Arg Gly Glu Ile Ala Lieu. Arg 1. 5 1O 15 Ile Lieu. Arg Ala Cys Lys Glu Lieu. Gly Ile Arg Thr Val Ala Val His 2O 25 3O Ser Lys Ala Asp Arg Glu Lieu Met His Val Arg Lieu Ala Asp Glu Ala 3 5 4 O 45 Val Cys Ile Gly Pro Ala Ser Ser Ala Glin Ser Tyr Lieu. Asn Ile Pro SO 55 6 O Ala Lieu. Ile Ser Ala Ala Glu Val Thir Asp Thir Ser Ala Ile His Pro 65 70 7s 8O Gly Tyr Gly Phe Lieu. Ser Glu Asn Ala Asp Phe Ala Glu Glin Val Glu 85 90 95 Arg Ser Gly Phe Thr Phe Ile Gly Pro Ser Ala Glu Thir Ile Arg Lieu. 1OO 105 11 O Met Gly Asp Llys Val Ser Ala Ile Asn Ala Met Lys Glu Ala Gly Val 115 12 O 125 Pro Thr Val Pro Gly Ser Asn Gly Pro Leu Gly Asp Asp Glu Gly Glu 13 O 135 14 O Ile Lieu Ala Thir Ala Arg Arg Ile Gly Tyr Pro Val Ile Ile Lys Ala 145 150 155 160 Ala Ala Gly Gly Gly Gly Arg Gly Met Arg Val Val His Ala Glu Gly 1.65 17O 17s His Lieu. Lieu. Ser Ala Val Asn Val Thr Arg Thr Glu Ala His Ser Ser 18O 185 19 O Phe Gly Asp Gly Thr Val Tyr Met Glu Lys Phe Lieu. Glu Asn Pro Arg 195 2OO 2O5 His Val Glu Val Glin Val Lieu Ala Asp Gly Glin Gly Asn Ala Ile His 21 O 215 22O Lieu. Tyr Asp Arg Asp Cys Ser Lieu. Glin Arg Arg His Glin Llys Val Lieu. 225 23 O 235 24 O Glu Glu Ala Pro Ala Pro Gly Lieu. Asp Glin Glin Ala Arg Glu Glin Val 245 250 255 Phe Lys Ala Cys Arg Asp Ala Cys Val Lys Ile Gly Tyr Arg Gly Ala 26 O 265 27 O Gly Thr Phe Glu Phe Lieu. Tyr Glu Asn Gly Glu Phe Phe Phe Ile Glu 27s 28O 285 Met Asn Thr Arg Val Glin Val Glu. His Pro Val Thr Glu Met Val Thr 29 O 295 3 OO

Gly Val Asp Ile Val Arg Glu Glin Lieu. Arg Ile Ala Ser Gly Lieu Pro 3. OS 310 315 32O Lieu. Ser Ile Arg Glin Glu Asp Val Glu Lieu. Ser Gly His Ala Phe Glu 3.25 330 335

Cys Arg Ile Asn Ala Glu Asp Ser Arg Thr Phe Met Pro Ser Pro Gly 34 O 345 35. O Arg Val Thr Lieu. Tyr His Pro Pro Gly Gly Lieu. Gly Val Arg Met Asp 355 360 365

US 2017/01 01638 A1 Apr. 13, 2017 45

- Continued <213> ORGANISM: Halomonas elongata <4 OO > SEQUENCE: 7 Met Ser Trp Lieu. Asp Llys Ile Val Pro Ser Val Gly Arg Ile Glin Arg 1. 5 1O 15 Lys Glu Arg Arg Thr Ser Val Pro Asp Gly Lieu. Trp Arg Lys Cys Pro 2O 25 3O Lys Cys Glu Ser Val Lieu. Tyr Lieu Pro Glu Lieu. Glu Lys His His Asn 35 4 O 45 Val Cys Pro Llys Cys Asp His His Lieu. Arg Lieu. Thir Ala Arg Lys Arg SO 55 6 O Lieu. Asp Trp Phe Lieu. Asp Llys Glu Gly Arg Glu Glu Ile Ala Ala Asp 65 70 7s 8O Lieu. Glu Pro Val Asp Arg Lieu Lys Phe Arg Asp Ser Llys Llys Tyr Lys 85 90 95 Asp Arg Lieu. Ser Ala Ala Glin Lys Ala Thr Gly Glu Lys Asp Gly Lieu 1OO 105 11 O Val Ala Met Arg Gly. Thir Lieu. Glu Gly Lieu Pro Val Val Ala Val Ala 115 12 O 125 Phe Glu Phe Thr Phe Met Gly Gly Ser Met Gly Ala Val Val Gly Glu 13 O 135 14 O Llys Phe Val Arg Ala Ala Thr Glin Ala Lieu. Asp Glu Gly Val Pro Lieu. 145 150 155 160 Val Cys Phe Ser Ala Ser Gly Gly Ala Arg Met Glin Glu Ala Lieu. Phe 1.65 17O 17s Ser Lieu Met Gln Met Ala Lys Thir Ser Ala Ala Lieu. Glu Lys Lieu Lys 18O 185 19 O Glin Ala Gly Val Pro Tyr Ile Ser Val Lieu. Thir Asp Pro Val Phe Gly 195 2OO 2O5 Gly Val Ser Ala Ser Lieu Ala Met Lieu. Gly Asp Lieu. Asn. Ile Ala Glu 21 O 215 22O Pro Asn Ala Lieu. Ile Gly Phe Ala Gly Pro Arg Val Ile Glu Glin Thr 225 23 O 235 24 O Val Arg Glu Glin Lieu Pro Glu Gly Phe Glin Arg Ser Glu Phe Lieu. Lieu 245 250 255 Glu. His Gly Ala Val Asp Met Ile Val His Arg Glin Glin Ile Arg Glu 26 O 265 27 O Arg Lieu. Gly Gly Val Lieu. Arg Llys Lieu. Thir His Glin Pro Ala Ser Gly 27s 28O 285 Pro Ala Val Val Glu Asn Asp Glu Pro Asp Lieu Val Asp Ala Ala Glu 29 O 295 3 OO

Glin Ala Glu Pro Glin Pro Glu Ala Pro Glu Ala Wall Glu Thir Ser Glu 3. OS 310 315 32O

Ser Glu Ala Pro Thr Glu Lys Gly Val Glu Ala Asp Ser Glu Glu Thir 3.25 330 335

Asp Glu Ser Pro Arg Ser Gly Asp Asn Arg 34 O 345

<210s, SEQ ID NO 8 &211s LENGTH: 1041 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE:

US 2017/01 01638 A1 Apr. 13, 2017 47

- Continued Pro Cys Val Pro Gly Ser Asp Gly Pro Lieu. Gly Asp Asp Met Asp Llys 13 O 135 14 O Asn Arg Ala Ile Ala Lys Arg Ile Gly Tyr Pro Val Ile Ile Lys Ala 145 150 155 160 Ser Gly Gly Gly Gly Gly Arg Gly Met Arg Val Val Arg Gly Asp Ala 1.65 17O 17s Glu Lieu Ala Glin Ser Ile Ser Met Thr Arg Ala Glu Ala Lys Ala Ala 18O 185 19 O Phe Ser Asn Asp Met Val Tyr Met Glu Lys Tyr Lieu. Glu Asn Pro Arg 195 2OO 2O5 His Val Glu Ile Glin Val Lieu Ala Asp Gly Glin Gly Asn Ala Ile Tyr 21 O 215 22O Lieu Ala Glu Arg Asp Cys Ser Met Glin Arg Arg His Glin Llys Val Val 225 23 O 235 24 O Glu Glu Ala Pro Ala Pro Gly Ile Thr Pro Glu Lieu. Arg Arg Tyr Ile 245 250 255 Gly Glu Arg Cys Ala Lys Ala Cys Val Asp Ile Gly Tyr Arg Gly Ala 26 O 265 27 O Gly Thr Phe Glu Phe Leu Phe Glu Asn Gly Glu Phe Tyr Phe Ile Glu 27s 28O 285 Met Asn Thr Arg Ile Glin Val Glu. His Pro Val Thr Glu Met Ile Thr 29 O 295 3 OO Gly Val Asp Lieu. Ile Lys Glu Gln Lieu. Arg Ile Ala Ala Gly Glin Pro 3. OS 310 315 32O Lieu. Ser Ile Lys Glin Glu Glu Val His Val Arg Gly His Ala Val Glu 3.25 330 335 Cys Arg Ile Asn Ala Glu Asp Pro Asn Thr Phe Leu Pro Ser Pro Gly 34 O 345 35. O Lys Ile Thr Arg Phe His Ala Pro Gly Gly Phe Gly Val Arg Trp Glu 355 360 365 Ser His Ile Tyr Ala Gly Tyr Thr Val Pro Pro Tyr Tyr Asp Ser Met 37 O 375 38O Ile Gly Lys Lieu. Ile Cys Tyr Gly Glu Asn Arg Asp Val Ala Ile Ala 385 390 395 4 OO Arg Met Lys Asn Ala Lieu. Glin Glu Lieu. Ile Ile Asp Gly Ile Llys Thr 4 OS 41O 415 Asn Val Asp Lieu. Glin Ile Arg Ile Met Asn Asp Glu Asn. Phe Gln His 42O 425 43 O Gly Gly. Thir Asn. Ile His Tyr Lieu. Glu Lys Llys Lieu. Gly Lieu. Glin Glu 435 44 O 445 Lys Asp Ile Arg Lys Ile Llys Llys Lieu. Ile Glu Lieu Val Glu Glu Ser 450 45.5 460

Gly Ile Ser Glu Lieu. Glu Ile Ser Glu Gly Glu Glu Ser Val Arg Ile 465 470 47s 48O

Ser Arg Ala Ala Pro Ala Ala Ser Phe Pro Val Met Glin Glin Ala Tyr 485 490 495

Ala Ala Pro Met Met Glin Glin Pro Ala Glin Ser Asn Ala Ala Ala Pro SOO 505 51O

Ala Thr Val Pro Ser Met Glu Ala Pro Ala Ala Ala Glu Ile Ser Gly 515 52O 525

His Ile Val Arg Ser Pro Met Val Gly Thr Phe Tyr Arg Thr Pro Ser

US 2017/01 01638 A1 Apr. 13, 2017 49

- Continued ccggcagcgg cc.gagattitc gggtcat atc gtgcgtagcc catggtggg Cacct tctat 162O cgcacgc.cgt. c9ccggacgc aaaagcct tc atcgaagttcg gcc agaaggt caatgtcggc 168O gacacgctgt gitatcgttga ggcaatgaaa atgatgalacc agattgaagc ggatalagagc 1740 ggtactgtta aag.cgatcct ggtggaatcc ggc.ca.gc.ctg. ittgagttcga tigaac.cgctg 18OO gttgttgat cq agtaa 1815

<210s, SEQ ID NO 11 &211s LENGTH: 647 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 11 Met Ser Trp Ile Glu Arg Ile Llys Ser Asn Ile Thr Pro Thr Arg Lys 1. 5 1O 15 Ala Ser Ile Pro Glu Gly Val Trp Thr Lys Cys Asp Ser Cys Gly Glin 2O 25 3O Val Lieu. Tyr Arg Ala Glu Lieu. Glu Arg Asn Lieu. Glu Val Cys Pro Llys 35 4 O 45 Cys Asp His His Met Arg Met Thr Ala Arg Asn Arg Lieu. His Ser Lieu. SO 55 6 O Lieu. Asp Glu Gly Ser Lieu Val Glu Lieu. Gly Ser Glu Lieu. Glu Pro Llys 65 70 7s 8O Asp Val Lieu Lys Phe Arg Asp Ser Lys Llys Tyr Lys Asp Arg Lieu Ala 85 90 95 Ser Ala Glin Lys Glu Thr Gly Glu Lys Asp Ala Lieu Val Val Met Lys 1OO 105 11 O Gly Thr Lieu. Tyr Gly Met Pro Val Val Ala Ala Ala Phe Glu Phe Ala 115 12 O 125 Phe Met Gly Gly Ser Met Gly Ser Val Val Gly Ala Arg Phe Val Arg 13 O 135 14 O Ala Val Glu Glin Ala Lieu. Glu Asp Asn. Cys Pro Lieu. Ile Cys Phe Ser 145 150 155 160 Ala Ser Gly Gly Ala Arg Met Glin Glu Ala Lieu Met Ser Lieu Met Glin 1.65 17O 17s Met Ala Lys Thir Ser Ala Ala Lieu Ala Lys Met Glin Glu Arg Gly Lieu. 18O 185 19 O Pro Tyr Ile Ser Val Lieu. Thr Asp Pro Thr Met Gly Gly Val Ser Ala 195 2OO 2O5 Ser Phe Ala Met Lieu. Gly Asp Lieu. Asn. Ile Ala Glu Pro Lys Ala Lieu. 21 O 215 22O

Ile Gly Phe Ala Gly Pro Arg Val Ile Glu Glin Thr Val Arg Glu Lys 225 23 O 235 24 O

Lieu Pro Pro Gly Phe Glin Arg Ser Glu Phe Lieu. Ile Glu Lys Gly Ala 245 250 255

Ile Asp Met Ile Val Arg Arg Pro Glu Met Arg Lieu Lys Lieu Ala Ser 26 O 265 27 O

Ile Lieu Ala Lys Lieu Met Asn Lieu Pro Ala Pro Asn Pro Glu Ala Pro 27s 28O 285 US 2017/01 01638 A1 Apr. 13, 2017 50

- Continued

Arg Glu Gly Val Val Val Pro Pro Val Pro Asp Gln Glu Pro Glu Ala 29 O 295 3 OO Lieu. Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 3. OS 310 315 32O Ser Gly Gly Gly Gly Ser Ala Ala Ala Ser Lieu. Asn. Phe Lieu. Asp Phe 3.25 330 335 Glu Gln Pro Ile Ala Glu Lieu. Glu Ala Lys Ile Asp Ser Lieu. Thir Ala 34 O 345 35. O Val Ser Arg Glin Asp Glu Lys Lieu. Asp Ile Asn. Ile Asp Glu Glu Val 355 360 365 His Arg Lieu. Arg Glu Lys Ser Val Glu Lieu. Thir Arg Lys Ile Phe Ala 37 O 375 38O Asp Lieu. Gly Ala Trp Glin Ile Ala Glin Lieu Ala Arg His Pro Glin Arg 385 390 395 4 OO Pro Tyr Thr Lieu. Asp Tyr Val Arg Lieu Ala Phe Asp Glu Phe Asp Glu 4 OS 41O 415 Lieu Ala Gly Asp Arg Ala Tyr Ala Asp Asp Lys Ala Ile Val Gly Gly 42O 425 43 O Ile Ala Arg Lieu. Asp Gly Arg Pro Val Met Ile Ile Gly His Glin Lys 435 44 O 445 Gly Arg Glu Thir Lys Glu Lys Ile Arg Arg Asn. Phe Gly Met Pro Ala 450 45.5 460 Pro Glu Gly Tyr Arg Lys Ala Lieu. Arg Lieu Met Gln Met Ala Glu Arg 465 470 47s 48O Phe Llys Met Pro Ile Ile Thr Phe Ile Asp Thr Pro Gly Ala Tyr Pro 485 490 495 Gly Val Gly Ala Glu Glu Arg Gly Glin Ser Glu Ala Ile Ala Arg Asn SOO 505 51O Lieu. Arg Glu Met Ser Arg Lieu. Gly Val Pro Val Val Cys Thr Val Ile 515 52O 525 Gly Glu Gly Gly Ser Gly Gly Ala Lieu Ala Ile Gly Val Gly Asp Llys 53 O 535 54 O Val Asn Met Leu Gln Tyr Ser Thr Tyr Ser Val Ile Ser Pro Glu Gly 5.45 550 555 560 Cys Ala Ser Ile Lieu. Trp Llys Ser Ala Asp Lys Ala Pro Lieu Ala Ala 565 st O sts Glu Ala Met Gly Ile Ile Ala Pro Arg Lieu Lys Glu Lieu Lys Lieu. Ile 58O 585 59 O Asp Ser Ile Ile Pro Glu Pro Lieu. Gly Gly Ala His Arg Asn Pro Glu 595 6OO 605 Ala Met Ala Ala Ser Lieu Lys Ala Glin Lieu. Lieu Ala Asp Lieu Ala Asp 610 615 62O

Lieu. Asp Val Lieu. Ser Thr Glu Asp Lieu Lys Asn Arg Arg Tyr Glin Arg 625 630 635 64 O

Lieu Met Ser Tyr Gly Tyr Ala 645

<210s, SEQ ID NO 12 &211s LENGTH: 1944 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic

US 2017/01 01638 A1 Apr. 13, 2017 52

- Continued

<4 OOs, SEQUENCE: 13 Met Glin Ile Thr Asn Lys Ile His Phe Arg Asn. Ile Arg Gly Asp Ile 1. 5 1O 15 Phe Gly Gly Lieu. Thir Ala Ala Val Ile Ala Lieu Pro Met Ala Lieu Ala 2O 25 3O Phe Gly Val Ala Ser Gly Ala Gly Ala Glu Ala Gly Lieu. Trp Gly Ala 35 4 O 45 Val Lieu Val Gly Phe Phe Ala Ala Leu Phe Gly Gly Thr Pro Thr Lieu. SO 55 6 O Ile Ser Glu Pro Thr Gly Pro Met Thr Val Val Met Thr Ala Val Ile 65 70 7s 8O Ala His Phe Thr Ala Ser Ala Ala Thr Pro Glu Glu Gly Lieu Ala Ile 85 90 95 Ala Phe Thr Val Val Met Met Ala Gly Val Phe Glin Ile Ile Phe Gly 1OO 105 11 O Ser Leu Lys Lieu. Gly Lys Tyr Val Thr Met Met Pro Tyr Thr Val Ile 115 12 O 125 Ser Gly Phe Met Ser Gly Ile Gly Ile Ile Leu Val Ile Leu Gln Leu 13 O 135 14 O Ala Pro Phe Leu Gly Glin Ala Ser Pro Gly Gly Gly Val Ile Gly Thr 145 150 155 160 Lieu. Glin Asn Lieu Pro Thr Lieu. Leu Ser Asn Ile Gln Pro Gly Glu Thr 1.65 170 175 Ala Lieu Ala Lieu. Gly. Thr Val Ala Ile Ile Trp Phe Met Pro Glu Lys 18O 185 19 O Phe Llys Llys Val Ile Pro Pro Glin Lieu Val Ala Lieu Val Lieu. Gly Thr 195 2OO 2O5 Val Ile Ala Phe Phe Val Phe Pro Pro Glu Val Ser Asp Lieu. Arg Arg 21 O 215 22O Ile Gly Glu Ile Arg Ala Gly Phe Pro Glu Lieu Val Arg Pro Ser Phe 225 23 O 235 24 O Ser Pro Val Glu Phe Glin Arg Met Ile Lieu. Asp Ala Ala Val Lieu. Gly 245 250 255 Met Lieu. Gly Cys Ile Asp Ala Lieu. Lieu. Thir Ser Val Val Ala Asp Ser 26 O 265 27 O Lieu. Thir Arg Thr Glu. His Asn. Ser Asn Lys Glu Lieu. Ile Gly Glin Gly 27s 28O 285 Lieu. Gly Asn Lieu. Phe Ser Gly Lieu. Phe Gly Gly Ile Ala Gly Ala Gly 29 O 295 3 OO Ala Thr Met Gly Thr Val Val Asn Ile Glin Ser Gly Gly Arg Thr Ala 3. OS 310 315 32O

Lieu. Ser Gly Lieu Val Arg Ala Phe Val Lieu. Lieu Val Val Ile Lieu. Gly 3.25 330 335

Ala Ala Ser Lieu. Thir Ala Thir Ile Pro Lieu Ala Val Lieu Ala Gly Ile 34 O 345 35. O

Ala Phe Llys Val Gly Val Asp Ile Ile Asp Trp Ser Phe Lieu Lys Arg 355 360 365

Ala His Glu Ile Ser Pro Lys Gly Ala Lieu. Ile Met Tyr Gly Val Ile 37 O 375 38O

Lieu. Lieu. Thr Val Lieu Val Asp Lieu. Ile Val Ala Val Gly Val Gly Val 385 390 395 4 OO

US 2017/01 01638 A1 Apr. 13, 2017 55

- Continued

<4 OOs, SEQUENCE: 16 Met Ser Arg Arg Thr Lieu Lys Ala Ala Ile Lieu. Gly Ala Thr Gly Lieu. 1. 5 1O 15 Val Gly Ile Glu Tyr Val Arg Met Leu Ser Asn His Pro Tyr Ile Llys 2O 25 3O Pro Ala Tyr Lieu Ala Gly Lys Gly Ser Val Gly Llys Pro Tyr Gly Glu 35 4 O 45 Val Val Arg Trp Glin Thr Val Gly Glin Val Pro Llys Glu Ile Ala Asp SO 55 6 O Met Glu Ile Llys Pro Thr Asp Pro Llys Lieu Met Asp Asp Wall Asp Ile 65 70 7s 8O Ile Phe Ser Pro Leu Pro Glin Gly Ala Ala Gly Pro Val Glu Glu Gln 85 90 95 Phe Ala Lys Glu Gly Phe Pro Val Ile Ser Asn Ser Pro Asp His Arg 1OO 105 11 O Phe Asp Pro Asp Val Pro Leu Lieu Val Pro Glu Lieu. Asn Pro His Thr 115 12 O 125 Ile Ser Lieu. Ile Asp Glu Glin Arg Lys Arg Arg Glu Trp Llys Gly Phe 13 O 135 14 O Ile Val Thir Thr Pro Leu. Cys Thr Ala Glin Gly Ala Ala Ile Pro Leu 145 150 155 160 Gly Ala Ile Phe Lys Asp Tyr Llys Met Asp Gly Ala Phe Ile Thir Thr 1.65 17O 17s Ile Glin Ser Leu Ser Gly Ala Gly Tyr Pro Gly Ile Pro Ser Lieu. Asp 18O 185 19 O Val Val Asp Asn. Ile Lieu Pro Lieu. Gly Asp Gly Tyr Asp Ala Lys Thr 195 2OO 2O5 Ile Lys Glu Ile Phe Arg Ile Lieu. Ser Glu Val Lys Arg Asn Val Asp 21 O 215 22O Glu Pro Llys Lieu. Glu Asp Wal Ser Lieu Ala Ala Thir Thr His Arg Ile 225 23 O 235 24 O Ala Thr Ile His Gly His Tyr Glu Val Lieu. Tyr Val Ser Phe Lys Glu 245 250 255 Glu Thir Ala Ala Glu Lys Wall Lys Glu Thir Lieu. Glu Asn. Phe Arg Gly 26 O 265 27 O Glu Pro Glin Asp Leu Lys Lieu Pro Thr Ala Pro Ser Lys Pro Ile Ile 27s 28O 285 Val Met Asn. Glu Asp Thr Arg Pro Glin Val Tyr Phe Asp Arg Trp Ala 29 O 295 3 OO Gly Asp Ile Pro Gly Met Ser Val Val Val Gly Arg Lieu Lys Glin Val 3. OS 310 315 32O

Asn Lys Arg Met Ile Arg Lieu Val Ser Lieu. Ile His Asn. Thr Val Arg 3.25 330 335

Gly Ala Ala Gly Gly Gly Ile Lieu Ala Ala Glu Lieu. Lieu Val Glu Lys 34 O 345 35. O

Gly Tyr Ile Glu Lys 355

<210s, SEQ ID NO 17 &211s LENGTH: 1098 &212s. TYPE: DNA

US 2017/01 01638 A1 Apr. 13, 2017 57

- Continued

Glu Thir Ser Met Pro Arg Ala Lieu. Glu Luell Glu Glu Ilie Pro Gly Ile 145 150 155 160

Val Asn Asp Phe Arg Glin Ala Ile Ala Asn Ala Arg Glu Ala Gly Phe 1.65 17O 17s

Asp Lieu Val Glu Lieu. His Ser Ala His Gly Lieu. Lieu. His Glin Phe 18O 185 19 O

Leul Ser Pro Ser Ser Asn His Arg Thir Asp Glin Tyr Gly Gly Ser Val 195

Glu Asn Arg Ala Arg Lieu Val Lieu. Glu Wall Wall Asp Ala Gly Ile Glu 21 O 215

Glu Trp Gly Ala Asp Arg Ile Gly Ile Arg Wall Ser Pro Ile Gly Thr 225 23 O 235 24 O

Phe Glin Asn Thr Asp Asn Gly Pro Asn Glu Glu Ala Asp Ala Leu Tyr 245 250 255

Lieu. Ile Glu Gln Lieu. Gly Lys Arg Gly Ile Ala Tyr Lieu. His Met Ser 26 O 265 27 O

Glu Pro Asp Trp Ala Gly Gly Glu Pro Thir Asp Ala Phe Arg Glu 27s 28O 285

Ala Arg Phe His Gly Pro Ile Ile Gly Ala Gly Ala Tyr 29 O 295 3 OO

Thir Wall Glu Lys Ala Glu Thir Lieu Ile Gly Lys Gly Lieu. Ile Asp Ala 3. OS 310 315 32O

Wall Ala Phe Gly Arg Asp Trp Ile Ala Asn Pro Asp Lieu Val Ala Arg 3.25 330 335

Lieu. Glin Arg Lys Ala Glu Lieu. Asn Pro Glin Arg Ala Glu Ser Phe Tyr 34 O 345 35. O

Gly Gly Gly Ala Glu Gly Tyr Thr Asp Pro Thir Lieu. 355 360 365

<210s, SEQ ID NO 19 &211s LENGTH: 591 212. TYPE : DNA <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 19 atgaacgaag ccgittagc cc aggtgcgctt agcaccctgt t caccgatgc cc.gcact cac 6 O aacggctggc gggaga cacc cgt.ca.gcgat gagacgttac gggagattta tgc cctgatg 12 O aaatgggggc cgacat cagc taactgttct ccggcacgga tcqtgtttac cc.gcacggca 18O gaaggaaaag cc.cggcactt tccagcggca atctgcaaaa aac cctdacc 24 O

cc.gctatogt cgc.ctgggac agtgaattitt atgaacggitt accact actg 3OO titt.ccc cacg Cagttggttt acctic cagoc cacaacttgc cgaagaalaca 360 gcgttt cqca acagttcc at gCagg.cggCC tat ctdatcg tcgc.ctg.ccg ggcgctggga

Ctggat accg gcc.cgatgtc. gggctittgac cgt caac acg tggacgacgc Ctttitttacg 48O ggcago acgc tgalaga.gcaa. tctgctgatt aatat cqgct atggcgatag cagcaa.gctt 54 O tatgcgc.gc.c tgccacgt.ct gtc.ctittgaa ggctgttgta a. 591

<210s, SEQ ID NO 2 O &211s LENGTH: 196 212. TYPE : PRT <213> ORGANISM: Escherichia coli

US 2017/01 01638 A1 Apr. 13, 2017 59

- Continued

<210s, SEQ ID NO 22 &211s LENGTH: 248 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 22 Met Val Val Lieu Val Thr Gly Ala Thr Ala Gly Phe Gly Glu. Cys Ile 1. 5 1O 15 Thir Arg Arg Phe Ile Glin Glin Gly His Llys Val Ile Ala Thr Gly Arg 2O 25 3O Arg Glin Glu Arg Lieu. Glin Glu Lieu Lys Asp Glu Lieu. Gly Asp Asn Lieu 35 4 O 45 Tyr Ile Ala Glin Lieu. Asp Val Arg Asn Arg Ala Ala Ile Glu Glu Met SO 55 6 O Lieu Ala Ser Lieu Pro Ala Glu Trp Cys Asn. Ile Asp Ile Lieu Val Asn 65 70 7s 8O Asn Ala Gly Lieu Ala Lieu. Gly Met Glu Pro Ala His Lys Ala Ser Val 85 90 95 Glu Asp Trp Glu Thir Met Ile Asp Thr Asn. Asn Lys Gly Lieu Val Tyr 1OO 105 11 O Met Thr Arg Ala Val Lieu Pro Gly Met Val Glu Arg Asn His Gly His 115 12 O 125 Ile Ile Asin Ile Gly Ser Thr Ala Gly Ser Trp Pro Tyr Ala Gly Gly 13 O 135 14 O Asn Val Tyr Gly Ala Thr Lys Ala Phe Val Arg Glin Phe Ser Lieu. Asn 145 150 155 160 Lieu. Arg Thr Asp Lieu. His Gly Thr Ala Val Arg Val Thr Asp Ile Glu 1.65 17O 17s Pro Gly Lieu Val Gly Gly Thr Glu Phe Ser Asn Val Arg Phe Lys Gly 18O 185 19 O Asp Asp Gly Lys Ala Glu Lys Thr Tyr Glin Asn Thr Val Ala Lieu. Thr 195 2OO 2O5 Pro Glu Asp Val Ser Glu Ala Val Trp Trp Val Ser Thr Lieu. Pro Ala 21 O 215 22O His Val Asn Ile Asn Thr Lieu. Glu Met Met Pro Val Thr Glin Ser Tyr 225 23 O 235 24 O Ala Gly Lieu. Asn. Wal His Arg Glin 245

<210s, SEQ ID NO 23 &211s LENGTH: 897 &212s. TYPE: DNA <213> ORGANISM: Pseudomonas aeruginosa

<4 OOs, SEQUENCE: 23 atggc.cgaca ttgcgtttct gggtctgggc aatatgggcg gtc.cgatggc cqc galacctg 6 O

Ctgaaag.ccg gccaccgtgt gaatgtgttc gacctgcaac Caaaag.cggit cctgggcttg 12 O gttgagcaag gcgc.gcaggg cqcagactict gct Ctgcaat gttgtgaggg toggaggit c 18O gtgatttcta totgc.ca.gc aggc.cago at gtggaaagcc titacctggg catgatggit 24 O

Ctgctggcac gcgtggcggg caa.gc.ctttg Ctgattgact gtagcac cat cqc accggaa 3OO acggcgcgta aggtggcgga ggcagcc.gca gcaaagggcc tacgctgct ggatgcc.ccg 360 US 2017/01 01638 A1 Apr. 13, 2017 60

- Continued gttt cqggcg gtgtcggtgg toccgtgca ggtacgctgt C9ttt at cqt gggtggit cog 42O gcggagggitt ttgcgc.gtgc gcgt.ccggitt Ctggagaata tdggit cqcala catttitccac 48O gcgggtgatc acggcgctgg to aggtggcg aaaatctgta acaac atgct gctgggt at C 54 O ttgatggcgg gCaccgc.cga agccttggcg Ctgggcgt.ca aaaacggtct ggacccggca 6OO gtgctgtc.cg aagtgatgala acagagcago ggtggtaact gggcgctgaa tictgtacaat 660 cc.gtggc.cgg gtgttgatgcc gcaggc.ccca gcct ctaatg gctacgcagg C9gct tccala 72 O gtgcgc.ctga tigaacaaaga Cctgggcctg gcgctggcga atgcgcaa.gc ggtccaa.gc.g 78O agcaccc.cgc tigggcgcact ggc.ccgtaac Ctgtttagcc tic acgctica agc.cgacgc.c 84 O gaggacgaag gtctggactt Cagct ctatt Caaaaactgt atcgcggtaa ggattag 897

<210s, SEQ ID NO 24 &211s LENGTH: 298 212. TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa

<4 OOs, SEQUENCE: 24 Met Ala Asp Ile Ala Phe Lieu. Gly Lieu. Gly Asn Met Gly Gly Pro Met 1. 5 1O 15 Ala Ala Asn Lieu Lleu Lys Ala Gly. His Arg Val Asn Val Phe Asp Lieu 2O 25 3O Glin Pro Lys Ala Val Lieu. Gly Lieu Val Glu Glin Gly Ala Glin Gly Ala 35 4 O 45 Asp Ser Ala Lieu. Glin Cys Cys Glu Gly Ala Glu Val Val Ile Ser Met SO 55 6 O Lieu Pro Ala Gly Glin His Val Glu Ser Lieu. Tyr Lieu. Gly Asp Asp Gly 65 70 7s 8O Lieu. Lieu Ala Arg Val Ala Gly Llys Pro Lieu. Lieu. Ile Asp Cys Ser Thr 85 90 95 Ile Ala Pro Glu Thir Ala Arg Llys Val Ala Glu Ala Ala Ala Ala Lys 1OO 105 11 O Gly Lieu. Thir Lieu. Lieu. Asp Ala Pro Val Ser Gly Gly Val Gly Gly Ala 115 12 O 125 Arg Ala Gly. Thir Lieu Ser Phe Ile Val Gly Gly Pro Ala Glu Gly Phe 13 O 135 14 O Ala Arg Ala Arg Pro Val Lieu. Glu Asn Met Gly Arg Asn. Ile Phe His 145 150 155 160 Ala Gly Asp His Gly Ala Gly Glin Val Ala Lys Ile Cys Asn. Asn Met 1.65 17O 17s Lieu. Lieu. Gly Ile Lieu Met Ala Gly Thr Ala Glu Ala Lieu Ala Lieu. Gly 18O 185 19 O

Val Lys Asn Gly Lieu. Asp Pro Ala Val Lieu. Ser Glu Val Met Lys Glin 195 2OO 2O5

Ser Ser Gly Gly Asn Trp Ala Lieu. Asn Lieu. Tyr Asn Pro Trp Pro Gly 21 O 215 22O

Val Met Pro Glin Ala Pro Ala Ser Asn Gly Tyr Ala Gly Gly Phe Glin 225 23 O 235 24 O

Val Arg Lieu Met Asn Lys Asp Lieu. Gly Lieu Ala Lieu Ala Asn Ala Glin 245 250 255

Ala Val Glin Ala Ser Thr Pro Lieu. Gly Ala Lieu Ala Arg Asn Lieu. Phe 26 O 265 27 O

US 2017/01 01638 A1 Apr. 13, 2017 62

- Continued

Ala Ala Ala Gly Thr Lieu. Thir Phe Met Val Gly Gly Asp Ala Glu Ala 13 O 135 14 O Lieu. Glu Lys Ala Arg Pro Lieu. Phe Glu Ala Met Gly Arg Asn. Ile Phe 145 150 155 160 His Ala Gly Pro Asp Gly Ala Gly Glin Val Ala Lys Val Cys Asn. Asn 1.65 17O 17s Glin Lieu. Lieu Ala Val Lieu Met Ile Gly. Thir Ala Glu Ala Met Ala Lieu. 18O 185 19 O Gly Val Ala Asn Gly Lieu. Glu Ala Lys Val Lieu Ala Glu Ile Met Arg 195 2OO 2O5 Arg Ser Ser Gly Gly Asn Trp Ala Lieu. Glu Val Tyr Asn Pro Trp Pro 21 O 215 22O Gly Val Met Glu Asn Ala Pro Ala Ser Arg Asp Tyr Ser Gly Gly Phe 225 23 O 235 24 O Met Ala Glin Lieu Met Ala Lys Asp Lieu. Gly Lieu Ala Glin Glu Ala Ala 245 250 255 Glin Ala Ser Ala Ser Ser Thr Pro Met Gly Ser Leu Ala Leu Ser Lieu. 26 O 265 27 O Tyr Arg Lieu. Lieu Lleu Lys Glin Gly Tyr Ala Glu Arg Asp Phe Ser Val 27s 28O 285 Val Glin Llys Lieu Phe Asp Pro Thr Glin Gly Glin 29 O 295

<210s, SEQ ID NO 27 &211s LENGTH: 262 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 27 Met Gly Phe Lieu. Ser Gly Lys Arg Ile Lieu Val Thr Gly Val Ala Ser 1. 5 1O 15 Llys Lieu. Ser Ile Ala Tyr Gly Ile Ala Glin Ala Met His Arg Glu Gly 2O 25 3O Ala Glu Lieu Ala Phe Thr Tyr Glin Asn Asp Llys Lieu Lys Gly Arg Val 35 4 O 45 Glu Glu Phe Ala Ala Glin Lieu. Gly Ser Asp Ile Val Lieu. Glin Cys Asp SO 55 6 O Val Ala Glu Asp Ala Ser Ile Asp Thr Met Phe Ala Glu Lieu. Gly Lys 65 70 7s 8O Val Trp Pro Llys Phe Asp Gly Phe Val His Ser Ile Gly Phe Ala Pro 85 90 95 Gly Asp Gln Lieu. Asp Gly Asp Tyr Val Asn Ala Val Thr Arg Glu Gly 1OO 105 11 O

Phe Lys Ile Ala His Asp Ile Ser Ser Tyr Ser Phe Val Ala Met Ala 115 12 O 125

Lys Ala Cys Arg Ser Met Lieu. Asn Pro Gly Ser Ala Lieu. Lieu. Thir Lieu. 13 O 135 14 O

Ser Tyr Lieu. Gly Ala Glu Arg Ala Ile Pro Asn Tyr Asn. Wal Met Gly 145 150 155 160

Lieu Ala Lys Ala Ser Lieu. Glu Ala Asn Val Arg Tyr Met Ala Asn Ala 1.65 17O 17s

Met Gly Pro Glu Gly Val Arg Val Asn Ala Ile Ser Ala Gly Pro Ile US 2017/01 01638 A1 Apr. 13, 2017 63

- Continued

18O 185 19 O Arg Thr Lieu Ala Ala Ser Gly Ile Lys Asp Phe Arg Llys Met Lieu Ala 195 2OO 2O5 His Cys Glu Ala Val Thr Pro Ile Arg Arg Thr Val Thr Ile Glu Asp 21 O 215 22O Val Gly Asn. Ser Ala Ala Phe Lieu. Cys Ser Asp Lieu. Ser Ala Gly Ile 225 23 O 235 24 O Phe Gly Glu Val Val His Val Asp Gly Gly Phe Ser Ile Ala Ala Met 245 25 O 255 Asn Glu Lieu. Glu Lieu Lys 26 O

<210s, SEQ ID NO 28 &211s LENGTH: 406 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 28 Met Lys Arg Ala Val Ile Thr Gly Lieu. Gly Ile Val Ser Ser Ile Gly 1. 5 1O 15 Asn Asn Glin Glin Glu Val Lieu Ala Ser Lieu. Arg Glu Gly Arg Ser Gly 2O 25 3O Ile Thr Phe Ser Glin Glu Lieu Lys Asp Ser Gly Met Arg Ser His Val 35 4 O 45 Trp Gly Asn. Wall Lys Lieu. Asp Thir Thr Gly Lieu. Ile Asp Arg Llys Val SO 55 6 O Val Arg Phe Met Ser Asp Ala Ser Ile Tyr Ala Phe Leu Ser Met Glu 65 70 7s 8O Glin Ala Ile Ala Asp Ala Gly Lieu. Ser Pro Glu Ala Tyr Glin Asn. Asn 85 90 95 Pro Arg Val Gly Lieu. Ile Ala Gly Ser Gly Gly Gly Ser Pro Arg Phe 1OO 105 11 O Glin Val Phe Gly Ala Asp Ala Met Arg Gly Pro Arg Gly Lieu Lys Ala 115 12 O 125 Val Gly Pro Tyr Val Val Thr Lys Ala Met Ala Ser Gly Val Ser Ala 13 O 135 14 O Cys Lieu Ala Thr Pro Phe Lys Ile His Gly Val Asn Tyr Ser Ile Ser 145 150 155 160 Ser Ala Cys Ala Thir Ser Ala His Cys Ile Gly Asn Ala Val Glu Glin 1.65 17O 17s Ile Glin Lieu. Gly Lys Glin Asp Ile Val Phe Ala Gly Gly Gly Glu Glu 18O 185 19 O Lieu. Cys Trp Glu Met Ala Cys Glu Phe Asp Ala Met Gly Ala Lieu. Ser 195 2OO 2O5

Thir Lys Tyr Asn Asp Thr Pro Glu Lys Ala Ser Arg Thr Tyr Asp Ala 21 O 215 22O His Arg Asp Gly Phe Val Ile Ala Gly Gly Gly Gly Met Val Val Val 225 23 O 235 24 O Glu Glu Lieu. Glu. His Ala Lieu Ala Arg Gly Ala His Ile Tyr Ala Glu 245 250 255

Ile Val Gly Tyr Gly Ala Thir Ser Asp Gly Ala Asp Met Val Ala Pro 26 O 265 27 O US 2017/01 01638 A1 Apr. 13, 2017 64

- Continued Ser Gly Glu Gly Ala Val Arg Cys Met Lys Met Ala Met His Gly Val 27s 28O 285 Asp Thr Pro Ile Asp Tyr Lieu. Asn Ser His Gly. Thir Ser Thr Pro Val 29 O 295 3 OO Gly Asp Wall Lys Glu Lieu Ala Ala Ile Arg Glu Val Phe Gly Asp Llys 3. OS 310 315 32O Ser Pro Ala Ile Ser Ala Thr Llys Val Met Thr Gly His Ser Leu Gly 3.25 330 335 Ala Ala Gly Val Glin Glu Ala Ile Tyr Ser Lieu Lleu Met Lieu. Glu. His 34 O 345 35. O Gly Phe Ile Ala Pro Ser Ile Asn. Ile Glu Glu Lieu. Asp Glu Glin Ala 355 360 365 Ala Gly Lieu. Asn Ile Val Thr Glu Thir Thr Asp Arg Glu Lieu. Thir Thr 37 O 375 38O Val Met Ser Asn Ser Phe Gly Phe Gly Gly Thr Asn Ala Thr Lieu Val 385 390 395 4 OO Met Arg Llys Lieu Lys Asp 4 OS

<210s, SEQ ID NO 29 &211s LENGTH: 309 212. TYPE: PRT <213> ORGANISM: Escherichia coli

< 4 OO SEQUENCE: 29 Met Thr Glin Phe Ala Phe Val Phe Pro Gly Glin Gly Ser Glin Thr Val 1. 5 1O 15 Gly Met Leu Ala Asp Met Ala Ala Ser Tyr Pro Ile Val Glu Glu Thr 2O 25 3O Phe Ala Glu Ala Ser Ala Ala Lieu. Gly Tyr Asp Lieu. Trp Ala Lieu. Thir 35 4 O 45 Gln Glin Gly Pro Ala Glu Glu Lieu. Asn Llys Thir Trp Gln Thr Glin Pro SO 55 6 O Ala Lieu. Lieu. Thir Ala Ser Val Ala Lieu. Tyr Arg Val Trp Glin Glin Glin 65 70 7s 8O Gly Gly Lys Ala Pro Ala Met Met Ala Gly His Ser Lieu. Gly Glu Tyr 85 90 95 Ser Ala Lieu Val Cys Ala Gly Val Ile Asp Phe Ala Asp Ala Val Arg 1OO 105 11 O Lieu Val Glu Met Arg Gly Llys Phe Met Glin Glu Ala Val Pro Glu Gly 115 12 O 125 Thr Gly Ala Met Ala Ala Ile Ile Gly Lieu. Asp Asp Ala Ser Ile Ala 13 O 135 14 O

Lys Ala Cys Glu Glu Ala Ala Glu Gly Glin Val Val Ser Pro Val Asn 145 150 155 160

Phe Asn. Ser Pro Gly Glin Val Val Ile Ala Gly His Lys Glu Ala Val 1.65 17O 17s

Glu Arg Ala Gly Ala Ala Cys Lys Ala Ala Gly Ala Lys Arg Ala Lieu. 18O 185 19 O

Pro Leu Pro Val Ser Val Pro Ser His Cys Ala Leu Met Llys Pro Ala 195 2OO 2O5

Ala Asp Llys Lieu Ala Val Glu Lieu Ala Lys Ile Thr Phe Asn Ala Pro 21 O 215 22O

US 2017/01 01638 A1 Apr. 13, 2017 66

- Continued

Arg Phe Ala Lys Glu Glin Arg Lieu Pro Lieu. Lieu. Phe Tyr Gly Val Ser 65 70 7s 8O Lieu. Gly Gly Met Asn Tyr Lieu Phe Tyr Lieu Ser Ile Glin Thr Val Pro 85 90 95 Lieu. Gly Ile Ala Val Ala Lieu. Glu Phe Thr Gly Pro Lieu Ala Val Ala 1OO 105 11 O Lieu. Phe Ser Ser Arg Arg Pro Val Asp Phe Val Trp Val Val Lieu Ala 115 12 O 125 Val Lieu. Gly Lieu. Trp Phe Lieu. Lieu Pro Lieu. Gly Glin Asp Val Ser His 13 O 135 14 O Val Asp Lieu. Thr Gly Cys Ala Lieu Ala Lieu. Gly Ala Gly Ala Cys Trip 145 150 155 160 Ala Ile Tyr Ile Lieu. Ser Gly Glin Arg Ala Gly Ala Glu. His Gly Pro 1.65 17O 17s Ala Thr Val Ala Ile Gly Ser Lieu. Ile Ala Ala Lieu. Ile Phe Val Pro 18O 185 19 O Ile Gly Ala Lieu. Glin Ala Gly Glu Ala Lieu. Trp His Trp Ser Val Ile 195 2OO 2O5 Pro Lieu. Gly Lieu Ala Val Ala Ile Lieu. Ser Thr Ala Lieu Pro Tyr Ser 21 O 215 22O Lieu. Glu Met Ile Ala Lieu. Thr Arg Lieu Pro Thr Arg Thr Phe Gly Thr 225 23 O 235 24 O Lieu Met Ser Met Glu Pro Ala Leu Ala Ala Val Ser Gly Met Ile Phe 245 250 255 Lieu. Gly Glu Thir Lieu. Thr Pro Ile Glin Lieu. Lieu Ala Lieu. Gly Ala Ile 26 O 265 27 O Ile Ala Ala Ser Met Gly Ser Thir Lieu. Thr Val Arg Lys Glu Ser Lys 27s 28O 285 Ile Lys Glu Lieu. Asp Ile Asn 29 O 295

<210s, SEQ ID NO 32 &211s LENGTH: 1269 &212s. TYPE: DNA <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 32 atgcgactico to cqgcaaaa C9gaagttta t cacttgttgc gttataacgg acaaatgcta 6 O cggtgcctgt acgctataac goacgaggtg actatgcgtc tdttittctat tcc to caccc 12 O acgctactgg C9gggtttct ggcgg tatta attggctacg C cagttcagc ggcaataatc 18O tggcaa.gcag cattgtc.gc cggagccacc actgcacaaa tict Ctggctg gatgacggcg 24 O

Ctggggctgg caatgggcgt cagtacgctg actctgacat tatggitatic cqtacctgtt 3OO

Ctcaccgcat ggt caacgcc tigcgcggct ttgttggtca ccggattgca gggactalaca 360 cittaacgaag ccatcgg.cgt ttittattgtc. accaacgc.gc taatagt cct c togcdgcata 42O acgggact ct ttgctcgt.ct gatgcgcatt attcc.gcact cqcttgcggc ggcaatgctt 48O gccgggattt tattacgctt ttt tacag gcgtttgcca gtctggacgg tdaatttacg 54 O ttgttgtggaa gtatgttgct gg tatggctg gcaaccalagg ccgttgcgcc gcgctatgcg 6OO gtaattgc.cg cgatgatt at tigatcgtg atcgt catcg cgcaaggtga C9ttgtcaca 660 US 2017/01 01638 A1 Apr. 13, 2017 67

- Continued actgatgttgtctittaaacc cqttct cocc actitat atta ccc.ctgattt titcgtttgct 72 O Cacagcctga gcgttgcact C cc cctttitt Ctggtgacga tiggcatcgca aaacgcaccg 78O ggitatcgcag caatgaaag.c agctggat at tcc.gctic ctd ttt cqccatt aattig tattt 84 O actggattgc tiggcactggit tttitt cocct titcggcgttt attic.cgt.cgg tattgcggca 9 OO at Caccgcgg ctatttgc.ca aagcc.cggaa gogcatc.cgg ataaagat.ca acgttggctg 96.O gcc.gctgc.cg ttgcaggc at titt ct atttg Ctc.gcagg to tdtttgg tag to catt acc 1 O2O gggatgatgg ctg.ccctgcc cqtaagttgg atccagatgc tiggcaggtot ggcgctgtta 108 O agtaccatcg gcggcagttt gtatic aggcg Ctgcataatg agcgtgagcg agacgcggcg 114 O gtggtggc at ttctggtaac ggcaa.gtgga ttgacgctgg togggattgg ttctg.cgttt 12 OO tggggattaa ttgc.cggagg cqtttgttac gtggtgttga atttaatcgc tigacagaaac 126 O cgatattga 1269

<210s, SEQ ID NO 33 &211s LENGTH: 422 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 33 Met Arg Lieu. Lieu. Arg Glin Asn Gly Ser Lieu. Ser Lieu Val Arg Tyr Asn 1. 5 1O 15 Gly Gln Met Lieu. Arg Cys Lieu. Tyr Ala Ile Thr His Glu Val Thr Met 2O 25 3O Arg Lieu. Phe Ser Ile Pro Pro Pro Thr Lieu. Leu Ala Gly Phe Leu Ala 35 4 O 45 Val Lieu. Ile Gly Tyr Ala Ser Ser Ala Ala Ile Ile Trp Glin Ala Ala SO 55 6 O Ile Val Ala Gly Ala Thir Thr Ala Glin Ile Ser Gly Trp Met Thr Ala 65 70 7s 8O Lieu. Gly Lieu Ala Met Gly Val Ser Thr Lieu. Thir Lieu. Thir Lieu. Trp Tyr 85 90 95 Arg Val Pro Val Lieu. Thir Ala Trp Ser Thr Pro Gly Ala Ala Lieu. Lieu 1OO 105 11 O Val Thr Gly Lieu. Glin Gly Lieu. Thir Lieu. Asn. Glu Ala Ile Gly Val Phe 115 12 O 125 Ile Val Thr Asn Ala Lieu. Ile Val Lieu. Cys Gly Ile Thr Gly Lieu Phe 13 O 135 14 O Ala Arg Lieu Met Arg Ile Ile Pro His Ser Lieu Ala Ala Ala Met Lieu 145 150 155 160 Ala Gly Ile Lieu. Lieu. Arg Phe Gly Lieu. Glin Ala Phe Ala Ser Lieu. Asp 1.65 17O 17s

Gly Glin Phe Thr Lieu. Cys Gly Ser Met Lieu. Leu Val Trp Leu Ala Thr 18O 185 19 O

Lys Ala Val Ala Pro Arg Tyr Ala Val Ile Ala Ala Met Ile Ile Gly 195 2OO 2O5

Ile Val Ile Val Ile Ala Glin Gly Asp Val Val Thr Thr Asp Val Val 21 O 215 22O

Phe Llys Pro Val Lieu Pro Thr Tyr Ile Thr Pro Asp Phe Ser Phe Ala 225 23 O 235 24 O

His Ser Lieu. Ser Wall Ala Lieu. Pro Leu Phe Lieu. Wall Thir Met Ala Ser US 2017/01 01638 A1 Apr. 13, 2017 68

- Continued

245 250 255 Glin Asn Ala Pro Gly Ile Ala Ala Met Lys Ala Ala Gly Tyr Ser Ala 26 O 265 27 O Pro Val Ser Pro Lieu. Ile Val Phe Thr Gly Lieu. Leu Ala Leu Val Phe 27s 28O 285 Ser Pro Phe Gly Val Tyr Ser Val Gly Ile Ala Ala Ile Thr Ala Ala 29 O 295 3 OO Ile Cys Glin Ser Pro Glu Ala His Pro Asp Lys Asp Glin Arg Trp Lieu. 3. OS 310 315 32O Ala Ala Ala Val Ala Gly Ile Phe Tyr Lieu. Lieu Ala Gly Lieu. Phe Gly 3.25 330 335 Ser Ala Ile Thr Gly Met Met Ala Ala Leu Pro Val Ser Trp Ile Glin 34 O 345 35. O Met Lieu Ala Gly Lieu Ala Lieu. Lieu. Ser Thir Ile Gly Gly Ser Lieu. Tyr 355 360 365 Glin Ala Lieu. His Asn. Glu Arg Glu Arg Asp Ala Ala Val Val Ala Phe 37 O 375 38O Lieu Val Thr Ala Ser Gly Lieu. Thir Lieu Val Gly Ile Gly Ser Ala Phe 385 390 395 4 OO Trp Gly Lieu. Ile Ala Gly Gly Val Cys Tyr Val Val Lieu. Asn Lieu. Ile 4 OS 41O 415 Ala Asp Arg Asn Arg Tyr 42O

<210s, SEQ ID NO 34 &211s LENGTH: 1037 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 34 Met Ala Asn. Phe Phe Ile Asp Arg Pro Ile Phe Ala Trp Val Lieu Ala 1. 5 1O 15 Ile Leu Lieu. Cys Lieu. Thr Gly Thr Lieu Ala Ile Phe Ser Leu Pro Val 2O 25 3O Glu Glin Tyr Pro Asp Lieu Ala Pro Pro Asn Val Arg Val Thir Ala Asn 35 4 O 45 Tyr Pro Gly Ala Ser Ala Glin Thr Lieu. Glu Asn Thr Val Thr Glin Val SO 55 6 O Ile Glu Glin Asn Met Thr Gly Lieu. Asp Asn Lieu Met Tyr Met Ser Ser 65 70 7s 8O Gln Ser Ser Gly Thr Gly Glin Ala Ser Val Thr Lieu Ser Phe Lys Ala 85 90 95 Gly. Thir Asp Pro Asp Glu Ala Val Glin Glin Val Glin Asn Glin Lieu. Glin 1OO 105 11 O

Ser Ala Met Arg Llys Lieu Pro Glin Ala Val Glin Asn Glin Gly Val Thr 115 12 O 125

Val Arg Llys Thr Gly Asp Thr Asn Ile Lieu. Thir Ile Ala Phe Val Ser 13 O 135 14 O

Thir Asp Gly Ser Met Asp Llys Glin Asp Ile Ala Asp Tyr Val Ala Ser 145 150 155 160

Asn. Ile Glin Asp Pro Lieu. Ser Arg Val Asin Gly Val Gly Asp Ile Asp 1.65 17O 17s US 2017/01 01638 A1 Apr. 13, 2017 69

- Continued Ala Tyr Gly Ser Glin Tyr Ser Met Arg Ile Trp Lieu. Asp Pro Ala Lys 18O 185 19 O Lieu. Asn. Ser Phe Glin Met Thr Ala Lys Asp Val Thr Asp Ala Ile Glu 195 2OO 2O5 Ser Glin Asn Ala Glin Ile Ala Val Gly Glin Leu Gly Gly Thr Pro Ser 21 O 215 22O Val Asp Llys Glin Ala Lieu. Asn Ala Thir Ile Asn Ala Glin Ser Lieu. Lieu 225 23 O 235 24 O Glin Thr Pro Glu Glin Phe Arg Asp Ile Thir Lieu. Arg Val Asin Glin Asp 245 250 255 Gly Ser Glu Val Arg Lieu. Gly Asp Wall Ala Thr Val Glu Met Gly Ala 26 O 265 27 O Glu Lys Tyr Asp Tyr Lieu. Ser Arg Phe Asin Gly Llys Pro Ala Ser Gly 27s 28O 285 Lieu. Gly Wall Lys Lieu Ala Ser Gly Ala Asn. Glu Met Ala Thir Ala Glu 29 O 295 3 OO Lieu Val Lieu. Asn Arg Lieu. Asp Glu Lieu Ala Glin Tyr Phe Pro His Gly 3. OS 310 315 32O Lieu. Glu Tyr Llys Val Ala Tyr Glu Thir Thr Ser Phe Val Lys Ala Ser 3.25 330 335 Ile Glu Asp Val Val Llys Thr Lieu. Lieu. Glu Ala Ile Ala Lieu Val Phe 34 O 345 35. O Lieu Val Met Tyr Lieu Phe Lieu. Glin ASn Phe Arg Ala Thr Lieu. Ile Pro 355 360 365 Thir Ile Ala Val Pro Val Val Lieu Met Gly Thr Phe Ser Val Lieu. Tyr 37 O 375 38O Ala Phe Gly Tyr Ser Val Asn Thr Lieu. Thr Met Phe Ala Met Val Lieu. 385 390 395 4 OO Ala Ile Gly Lieu. Lieu Val Asp Asp Ala Ile Val Val Val Glu Asn Val 4 OS 41O 415 Glu Arg Ile Met Ser Glu Glu Gly Lieu. Thr Pro Arg Glu Ala Thr Arg 42O 425 43 O Llys Ser Met Gly Glin Ile Glin Gly Ala Lieu Val Gly Ile Ala Met Val 435 44 O 445 Lieu. Ser Ala Val Phe Val Pro Met Ala Phe Phe Gly Gly Thr Thr Gly 450 45.5 460 Ala Ile Tyr Arg Glin Phe Ser Ile Thr Ile Val Ala Ala Met Val Lieu. 465 470 47s 48O Ser Val Lieu Val Ala Met Ile Lieu. Thr Pro Ala Lieu. Cys Ala Thr Lieu. 485 490 495 Lieu Lys Pro Lieu Lys Lys Gly Glu. His His Gly Glin Lys Gly Phe Phe SOO 505 51O

Ala Trp Phe Asin Gln Met Phe Asn Arg Asn Ala Glu Arg Tyr Glu Lys 515 52O 525 Gly Val Ala Lys Ile Lieu. His Arg Ser Lieu. Arg Trp Ile Val Ile Tyr 53 O 535 54 O

Val Lieu. Lieu. Lieu. Gly Gly Met Val Phe Lieu. Phe Lieu. Arg Lieu Pro Thr 5.45 550 555 560

Ser Phe Leu Pro Leu Glu Asp Arg Gly Met Phe Thr Thr Ser Val Glin 565 st O sts

Lieu Pro Ser Gly Ser Thr Glin Glin Gln Thr Lieu Lys Val Val Glu Gln US 2017/01 01638 A1 Apr. 13, 2017 70

- Continued

585 59 O

Ile Glu Lys Phe Thir His Glu Asp Asn Ile Met Ser Wall 595 605

Phe Ala Thir Wall Gly Ser Gly Pro Gly Gly ASn Gly Glin Asn Wall Ala 610 615 62O

Arg Met Phe Ile Arg Lell Asp Trp Ser Glu Arg Asp Ser Thir 625 630 635 64 O

Gly Thir Ser Phe Ala Ile Ile Glu Arg Ala Thir Ala Phe Asn Glin 645 650 655

Ile Glu Ala Arg Wall Ile Ala Ser Ser Pro Pro Ala Ile Ser Gly 660 665 67 O

Lell Gly Ser Ser Ala Gly Phe Asp Met Glu Luell Glin Asp His Ala Gly 675 685

Ala Gly His Asp Ala Lell Met Ala Ala Arg ASn Glin Lell Luell Ala Luell 69 O. 695 7 OO

Ala Ala Glu Asn Pro Glu Lell Thir Arg Wall Arg His Asn Gly Luell Asp 7 Os

Asp Ser Pro Glin Lell Glin Ile Asp Ile Asp Glin Arg Ala Glin Ala 72 73 O 73

Lell Gly Wall Ala Ile Asp Asp Ile Asn Asp Thir Lell Glin Thir Ala Trp 740 74. 7 O

Gly Ser Ser Wall Asn Asp Phe Met Asp Arg Gly Arg Wall 755 76 O 76.5

Wall Tyr Wall Glin Ala Ala Ala Pro Arg Met Lell Pro Asp Asp Ile 770 775

Asn Luell Trp Wall Arg Asn Asp Gly Gly Met Wall Pro Phe Ser 79 O 79.

Ala Phe Ala Thir Ser Arg Trp Glu Thir Gly Ser Pro Arg Luell Glu Arg 805 810 815

Asn Gly Tyr Ser Ala Wall Glu Ile Wall Gly Glu Ala Ala Pro Gly 825 83 O

Wall Ser Thir Gly Thir Ala Met Asp Ile Met Glu Ser Lell Wall Glin 835 84 O 845

Lell Pro Asn Gly Phe Gly Lell Glu Trp Thir Ala Met Ser Glin Glu 850 855 860

Arg Luell Ser Gly Ala Glin Ala Pro Ala Luell Tyr Ala Ile Ser Luell Luell 865

Wall Wall Phe Luell Cys Lell Ala Ala Luell Tyr Glu Ser Trp Ser Wall Pro 885 890 895

Phe Ser Wall Met Lell Wall Wall Pro Luell Gly Wall Ile Gly Ala Luell Luell 9 OO 905 91 O

Ala Thir Trp Met Arg Gly Lell Glu Asn Asp Wall Phe Glin Wall Gly 915 92 O 925

Lell Luell Thir Wall Ile Gly Lell Ser Ala ASn Ala Ile Luell Ile Wall 93 O 935 94 O

Glu Phe Ala Asn Glu Met Asn Glin Gly His Asp Lell Phe Glu Ala 945 950 955 96.O

Thir Luell His Ala Cys Arg Glin Arg Luell Arg Pro Ile Lell Met Thir Ser 965 97O 97.

Lell Ala Phe Ile Phe Gly Wall Luell Pro Met Ala Thir Ser Thir Gly Ala 98O 985 99 O US 2017/01 01638 A1 Apr. 13, 2017 71

- Continued

Gly Ser Gly Gly Gln His Ala Val Gly Thr Gly Val Met Gly Gly Met 995 1005

Ile Ser Ala Thir Ile Lieu Ala Ile Tyr Phe Val Pro Lieu. Phe Phe 1010 1015

Wall Lieu Val Arg Arg Arg Phe Pro Lieu Lys Pro Arg Pro Glu 1025 1035

<210s, SEQ ID NO 35 &211s LENGTH: 591 212. TYPE : PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 35

Met Ala His Pro Pro Lell Lell His Luell Glin Asp Ile Thir Luell Ser Luell 1. 5 15

Gly Gly Asn Pro Lell Lell Asp Gly Ala Gly Phe Ala Wall Gly Arg Gly 2O 25

Glu Arg Luell Lell Wall Gly Arg Asn Gly Ser Gly Lys Ser Thir Luell 35 4 O 45

Lell Lys Ile Ala Gly Wall Ile Glin Pro Asp Ser Gly Ser Wall Phe SO 55 6 O

Wall Glin Pro Ala Ser Lell Arg Luell Pro Glin Glu Pro Asp Luell 65 70

Ser Ala Thr Thr Ala Asp Wall Wall Gly Gln Ile Gly Asp 85 90 95

Pro Asp Met Trp Arg Ala Thir Pro Luell Luell Asp Ala Luell Gly Luell 105 11 O

Thir Gly Arg Ser Thir Glin Asn Luell Ser Gly Gly Glu Gly Arg Arg 115 12 O 125

Ala Ile Gly Wall Lell Ala Ala Ala Pro Asp Wall Luell Luell Luell 13 O 135 14 O

Asp Glu Pro Thir Asn His Lell Asp Met Pro Thir Ile Glu Trp Luell Glu 145 150 155 160

Arg Glu Luell Luell Ser Lell Gly Ala Met Wall Ile Ile Ser His Asp Arg 1.65 17O 17s

Arg Luell Luell Ser Thir Lell Ser Arg Ser Wall Wall Trp Lell Asp Arg Gly 18O 185 19 O

Wall Thir Arg Arg Lell Asp Glu Gly Phe Gly Arg Phe Glu Ala Trp Arg 195 2O5

Glu Glu Wall Luell Glu Glin Glu Glu Arg Asp Ala His Luell Asp Arg 21 O 215 22O

Lys Ile Ala Arg Glu Glu Asp Trp Met Arg Tyr Gly Wall Thir Ala Arg 225 23 O 235 24 O

Arg Arg Asn Wall Arg Arg Wall Arg Glu Luell Ala Asp Luell Arg Thir 245 250 255

Ala Arg Glu Ala Ile Arg Ala Pro Gly Thir Lell Thir Luell Asn Thir 26 O 265 27 O

Glin Luell Arg Pro His Arg Luell Wall Ala Wall Ala Glu Asp Ile Ser 27s 28O 285

Ala Trp Gly Glu Glin Wall Wall Arg His Lell Asp Luell Arg Ile 29 O 295 3 OO

Lell Arg Gly Asp Arg Lell Gly Ile Wall Gly Ala Asn Gly Ala Gly US 2017/01 01638 A1 Apr. 13, 2017 72

- Continued

3. OS 310 315 32O Thir Thr Lieu. Lieu. Arg Met Lieu. Thr Gly Lieu. Asp Glin Pro Asp Ser Gly 3.25 330 335 Thir Ile Ser Leu Gly Pro Ser Lieu. Asn Met Val Thr Lieu. Asp Glin Glin 34 O 345 35. O Arg Arg Thir Lieu. ASn Pro Glu Arg Thr Lieu Ala Asp Thir Lieu. Thr Glu 355 360 365 Gly Gly Gly Asp Met Val Glin Val Gly. Thr Glu Lys Arg His Val Val 37 O 375 38O Gly Tyr Met Lys Asp Phe Leu Phe Arg Pro Glu Glin Ala Arg Thr Pro 385 390 395 4 OO Val Ser Ala Lieu. Ser Gly Gly Glu Arg Gly Arg Lieu Met Lieu Ala Cys 4 OS 41O 415 Ala Lieu Ala Lys Pro Ser Asn Lieu. Lieu Val Lieu. Asp Glu Pro Thir Asn 42O 425 43 O Asp Lieu. Asp Lieu. Glu Thir Lieu. Asp Ile Lieu. Glin Asp Met Lieu Ala Ser 435 44 O 445 Cys Glu Gly. Thr Val Lieu. Lieu Val Ser His Asp Arg Asp Phe Lieu. Asp 450 45.5 460 Arg Val Ala Thir Ser Val Lieu Ala Thr Glu Gly Asp Gly Asn Trp Ile 465 470 47s 48O Glu Tyr Ala Gly Gly Tyr Ser Asp Met Lieu Ala Glin Arg His Gln Lys 485 490 495 Pro Leu. Thir Thr Ala Ser Val Val Glu Asin Glu Pro Thr Llys Pro Llys SOO 505 51O Glu Thir Thr Ala Ala Arg Gly Pro Thir Lys Llys Lieu. Ser Tyr Lys Asp 515 52O 525 Glin Phe Ala Lieu. Asp Asn Lieu Pro Lys Glu Met Glu Lys Lieu. Glu Ala 53 O 535 54 O Glin Ala Ala Asn. Cys Wall Lys Asn Trp Glin Ile Glin Ile Tyr Met Glu 5.45 550 555 560 Llys Thr Pro Arg Ser Lieu. Arg Asin Phe Arg Lieu. Ile Tyr Arg Ser Ser 565 st O sts Lys Glin Ser Trp Glin Asn Lieu Lys Asn Ala Gly Trp Asn Trp Llys 58O 585 59 O

<210s, SEQ ID NO 36 &211s LENGTH: 396 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 36 Met Thr Thr Arg Gln His Ser Ser Phe Ala Ile Val Phe Ile Leu Gly 1. 5 1O 15

Lieu. Lieu Ala Met Lieu Met Pro Lieu. Ser Ile Asp Met Tyr Lieu Pro Ala 2O 25 3O

Lieu Pro Val Ile Ser Ala Glin Phe Gly Val Pro Ala Gly Ser Thr Glin 35 4 O 45

Met Thr Lieu Ser Thr Tyr Ile Leu Gly Phe Ala Lieu. Gly Glin Lieu. Ile SO 55 6 O

Tyr Gly Pro Met Ala Asp Ser Phe Gly Arg Llys Pro Val Val Lieu. Gly 65 70 7s 8O US 2017/01 01638 A1 Apr. 13, 2017 73

- Continued

Gly. Thir Lieu Val Phe Ala Ala Ala Ala Val Ala Cys Ala Lieu Ala Asn 85 90 95 Thir Ile Asp Gln Lieu. Ile Val Met Arg Phe Phe His Gly Lieu Ala Ala 1OO 105 11 O Ala Ala Ala Ser Val Val Ile Asn Ala Lieu Met Arg Asp Ile Tyr Pro 115 12 O 125 Lys Glu Glu Phe Ser Arg Met Met Ser Phe Val Met Leu Val Thir Thr 13 O 135 14 O Ile Ala Pro Leu Met Ala Pro Ile Val Gly Gly Trp Val Lieu Val Trp 145 150 155 160 Lieu. Ser Trp His Tyr Ile Phe Trp Ile Lieu Ala Lieu Ala Ala Ile Lieu. 1.65 17O 17s Ala Ser Ala Met Ile Phe Phe Lieu. Ile Lys Glu Thir Lieu Pro Pro Glu 18O 185 19 O Arg Arg Gln Pro Phe His Ile Arg Thr Thr Ile Gly Asn Phe Ala Ala 195 2OO 2O5 Lieu. Phe Arg His Lys Arg Val Lieu. Ser Tyr Met Lieu Ala Ser Gly Phe 21 O 215 22O Ser Phe Ala Gly Met Phe Ser Phe Leu Ser Ala Gly Pro Phe Val Tyr 225 23 O 235 24 O Ile Glu Ile Asn His Val Ala Pro Glu Asn Phe Gly Tyr Tyr Phe Ala 245 250 255 Lieu. ASn Ile Val Phe Lieu Phe Val Met Thir Ile Phe ASn Ser Arg Phe 26 O 265 27 O Val Arg Arg Ile Gly Ala Lieu. Asn Met Phe Arg Ser Gly Lieu. Trp Ile 27s 28O 285 Glin Phe Ile Met Ala Ala Trp Met Val Ile Ser Ala Lieu. Lieu. Gly Lieu. 29 O 295 3 OO Gly Phe Trp Ser Lieu Val Val Gly Val Ala Ala Phe Val Gly Cys Val 3. OS 310 315 32O Ser Met Val Ser Ser Asn Ala Met Ala Val Ile Lieu. Asp Glu Phe Pro 3.25 330 335 His Met Ala Gly Thr Ala Ser Ser Leu Ala Gly Thr Phe Arg Phe Gly 34 O 345 35. O Ile Gly Ala Ile Val Gly Ala Lieu. Lieu. Ser Lieu Ala Thr Phe Asin Ser 355 360 365 Ala Trp Pro Met Ile Trp Ser Ile Ala Phe Cys Ala Thr Ser Ser Ile 37 O 375 38O Lieu. Phe Cys Lieu. Tyr Ala Ser Arg Pro Llys Lys Arg 385 390 395

<210s, SEQ ID NO 37 &211s LENGTH: 1047 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OO > SEQUENCE: 37 Met Ile Glu Trp Ile Ile Arg Arg Ser Val Ala Asn Arg Phe Lieu Val 1. 5 1O 15

Lieu Met Gly Ala Lieu. Phe Leu Ser Ile Trp Gly. Thir Trp Thir Ile Ile 2O 25 3O

Asn Thr Pro Val Asp Ala Lieu Pro Asp Lieu. Ser Asp Val Glin Val Ile 35 4 O 45 US 2017/01 01638 A1 Apr. 13, 2017 74

- Continued

Ile Lys Thir Ser Pro Gly Glin Ala Pro Glin Ile Wall Glu Asn Glin SO 55 6 O

Wall Thir Pro Lell Thir Thir Thir Met Luell Ser Wall Pro Gly Ala Lys 65 70

Thir Wall Arg Gly Phe Ser Glin Phe Gly Asp Ser Wall Wall Ile 85 90 95

Phe Glu Asp Gly Thir Asp Pro Trp Ala Arg Ser Arg Wall Luell Glu 105 11 O

Luell Asn Glin Wall Glin Gly Lys Luell Pro Ala Gly Wall Ser Ala Glu 115 12 O 125

Lell Gly Pro Asp Ala Thir Gly Wall Gly Trp Ile Tyr Glu Tyr Ala Luell 13 O 135 14 O

Wall Asp Arg Ser Gly Lys His Asp Luell Ala Asp Lell Arg Ser Luell Glin 145 150 155 160

Asp Trp Phe Luell Lys Tyr Glu Luell Thir Ile Pro Asp Wall Ala Glu 1.65

Wall Ala Ser Wall Gly Gly Wall Wall Lys Glu Tyr Glin Wall Wall Ile Asp 18O 185 19 O

Pro Glin Arg Luell Ala Glin Tyr Gly Ile Ser Luell Ala Glu Wall Ser 195

Ala Luell Asp Ala Ser Asn Glin Glu Ala Gly Gly Ser Ser Glu Luell 21 O 215 22O

Ala Glu Ala Glu Tyr Met Wall Arg Ala Ser Gly Tyr Lell Thir Luell 225 23 O 235 24 O

Asp Asp Phe Asn His Ile Wall Luell Ala Ser Glu Asn Wall Pro 245 250 255

Wall Luell Arg Asp Wall Ala Wall Glin Gly Pro Met Arg 26 O 265

Arg Gly Ile Ala Glu Lell Asn Gly Glu Gly Wall Ala Gly Wall 28O 285

Wall Ile Luell Arg Ser Gly Lys Asn Ala Arg Wall Ile Ala Wall 29 O 295 3 OO

Lys Asp Luell Glu Thir Lell Ser Ser Pro Glu Wall Glu 3. OS 310

Ile Wall Thir Thir Tyr Asp Arg Ser Glin Luell Asp Arg Ile Asp 3.25 330 335

Asn Luell Ser Gly Lys Lell Lell Glu Glu Phe Wall Wall Wall Wall 34 O 345

Ala Luell Phe Lell Trp His Wall Arg Ser Lell Wall Ile Ile 355 360 365

Ser Luell Pro Luell Gly Lell Cys Ile Ala Phe Wall Met His Phe Glin 37 O 375

Gly Luell Asn Ala Asn Ile Met Ser Luell Gly Ile Ala Ile Ala Wall 385 390 4 OO

Gly Ala Met Wall Asp Ala Ala Ile Wall Met Glu Asn Ala His 4 OS 415

Arg Luell Glu Glu Trp Glin His Glin His Pro Asp Ala Thir Luell Asp Asn 425 43 O

Thir Arg Trp Glin Wall Ile Thir Asp Ala Ser Wall Glu Wall Gly Pro 435 44 O 445 US 2017/01 01638 A1 Apr. 13, 2017 75

- Continued

Ala Lieu. Phe Ile Ser Lieu Lleu. Ile Ile Thir Lieu. Ser Phe Ile Pro Ile 450 45.5 460 Phe Thr Lieu. Glu Gly Glin Glu Gly Arg Lieu Phe Gly Pro Leu Ala Phe 465 470 47s 48O Thir Lys Thr Tyr Ala Met Ala Gly Ala Ala Lieu. Lieu Ala Ile Val Val 485 490 495 Ile Pro Ile Leu Met Gly Tyr Trp Ile Arg Gly Lys Ile Pro Pro Glu SOO 505 51O Ser Ser Asn Pro Lieu. Asn Arg Phe Lieu. Ile Arg Val Tyr His Pro Lieu. 515 52O 525 Lieu. Lieu Lys Val Lieu. His Trp Pro Llys Thir Thr Lieu. Lieu Val Ala Ala 53 O 535 54 O Lieu. Ser Val Lieu. Thr Val Lieu. Trp Pro Lieu. Asn Llys Val Gly Gly Glu 5.45 550 555 560 Phe Leu Pro Glin Ile Asn Glu Gly Asp Leu Lleu Tyr Met Pro Ser Thr 565 st O sts Lieu Pro Gly Ile Ser Ala Ala Glu Ala Ala Ser Met Lieu Gln Lys Thr 58O 585 59 O Asp Llys Lieu. Ile Met Ser Val Pro Glu Val Ala Arg Val Phe Gly Lys 595 6OO 605 Thr Gly Lys Ala Glu Thir Ala Thr Asp Ser Ala Pro Leu Glu Met Val 610 615 62O Glu Thir Thir Ile Gln Lieu Lys Pro Gln Glu Gln Trp Arg Pro Gly Met 625 630 635 64 O Thir Met Asp Llys Ile Ile Glu Glu Lieu. Asp Asn. Thr Val Arg Lieu Pro 645 650 655 Gly Lieu Ala Asn Lieu. Trp Val Pro Pro Ile Arg Asn Arg Ile Asp Met 660 665 67 O Lieu. Ser Thr Gly Ile Llys Ser Pro Ile Gly Ile Llys Val Ser Gly Thr 675 68O 685 Val Lieu Ala Asp Ile Asp Ala Met Ala Glu Glin Ile Glu Glu Val Ala 69 O. 695 7 OO Arg Thr Val Pro Gly Val Ala Ser Ala Lieu Ala Glu Arg Lieu. Glu Gly 7 Os 71O 71s 72O Gly Arg Tyr Ile Asn Val Glu Ile Asn Arg Glu Lys Ala Ala Arg Tyr 72 73 O 73 Gly Met Thr Val Ala Asp Val Glin Leu Phe Val Thr Ser Ala Val Gly 740 74. 7 O Gly Ala Met Val Gly Glu Thr Val Glu Gly Ile Ala Arg Tyr Pro Ile 7ss 760 765 Asn Lieu. Arg Tyr Pro Glin Ser Trp Arg Asp Ser Pro Glin Ala Lieu. Arg 770 775 78O

Gln Leu Pro Ile Lieu. Thr Pro Met Lys Glin Glin Ile Thr Lieu Ala Asp 78s 79 O 79. 8OO

Val Ala Asp Ile Llys Val Ser Thr Gly Pro Ser Met Leu Lys Thr Glu 805 810 815

Asn Ala Arg Pro Thir Ser Trp Ile Tyr Ile Asp Ala Arg Asp Arg Asp 82O 825 83 O

Met Val Ser Val Val His Asp Lieu Gln Lys Ala Ile Ala Glu Lys Val 835 84 O 845

Gln Leu Lys Pro Gly Thr Ser Val Ala Phe Ser Gly Glin Phe Glu Lieu. US 2017/01 01638 A1 Apr. 13, 2017 76

- Continued

850 855 860 Lieu. Glu Arg Ala Asn His Llys Lieu Lys Lieu Met Val Pro Met Thr Lieu. 865 87O 87s 88O Met Ile Ile Phe Val Lieu. Lieu. Tyr Lieu Ala Phe Arg Arg Val Gly Glu 885 890 895 Ala Lieu. Lieu. Ile Ile Ser Ser Val Pro Phe Ala Lieu Val Gly Gly Ile 9 OO 905 91 O Trp Lieu. Leu Trp Trp Met Gly Phe His Leu Ser Val Ala Thr Gly Thr 915 92 O 925 Gly Phe Ile Ala Lieu Ala Gly Val Ala Ala Glu Phe Gly Val Val Met 93 O 935 94 O Lieu Met Tyr Lieu. Arg His Ala Ile Glu Ala Val Pro Ser Lieu. Asn. Asn 945 950 955 96.O Pro Glin Thr Phe Ser Glu Gln Lys Lieu. Asp Glu Ala Lieu. Tyr His Gly 965 97O 97. Ala Val Lieu. Arg Val Arg Pro Lys Ala Met Thr Val Ala Val Ile Ile 98O 985 99 O Ala Gly Lieu. Lieu Pro Ile Lieu. Trp Gly Thr Gly Ala Gly Ser Glu Val 995 1OOO 1005 Met Ser Arg Ile Ala Ala Pro Met Ile Gly Gly Met Ile Thr Ala 1010 1015 1 O2O Pro Lieu. Lieu. Ser Lieu. Phe Ile Ile Pro Ala Ala Tyr Lys Lieu Met 1025 103 O 1035 Trp Lieu. His Arg His Arg Val Arg Llys 104 O 1045

<210s, SEQ ID NO 38 &211s LENGTH: 384 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 38 Met Lieu. Lieu Val Lieu Val Lieu. Ile Gly Lieu. Asn Met Arg Pro Lieu. Lieu. 1. 5 1O 15 Thir Ser Val Gly Pro Leu Lleu Pro Gln Leu Arg Glin Ala Ser Gly Met 2O 25 3O

Ser Phe Ser Wall Ala Ala Leu Lleu. Thir Ala Lieu. Pro Wal Wall. Thir Met 35 4 O 45 Gly Gly Lieu Ala Lieu Ala Gly Ser Trp Lieu. His Glin His Val Ser Glu SO 55 6 O Arg Arg Ser Val Ala Ile Ser Lieu. Lieu. Lieu. Ile Ala Val Gly Ala Lieu 65 70 7s 8O Met Arg Glu Lieu. Tyr Pro Glin Ser Ala Lieu. Lieu Lleu Ser Ser Ala Lieu. 85 90 95

Lieu. Gly Gly Val Gly Ile Gly Ile Ile Glin Ala Val Met Pro Ser Val 1OO 105 11 O

Ile Lys Arg Arg Phe Glin Glin Arg Thr Pro Lieu Val Met Gly Lieu. Trp 115 12 O 125

Ser Ala Ala Lieu Met Gly Gly Gly Gly Lieu. Gly Ala Ala Ile Thr Pro 13 O 135 14 O Trp Leu Val Gln His Ser Glu Thir Trp Tyr Glin Thr Lieu Ala Trp Trp 145 150 155 160 US 2017/01 01638 A1 Apr. 13, 2017 77

- Continued

Ala Lieu Pro Ala Val Val Ala Lieu. Phe Ala Trp Trp Trp Glin Ser Ala 1.65 17O 17s Arg Glu Val Ala Ser Ser His Llys Thr Thr Thr Thr Pro Val Arg Val 18O 185 19 O Val Phe Thr Pro Arg Ala Trp Thr Lieu. Gly Val Tyr Phe Gly Lieu. Ile 195 2OO 2O5 Asn Gly Gly Tyr Ala Ser Lieu. Ile Ala Trp Lieu Pro Ala Phe Tyr Ile 21 O 215 22O Glu Ile Gly Ala Ser Ala Glin Tyr Ser Gly Ser Lieu. Lieu Ala Lieu Met 225 23 O 235 24 O Thir Lieu. Gly Glin Ala Ala Gly Ala Lieu. Lieu Met Pro Ala Met Ala Arg 245 250 255 His Glin Asp Arg Arg Llys Lieu. Lieu Met Lieu Ala Lieu Val Lieu. Glin Lieu. 26 O 265 27 O Val Gly Phe Cys Gly Phe Ile Trp Leu Pro Met Gln Leu Pro Val Lieu. 27s 28O 285 Trp Ala Met Val Cys Gly Lieu. Gly Lieu. Gly Gly Ala Phe Pro Lieu. Cys 29 O 295 3 OO Lieu. Lieu. Lieu Ala Lieu. Asp His Ser Val Glin Pro Ala Ile Ala Gly Lys 3. OS 310 315 32O Lieu Val Ala Phe Met Glin Gly Ile Gly Phe Ile Ile Ala Gly Lieu Ala 3.25 330 335 Pro Trp Phe Ser Gly Val Lieu. Arg Ser Ile Ser Gly ASn Tyr Lieu Met 34 O 345 35. O Asp Trp Ala Phe His Ala Lieu. Cys Val Val Gly Lieu Met Ile Ile Thr 355 360 365 Lieu. Arg Phe Ala Pro Val Arg Phe Pro Glin Lieu. Trp Val Lys Glu Ala 37 O 375 38O

<210s, SEQ ID NO 39 &211s LENGTH: 219 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 39 Met Asp Lieu. Ile Tyr Phe Lieu. Ile Asp Phe Ile Lieu. His Ile Asp Wall 1. 5 1O 15 His Lieu Ala Glu Lieu Val Ala Glu Tyr Gly Val Trp Val Tyr Ala Ile 2O 25 3O Lieu. Phe Lieu. Ile Leu Phe Cys Glu Thr Gly Lieu Val Val Thr Pro Phe 35 4 O 45 Lieu Pro Gly Asp Ser Lieu. Lieu. Phe Val Ala Gly Ala Lieu Ala Ser Lieu. SO 55 6 O Glu Thir Asn Asp Lieu. Asn Val His Met Met Val Val Lieu Met Lieu. Ile 65 70 7s 8O

Ala Ala Ile Val Gly Asp Ala Val Asn Tyr Thir Ile Gly Arg Lieu. Phe 85 90 95

Gly Glu Lys Lieu. Phe Ser Asn. Pro Asn. Ser Lys Ile Phe Arg Arg Ser 1OO 105 11 O Tyr Lieu. Asp Llys Thr His Glin Phe Tyr Glu Lys His Gly Gly Lys Thr 115 12 O 125

Ile Ile Leu Ala Arg Phe Val Pro Ile Val Arg Thr Phe Ala Pro Phe 13 O 135 14 O US 2017/01 01638 A1 Apr. 13, 2017 78

- Continued

Val Ala Gly Met Gly His Met Ser Tyr Arg His Phe Ala Ala Tyr Asn 145 150 155 160 Val Ile Gly Ala Lieu. Lieu. Trp Val Lieu. Lieu. Phe Thr Tyr Ala Gly Tyr 1.65 17O 17s Phe Phe Gly Thr Ile Pro Met Val Glin Asp Asn Lieu Lys Lieu. Lieu. Ile 18O 185 19 O Val Gly Ile Ile Val Val Ser Ile Leu Pro Gly Val Ile Glu Ile Ile 195 2OO 2O5 Arg His Lys Arg Ala Ala Ala Arg Ala Ala Lys 21 O 215

<210s, SEQ ID NO 4 O &211s LENGTH: 299 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 4 O Met Ser Arg Lys Asp Gly Val Lieu Ala Lieu. Lieu Val Val Val Val Trp 1. 5 1O 15 Gly Lieu. Asn Phe Val Val Ile Llys Val Gly Lieu. His Asn Met Pro Pro 2O 25 3O Lieu Met Lieu Ala Gly Lieu. Arg Phe Met Lieu Val Ala Phe Pro Ala Ile 35 4 O 45 Phe Phe Val Ala Arg Pro Llys Val Pro Lieu. ASn Lieu Lleu Lieu. Gly Tyr SO 55 6 O Gly Lieu. Thir Ile Ser Phe Ala Glin Phe Ala Phe Leu Phe Cys Ala Ile 65 70 7s 8O Asn Phe Gly Met Pro Ala Gly Lieu Ala Ser Lieu Val Lieu. Glin Ala Glin 85 90 95 Ala Phe Phe Thr Ile Met Leu Gly Ala Phe Thr Phe Gly Glu Arg Lieu. 1OO 105 11 O His Gly Lys Glin Lieu Ala Gly Ile Ala Lieu Ala Ile Phe Gly Val Lieu. 115 12 O 125 Val Lieu. Ile Glu Asp Ser Lieu. Asn Gly Glin His Val Ala Met Lieu. Gly 13 O 135 14 O Phe Met Lieu. Thir Lieu Ala Ala Ala Phe Ser Trp Ala Cys Gly Asn. Ile 145 150 155 160 Phe Asn Llys Lys Ile Met Ser His Ser Thr Arg Pro Ala Val Met Ser 1.65 17O 17s Lieu Val Ile Trp Ser Ala Lieu. Ile Pro Ile Ile Pro Phe Phe Val Ala 18O 185 19 O Ser Lieu. Ile Lieu. Asp Gly Ser Ala Thr Met Ile His Ser Leu Val Thr 195 2OO 2O5

Ile Asp Met Thr Thr Ile Leu Ser Leu Met Tyr Lieu Ala Phe Val Ala 21 O 215 22O

Thir Ile Val Gly Tyr Gly Ile Trp Gly Thr Lieu. Leu Gly Arg Tyr Glu 225 23 O 235 24 O

Thir Trp Arg Val Ala Pro Lieu. Ser Lieu. Lieu Val Pro Val Val Gly Lieu. 245 250 255

Ala Ser Ala Ala Lieu. Lieu. Lieu. Asp Glu Arg Lieu. Thr Gly Lieu. Glin Phe 26 O 265 27 O

Lieu. Gly Ala Val Lieu. Ile Met Thr Gly Lieu. Tyr Ile Asn Val Phe Gly US 2017/01 01638 A1 Apr. 13, 2017 79

- Continued

27s 28O 285 Lieu. Arg Trp Arg Lys Ala Wall Lys Val Gly Ser 29 O 295

<210s, SEQ ID NO 41 &211s LENGTH: 195 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 41 Met Thr Pro Thr Lieu Lleu Ser Ala Phe Trp Thr Tyr Thr Lieu. Ile Thr 1. 5 1O 15 Ala Met Thr Pro Gly Pro Asn Asn Ile Leu Ala Leu Ser Ser Ala Thr 2O 25 3O Ser His Gly Phe Arg Glin Ser Thr Arg Val Lieu Ala Gly Met Ser Lieu. 35 4 O 45 Gly Phe Lieu. Ile Val Met Leu Lieu. Cys Ala Gly Ile Ser Phe Ser Lieu. SO 55 6 O Ala Val Ile Asp Pro Ala Ala Val His Lieu. Lieu. Ser Trp Ala Gly Ala 65 70 7s 8O Ala Tyr Ile Val Trp Lieu Ala Trp Lys Ile Ala Thr Ser Pro Thr Lys 85 90 95 Glu Asp Gly Lieu. Glin Ala Lys Pro Ile Ser Phe Trp Ala Ser Phe Ala 1OO 105 11 O Lieu. Glin Phe Val Asn Val Lys Ile Ile Lieu. Tyr Gly Val Thir Ala Lieu. 115 12 O 125 Ser Thr Phe Val Lieu Pro Gln Thr Glin Ala Leu Ser Trp Val Val Gly 13 O 135 14 O Val Ser Val Lieu. Leu Ala Met Ile Gly Thr Phe Gly Asn Val Cys Trp 145 150 155 160 Ala Lieu Ala Gly His Lieu. Phe Glin Arg Lieu. Phe Arg Glin Tyr Gly Arg 1.65 17O 17s Glin Lieu. Asn. Ile Val Lieu Ala Lieu. Lieu. Lieu Val Tyr Cys Ala Val Arg 18O 185 19 O Ile Phe Tyr 195

<210s, SEQ ID NO 42 &211s LENGTH: 512 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 42 Met Glin Glin Gln Lys Pro Leu Glu Gly Ala Gln Leu Val Ile Met Thr 1. 5 1O 15

Ile Ala Leu Ser Leu Ala Thr Phe Met Glin Val Lieu. Asp Ser Thr Ile 2O 25 3O

Ala Asn. Wall Ala Ile Pro Thir Ile Ala Gly Asn Lieu. Gly Ser Ser Lieu 35 4 O 45

Ser Glin Gly. Thir Trp Val Ile Thr Ser Phe Gly Val Ala Asn Ala Ile SO 55 6 O

Ser Ile Pro Lieu. Thr Gly Trp Lieu Ala Lys Arg Val Gly Glu Val Lys 65 70 7s 8O

Lieu. Phe Lieu. Trp Ser Thir Ile Ala Phe Ala Ile Ala Ser Trp Ala Cys US 2017/01 01638 A1 Apr. 13, 2017 80

- Continued

85 90 95 Gly Val Ser Ser Ser Lieu. Asn Met Lieu. Ile Phe Phe Arg Val Ile Glin 1OO 105 11 O Gly Ile Val Ala Gly Pro Lieu. Ile Pro Lieu. Ser Glin Ser Lieu. Lieu. Lieu. 115 12 O 125 Asn Asn Tyr Pro Pro Ala Lys Arg Ser Ile Ala Lieu Ala Lieu. Trp Ser 13 O 135 14 O Met Thr Val Ile Val Ala Pro Ile Cys Gly Pro Ile Leu Gly Gly Tyr 145 150 155 160 Ile Ser Asp Asn Tyr His Trp Gly Trp Ile Phe Phe Ile Asin Val Pro 1.65 17O 17s Ile Gly Val Ala Val Val Lieu Met Thr Lieu. Glin Thr Lieu. Arg Gly Arg 18O 185 19 O Glu Thir Arg Thr Glu Arg Arg Arg Ile Asp Ala Val Gly Lieu Ala Lieu. 195 2OO 2O5 Lieu Val Ile Gly Ile Gly Ser Lieu. Glin Ile Met Lieu. Asp Arg Gly Lys 21 O 215 22O Glu Lieu. Asp Trp Phe Ser Ser Glin Glu Ile Ile Ile Lieu. Thr Val Val 225 23 O 235 24 O Ala Val Val Ala Ile Cys Phe Lieu. Ile Val Trp Glu Lieu. Thir Asp Asp 245 250 255 Asn Pro Ile Val Asp Lieu. Ser Lieu. Phe Llys Ser Arg Asn. Phe Thir Ile 26 O 265 27 O Gly Cys Lieu. Cys Ile Ser Leu Ala Tyr Met Leu Tyr Phe Gly Ala Ile 27s 28O 285 Val Lieu. Leu Pro Gln Lieu. Leu Gln Glu Val Tyr Gly Tyr Thr Ala Thr 29 O 295 3 OO Trp Ala Gly Lieu Ala Ser Ala Pro Val Gly Ile Ile Pro Val Ile Lieu. 3. OS 310 315 32O Ser Pro Ile Ile Gly Arg Phe Ala His Llys Lieu. Asp Met Arg Arg Lieu. 3.25 330 335 Val Thr Phe Ser Phe Ile Met Tyr Ala Val Cys Phe Tyr Trp Arg Ala 34 O 345 35. O Tyr Thr Phe Glu Pro Gly Met Asp Phe Gly Ala Ser Ala Trp Pro Glin 355 360 365 Phe Ile Glin Gly Phe Ala Val Ala Cys Phe Phe Met Pro Leu. Thir Thr 37 O 375 38O Ile Thr Lieu. Ser Gly Lieu Pro Pro Glu Arg Lieu Ala Ala Ala Ser Ser 385 390 395 4 OO Lieu. Ser Asn Phe Thr Arg Thr Lieu Ala Gly Ser Ile Gly Thr Ser Ile 4 OS 41O 415 Thir Thir Thr Met Trp Thr Asn Arg Glu Ser Met His His Ala Gln Leu 42O 425 43 O

Thr Glu Ser Val Asn Pro Phe Asin Pro Asn Ala Glin Ala Met Tyr Ser 435 44 O 445

Gln Leu Glu Gly Lieu. Gly Met Thr Glin Glin Glin Ala Ser Gly Trp Ile 450 45.5 460

Ala Glin Glin Ile Thr Asn Glin Gly Lieu. Ile Ile Ser Ala Asn. Glu Ile 465 470 47s 48O

Phe Trp Met Ser Ala Gly Ile Phe Leu Val Lieu. Leu Gly Lieu Val Trp 485 490 495 US 2017/01 01638 A1 Apr. 13, 2017 81

- Continued

Phe Ala Lys Pro Pro Phe Gly Ala Gly Gly Gly Gly Gly Gly Ala His SOO 505 51O

<210s, SEQ ID NO 43 &211s LENGTH: 394 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 43 Met Lys Arg Glin Arg Asn. Wall Asn Lieu. Lieu. Lieu Met Lieu Val Lieu. Lieu. 1. 5 1O 15 Val Ala Val Gly Gln Met Ala Glin Thr Ile Tyr Ile Pro Ala Ile Ala 2O 25 3O Asp Met Ala Arg Asp Lieu. Asn Val Arg Glu Gly Ala Val Glin Ser Val 35 4 O 45 Met Gly Ala Tyr Lieu Lleu. Thir Tyr Gly Val Ser Gln Leu Phe Tyr Gly SO 55 6 O Pro Ile Ser Asp Arg Val Gly Arg Arg Pro Val Ile Lieu Val Gly Met 65 70 7s 8O

Ser Ile Phe Met Leu Ala Thir Lieu. Wall Ala Wall. Thir Thir Ser Ser Lieu. 85 90 95 Thr Val Lieu. Ile Ala Ala Ser Ala Met Glin Gly Met Gly Thr Gly Val 1OO 105 11 O Gly Gly Wal Met Ala Arg Thr Lieu Pro Arg Asp Lieu. Tyr Glu Arg Thr 115 12 O 125 Glin Lieu. Arg His Ala Asn. Ser Lieu. Lieu. Asn Met Gly Ile Lieu Val Ser 13 O 135 14 O Pro Lieu. Lieu Ala Pro Lieu. Ile Gly Gly Lieu. Lieu. Asp Thr Met Trp Asn 145 150 155 160 Trp Arg Ala Cys Tyr Lieu. Phe Lieu. Lieu Val Lieu. Cys Ala Gly Val Thr 1.65 17O 17s Phe Ser Met Ala Arg Trp Met Pro Glu Thr Arg Pro Val Asp Ala Pro 18O 185 19 O Arg Thr Arg Lieu Lleu. Thir Ser Tyr Lys Thr Lieu Phe Gly Asn Ser Gly 195 2OO 2O5 Phe Asn. Cys Tyr Lieu Lleu Met Lieu. Ile Gly Gly Lieu Ala Gly Ile Ala 21 O 215 22O Ala Phe Glu Ala Cys Ser Gly Val Lieu Met Gly Ala Val Lieu. Gly Lieu 225 23 O 235 24 O

Ser Ser Met Thir Wal Ser Ile Lieu. Phe Ile Lieu. Pro Ile Pro Ala Ala 245 250 255 Phe Phe Gly Ala Trp Phe Ala Gly Arg Pro Asn Lys Arg Phe Ser Thr 26 O 265 27 O

Lieu Met Trp Glin Ser Val Ile Cys Cys Lieu. Lieu Ala Gly Lieu. Lieu Met 27s 28O 285

Trp Ile Pro Asp Trp Phe Gly Val Met Asin Val Trp Thr Lieu. Leu Val 29 O 295 3 OO

Pro Ala Ala Leu Phe Phe Phe Gly Ala Gly Met Leu Phe Pro Leu Ala 3. OS 310 315 32O

Thir Ser Gly Ala Met Glu Pro Phe Pro Phe Leu Ala Gly Thr Ala Gly 3.25 330 335

Ala Lieu Val Gly Gly Lieu. Glin Asn. Ile Gly Ser Gly Val Lieu Ala Ser US 2017/01 01638 A1 Apr. 13, 2017 82

- Continued

34 O 345 35. O Lieu. Ser Ala Met Lieu Pro Glin Thr Gly Glin Gly Ser Lieu. Gly Lieu. Lieu. 355 360 365 Met Thr Lieu Met Gly Lieu. Lieu. Ile Val Lieu. Cys Trp Lieu Pro Lieu Ala 37 O 375 38O Thr Arg Met Ser His Glin Gly Glin Pro Val 385 390

<210s, SEQ ID NO 44 &211s LENGTH: 512 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 44 Met Ala Ile Thr Lys Ser Thr Pro Ala Pro Leu. Thr Gly Gly Thr Lieu. 1. 5 1O 15 Trp. Cys Val Thir Ile Ala Leu Ser Leu Ala Thr Phe Met Glin Met Leu 2O 25 3O Asp Ser Thir Ile Ser Asn Val Ala Ile Pro Thir Ile Ser Gly Phe Leu 35 4 O 45 Gly Ala Ser Thr Asp Glu Gly. Thir Trp Val Ile Thr Ser Phe Gly Val SO 55 6 O Ala Asn Ala Ile Ala Ile Pro Val Thr Gly Arg Lieu Ala Glin Arg Ile 65 70 7s 8O Gly Glu Lieu. Arg Lieu Phe Lieu. Leu Ser Val Thr Phe Phe Ser Leu Ser 85 90 95 Ser Leu Met Cys Ser Leu Ser Thr Asn Lieu. Asp Val Lieu. Ile Phe Phe 1OO 105 11 O Arg Val Val Glin Gly Lieu Met Ala Gly Pro Lieu. Ile Pro Lieu. Ser Glin 115 12 O 125 Ser Lieu. Lieu. Lieu. Arg Asn Tyr Pro Pro Glu Lys Arg Thr Phe Ala Lieu. 13 O 135 14 O Ala Leu Trp Ser Met Thr Val Ile Ile Ala Pro Ile Cys Gly Pro Ile 145 150 155 160 Lieu. Gly Gly Tyr Ile Cys Asp Asin Phe Ser Trp Gly Trp Ile Phe Leu 1.65 17O 17s Ile Asin Val Pro Met Gly Ile Ile Val Lieu. Thir Lieu. Cys Lieu. Thir Lieu. 18O 185 19 O Lieu Lys Gly Arg Glu Thr Glu Thir Ser Pro Val Lys Met Asn Lieu Pro 195 2OO 2O5 Gly Lieu. Thir Lieu. Lieu Val Lieu. Gly Val Gly Gly Lieu. Glin Ile Met Lieu. 21 O 215 22O Asp Llys Gly Arg Asp Lieu. Asp Trp Phe Asin Ser Ser Thir Ile Ile Ile 225 23 O 235 24 O

Lieu. Thr Val Val Ser Val Ile Ser Lieu. Ile Ser Lieu Val Ile Trp Glu 245 250 255

Ser Thir Ser Glu Asn Pro Ile Lieu. Asp Lieu. Ser Lieu. Phe Llys Ser Arg 26 O 265 27 O

Asn Phe Thir Ile Gly Ile Val Ser Ile Thr Cys Ala Tyr Lieu. Phe Tyr 27s 28O 285

Ser Gly Ala Ile Val Lieu Met Pro Gln Leu Lleu Gln Glu Thr Met Gly 29 O 295 3 OO US 2017/01 01638 A1 Apr. 13, 2017 83

- Continued Tyr Asn Ala Ile Trp Ala Gly Lieu Ala Tyr Ala Pro Ile Gly Ile Met 3. OS 310 315 32O Pro Lieu. Lieu. Ile Ser Pro Lieu. Ile Gly Arg Tyr Gly Asn Lys Ile Asp 3.25 330 335 Met Arg Lieu. Leu Val Thr Phe Ser Phe Leu Met Tyr Ala Val Cys Tyr 34 O 345 35. O Tyr Trp Arg Ser Val Thr Phe Met Pro Thr Ile Asp Phe Thr Gly Ile 355 360 365 Ile Leu Pro Glin Phe Phe Glin Gly Phe Ala Val Ala Cys Phe Phe Leu 37 O 375 38O Pro Leu. Thir Thr Ile Ser Phe Ser Gly Leu Pro Asp Asn Llys Phe Ala 385 390 395 4 OO Asn Ala Ser Ser Met Ser Asn Phe Phe Arg Thr Lieu Ser Gly Ser Val 4 OS 41O 415 Gly. Thir Ser Lieu. Thr Met Thr Lieu. Trp Gly Arg Arg Glu Ser Lieu. His 42O 425 43 O His Ser Glin Lieu. Thir Ala Thir Ile Asp Glin Phe Asn Pro Val Phe Asn 435 44 O 445 Ser Ser Ser Glin Ile Met Asp Llys Tyr Tyr Gly Ser Leu Ser Gly Val 450 45.5 460

Lieu. Asn. Glu Ile ASn Asn. Glu Ile Thr Glin Glin Ser Lieu. Ser Ile Ser 465 470 47s 48O Ala ASn Glu Ile Phe Arg Met Ala Ala Ile Ala Phe Ile Lieu. Lieu. Thir 485 490 495 Val Lieu Val Trp Phe Ala Lys Pro Pro Phe Thr Ala Lys Gly Val Gly SOO 505 51O

<210s, SEQ ID NO 45 &211s LENGTH: 512 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 45 Met Ala Ile Thr Lys Ser Thr Pro Ala Pro Leu. Thr Gly Gly Thr Lieu. 1. 5 1O 15 Trp. Cys Val Thir Ile Ala Leu Ser Leu Ala Thr Phe Met Glin Met Leu 2O 25 3O Asp Ser Thir Ile Ser Asn Val Ala Ile Pro Thir Ile Ser Gly Phe Leu 35 4 O 45 Gly Ala Ser Thr Asp Glu Gly. Thir Trp Val Ile Thr Ser Phe Gly Val SO 55 6 O Ala Asn Ala Ile Ala Ile Pro Val Thr Gly Arg Lieu Ala Glin Arg Ile 65 70 7s 8O

Gly Glu Lieu. Arg Lieu Phe Lieu. Leu Ser Val Thr Phe Phe Ser Leu Ser 85 90 95

Ser Leu Met Cys Ser Leu Ser Thr Asn Lieu. Asp Val Lieu. Ile Phe Phe 1OO 105 11 O

Arg Val Val Glin Gly Lieu Met Ala Gly Pro Lieu. Ile Pro Lieu. Ser Glin 115 12 O 125

Ser Lieu. Lieu. Lieu. Arg Asn Tyr Pro Pro Glu Lys Arg Thr Phe Ala Lieu. 13 O 135 14 O

Ala Leu Trp Ser Met Thr Val Ile Ile Ala Pro Ile Cys Gly Pro Ile 145 150 155 160 US 2017/01 01638 A1 Apr. 13, 2017 84

- Continued

Lieu. Gly Gly Tyr Ile Cys Asp Asin Phe Ser Trp Gly Trp Ile Phe Leu 1.65 17O 17s Ile Asin Val Pro Met Gly Ile Ile Val Lieu. Thir Lieu. Cys Lieu. Thir Lieu. 18O 185 19 O Lieu Lys Gly Arg Glu Thr Glu Thir Ser Pro Val Lys Met Asn Lieu Pro 195 2OO 2O5 Gly Lieu. Thir Lieu. Lieu Val Lieu. Gly Val Gly Gly Lieu. Glin Ile Met Lieu. 21 O 215 22O Asp Llys Gly Arg Asp Lieu. Asp Trp Phe Asin Ser Ser Thir Ile Ile Ile 225 23 O 235 24 O Lieu. Thr Val Val Ser Val Ile Ser Lieu. Ile Ser Lieu Val Ile Trp Glu 245 250 255 Ser Thir Ser Glu Asn Pro Ile Lieu. Asp Lieu. Ser Lieu. Phe Llys Ser Arg 26 O 265 27 O Asn Phe Thir Ile Gly Ile Val Ser Ile Thr Cys Ala Tyr Lieu. Phe Tyr 27s 28O 285 Ser Gly Ala Ile Val Lieu Met Pro Gln Leu Lleu Gln Glu Thr Met Gly 29 O 295 3 OO Tyr Asn Ala Ile Trp Ala Gly Lieu Ala Tyr Ala Pro Ile Gly Ile Met 3. OS 310 315 32O Pro Lieu. Lieu. Ile Ser Pro Lieu. Ile Gly Arg Tyr Gly Asn Lys Ile Asp 3.25 330 335 Met Arg Lieu. Leu Val Thr Phe Ser Phe Leu Met Tyr Ala Val Cys Tyr 34 O 345 35. O Tyr Trp Arg Ser Val Thr Phe Met Pro Thr Ile Asp Phe Thr Gly Ile 355 360 365 Ile Leu Pro Glin Phe Phe Glin Gly Phe Ala Val Ala Cys Phe Phe Leu 37 O 375 38O Pro Leu. Thir Thr Ile Ser Phe Ser Gly Leu Pro Asp Asn Llys Phe Ala 385 390 395 4 OO Asn Ala Ser Ser Met Ser Asn Phe Phe Arg Thr Lieu Ser Gly Ser Val 4 OS 41O 415 Gly. Thir Ser Lieu. Thr Met Thr Lieu. Trp Gly Arg Arg Glu Ser Lieu. His 42O 425 43 O His Ser Glin Lieu. Thir Ala Thir Ile Asp Glin Phe Asn Pro Val Phe Asn 435 44 O 445 Ser Ser Ser Glin Ile Met Asp Llys Tyr Tyr Gly Ser Leu Ser Gly Val 450 45.5 460

Lieu. Asn. Glu Ile ASn Asn. Glu Ile Thr Glin Glin Ser Lieu. Ser Ile Ser 465 470 47s 48O Ala Asn. Glu Ile Phe Arg Met Ala Ala Ile Ala Phe Ile Lieu. Lieu. Thr 485 490 495

Val Lieu Val Trp Phe Ala Lys Pro Pro Phe Thr Ala Lys Gly Val Gly SOO 505 51O

<210s, SEQ ID NO 46 &211s LENGTH: 444 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 46 Met Ile Lieu. Asp Thr Val Asp Glu Lys Llys Lys Gly Val His Thr Arg US 2017/01 01638 A1 Apr. 13, 2017 85

- Continued

1. 5 1O 15 Tyr Lieu. Ile Lieu. Lieu. Ile Ile Phe Ile Val Thr Ala Val Asn Tyr Ala 2O 25 3O Asp Arg Ala Thr Lieu. Ser Ile Ala Gly Thr Glu Val Ala Lys Glu Lieu 35 4 O 45 Gln Leu Ser Ala Val Ser Met Gly Tyr Ile Phe Ser Ala Phe Gly Trp SO 55 6 O Ala Tyr Lieu. Lieu Met Glin Ile Pro Gly Gly Trp Lieu. Lieu. Asp Llys Phe 65 70 7s 8O Gly Ser Lys Llys Val Tyr Thr Tyr Ser Leu Phe Phe Trp Ser Leu Phe 85 90 95 Thr Phe Leu Gln Gly Phe Val Asp Met Phe Pro Leu Ala Trp Ala Gly 1OO 105 11 O Ile Ser Met Phe Phe Met Arg Phe Met Leu Gly Phe Ser Glu Ala Pro 115 12 O 125 Ser Phe Pro Ala Asn Ala Arg Ile Val Ala Ala Trp Phe Pro Thr Lys 13 O 135 14 O Glu Arg Gly Thr Ala Ser Ala Ile Phe Asin Ser Ala Glin Tyr Phe Ser 145 150 155 160 Lieu Ala Lieu. Phe Ser Pro Lieu. Lieu. Gly Trp Lieu. Thir Phe Ala Trp Gly 1.65 17O 17s Trp. Glu. His Val Phe Thr Val Met Gly Val Ile Gly Phe Val Lieu. Thr 18O 185 19 O Ala Lieu. Trp Ile Llys Lieu. Ile His Asn Pro Thr Asp His Pro Arg Met 195 2OO 2O5 Ser Ala Glu Glu Lieu Lys Phe Ile Ser Glu Asn Gly Ala Val Val Asp 21 O 215 22O Met Asp His Llys Llys Pro Gly Ser Ala Ala Ala Ser Gly Pro Llys Lieu. 225 23 O 235 24 O His Tyr Ile Lys Glin Lieu Lleu Ser Asn Arg Met Met Lieu. Gly Val Phe 245 250 255 Phe Gly Glin Tyr Phe Ile Asn. Thir Ile Thr Trp Phe Phe Lieu. Thir Trp 26 O 265 27 O Phe Pro Ile Tyr Lieu Val Glin Glu Lys Gly Met Ser Ile Leu Lys Val 27s 28O 285 Gly Lieu Val Ala Ser Ile Pro Ala Lieu. Cys Gly Phe Ala Gly Gly Val 29 O 295 3 OO Lieu. Gly Gly Val Phe Ser Asp Tyr Lieu. Ile Lys Arg Gly Lieu. Ser Lieu. 3. OS 310 315 32O Thir Lieu Ala Arg Llys Lieu Pro Ile Val Lieu. Gly Met Lieu. Lieu Ala Ser 3.25 330 335

Thir Ile Ile Lieu. Cys Asn Tyr Thr Asn Asn Thr Thr Lieu Val Val Met 34 O 345 35. O

Lieu Met Ala Lieu Ala Phe Phe Gly Lys Gly Phe Gly Ala Lieu. Gly Trp 355 360 365

Pro Val Ile Ser Asp Thr Ala Pro Lys Glu Ile Val Gly Lieu. Cys Gly 37 O 375 38O

Gly Val Phe Asin Val Phe Gly Asn Val Ala Ser Ile Val Thr Pro Leu 385 390 395 4 OO

Val Ile Gly Tyr Lieu Val Ser Glu Lieu. His Ser Phe Asn Ala Ala Lieu 4 OS 41O 415 US 2017/01 01638 A1 Apr. 13, 2017 86

- Continued

Val Phe Val Gly Cys Ser Ala Leu Met Ala Met Val Cys Tyr Lieu. Phe 42O 425 43 O Val Val Gly Asp Ile Lys Arg Met Glu Lieu. Glin Llys 435 44 O

<210s, SEQ ID NO 47 &211s LENGTH: 450 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 47 Met Ser Ser Lieu. Ser Glin Ala Ala Ser Ser Val Glu Lys Arg Thr Asn 1. 5 1O 15 Ala Arg Tyr Trp Ile Val Val Met Leu Phe Ile Val Thr Ser Phe Asn 2O 25 3O Tyr Gly Asp Arg Ala Thr Lieu. Ser Ile Ala Gly Ser Glu Met Ala Lys 35 4 O 45 Asp Ile Gly Lieu. Asp Pro Val Gly Met Gly Tyr Val Phe Ser Ala Phe SO 55 6 O Ser Trp Ala Tyr Val Ile Gly Glin Ile Pro Gly Gly Trp Lieu. Lieu. Asp 65 70 7s 8O Arg Phe Gly Ser Lys Arg Val Tyr Phe Trp Ser Ile Phe Ile Trp Ser 85 90 95 Met Phe Thr Lieu Lieu. Glin Gly Phe Val Asp Ile Phe Ser Gly Phe Gly 1OO 105 11 O Ile Ile Val Ala Lieu. Phe Thr Lieu. Arg Phe Lieu Val Gly Lieu Ala Glu 115 12 O 125 Ala Pro Ser Phe Pro Gly Asn Ser Arg Ile Val Ala Ala Trp Phe Pro 13 O 135 14 O Ala Glin Glu Arg Gly. Thir Ala Val Ser Ile Phe Asn. Ser Ala Glin Tyr 145 150 155 160 Phe Ala Thr Val Ile Phe Ala Pro Ile Met Gly Trp Lieu. Thr His Glu 1.65 17O 17s Val Gly Trp Ser His Val Phe Phe Phe Met Gly Gly Lieu. Gly Ile Val 18O 185 19 O Ile Ser Phe Ile Trp Leu Lys Val Ile His Glu Pro Asn Gln His Pro 195 2OO 2O5 Gly Val Asn Llys Lys Glu Lieu. Glu Tyr Ile Ala Ala Gly Gly Ala Lieu. 21 O 215 22O Ile Asn Met Asp Glin Glin Asn Thr Llys Val Llys Val Pro Phe Ser Val 225 23 O 235 24 O Llys Trp Gly Glin Ile Lys Glin Lieu. Lieu. Gly Ser Arg Met Met Ile Gly 245 250 255

Val Tyr Ile Gly Glin Tyr Cys Ile Asn Ala Lieu. Thr Tyr Phe Phe Ile 26 O 265 27 O

Thir Trp Phe Pro Val Tyr Lieu Val Glin Ala Arg Gly Met Ser Ile Leu 27s 28O 285

Lys Ala Gly Phe Val Ala Ser Val Pro Ala Val Cys Gly Phe Ile Gly 29 O 295 3 OO

Gly Val Lieu. Gly Gly Ile Ile Ser Asp Trp Lieu Met Arg Arg Thr Gly 3. OS 310 315 32O

Ser Lieu. Asn. Ile Ala Arg Llys Thr Pro Ile Val Met Gly Met Lieu. Lieu. US 2017/01 01638 A1 Apr. 13, 2017 87

- Continued

3.25 330 335 Ser Met Val Met Val Phe Cys Asn Tyr Val Asn Val Glu Trp Met Ile 34 O 345 35. O Ile Gly Phe Met Ala Lieu Ala Phe Phe Gly Lys Gly Ile Gly Ala Lieu. 355 360 365 Gly Trp Ala Wal Met Ala Asp Thir Ala Pro Lys Glu Ile Ser Gly Lieu. 37 O 375 38O Ser Gly Gly Lieu Phe Asn Met Phe Gly Asin Ile Ser Gly Ile Val Thr 385 390 395 4 OO Pro Ile Ala Ile Gly Tyr Ile Val Gly Thr Thr Gly Ser Phe Asin Gly 4 OS 41O 415 Ala Lieu. Ile Tyr Val Gly Val His Ala Lieu. Ile Ala Val Lieu. Ser Tyr 42O 425 43 O Lieu Val Lieu Val Gly Asp Ile Lys Arg Ile Glu Lieu Lys Pro Val Ala 435 44 O 445 Gly Glin 450

<210s, SEQ ID NO 48 &211s LENGTH: 475 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 48 Met Ser Asp Llys Llys Lys Arg Ser Met Ala Gly Lieu Pro Trp Ile Ala 1. 5 1O 15 Ala Met Ala Phe Phe Met Glin Ala Lieu. Asp Ala Thir Ile Lieu. Asn Thr 2O 25 3O Ala Lieu Pro Ala Ile Ala His Ser Lieu. Asn Arg Ser Pro Lieu Ala Met 35 4 O 45 Gln Ser Ala Ile Ile Ser Tyr Thr Lieu. Thr Val Ala Met Lieu. Ile Pro SO 55 6 O Val Ser Gly Trp Lieu Ala Asp Arg Phe Gly Thr Arg Arg Ile Phe Thr 65 70 7s 8O Lieu Ala Val Ser Lieu. Phe Thr Lieu. Gly Ser Lieu Ala Cys Ala Lieu. Ser 85 90 95 Asn Ser Lieu Pro Gln Lieu Val Val Phe Arg Val Ile Glin Gly Ile Gly 1OO 105 11 O Gly Ala Met Met Met Pro Wall Ala Arg Lieu Ala Lieu. Lieu. Arg Ala Tyr 115 12 O 125 Pro Arg Asn. Glu Lieu Lleu Pro Val Lieu. Asn. Phe Val Ala Met Pro Gly 13 O 135 14 O Lieu Val Gly Pro Ile Leu Gly Pro Val Lieu. Gly Gly Val Lieu Val Thr 145 150 155 160 Trp Ala Thr Trp His Trp Ile Phe Lieu. Ile Asn Ile Pro Ile Gly Ile 1.65 17O 17s Ala Gly Lieu. Lieu. Tyr Ala Arg Llys His Met Pro Asn. Phe Thir Thr Ala 18O 185 19 O

Arg Arg Arg Phe Asp Ile Thr Gly Phe Lieu. Lieu. Phe Gly Lieu. Ser Lieu 195 2OO 2O5

Val Lieu. Phe Ser Ser Gly Ile Glu Lieu. Phe Gly Glu Lys Ile Val Ala 21 O 215 22O US 2017/01 01638 A1 Apr. 13, 2017 88

- Continued

Ser Trp Ile Ala Lieu. Thr Val Ile Val Thr Ser Ile Gly Lieu. Leu Lieu. 225 23 O 235 24 O Lieu. Tyr Ile Lieu. His Ala Arg Arg Thr Pro Asn. Pro Lieu. Ile Ser Lieu. 245 250 255 Asp Leu Phe Llys Thr Arg Thr Phe Ser Ile Gly Ile Val Gly Asn Ile 26 O 265 27 O Ala Thr Arg Lieu. Gly Thr Gly Cys Val Pro Phe Leu Met Pro Leu Met 27s 28O 285 Lieu. Glin Val Gly Phe Gly Tyr Glin Ala Phe Ile Ala Gly Cys Met Met 29 O 295 3 OO

Ala Pro Thir Ala Lieu G Ser Ile Ile Ala Lys Ser Met Val Thr Glin 3. OS 3 315 32O Val Lieu. Arg Arg Lieu. Gly Tyr Arg His Thr Lieu Val Gly Ile Thr Val 3.25 330 335 Ile Ile Gly Lieu Met Ile Ala Glin Phe Ser Leu Gln Ser Pro Ala Met 34 O 345 35. O Ala Ile Trp Met Lieu. Ile Leu Pro Leu Phe Ile Leu Gly Met Ala Met 355 360 365 Ser Thr Glin Phe Thr Ala Met Asn. Thir Ile Thr Lieu Ala Asp Lieu. Thr 37 O 375 38O Asp Asp Asn Ala Ser Ser Gly Asn. Ser Val Lieu Ala Val Thr Glin Glin 385 390 395 4 OO Lieu Ser Ile Ser Lieu. Gly Val Ala Val Ser Ala Ala Val Lieu. Arg Val 4 OS 41O 415 Tyr Glu Gly Met Glu Gly. Thir Thr Thr Val Glu Glin Phe His Tyr Thr 42O 425 43 O Phe Ile Thr Met Gly Ile Ile Thr Val Ala Ser Ala Ala Met Phe Met 435 44 O 445 Lieu. Lieu Lys Thir Thr Asp Gly Asn. Asn Lieu. Ile Lys Arg Glin Arg Llys 450 45.5 460 Ser Llys Pro Asn Arg Val Pro Ser Glu Ser Glu 465 470 47s

<210s, SEQ ID NO 49 &211s LENGTH: 212 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 49 Met Phe Ala Glu Tyr Gly Val Lieu. Asn Tyr Trp Thr Tyr Lieu Val Gly 1. 5 1O 15 Ala Ile Phe Ile Val Lieu Val Pro Gly Pro Asn Thr Lieu Phe Val Lieu. 2O 25 3O Lys Asn. Ser Val Ser Ser Gly Met Lys Gly Gly Tyr Lieu Ala Ala Cys 35 4 O 45

Gly Val Phe Ile Gly Asp Ala Val Lieu Met Phe Lieu Ala Trp Ala Gly SO 55 6 O

Val Ala Thr Lieu. Ile Llys Thir Thr Pro Ile Leu Phe Asn Ile Val Arg 65 70 7s 8O

Tyr Lieu. Gly Ala Phe Tyr Lieu. Lieu. Tyr Lieu. Gly Ser Lys Ile Lieu. Tyr 85 90 95

Ala Thr Lieu Lys Gly Lys Asn. Ser Glu Ala Lys Ser Asp Glu Pro Glin 1OO 105 11 O US 2017/01 01638 A1 Apr. 13, 2017 89

- Continued

Tyr Gly Ala Ile Phe Lys Arg Ala Lieu. Ile Lieu. Ser Lieu. Thir Asn Pro 115 12 O 125 Lys Ala Ile Leu Phe Tyr Val Ser Phe Phe Val Glin Phe Ile Asp Val 13 O 135 14 O Asn Ala Pro His Thr Gly Ile Ser Phe Phe Ile Leu Ala Ala Thr Lieu. 145 150 155 160 Glu Lieu Val Ser Phe Cys Tyr Lieu Ser Phe Lieu. Ile Ile Ser Gly Ala 1.65 17O 17s Phe Val Thr Glin Tyr Ile Arg Thr Lys Llys Llys Lieu Ala Lys Val Gly 18O 185 19 O Asn Ser Lieu. Ile Gly Lieu Met Phe Val Gly Phe Ala Ala Arg Lieu Ala 195 2OO 2O5

Thir Lieu. Glin Ser 21 O

<210s, SEQ ID NO 50 &211s LENGTH: 593 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 50 Met Arg Ser Phe Ser Glin Lieu. Trp Pro Thir Lieu Lys Arg Lieu. Lieu Ala 1. 5 1O 15 Tyr Gly Ser Pro Trp Arg Llys Pro Lieu. Gly Ile Ala Val Lieu Met Met 2O 25 3O Trp Val Ala Ala Ala Ala Glu Val Ser Gly Pro Lieu. Lieu. Ile Ser Tyr 35 4 O 45 Phe Ile Asp Asn Met Val Ala Lys Asn. Asn Lieu Pro Lieu Lys Val Val SO 55 6 O Ala Gly Lieu Ala Ala Ala Tyr Val Gly Lieu. Glin Lieu. Phe Ala Ala Gly 65 70 7s 8O Lieu. His Tyr Ala Glin Ser Lieu. Lieu. Phe Asn Arg Ala Ala Val Gly Val 85 90 95 Val Glin Glin Lieu. Arg Thr Asp Wal Met Asp Ala Ala Lieu. Arg Glin Pro 1OO 105 11 O Lieu. Ser Glu Phe Asp Thr Gln Pro Val Gly Glin Val Ile Ser Arg Val 115 12 O 125 Thir Asn Asp Thr Glu Val Ile Arg Asp Lieu. Tyr Val Thr Val Val Ala 13 O 135 14 O Thr Val Lieu. Arg Ser Ala Ala Lieu Val Gly Ala Met Lieu Val Ala Met 145 150 155 160 Phe Ser Lieu. Asp Trp Arg Met Ala Leu Val Ala Ile Met Ile Phe Pro 1.65 17O 17s

Val Val Lieu Val Val Met Val Ile Tyr Glin Arg Tyr Ser Thr Pro Ile 18O 185 19 O

Val Arg Arg Val Arg Ala Tyr Lieu Ala Asp Ile Asn Asp Gly Phe Asn 195 2OO 2O5

Glu Ile Ile Asin Gly Met Ser Val Ile Glin Glin Phe Arg Glin Glin Ala 21 O 215 22O Arg Phe Gly Glu Arg Met Gly Glu Ala Ser Arg Ser His Tyr Met Ala 225 23 O 235 24 O

Arg Met Glin Thr Lieu. Arg Lieu. Asp Gly Phe Lieu. Lieu. Arg Pro Lieu. Lieu US 2017/01 01638 A1 Apr. 13, 2017 90

- Continued

245 250 255 Ser Lieu. Phe Ser Ser Lieu. Ile Lieu. Cys Gly Lieu Lleu Met Lieu. Phe Gly 26 O 265 27 O Phe Ser Ala Ser Gly. Thir Ile Glu Val Gly Val Lieu. Tyr Ala Phe Ile 27s 28O 285 Ser Tyr Lieu. Gly Arg Lieu. Asn. Glu Pro Lieu. Ile Glu Lieu. Thir Thr Glin 29 O 295 3 OO Glin Ala Met Lieu. Glin Glin Ala Val Val Ala Gly Glu Arg Val Phe Glu 3. OS 310 315 32O Lieu Met Asp Gly Pro Arg Glin Glin Tyr Gly Asn Asp Asp Arg Pro Lieu. 3.25 330 335 Glin Ser Gly. Thir Ile Glu Val Asp Asn Val Ser Phe Ala Tyr Arg Asp 34 O 345 35. O Asp Asn Lieu Val Lieu Lys Asn. Ile Asn Lieu. Ser Val Pro Ser Arg Asn 355 360 365 Phe Val Ala Leu Val Gly His Thr Gly Ser Gly Lys Ser Thr Lieu Ala 37 O 375 38O Ser Lieu. Leu Met Gly Tyr Tyr Pro Leu. Thr Glu Gly Glu Ile Arg Lieu. 385 390 395 4 OO Asp Gly Arg Pro Lieu. Ser Ser Lieu. Ser His Ser Ala Lieu. Arg Glin Gly 4 OS 41O 415 Val Ala Met Val Glin Glin Asp Pro Val Val Lieu Ala Asp Thr Phe Lieu 42O 425 43 O. Ala Asn Val Thir Lieu. Gly Arg Asp Ile Ser Glu Glu Arg Val Trp Glin 435 44 O 445 Ala Lieu. Glu Thr Val Glin Lieu Ala Glu Lieu Ala Arg Ser Met Ser Asp 450 45.5 460 Gly Ile Tyr Thr Pro Leu Gly Glu Gln Gly Asn Asn Lieu Ser Val Gly 465 470 47s 48O Glin Lys Glin Lieu. Lieu Ala Lieu Ala Arg Val Lieu Val Glu Thr Pro Glin 485 490 495 Ile Lieu. Ile Lieu. Asp Glu Ala Thir Ala Ser Ile Asp Ser Gly Thr Glu SOO 505 51O Glin Ala Ile Glin His Ala Lieu Ala Ala Val Arg Glu. His Thir Thr Lieu. 515 52O 525 Val Val Ile Ala His Arg Lieu. Ser Thir Ile Val Asp Ala Asp Thir Ile 53 O 535 54 O Lieu Val Lieu. His Arg Gly Glin Ala Val Glu Glin Gly Thr His Glin Glin 5.45 550 555 560 Lieu. Lieu Ala Ala Glin Gly Arg Tyr Trp Gln Met Tyr Glin Lieu. Glin Lieu. 565 st O sts

Ala Gly Glu Glu Lieu Ala Ala Ser Val Arg Glu Glu Glu Ser Lieu. Ser 58O 585 59 O

Ala

<210s, SEQ ID NO 51 &211s LENGTH: 471 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 51 Met Thr Asp Lieu Pro Asp Ser Thr Arg Trp Glin Lieu. Trp Ile Val Ala