US 2014.0171683A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0171683 A1 Sieber et al. (43) Pub. Date: Jun. 19, 2014

(54) PROCESS FOR THE ENZYMATIC Publication Classification PRODUCTION OF C4 COMPOUNDS FROM C6 SUBSTRATES (51) Int. Cl. CI2P 7/18 (2006.01) (52) U.S. Cl. (76) Inventors: Volker Sieber, Nandlstadt (DE); André CPC ...... CI2P 7/18 (2013.01) Pick, Bebra-Breitenbach (DE); Broder USPC ...... 562/577; 435/155; 435/128; 435/158; Rihmann, Straubing (DE) 435/146; 435/189:568/852:564/511 (21) Appl. No.: 14/006,847 (57) ABSTRACT The present invention relates to a novel process for converting (22) PCT Filed: Mar. 26, 2012 a of formula (III) and/or (IV) into a of formula (I) or (II) comprising the following reactions: a) (86). PCT No.: PCT/EP2012/055.308 oxidation of at least one terminal C-atom, b) dehydratation, c) decarboxylation and d) reduction and/or amination. At least S371 (c)(1), step b is -catalyzed. In a preferred embodiment, all (2), (4) Date: Dec. 9, 2013 reactions are enzymatically catalyzed. The cata lyzing the reactions are selected from , decar (30) Foreign Application Priority Data boxylases, dehydratases and/or aminotransferases. The pro cess may be performed in a cell-free in vitro production Mar. 24, 2011 (EP) ...... 11159592.2 system or in an improved fermentative production system. Patent Application Publication Jun. 19, 2014 Sheet 1 of 23 US 2014/0171683 A1

Figure 1 H O OH H OH O OH H

R O r R y R

OH OH OH OH

oxidation oxidation OH O OH O R 11. OH R 11. OH O O O dehydration O dehydration

O decarboxylation-- ---.O ana) via: NH2 reduction decarboxylation Patent Application Publication Jun. 19, 2014 Sheet 2 of 23 US 2014/0171683 A1

Figure 2

OH OH OH O

OH OH OH OH OH OH Glucose OH O OH

O R R OH

OH OH OH O

O O R -l sh- R O OH

O O R N N \, lu r N No

OH OH su" < . -> r OH OH 1,4-Butandiol Patent Application Publication Jun. 19, 2014 Sheet 3 of 23 US 2014/0171683 A1

Figure 3

Glucose

O 2 HO: Hexose 6-oxidase 1. D-gluco-Hexodialdo-1,5-pyranose NAD Aldehyde dehydrogenase NADH + H 2 D-Glucuronic acid NAD Uronate dehydrogenase NADH -- H D-Glucaric acid Glucarate dehydratase 5-keto-4-deoxy-glucarate HO + CO2 Keto-deoxy-glucarate dehydratase 2,5-Dioxopentanoate (g)NADH -- H s 5-hydroxy-2-oxo-Pentanoate CO Decarboxylase 4-hydroxy-Butyraldehyde NADH + H NAD Alcohol dehydrogenase 1,4-Butanediol Patent Application Publication Jun. 19, 2014 Sheet 4 of 23 US 2014/0171683 A1

Figure 4

Glucose O Catalase 2 Hexose 6-oxidase HO +% O, (e- H,0; Aldehyde D-gluco-Hexodialdo-1,5-pyranose dehydrogenase NAD' Uronate dehydrogenase NADH + H D-Glucuronic acid NAD' Uronate dehydrogenase NADH. H. D-Glucaric acid HO Glucarate dehydratase 5-keto-4-deoxy-glucarate HO + CO, Keto-deoxy-glucarate dehydratase 2,5-Dioxopentanoate CO, Decarboxylase Succinaldehyde

2NAD 2NADH + 2H NADH-2H 2NAD Alcohol dehydrogenase Transaminase/Amino acid dehydrogenase Aldehyde 1,4-Butanediol Annine dehydrogenase dehydrogenase NADH + 2H

2NAD

1,4-Diaminobutane Patent Application Publication Jun. 19, 2014 Sheet 5 of 23 US 2014/0171683 A1

Figure 5

Glucose NAD'? O, Glucose dehydrogenase/ NADH + H/H,0, H0 +% O, -Goldatalase D-1,5-glucono-lactone/D-Gluconate D-Gluconate dehydratase HO 2-keto-3-deoxy-D-gluconate NAD' 2-keto-3-deoxy-D-gluconate-6-dehydrogenase NADH - 4,5-dihydroxy-2,5-dioxohexanoate NADt Uronate dehydrogenase NADH -- H 2-keto-3-deoxy-glucarate HO + CO Keto-deoxy-glucarate dehydratase 2,5-Dioxopentanoate CO Decarboxylase Succinaldehyde

2NAD NADH + 2 Alcoholdehydrogenase 2NADH -- 2H 2NAD Aldehyde dehydrogenase 1,4-Butanediol

Transaminase/Anino acid dehydrogenase Amine dehydrogenase Succinc acid

1,4-Diaminobutane Patent Application Publication Jun. 19, 2014 Sheet 6 of 23 US 2014/0171683 A1

Figure 6

A.

- -- m

s:

st)

-:) ------2: -...------

-50 -

2COOOC

000

O-- --- : 3. . Patent Application Publication Jun. 19, 2014 Sheet 7 of 23 US 2014/0171683 A1

Figure 7

Patent Application Publication Jun. 19, 2014 Sheet 8 of 23 US 2014/0171683 A1

Figure 8

Patent Application Publication Jun. 19, 2014 Sheet 9 of 23 US 2014/0171683 A1

Figure 9

Activity Assay for slAIDH using Acetaldehyde as substrate

E ed t fy d s c e -- NHSIADH es o As so

time (minutes) Patent Application Publication Jun. 19, 2014 Sheet 10 of 23 US 2014/0171683 A1

Figure C

- ificatiof of C at eier df. erase f : 'Agioitacierun snai acters C58 Patent Application Publication Jun. 19, 2014 Sheet 11 of 23 US 2014/0171683 A1

Figure 11

Uronate-Dehydrogenase Acitivity against Glucuronate

1. 0.9 0,8 0.7 0,6 0.5 0,4 -0-NH Udh A. tumefaciens 0,3 1:250 dilution 0.2 0,1

time (minutes) Patent Application Publication Jun. 19, 2014 Sheet 12 of 23 US 2014/0171683 A1

Figure 12

Furific:- 2 - ; ; ; Giscaratear. 2 8hydratase if it, Aiii,3. rivecii.. . . . , ; 3, Slic, ; resis-, insigeries -, *.*.*.*, * E.if: - Patent Application Publication Jun. 19, 2014 Sheet 13 of 23 US 2014/0171683 A1

Figure 13

Glucarate conversion with glucarate-dehydratase from A. Succinogenes 130Z O,6 O,5 0,4

0.2 -0-NH GlucDA.S. 1:100 0,1 1 Enzyme Dilution 1:100 O O 2 4. 6 8 Time (minutes) Patent Application Publication Jun. 19, 2014 Sheet 14 of 23 US 2014/0171683 A1

Figure 14

Purificatio F if ket-dec xy-Glucarate Dahydratasa frary Acipate:3cts,. :-3 is isi?, AEP 1 Patent Application Publication Jun. 19, 2014 Sheet 15 of 23 US 2014/0171683 A1

Figure 15 Coupled Assay using aldehyde dehydrogenase for activity determination of NHKdgD A.baylyi

O,35 0,3 0.25 0,2 0,15 --NHKdgD A.baylyi 0,1 1:500 dilution O,05 O 20 40 time (minutes) Patent Application Publication Jun. 19, 2014 Sheet 16 of 23 US 2014/0171683 A1

Figure 16

Patent Application Publication Jun. 19, 2014 Sheet 17 of 23 US 2014/0171683 A1

Figure 17

Coupled assay for YigB using KdgD for substrate generation 1,

0,8 0,6 --CHYigB E.coli K-12 0,4 -- Blank 0,2

O 2 4. 6 8 time (minutes)

Patent Application Publication Jun. 19, 2014 Sheet 18 of 23 US 2014/0171683 A1

Figure 18

Patent Application Publication Jun. 19, 2014 Sheet 19 of 23 US 2014/0171683 A1

Figure 19

O ------2 3 4. 5 6 7 8 9 Time Emir - EC540.0 AMS Ints, +MS, 2.7min #274 x10 539.1 541.1

3

2

40.

1

43.

O Y------r ------530 535 540 545 550 555 56Oz Patent Application Publication Jun. 19, 2014 Sheet 20 of 23 US 2014/0171683 A1

Figure 20

intens. x07

O a 2 3 4. s 8 7 8 9 Time min - EICS37.O-AIMS intens. -MS, 3.1min:319

x107 537.1 539.1

1.25

1.00

0.75

0.50

0.25

0.30 H-H --all--a- w T 530 S35 50 54s 55 555 560 m Patent Application Publication Jun. 19, 2014 Sheet 21 of 23 US 2014/0171683 A1

Figure 21

2 3 4 5 6 7 8 9 Time min) EIC 442. AllMS

430 435 440 445 450 455 m/2 Patent Application Publication Jun. 19, 2014 Sheet 22 of 23 US 2014/0171683 A1

Figure 22

intens. x107

O Aas , , , , , , , , , , , , - 2 3 4. 5 s 7 B 9 Time (min) EC 475. AMS EC 433.2+AIMS Intens. MS, 4.7min A99 x105 s

- --s-s 470 472 474 476 478 480 482 484 niz Patent Application Publication Jun. 19, 2014 Sheet 23 of 23 US 2014/0171683 A1

Figure 23

intens. x107

0 ------fer 2 3 4. 5 6 7 8 Time min - EC 477.1 AIIMS

hisxOO +MS, 4.9min its 6

2.5-

5

0.5-

d --a- --al 480 465 470 US 2014/0171683 A1 Jun. 19, 2014

PROCESS FOR THE ENZYMATIC Presently this family has a market opportunity that exceeds PRODUCTION OF C4 COMPOUNDS FROM €3.000 M. Approximately 1.4 Mt BDO is produced by C6 SUBSTRATES chemical catalyst 8. The demand for BDO stems largely from its use as an intermediate for polybutylene terephthalate TECHNICAL FIELD (PBT) plastic resins, polyurethane thermoplastics and co 0001. The present invention relates to a novel process that polyester ethers. BDO also serves as a primary precursor to effectively reduces the functionalization of polyol com THF, which is employed as an intermediate for poly(tetram pounds and comprises the following reactions: a) oxidation of ethylene glycol) PTMEG copolymers required for lycra and at least one terminal C-atom, b) dehydration, c) decarboxy spandex production. Approximately 0.32 Mt of THF is pro lation and d) reduction and/or amination. At least step b is duced globally per year with an annual growth rate over 6%. enzyme-catalyzed. Preferably, all of the reactions may be A significant percentage of growth (>30%) for both BDO and enzyme-catalyzed combining the activity of 4 types of THF is occurring in Asia (China and India). GBL currently is enzymes: oxidoreductases, e.g. dehydrogenases or oxidases, a smaller volume (0.18 Mt/year) product which has numer dehydratases, decarboxylases, and aminotransferases. This ous applications as a solvent, as an additive for inks, paints, inexpensive process can be used to converthexoses or hexi and dyes, as well as the primary precursor to pyrrolidone tols into C4-chemicals such as 1,4-butanediol. 1,4-butane derivatives such as NMP. dial, 1,4-diaminobutane, 4-hydroxybutyric acid or Succinic 0005. However, the replacement of fossil raw materials by acid at mild conditions, using only enzymes, water, biogenic resources is still one of the major obstacles prevent (plus an amino donor like ammonium in case of the produc ing widespread commercialization of Such devices. tion of amines) and leaving the inner structure of the carbon 0006 Enzymes exhibit a great advantage compared to chain intact. chemical catalysts because they are accepting a wide array of complex molecules as Substrates, catalyzing reactions with BACKGROUND OF THE INVENTION unparalleled chiral (enantio-) and positional (regio-) selec tivities. For this reason, the need of tedious blocking and 0002 Climate change and the eventual depletion of the deblocking steps known in traditional organic synthesis is world's fossil raw materials reserves are threatening Sustain dispensable 9. Biological catalysts allow the development able development 1, 2. Renewable resources display a large of Sustainable technologies for the production of chemicals potential for the substitution of chemical compounds derived by waste reduction using solvent-free reaction media and from petrochemicals. They allow a more sustainable chemis minimizing the amount of unrequested by-products compli try with the attempt to design chemical products and pro cating the downstream processing 10. Biocatalyst can be cesses that reduce or eliminate the use and generation of used either as isolated enzymes or in the form of whole cell hazardous Substances, minimize waste and energy consump preparations. The use depends on the requirements of the tion, favor renewable resources and integrate aspects of recy production process like the half-life of the biocatalyst or the cling 3. Besides, nature offers a wide range of resources dependency on co-factors. Reaction processes regarding co mainly from plants due to fast biomass building with low factors, especially NAD(P)", which are utilized in stochimet efforts. At the moment only a few industries are using this ric quantities whole-cell fermentation is favored. The use of immense reservoir of resources 4. Additionally there have isolated co-factor depending enzymes for establishing a been 12 principles postulated for a Green Engineering con multi-step Substrate conversion requires an additional co cerning new processes or the displacement of antiquated pro factor recycling system for a continuous reaction. Presently, cesses to engage in a more Sustainable development 5. Sev there are some co-factor recycling systems like glucose dehy eral programs were initiated by the European Union and drogenase/glucose established which allow TTN's (total miscellaneous German institutions that promote the research turnover number) from 10 to 10° or higher for an economical and development concerning this topic. efficient reaction process. It is, however, preferred that cofac 0003. Succinic acid is a chemical substance with a broad tor recycling can be achieved without additional Substrates. area of application in the chemical industry. Succinic acid 0007 Carbohydrates represent 95% of the annually represents an important building block that can be converted renewable biomass. Being renewable carbohydrates such as into various valuable compounds 6. Beyond fossil based glucose or other monosaccharides have the potential to com chemistry, derivatives of Succinic acid are announced to have pensate the emerging lack of petroleum for the production of a potential of hundreds of thousands tons 7. Succinic acid is bulk chemicals or biofuels. For use as chemicals or fuel an intermediate of the TCA cycle (tricarboxylic acid cycle) carbohydrates contain too many polar functional groups. In and one of the fermentation end-products of anaerobic the past this was the reason they were disqualified as well metabolism. The research for biotechnological production Suited precursors for applications in organic chemistry 11. processes mainly focused on a whole-cell approach using The use of low molecular weight carbohydrates as well as natural overproducers or recombinant producers. The down high molecular weight carbohydrates as the C-source for stream purification cost for fermentation-based processes fermentation processes to produce industrial important normally amounts to more than 60% of the total production chemical compounds is well known. Succinic acid or 2.3- costs. For Succinic acid purification, the separation of byprod butanediol are two examples of compounds produced by fer ucts has a crucial effect on process cost 4. mentation from carbohydrates. In contrast, the specific con 0004 1,4-Butanediol (BDO) is a four carbon dialcohol version of glucose with a multi-step cell free biocatalytic or that is at the moment manufactured exclusively through Vari catalytic process into chemical intermediates is mostly unde ous petrochemical routes. BDO represents a chemical build veloped. The only economically viable examples are the ing block which can be used for production of gamma-buty hydrogenation of glucose to sorbitol followed by the conver rolactone (GBL), tetrahydrofuran (THF), pyrrolidone, sion to isosorbide and the oxidation of glucose to gluconate. N-methylpyrrolidone (NMP) and N-vinyl-pyrrolidone 8. For the production of C4-compounds from hexoses to date US 2014/0171683 A1 Jun. 19, 2014 only fermentative processes have been developed, mostly trum of industrial applications. It provides a novel route to aiming at succinic acid. The Department of Energy of the US produce the above mentioned chemicals in a cell-free produc has proposed 1,4-diacids, and particularly Succinic acid, as tion system or in an improved fermentative production sys key biologically-produced intermediates for the manufacture tem of the butanediol family of products 6. However, using 0012. The invention can be described due to the reactions fermentation processes, always side products are formed due that are applied: (a) oxidation of a terminal C-atom (alcohol to the presence of many different enzymes within the organ or aldehyde) to carboxylic acid; (b) dehydration of an internal isms. In addition the conditions of the production process carbonatom to produce a methylen group (deoxy-group) and (temperature, pH, salt etc.) are limited by the viability of the adjacent to it a carbonyl group; (c) removal of the terminal cells. Product purification often is the most costly process carboxylic acid by forming carbon dioxide; (d) conversion of step in a fermentative production system. All these difficulties a now terminal carbonyl group to a hydroxyl group (reduc can be diminished when a cell free production process can be tion), amino group (transamination or reductive amination) or used. By limiting the number of enzymes in such a cell free carboxyl group (oxidation). production to only those essential for the targeted conversion, fewer side products are formed. By applying conditions far 0013 At least the dehydration reaction is enzymatically from being ambient (e.g. high temperatures, co-solvents) catalyzed. In a preferred embodiment, all reactions are enzy product purification can be more easily integrated into the matically catalyzed. The enzymes catalyzing the reactions are conversion process. The known pathways from glucose to selected from oxidoreductases, decarboxylases, dehydratases bifunctional C4-compounds, modified at position 1 and 4, all and/or aminotransferases. In a preferred embodiment, the go via Succinate and have never been used in cell free pro is an alcohol dehydrogenase, aldehyde dehy duction systems and are probably too difficult to handle drogenase, amino acid dehydrogenase, and/ (>10 enzymes) to ever be used in a cell free production or aldehyde oxidase. system. There is a need for a production process lacking live 0014. During the process, redox reactions take place. To organisms using just enzymes or other catalysts to cheaply take up and to deliver electrons, a co-factor may be employed. converthexoses to bifunctional C4 compounds and therefore, This can be, for example, NAD"/NADH. Alternatively, it is there is a need of new and simpler enzymatic pathways, possible to use NADP"/NADPH or FAD"/FADH or even requiring fewer enzymes than existing natural pathways. other molecules as co-factors for the process; however, it is There is a demand for a new enzymatic pathway that can be advantageous that every enzyme can utilize the identical applied using purified enzymes or enzymes in cell lysates for pair. Preferably, only one free co-factor is employed a completely cell free in vitro production process or in whole during the process. cells containing the enzymes. In addition, it would be benefi 0015 Thus, in an exemplary embodiment, the process can cial if a new enzymatic pathway could help to improve the comprise: (a) providing a composition (e.g. mixture) com yield and productivity of a fermentation process when the prising at least onehexose, water, oxygen and when necessary enzymes of the pathway are recombinantly expressed in ammonia; (b) providing one (or more) enzymes or catalysts microorganisms. able to oxidize the C1 carbon atom; (c) providing an agent 0008. It is desirable to have such a synthetic pathway for (acid, base, enzyme) for ring-opening of lactones; (d) provid the production of C4 chemicals by alternative means not only ing one (or more) enzymes able to oxidize the terminal group to substitute petroleum-based feedstocks but also to facilitate at C-6 to generate a diacid compound; (e) providing one (or a Sustainable process with less waste. more) enzymes having a dehydratase activity for deoxygen 0009 All previously described microbial routes for the ation of the internal carbon atoms of dihydroxy acids by production of bifunctional C4-chemicals like 1,4-butanediol removal of water; (e) providing one or more agents (acid, or 1,4-aminobutane from C6 polyols and hexoses use more base, enzyme) for the decarboxylation of carboxylic acids; (f) than 10 enzymes in complex metabolic pathways (glycolysis, providing one (or more) enzymes for the reduction of alde TCA cycles) requiring a multitude of cofactors (at least hydes to alcohols or for the oxidation of aldehydes to car NAD"/NADH, ATP/ADP, Coenzyme A) and break down the boxylic acids or for conversion of aldehydes into amines. C6-molecules in two C3-molecules like 3-phospho-glycerate 0016. The process of the invention, in its general, basic to then reconstruct the C4 entity. form or as described in detailed embodiments, can be per formed in any convenient manner. Thus, all of the chemical or SUMMARY OF THE INVENTION biochemical reaction steps may be performed in a single 0010. The present invention provides a novel synthetic reaction vessel. Alternatively, one or more of the reactions enzymatic pathway fulfilling the requirements mentioned may be performed separately. The process may be performed above. In the process according to the present invention the as a batch process or as a continuous process, with products carbon chain of the substrate, e.g. a C carbon chain, is inter being removed continuously and new Substrates being intro nally left intact, the functionalities (hydroxyl groups) are duced. moved from the inside of the molecule to the terminal ends 0017 Advantageously, the process can be conducted at and are removed from the molecule by release of the two low to moderate temperatures, such as between 10° C. and terminal carbon atoms in the form of CO. This elegant reac 100° C. It is also possible to operate the reaction attempera tion cascade requires less than ten enzymes and has the poten tures below 10°C. if enzymes from psychrophilic organisms tial to be applied in an in vitro enzyme system, and can also be are used. In some embodiments, no external chemical energy used inside a microbial cell. source is added, and the only energy added is heat. Preferably, 0011. The process according to the present invention pro the system is maintained at a constant temperature, taking vides a non-naturally occurring pathway, which for example into consideration that the temperature is a function of Sub allows the production of C4-chemicals from hexoses. The strate concentration, net heats of the reaction and heat losses resulting chemical compounds can be used for a broad spec of the particular system. US 2014/0171683 A1 Jun. 19, 2014

0018 Referring to the present invention, the process yield carate, Dotted line=Glucarate, Dashed-dotted line=NADH: of the different products is typically one mol of product per B. Sample containing all enzymes after 24h. The peak at 26.2 mol of Substrate, in the case that the Substrates are monosac min retention time indicates the formation of 1,4-butandiole. charides or derivates of them. 0030 FIG. 7 shows the analysis of the product of the 0019. An exemplary embodiment of the process can con purification of galactose oxidase from Fusarium tain: (a) glucose as Substrate source, water and oxygen (b) one graminearum. or more enzymes and cofactors capable of oxidizing both 0031 FIG. 8 shows the analysis of the product of the terminal carbonatoms to get analdaric acid (c) dehydrating of purification of aldehyde dehydrogenase from Ovies aries. the internal carbon atoms (d) decarboxylation for removal of 0032 FIG. 9 shows the results of the activity assay for the two terminal C-atoms (e) reduction of the new terminal slAlDH using acetaldehyde as substrate. carbonyl groups to form a diol and (f) oxidation of excess 0033 FIG. 10 shows the analysis of the product of the cofactors that are in the reduced form. purification of uronate dehydrogenase from Agrobacterium 0020. A different exemplary embodiment of the process tumefaciens C58. can contain: (a) glucose as Substrate source, water and oxygen 0034 FIG. 11 shows the results of the activity assay for (b) one or more enzymes and cofactors capable of oxidizing uronate dehydrogenase using glucuronate as Substrate. both terminal carbon atoms to get an aldaric acid (c) dehy 0035 FIG. 12 shows the analysis of the product of the drating of the internal carbon atoms (d) decarboxylation for purification of glucarate dehydratase from Actinobacillus removal of the two terminal C-atoms (e) oxidation of the new succinogenes 130 Z. terminal carbonyl groups to form a diacid and (f) oxidation of 0036 FIG. 13 shows the results of the activity assay for excess cofactors that are in the reduced form. glucarate dehydratase using glucarate as Substrate. 0021. In particular, the oxidation of hexoses at the terminal 0037 FIG. 14 shows the analysis of the product of the hydroxylated carbon atom (C-6) can be accomplished by any purification of Keto-deoxy-Glucarate-Dehydratase from Suitable means, preferably by a chemical or biochemical cata Acinetobacter baylvi ADP1. lyst. However, more typically, when a biocatalyst is used the 0038 FIG. 15 shows the results of the activity assay for oxidation will be in two steps by generating an aldehyde, Keto-deoxy-Glucarate-Dehydratase using keto-deoxy-glu which is oxidized to a carboxylic acid. carate as Substrate. 0022. The present invention further provides the alcohol 0039 FIG. 16 shows the analysis of the product of the dehydrogenase YigB from E. coli, which may be employed in purification of alcohol-dehydrogenase from Escherichia coli the process according to the present invention and is capable K-12 (YigB). of catalyzing the conversion of 2,5-dioxopentanoate into 0040 FIG. 17 shows the results of the activity assay for 5-hydroxy-2-oxo-pentanoate. This step is depicted in step 6 alcohol-dehydrogenase from Escherichia coli K-12 (YigB) of FIG. 3. No previously isolated enzyme is capable of cata using 2,5-dioxo-pentanoate as Substrate. lyzing this reaction. 0041 FIG. 18 shows the analysis of the product of the 0023 The present invention also provides an enzyme mix purification of alcohol dehydrogenase from Escherichia coli ture comprising less than 10 enzymes and optionally cofac K-12. tors required by the enzymes, wherein the enzymes are 0042 FIG. 19 shows the analysis of intermediates using selected from oxidoreductases, dehydratases, decarboxy HPLC-MS (Example 3): D-glucuronic derivative, upper lases, and aminotransferases. panel: elution time, lower panel: mass values detected 0043 FIG. 20 shows the analysis of intermediates using BRIEF DESCRIPTION OF THE FIGURES HPLC-MS (Example 3): D-glucaric acid derivative, upper 0024 FIG. 1 shows the reaction sequence leading to a panel: elution time, lower panel: mass values detected reduced number of functional groups of polyols. 0044 FIG. 21 shows the analysis of intermediates using 0025 FIG. 2 shows the example for the application of a HPLC-MS (Example 3): 5-keto-4-deoxy-glucuronic acid reaction sequence to convert glucose into 1,4-butandiol. derivative, upper panel: elution time, lower panel: mass Val 0026 FIG.3 shows a possible reaction sequence including ues detected (m/Z-2) the different enzymes involved. NAD"/NADH represent 0045 FIG. 22 shows the analysis of intermediates using cofactors carrying redox potential. In reality NADP/ HPLC-MS (Example3): 2,5-dioxo-pentanoic acid derivative, NADPH or FAD/FADH, could be applied. upper panel: elution time, lower panel: mass values detected 0027 FIG. 4 is a schematic diagram showing one possible 0046 FIG. 23 shows the analysis of intermediates using synthetic pathway for converting hexoses into different pos HPLC-MS (Example 3): 5-hydroxy-2-oxo-pentanoic acid sible products starting with the oxidation at position C-6. derivative, upper panel: elution time, lower panel: mass Val Solid black arrows show the different enzymatic steps. ues detected Circles illustrate the co-factor recycling system which depends on the produced product. The NADH production per DETAILED DESCRIPTION OF EMBODIMENTS mol glucose depends on the enzymes used. OF THE INVENTION 0028 FIG. 5 shows a synthetic pathway for converting 0047. The following description provides a detailed dis hexoses into different possible products starting with the oxi cussion of certain embodiments and features of the pathway, dation at position C-1. Solid black arrows show the different co-factor recycling and compositions of the invention. It is enzymatic steps. Circles illustrate the co-factor recycling sys not meant to be exhaustive of all such embodiments and tem which depends on the produced product. The NADH features, but rather is presented to give the reader a better production per mol glucose depends on the enzymes used. understanding of selected exemplary embodiments and fea 0029 FIG. 6 shows chromatograms representing the tures. results of Example 1. A. Standards, Solid line=1,4-Butandi 0048. To give a better understanding of the invention, cer ole, retention time at 26.2 min, Dashed line-keto-deoxy Glu tain terms are now defined and for discussed. Terms not US 2014/0171683 A1 Jun. 19, 2014

discussed or defined herein are to be understood as being used inside of the molecule to the terminal ends and are removed in their normal and customary way in the art. By “hexose' it from the molecule by release of the two terminal carbon is meant any monosaccharide as the basic unit of carbohy atoms in the form of CO. The process according to the drates. Hexoses can include allose, altrose, glucose, mannose, present invention may be employed for the production of gulose, idose, galactose, talose, psicose, fructose, Sorbose, bifunctional C4 molecules, e.g. 1,4-butandiol, from C 6 sub tagatose). strates, e.g. hexoses, wherein the bond between the C3 and the 0049 C4-chemicals generated in the pathway are C4 is left intact. described briefly. The term 1,4-butanediol is intended to 0055. In particular, the present invention relates to a pro mean an alcohol derivative of the alkane butane, carrying two cess for the conversion of a substrate of the chemical structure hydroxyl groups having the chemical formula C4H10O2 (III) and/or (IV) into an alcohol oramine of structure (I) or (II) molecular mass of 90.12 g/mol. Succinic acid is a derivative of the comprising the following reactions: (a) oxidation of at least alkane butane, carrying two carboxylic acid groups having one terminal C-atom; (b) dehydration; (c) decarboxylation; the chemical formula CHO and a molecular mass of 118. (d) reduction and/or amination. 09 g/mol. 1,4-Butanedial is another derivative of the alkane butane, carrying two carbonyl groups having the chemical formula CHO, and a molecular mass of 86.09 g/mol. 1,4- (I) Diaminobutane is a derivative of the alkane butane, carrying two amino groups having the chemical formula CHN and r a molecular mass of 88.15 g/mol. OH 0050. As used herein, “enzymes' are protein catalysts that (II) catalyze (i.e., accelerate) chemical and biochemical reac tions. As used herein “enzyme” is meant to encompass a r single enzyme, mixtures comprising one or more enzymes, or NH2 enzyme complexes. OH t (III) 0051. The (EC number) is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. For the purposes of the present invention, an EC number will also be used to specify - OH enzymes. When an enzyme is characterized by an EC number (IV) herein, it is understood that there can be multiple enzymes OH OH from different sources or organisms that all catalyze the same reaction. The invention is not limited to any particular enzyme or source of enzymes, but rather to certain enzyme - OH catalyzed reactions in the pathway, as will be described below. The language “an enzyme that is characterized by EC 1.1.1.1, for example, means any amino acid sequence that 0056 R is a C- alkyl, preferably a Cls alkyl, more has the EC number 1.1.1.1 according to at least one art preferably a C- alkyl, wherein the alkyl may be substituted recognized enzyme information system (such as BRENDA or with one or more hydroxyl groups. Preferably, R may be the KEGG) as of the filing date of this application. following residues but is not limited to these: —CHOH, 0052. As is known in the art, “identity” between two CH(OH) CH(OH), -CH(OH) CH(OH) CH(OH). enzymes is determined by comparing the amino acid In a preferred embodiment of the invention, polyols of the sequence of one enzyme to the sequence of a second enzyme. general formula R-CH(OH)-CH(OH)-CH(OH) are Identity may be determined by procedures which are well used. The substrate may preferably be a C6-polyol, C6-sugar known in the art, for example, by utilizing BLAST (Basic or C6-Sugar acid. Local Alignment Search Tool at the National Center for Bio 0057. At least reaction b is enzyme-catalyzed. In a pre logical Information). When enzyme identity is recited in con ferred embodiment, all reactions a-d are enzyme-catalyzed. junction with an enzyme EC number, according to the present Reaction a may be catalyzed by an oxidoreductase, reaction b description it is to be understood that there can be many by a dehydratase, reaction c by a decarboxylase and reaction different amino acid sequences that all have the same EC d by an aminotransferase or an oxidoreductase. The oxi number. Thus, for example, the language “an enzyme that is doreductase may preferably be an alcohol dehydrogenase, at least 90% identical to EC 1.1.1.1' means an amino acid aldehyde dehydrogenase, amino acid dehydrogenase, alcohol sequence that is computed to have 90% or better sequence oxidase and/or aldehyde oxidase. identity to at least one amino acid sequence that has the EC 0058. The process according to the present invention may number 1.1.1.1 according to at least one art-recognized be performed in the presence of one or more cofactors for enzyme information system (such as BRENDA or KEGG) as transfer of reduction equivalents. The cofactor(s) may be of the filing date of the present application. selected from NAD"/NADH, NADP/NADPH and FAD"/ 0053. The invention is directed to the design of a synthetic FADH. Alternatively, NAD(P)"/NAD(P)H-mimicking pathway to enable the production of different products with agents as described in US2003/0022266 may be employed as isolated enzymes or whole cells in a multi-step enzymatic cofactor. reaction or in a fermentation process employing microbial 0059. The production of an alcohol of formula (I) is cells. achieved by the sequence of the following four reactions. 0054. In the process according to the present invention the These reactions may be performed simultaneously in one carbon chain of the Substrate, e.g. a C carbon chain, is inter reaction mixture (one-pot synthesis) or can also be performed nally left intact, the functional groups are moved from the separately in separate reaction vessels. US 2014/0171683 A1 Jun. 19, 2014

0060 a) Oxidation (preferably catalyzed by oxi acids preferably using an amino acid dehydrogenase. In case doreductases, EC 1.1.x.x, EC 1.2.x.x), then: this step is catalyzed by a dehydrogenase, cofactors such as 0061 b) Dehydration (preferably catalyzed by dehy NADPH or NADH are needed. In another preferred embodi dratases, EC 4.2.1.X), after that or simultaneously: ment, the conversion of the carbonyl function in C-position to 0062 c) Decarboxylation (catalyzed preferably by the carboxylic acid into an O-amino carboxylic acids may be decarboxylases, EC 4.1.1.x), then: achieved via a transaminase and an amino donor, which itself 0063 d) Reduction (preferably catalyzed by dehydro may be regenerated by an amino acid dehydrogenase. genases, EC 1.1.1.X) 0080 Reaction d1 is a decarboxylation reaction, which 0064. This is illustrated in the following figure (FIG. 1, leads to a shortening of the carbon chain by one carbon atom left). by the release of CO. This reaction may preferably be cata 0065 Reaction a is an oxidation of the terminal hydroxyl lyzed by a decarboxlyase that recognizes C.-amino carboxylic function to an aldehyde function. This step may preferably be acids as a Substrate. Finally, a product is formed that contains performed using an oxidase or a dehydrogenase. In the case of a terminal amino function. using a dehydrogenase, cofactors such as NADP" or NAD" are needed. In a further reaction a carboxyl function is gen I0081 Reaction c2 is a decarboxylation reaction, which erated analogously from the aldehyde function. leads to a shortening of the carbon chain by one carbon atom 0066 Reaction b is a dehydration reaction at two internal by the release of CO. This reaction may preferably be cata hydroxyl functions, resulting in the elimination of water and lyzed by a decarboxlyase that recognizes C.-keto carboxylic the generation of a carbonyl function in a-position to the acids as a Substrate. carboxyl function. This reaction is catalyzed by a dehy I0082 Reaction d2 is a conversion of a terminal carbonyl dratase. function into a primary amine preferably using an amin dehy 0067. Reaction c is a decarboxylation reaction, which drogenase. In case this step is catalyzed by a dehydrogenase, leads to a shortening of the carbon chain by one carbonatom cofactors such as NADPH or NADH are needed. In another by the release of CO. This reaction may preferably be cata preferred embodiment, the conversion of the carbonyl func lyzed by a decarboxylase that recognizes C.-keto carboxylic tion in C-position to the carboxylic acid into an O-amino acids as a Substrate. Such a product is formed that contains a carboxylic acids may be achieved viaan w-transaminase and terminal carbonyl function (aldehyde). an amino donor, which itselfmay be regenerated by an amino 0068 Reaction d is a reduction of the terminal carbonyl acid dehydrogenase. Finally, a product is formed that con function, preferably using a dehydrogenase. In case this step tains a terminal amino function. This is illustrated by the is catalyzed by a dehydrogenase, cofactors such as NADPH following figure (FIG. 1, right). or NADH are needed. 0083. In case the substrate contains several of the structure 0069. The production of an amine of formula (II) is elements (III) and/or (IV), then the described reaction achieved by the sequence of the following four reactions. sequence can occurat all these structure elements. Examples These reactions may be performed simultaneously in one of Such molecules include hexoses such as glucose (V) and reaction mixture (one-pot synthesis) or can also be performed their alcohol derivatives such as sorbitol (VI): separately in separate reaction vessels. 0070 a) Oxidation (preferably catalyzed by oxi doreductases, EC 1.1.x.x, EC 1.2.x.x), then: (V) 0071 b) Dehydration (preferably catalyzed by dehy OH OH O dratases, EC 4.2.1.x), then either: 0072 c1) Amination (catalyzed preferably by an amino acid dehydrogenases, EC 1.4.X.X or by a transaminases, OH OH OH EC 2.6.1.x), then: (VI) (0073 d1) Decarboxylation (preferably catalyzed by OH OH OH amino acid decarboxylases, EC 4.1.1.X) 0074 or (0075 c2) Decarboxylation (preferably catalyzed by C.-keto-decarboxylases, EC 4.1.1.X), then: OH OH OH 0076 d2) Amination (catalyzed preferably by an amino acid dehydrogenases, EC 1.4.X.X or by a transaminases, I0084. When these molecules are converted by the EC 2.6.1.x) described sequence of reactions, 1,4-Butandiol is formed as 0077 Reaction a is an oxidation of the terminal hydroxyl shown in FIG. 2. function to an aldehyde function. This step may preferably be performed using an oxidase or a dehydrogenase. In the case of I0085. In a preferred embodiment, the present invention using a dehydrogenase cofactors such as NADP" or NAD" are therefore relates to an enzymatic/chemical process for the needed. In a further reaction a carboxyl function is generated production of bifunctional C4 chemicals from polyols or analogously from the aldehyde function. partially oxidized polyols. 0078 Reaction b is a dehydration reaction at two internal 0.086 Bifunctional C4 chemicals, such as 1,4-butandiol hydroxyl functions, resulting in the elimination of water and and 1,4-diaminobutane, are valuable building blocks for pro the generation of a carbonyl function in C-position to the ducing a wide range of polymers that meet the requirements carboxyl function. This reaction is catalyzed by a dehy for a variety of applications. dratase. I0087 Polyols are chemical compounds containing at least 0079 Reaction c1 is a conversion of a carbonyl function in 4 hydroxyl functions (—OH). Classical chemical methods C-position to a carboxylic acid into an O-amino carboxylic are not suitable for functionalization or defunctionalization of US 2014/0171683 A1 Jun. 19, 2014 compounds with several functional groups, since the reac process according to the present invention may be performed tions are not specific and single functional groups cannot be separately in separate reaction vessels. The reactions may all changed selectively. be carried out separately or they may be individually com 0088. The precise sequence of reactions at a structural bined in various combinations. The enzymes used in the element (III or IV) is exactly defined as described above reactions may be immobilized, e.g. on a carrier. In another (oxidation, dehydration, decarboxylation and reduction for embodiment, the enzymes may be genetically-engineered the production of alcohols or oxidation, dehydration, amina enzymes or enzyme-complexes, which may preferably tion and decarboxylation or decarboxylation and amination exhibit several enzymatic activities. In a further embodiment, for the production of amines). When two structural units are the process according to the present invention may be per combined in one Substrate, e.g. glucose, the sequence of formed in a bioreactor. reactions can proceed in various combinations. Thus, for example with glucose both terminal carbon atoms are first 0100. The process according to the present invention may oxidized to the carboxylic acid (formation of glucaric acid), also be exploited in microorganisms, wherein the microor followed by the dehydration at both sides (formation of 2.5- ganism recombinantly expresses, preferably overexpresses, dioxo 3.4 dideoxy glucaric acid), then both are decarboxy the enzymes catalyzing the reactions of the process according lated (succinaldehyd) and finally both ends are reduced (1.4 to the present invention. For example, 1.4 butanediol may be butandiol). Alternatively, glucose could initially be oxidized produced from glucose or other hexoses by fermentation or on one side (C1) (formation of gluconate) followed by the by a whole cell biotransformation. 1,4-butanediol is not a dehydration (2-oxo 3-deoxygluconate), then followed by the natural metabolite. A synthesis from glucose through this oxidation at the other side of the molecule (C6) (2-oxo pathway has not yet been demonstrated. 3-deoxyglucaric acid), followed by the decarboxylation and 0101 Implementing further enzymatic activities or reduction at C1 (2,4-dihydroxybutanoic acid), and finally at replacing selected enzymatic activities in the present reaction the former C6-end dehydration, decarboxylation and reduc sequence additional reaction schemes are possible and addi tion take place. These reaction sequences can be presented tional products can be produced (like Succinic acid or 4-hy schematically the following way, for the production of diols: droxybutyric acid). This is shown schematically in FIGS. 4 I0089. 1.) O1-O6-DH1-DH6-DC1-DC6-R1-R6 (oxida and 5. tion at C1, oxidation at C6, dehydration at C2 and C3, dehydration at C4 and C5, decarboxylation at C1, decar 0102 The present invention further provides the alcohol boxylation at former C6, reduction at former C2, reduc dehydrogenase YigB from E. coli, which is capable of cata tion at former C5) lyzing the conversion of 2,5-dioxopentanoate into 5-hy 0090. 2.) O1-DH1-O6-DC1-R1-DH6-DC6-R6 droxy-2-oxo-pentanoate. This step is depicted in step 6 of 0091 All together more than 70 different combinations FIG. 3. No previously isolated enzyme is capable of cata are possible that fall into the constraint presented above, like lyzing this reaction. In Example 2.6, the production and char for example: acterization of this enzyme is described. 0092. 3.) O1-DH1-DC1-R1-O6-DH6-DC6-R6 0103) The alcohol dehydrogenase YigE from E. coli may 0093. 4.) O6-DH6-DC6-R6-O1-DH1-DC1-R1 be used in the process according to the present invention. 0094 5.) etc. 0095. The production of 1,4-diaminobutan can analo 0104. The present invention also provides an enzyme mix gously be achieved by many different combination of reac ture comprising less than 10 enzymes and optionally cofac tion routes. tors required by the enzymes, wherein the enzymes are selected from oxidoreductases, dehydratases, decarboxy 0096. In FIG.3, an example is presented with the success lases, and aminotransferases. The oxidoreductase may pref ful implementation of the conversion of glucose to 1,4-bu erably be an alcohol dehydrogenase, aldehyde dehydroge tanediol. According to the above-described nomenclature this nase, amino acid dehydrogenase, alcohol oxidase and/or example relates to the order: O1-O6-DH1-DH6-DC6-R6 aldehyde oxidase. DC1-R1, wherein O6-reaction 1 and 2, Ol=reaction 3, DH6-reaction 4, DH1 and DC6-reaction 5, R6-reaction 6, 0105 Said enzyme mixture may be used in the process DC1 reaction 7, R1 =reaction 8. according to the present invention. 0097 Further embodiments are possible. 0106 The enzyme mixture according to the present inven 0.098 Inafurther embodiment, substrates can be used and tion may comprise one or more cofactors for transfer of introduced at any reaction step in the procedure, for example reduction equivalents. The cofactor(s) may be selected from for the possible use of already oxidized or modified polyols. NAD"/NADH, NADP/NADPH and FAD"/FADH. Alterna For example, glucuronic acid can be used as Substrate, which tively, NAD(P)"/NAD(P)H-mimicking agents as described in reduces the reaction sequence in the above-mentioned US2003/0022266 may be employed as cofactor. embodiment to O1-DH1-DH6-DC6-R6-DC1-R1. When glu 0107 Exemplary enzymes suitable for use in the process caric acid is used, it is reduced to DH1-DH6-DC6-R6-DC1 according to the present invention are listed in table 1. They R1 provide possible solutions and are described in the following 0099. The one-pot synthesis can be performed in vitro paragraphs as particular embodiments for a better under using isolated or not isolated enzymes, e.g. in enzymes con standing. The replacement of individual enzymes or more is tained in crude cell extracts. Alternatively, the reactions of the within the skill of an ordinary artisan. US 2014/0171683 A1 Jun. 19, 2014

TABELLE 1. Exemplary for the embodiments discussed in the text and FIGURES Enzymes and Catalysed Reactions EC Enzyme Name Reaction 1.1.3.9 Hexose-6-oxidase D-glucopyranose + O2 €s D-gluco-dialdose + H2O2 1.1.3.4 Glucose oxidase D-glucopyranose + O2 €s D-glucono-1,5-lactOne + H2O2 1.1.1.118. Glucose D-glucopyranose + NAD" (s D-glucono-1,5-lactone + NADH ehydrogenase 4.2.1.39 Gluconate D-gluconate €s 2-keto-3-deoxy-D-gluconate + H2O ehydratase 1.1.1.126 2-keto-3-deoxy-D- 2-keto-3-deoxy-D-gluconate + NAD' €s 4,5-dihydroxy-2,6- gluconate-6- dioxohexanoate + NADH enydrogenase 1.2.1.3 Aldehyde D-glucopyranose + NAD' + H2O €s D-glucuronic acid + NADH enydrogenase 1.1.1.203 Uronate D-gluco-dialdose + NAD+ H2O €s D-glucuronic acid + NADH enydrogenase D-glucuronate + NAD+ H2O €s D-glucaric acid + NADH 4.2.1.40 Glucarate D-glucaric acid €s 5-dehydro-4-deoxy-D-glucarate + H2O ehydratase 4.2.1.41 Keto-deoxy- 5-dehydro-4-deoxy-D-glucarate €s 2,5-dioxopentanoate + H2O + CO2 glucarate ehydratase 4.1.1.1 Pyruvate 2,5-dioxopentanoatees succinaldehyde + CO2 ecarboxylase 4.1.1.72 branched-chain-2- 2,5-dioxopentanoate €s succinaldehyde + CO2 oxoacid ecarboxylase 1.1.1.1 Alcohol Succinaldehyde + 2 NADH €s 1,4-butanediol + 2 NAD+ 2 H2O ehydrogenase 14.99.3 Amine succinaldehyde + 2 NADH + 2 H* + 2 NH €s 1,4-diaminobutane + 2 ehydrogenase NAD+ 2 H2O 2.6.1.18 Transaminase Succinaldehyde +2 L-alanine €s 1,4-diaminobutane + 2 pyruvate

0108. In the following, a detailed description of single at position C-5 ("keto-enol-tautomerie') occurs leading to a conversion steps within the scope of this invention is given. carbonyl group at position C-5 (Eq. 5) and a methylen-group The following paragraphs will describe illustrative enzyme at C4. selections that demonstrate one or more embodiments of the invention. CHOOsés CH3O+H2O Eq. 5 0109. Initially the oxidation of the C-6 is achieved with 0113. An additional dehydration is achieved by keto hexose-6-oxidase (Eq. 1) followed by the oxidation of the C-1 deoxy-glucarate dehydratase (EC 4.2.1.41) which not only carbon to a carboxylic group (Eq. 2) using uronate dehydro catalyzes the dehydration of the substrate but also a first genase (EC 1.1.1.203). This step requires a ring-opening decarboxylation (Eq. 6). The product is a C-5 compound. (lactone hydrolysis) which could be achieved by acid/base , enzymes or other ways. I0114. When CO, is continuously removed from liquid reaction solution, the net reaction becomes favorable (in the forward direction) according to Le Chatelier's principle. Therefore, it is preferable to remove the gaseous products as it is formed. 0110. As an alternative route it is possible to use an alde hyde dehydrogenase (Eq. 3) (EC 1.2.1.3) for the oxidation of 0115 For the production of C-4 compounds a further the C-6. Using aldehyde dehydrogenase the C-1 carbonyl enzymatic step is required. Using a decarboxylase (EC 4.1. group is simultaneously oxidized to the carboxyl group. 1.X) the other carboxyl group is removed as carbon dioxide (Eq. 7). CHO+2NAD+HOesCHO,--2NADH-2H- Eq. 3 CHOesCHO+CO Eq. 7 0111. To achieve the complete oxidation of both terminal 0116. If the desired product is 1,4-butanediol both carbo carbon atoms the uronate dehydro-genase (EC 1.1.1.203) nyl groups are reduced to hydroxyl groups (Eq. 8). catalyzes in a second step the oxidation of the aldehyde group at C-6 to the corresponding carboxylic group (Eq. 4). CHO+2NADH-2H'esCHO2+2NAD Eq. 8 CHO,--NAD+HOesCHOs:-NADH--H" Eq. 4 0117 If the desired product is succinic acid both carbonyl groups are oxidized to carboxyl groups (Eq. 9). 0112 The next step is the defunctionalization (deoxygen ation) of the internal carbons. Removal of the hydroxyl group Eq. 9 at position C-4 is achieved by using the enzyme glucarate 0118. If the desired product is 4-Hydroxybutyric acid one dehydratase (EC 4.2.1.40) or other suitable dihydroxy acid carbonyl group is reduced to a hydroxyl group and the other dehydratase. After the elimination of water a rearrangement carbonyl group is oxidized to a carboxyl group (Eq. 10). US 2014/0171683 A1 Jun. 19, 2014

CHO+H2OesCHO Eq. 10 produced the enzymes in a previous fermentation process. In 0119. If the desired product is 1.4 diaminobutane both this case, there could also be cell fragments added to the carbonyl groups are reduced to amino groups. This step can reactOr. be done by different enzymes, e.g. amine dehydrogenase (Eq. I0128. The advantage of using a cell-free system is that no unwanted side-reactions occur. The co-factor (NAD") is con 11) (EC 1.4.99.3) or transaminase (Eq. 12) (2.6.1.18). tinuously recycled in the system. That is, these Substances are produced and consumed in equal rates. Preferably, it is pos sible to use a co-factor recycling system, e.g. NADHOxidase, for the generating NAD" (Eq. 17) in case that excess NADH Eq. 12 is developed during the reaction cascade. 0120 In another example the conversion of glucose is initiated by the oxidation of C-1 by glucose dehydrogenase NADH--H+Oes NAD+HO, Eq. 17 (Eq. 13) (EC 1.1.1.118) or glucose oxidase (Eq. 14) (EC I0129. If necessary, accumulated hydrogen peroxide can be 1.1.3.4) to yield gluconolactone, which is hydrolyzed to glu efficiently eliminated by catalase (Eq. 18). COnate. Eq. 18 CH2O+NAD'esCHO-NADH--H' Eq. 13 0.130 By the use of close to irreversible reactions, like decarboxylation, the equilibrium of the process is continu Eq. 14 ously shifted to the desired end product. 0121 The oxidation of C-1 is followed by the dehydration I0131. In another embodiment the enzymes are expressed using gluconate dehydratase (Eq. 15) (EC 4.2.1.39). recombinantly in a suitable microorganism and the conver sion of the Substrate is achieved using whole cell catalysis or Eq. 15 in a fermentation process. 0122) Subsequently, the oxidation of C-6 of 2-keto-3- 0.132. The disadvantage of using a fermentation process deoxy-gluconate is achieved using 2-keto-3-deoxy-gluconate for generation of chemical compounds lies in the difficult 6-dehydrogenase (Eq. 16) (EC 1.1.1.126). Alternatively, downstream product purification. The separation of byprod 2-keto-3-deoxy-gluconate can also first be decarboxylated ucts has a crucial effect on process costs. In a cell-free pro using a decarboxylase. duction system using isolated enzymes only those reactions are catalyzed that lead to the desired intermediates and final CHO-NAD'esCHO-NADH--H" Eq. 16 products and the amount of byproducts is reduced drastically. 0123. The enzymatic process for the conversion of hex I0133. The pH of the solution is not regarded as particularly oses, like glucose, may be summarized into three main steps critical, but pH will impact activity of each enzyme in a as follows: (a) the oxidation of the terminal carbon atoms and potentially different way. A person of ordinary skill in the art their/its removal as carbon dioxide; (b) removal of the can readily perform routine experimentation, given a specific hydroxyl-groups at position 3 and 4; (c) the conversion of the selection of enzymes, to determine the optimum pH, or to terminal carbonyl groups into the desired functionality by determine a range of preferred pH values, with respect to oxidation, reduction or transfunctionalization. product yield and production rate. In other words, the process 0.124. In a preferred embodiment these three main steps of invention can comprise adjusting one or more parameters are catalyzed by in total only four different enzymes (e.g. during the reaction to maintain parameter or optimize param aldehyde dehydrogenase, alcohole dehydrogenase, dihy eter. An illustrative range of preferred pH values for some droxy acid dehydratase, decarboxylase) having Substrate embodiments is pH 2-12, more preferably 4-9, and most preferably a neutral pH, such as pH 6-8. specificities wide enough to be active on both sides of the C6, I0134) Temperature is not regarded as being critical to the C5 or C4 chemical. present invention. Low to moderate temperatures are appro 0.125. In general, selection of a plurality of enzymes that priate, especially when mesophilic enzymes are chosen. The lead to glucaric acid is within the skill of an ordinary artisan. process can generally be practiced conveniently at one or One particular embodiment is discussed in this invention more temperatures from 10° C. to about 100° C., enzymes using glucose as starting substrate (FIG. 4, FIG. 5 and Table selected will have its own respective function of the specific 1). Other embodiments employ similar enzymes, such as enzymes chosen. One skilled in the art will recognize that enzymes with at least 80% preferably at least 90% sequence temperatures outside the range of 10°C. to 100° C. could even identity to the enzymes listed in Table 1. be employed, such as when thermophilic or psychrophilic 0126. In some embodiments, enzymes are added directly enzymes are selected. In some embodiments, no external together with the required co-factors into the aqueous solu energy is added, and the temperature will be a function of tion of Substrate(s). The quantity of enzymes to add depends Substrate concentration, netheats of reactions and heat losses on the desired reaction temperature and residence time and in the system. the determination of optimal enzyme quantity or concentra 0.135 Sources of enzymes can be any organism in which tion lies within the ordinary skill of the person skilled in the the enzyme (encoded gene product) is capable of catalyzing art. In general, there will be concentration of each particular the referenced reaction. Including both prokaryotic and enzyme above which no further enhancement in reaction rate eukaryotic organisms, this includes, but is not limited to, occurs. The optimal quantity of enzyme will be dictated by bacteria like archaea and eubacteria and eukaryotes like overall economics. yeast, plant, insects, animal and mammal. The recombinant 0127. The enzymes may be purified (but are not necessar expression or natural expression of the enzyme can be applied ily purified), and they can exist in the form of mixtures of to different kinds of expression systems. enzymes or enzyme complexes with the desired functions. 0.136 Pressure is also not critical to the present invention, Enzymes can be added in the form of lysed cells which but a skilled artisan will appreciate that the reaction pressure US 2014/0171683 A1 Jun. 19, 2014

can impact the equilibrium distribution of species. A high -continued pressure, such as several atmospheres, would tend to inhibit as the CO should be removed. Concentration Concentration 0.137 The process can be conducted in a batch reactor, Component stock solution Sample in test continuous reactor, membrane reactor or combination of NAD 100 nM 1000 ul 10 mM these. A variety of means for agitation (mixing) can be NADPH 50 nM 500 ul 2.5 mM Na-Glucuronate SOO mM 400 ul 20 mM employed, or plug-flow reactor without internal mixing can Buffer 4958.01 ul be effective. Unconverted reactants can be recycled to the Total Volume 10.000 ul reactor inlet, as in known in the art. 0138 Optimization can also be carried out to improve the overall reaction rate the stability of some or all of the 0.142 Buffer-Content: enzymes. Such optimization can include, for example, 0143 100 mM HEPES/NaOH pH 7.5 enzyme component optimization via metabolic engineering and modeling; Substitution of mesophilic enzymes by recom 0144 100 mM. NaCl binant thermophilic or even hyperthermophilic enzymes; (0145) 5 mM MgCl, protein engineering to improve enzyme activity and/or selec 0146 0.1 mMTPP tivity; higher concentrations of enzymes and Substrates; 0147 10% Glycerol variation of process parameters such as pH and temperature; 0148 NAD" was dissolved in assay-buffer and the pH was stabilization of enzymes through additives; enzyme immobi adjusted to 6.5 to prevent fluctuations during the test. Na lization; and development of minimal microorganism to cre glucuronate was dissolved as a stock solution with a concen ate an in vivo enzyme system that produces the different tration of 500 mM in the assay buffer. The enzymes were products. It is within the ordinary skill of the person skilled in taken from glycerol stocks: Uronate dehydrogenase A. tume the enzyme art to conduct such optimization, and the present faciens C58, E. coli dehydrogenase YigB and E. coli dehy invention is intended to include this type of experimentation. drogenase YohD (25% glycerol, 25 mM Tris-HCl pH 8.0), Statistical experimental design can be employed to explore glucarate dehydratase A. Succinogenes 130Z and keto-deoxy global response Surfaces and establish models of product glucarate dehydratase A. baylvi ADP1 (27.5% glycerol, 25 yield and rate versus process and enzyme factors as well as interaction effects. mM Tris-HCl pH 8.0, 25 mM NaCl, 0.5 mM DTT). 014.9 The required amount of NADPH was weighed, dis 0139. In yet another embodiment of this invention pen solved directly in buffer and added to the reaction mixture. toses are used instead of hexoses for example for the produc The decarboxylase KdcA from L. lactis was directly weighed tion of 1,2,4-butantriol using the same mechanism of a) oxi as lyophilisate and added. The test mixture was incubated for dation of terminal C1, b) dehydration of internal C3, c) 24h at 30°C. After 24h samples (sample volume 500 ul) were decarboxylation of C1 and d) reduction of C2. taken and analyzed by HPLC for the enzymatic reaction. The 0140. The present invention is not limited in scope by results are shown in the chromatograms in FIG. 6. The peak at specific embodiments described herein. Indeed, various 26.2 min retention time in FIG. 6 Bindicates the formation of modifications of the invention in addition to those described herein will become apparent to those skilled in the art in view 1,4-butandiole. According to the peak area more than 2 mM of the foregoing description and the accompanying figures. 1,4-Butanediol was produced. Such modifications are intended to fall within the scope of the appended claims. Example 2 EXAMPLES Enzyme Preparation and Activity Tests Example 1 O150 2.1 Galactose-Oxidase from Fusarium graminearum (GaoA-M-RQWY) Enzymatic Production of 1,4-butandiole 0151. The gene gaoA-M-ROWY for the enzyme galac tose-oxidase from Fusarium graminearum was a synthetic 0141. The genes encoding the following enzymes were gene codon-optimized for expression in Escherichia coli. It cloned from their host or synthesized and recombinantly was cloned into a derivative of plT28a with an alternative expressed in E. coli. MCS carrying additionally the recognition sites for the two restriction-endonucleases Bsal and BfuAl. 0152 The enzyme expression was done with an autoin Concentration Concentration duction-media developed by F. W. Studier and colleagues. Component stock solution Sample in test The method is based upon a buffered medium that contains a Uronate-Dehydro-genase 300 Uml 11.79 ul 3.75 U. mixture of carbon sources, including lactose. The medium (Udh) allows the recombinant protein expression without any addi Glucarate-Dehydratase 289 Uml 13 Jul 3.75 U. tional inducer Substances. In the following section the (GlucD) Keto-deoxy-Glucarate- 32 Uml 117.2 ul 3.75 U. reagents and stock solutions are described: ZY. 20xNPS, Dehydratase (KodgD) 50x5052, MgSO, Antibiotic. Alcohol Dehydrogenase 1.7 Uml 1000 ul 1.7 U. (YigB) 0153. ZY Decarboxylase (KodcA) 75 U/mg 5 mg 375 U. 0154) 10 g tryptone Alcohol Dehydrogenase 2000 ul N.a. (YahD) (O155 5g yeast extract 0156 925 ml water US 2014/0171683 A1 Jun. 19, 2014 10

O157 20xNPS column (GE Health Care Europe) with a mobile phase com posed of 100 mM NaPi, pH 7.0. After this the protein was tested in an activity assay and stored at -20°C. The analysis of the product is shown in FIG. 7. Component 1 liter molliter 0.167 Enzyme activity on glucose as substrate of the puri did H2O fied enzyme was measured with a coupled assay using horse (NH4)2SO 66 g O.SM radish peroxidase and ABTS at 445 nm at 25°C. The assay KH2PO 136 g 1M was done in 96-well microtiter plates containing the follow NaHPO. 142g 1M ing components: 0158 50x5052 Stock Assay Stock solution Solution pro Component 1 liter Component conc. Unit COC. Unit well (ul) NaPi (pH 7.0) 50.00 nM SOO.O mM 2O.OO Glycerol (weigh in beaker) 250 g Glucose 250.0 mM 1OOO.O mM SO.OO HO 730 in Glucose 25g Catalase 850 Uml 609131.00 mM O.28 Cu2SO O.5 mM 100 1 C-Lactose 100 g Horse radish 0.01 Uml 10 O.2 peroxidase 0159) 1 M MgSO, dd H2O 88.52 (0160 24.65 g MgSO.7HO 160.00 (0161 Water to make 100 ml Purified enzyme 40.00 (0162 ZYP-5052 Rich Medium for Auto-Induction Assay volume 200.00 (0163 Add 1 MMgSO before adding 20xNPS to avoid precipitate (0168 The enzymatic assay was used to define the enzyme 0.164 Kanamycin is used at significantly higher con activity. centrations (100 ug/ml) than is normally (25-40 ug/ml). (0169. 2.2 Cytosolic Sheep Liver Aldehyde-Dehydroge Studier has found that in the T7 expression strains in nase (slAlDH) these rich media, it does not provide adequate selection 0170 The gene slalDH for the cytosolic liver enzyme at the lower concentration aldehyde-dehydrogenase from Ovies aries was a synthetic gene codon-optimized for expression in Escherichia coli. It was cloned into a derivative of plT28a with an alternative Component 200 ml MCS carrying additionally the recognition sites for the two restriction-endonucleases Bsal and BfuAl. ZY 186 ml 1MMgSO 0.2 ml 0171 The enzyme expression was done with an autoin SOX SOS2 4 ml duction-media developed by F. W. Studier and colleagues. 2Ox NPS 10 ml The method is based upon a buffered medium that contains a Kanamycin (30 mg/ml) 0.667 ml mixture of carbon sources, including lactose. The medium allows the recombinant protein expression without any addi (0165. The plasmid pCBR-NH-gaoA-M-RQWY-F.g. car tional inducer Substances. In the following section the rying the galactose-oxidase from Fusarium graminearum reagents and stock Solutions are described: was used to transform E. coli BL21 (DE3) for use for protein 0172 ZY expression. The recombinant E. coli BL21 strain was culti (0173 10 g tryptone vated in auto-induction media described above with the fol 0.174 5 g yeast extract lowing procedure. First, the bacteria culture was cultivated at (0175 925 ml water 37° C. and 150 rpm for 3 h, after that the culture was trans 0176) 20xNPS ferred to 16° C. at 150 rpm for additionally 21 h. 0166. After centrifugation, cells were frozen or directly used and suspended in Lysis/Wash Buffer (50 mM phosphate, pH 8.0, 500 mMNaCl, 10% glycerol, 20 mMimidazole) and Component 1 liter molliter for that 1 g of cells were resuspended in 10 ml Buffer. Forcell did H2O disruption a cell-disrupter was used. After this step 10 ul (NH4)2SO 66 g DNase Stock-Solution (10 mg/ml DNase) per 10 ml and 25ul KH2PO 136 g of 1 MMgSO were added and incubated for 20 min at room Na HPO. 142g temperature for DNA cleavage. After centrifugation 45 minat 40.000 g at 4°C. to clarify the cell extract, the supernatant was 0177 50x5052 loaded on a 5 ml HisTrap FF column (GE Health Care Europe) using a Akta Purifier 100 (GE Health Care Europe). After sample loading, the column was washed with 5 volumes Component 1 liter of Lysis/Wash Buffer and the protein was eluted with the Glycerol (weigh in beaker) 250 g same buffer containing 500 mM imidazole. In the final step HO 730 in the buffer exchange was done using a HiPrep26/10 desalting US 2014/0171683 A1 Jun. 19, 2014 11

-continued

Component 1 liter Stock Stock Solution Solution pro Glucose 25g Component Assay conc. Unit conc. Unit well (ul) C-Lactose 100 g TRIS (pH 8.0) 25.00 mM 25O.O mM 2O.OO NAD 1.O mM 2O.O mM 10.00 D-Glucuronate 2O.OO mM 1 OOOO mM 40.00 0178) 1 M MgSO did H2O 125.00

(0179 24.65 g MgSO.7HO 195.00 0180 Water to make 100 ml Purified enzyme S.OO 0181 ZYP-5052 Rich Medium for Auto-Induction Assay volume 200.00 0182. Add 1 MMgSO before adding 20xNPS to avoid precipitate 0187. The enzymatic assay was used to define the enzyme activity. The results are shown in FIG. 9. 0183 Kanamycin is used at significantly higher con 0188 2.3 Uronate-Dehydrogenase from Agrobacterium centrations (100 g/ml) than is normally (25-40Ng/ml). tumefaciens C58 (Udh) Studier has found that in the T7 expression strains in 0189 The gene udh for the enzyme uronate-dehydroge these rich media, it does not provide adequate selection nase from Agrobacterium tumefaciens C58 was a synthetic at the lower concentration gene codon-optimized for expression in Escherichia coli. It was cloned into a derivative of plT28a with an alternative MCS carrying additionally the recognition sites for the two restriction-endonucleases Bsal and BfuAl. Component 200 ml 0190. The enzyme expression was done with an autoin ZY 186 ml duction-media developed by F. W. Studier and colleagues. 1MMgSO 0.2 ml The method is based upon a buffered medium that contains a SOX SOS2 4 ml 2Ox NPS 10 ml mixture of carbon sources, including lactose. The medium Kanamycin (30 mg/ml) 0.667 m allows the recombinant protein expression without any addi tional inducer Substances. In the following section the reagents and stock Solutions are described: 0184 The plasmid pCBR-NH-slaldh-O.a. carrying the 0191 ZY cytosolic liveraldehyde-dehydrogenase from Ovies aries was (0192 10 g tryptone used to transform E. coli BL21 (DE3) for use for protein (0193 5g yeast extract expression. The recombinant E. coli BL21 strain was culti 0194 925 ml water vated in auto-induction media described above with the fol 0.195 20xNPS lowing procedure. First the bacteria culture was cultivated at 37° C. and 150 rpm for 3 h, after that the culture was trans ferred to 16° C. at 150 rpm for additionally 21 h. Component 1 liter molliter 0185. After centrifugation, cells were frozen or directly did H2O used and suspended in Lysis/Wash Buffer (50 mM phosphate, (NH4)2SO 66 g O.SM pH 8.0, 500 mMNaCl, 10% glycerol, 20 mMimidazole) and KHPO. 136 g 1M for that 1 g of cells were resuspended in 10 ml Buffer. Forcell NaHPO. 142g 1M disruption a cell-disrupter was used. After this step 10 ul DNase Stock-Solution (10 mg/ml DNase) per 10 ml and 25ul of 1 MMgSO were added and incubated for 20 minat room 0196) 50x5052 temperature for DNA cleavage. After centrifugation 45 minat 40.000 g at 4°C. to clarify the cell extract, the supernatant was loaded on a 5 ml HisTrap FF column (GE Health Care Europe) using a Akta Purifier 100 (GE Health Care Europe). Component 1 liter After sample loading, the column was washed with 5 volumes Glycerol (weigh in beaker) 250 g HO 730 in of Lysis/Wash Buffer and the protein was eluted with the Glucose 25 g same buffer containing 500 mM imidazole. In the final step C-Lactose 100 g the buffer exchange was done using a HiPrep26/10 desalting column (GE Health Care Europe) with a mobile phase com posed of 50 mM TRIS, pH 8.0. After this the protein was (0197) 1 M MgSO tested in an activity assay and stored preparing glycerol (0198 24.65 g MgSO.7HO stocks (1:1 dilution with 50% glycerol). The analysis of the (0199 Water to make 100 ml product is shown in FIG. 8. 0200 ZYP-5052 rich medium for auto-induction 0186 Enzyme activity on acetaldehyde as substrate of the 0201 Add 1 MMgSO before adding 20xNPS to avoid purified enzyme was measured by monitoring initial NADH precipitate Generation at 340 nm at 25°C. The assay was done in 96-well 0202 Kanamycin is used at significantly higher con microtiter plates containing the following components: centrations (100 ug/ml) than is normally (25-40 ug/ml). US 2014/0171683 A1 Jun. 19, 2014 12

Studier has found that in the T7 expression strains in 0208 2.4 Glucarate-Dehydratase from Actinobacillus these rich media, it does not provide adequate selection succinogenes 130 Z (GlucD) at the lower concentration 0209. The gene glucD for the enzyme glucarate-dehy dratase from Actinobacillus succinogenes 130 Z was a syn thetic gene codon-optimized for expression in Escherichia coli. It was cloned into a derivative of plT28a with an alter Component 200 ml native MCS carrying additionally the recognition sites for the ZY 186 ml two restriction-endonucleases Bsal and BfuAl. 1MMgSO 0.2 ml SOX SOS2 4 ml 0210. The enzyme expression was done with a modified 2Ox NPS 10 ml terrific broth medium containing additionally 1 M sorbitol Kanamycin (30 mg/ml) 0.667 ml and 5 mM betaine. Terrific-Broth 0203 The plasmid pCBR-NH-udh-A.t. carrying the uronate-dehydrogenase from Agrobacterium tumefaciens 0211 C58 was used to transform E. coli BL21 (DE3) for use for protein expression. The recombinant E. coli BL21 strain was cultivated in auto-induction media described above with the following procedure. First the bacteria culture was cultivated Component 1 liter at 37° C. and 150 rpm for 3 h, after that the culture was Casein 12g transferred to 16° C. at 150 rpm for additionally 21 h. Yeast extract 24g Sorbitol 182g 0204 After centrifugation, cells were frozen or directly KHPO. 12.5g used and suspended in Lysis/Wash Buffer (50 mM phosphate, pH 8.0, 500 mMNaCl, 10% glycerol, 20 mMimidazole) and KH2PO 2.3g for that 1 g of cells were resuspended in 10 ml Buffer. Forcell disruption a cell-disrupter was used. After this step 10 ul DNase Stock-Solution (10 mg/ml DNase) per 10 ml and 25ul 0212 5 M Betaine Stock Solution of 1 MMgSO were added and incubated for 20 min at room 0213) 58.575g temperature for DNA cleavage. After centrifugation 45 minat 0214) ad. 100 ml HO 40.000 g at 4°C. to clarify the cell extract, the supernatant was 0215. The plasmid pCBR-NH-glucD-A.S. carrying the loaded on a 5 ml HisTrap FF column (GE Health Care glucarate-dehydratase from Actinobacillus succinogenes 130 Europe) using a Akta Purifier 100 (GE Health Care Europe). Z was used to transform E. coli BL21 (DE3) for use for After sample loading, the column was washed with 5 volumes protein expression. The recombinant E. coli BL21 strain was of Lysis/Wash Buffer and the protein was eluted with the cultivated in terrific broth medium containing 1 M sorbitol, 5 same buffer containing 500 mM imidazole. In the final step mM betaine and 90 g/ml kanamycin. The bacteria culture the buffer exchange was done using a HiPrep26/10 desalting was cultivated at 37° C. at 150 rpm until reaching an Aeo of column (GE Health Care Europe) with a mobile phase com 1. Isopropyl B-D-thiogalactopyranoside was added at a con posed of 50 mMTRIS, pH 8.0. The analysis of the product is centration of 250 uM to induce protein production, and the shown in FIG. 10. culture was transferred to 16° C. at 150 rpm for additionally 0205. After this the protein was tested in an activity assay 16 h. and stored preparing glycerol stocks (1:1 dilution with 50% 0216. After centrifugation, cells were frozen or directly glycerol). used and suspended in Lysis/Wash Buffer (50 mM phosphate, 0206 Enzyme activity on glucuronate as substrate of the pH 8.0, 500 mMNaCl, 10% glycerol, 20 mMimidazole) and purified enzyme was measured by monitoring initial NADH for that 1 g of cells were resuspended in 10 ml Buffer. Forcell Generation at 340 nm at 25°C. The assay was done in 96-well disruption a cell-disrupter was used. After this step 10 ul microtiter plates containing the following components: DNase Stock-Solution (10 mg/ml DNase) per 10 ml and 25ul of 1 M MgSO were added and incubated for 20 min at room temperature for DNA cleavage. After centrifugation 45 minat 40.000 g at 4°C. to clarify the cell extract, the supernatant was Stock Stock loaded on a 5 ml HisTrap FF column (GE Health Care solution Solution pro Europe) using a Akta Purifier 100 (GE Health Care Europe). Component Assay conc. Unit conc. Unit well (ul) After sample loading, the column was washed with 5 volumes TRIS (pH 8.0) 1OOOO mM 250.0 mM 80.00 of Lysis/Wash Buffer and the protein was eluted with the NAD 1.O mM 20.0 mM 1O.OO same buffer containing 500 mM imidazole. In the final step D-Glucuronate 1O.OO mM 1OOOO mM 2O.OO the buffer exchange was done using a HiPrep26/10 desalting did H2O 85.00 column (GE Health Care Europe) with a mobile phase com 195.00 posed of 50 mM TRIS, pH 8.0, 50 mM. NaCl, 10% glycerol Purified enzyme S.OO and 1 mM dithiothreitol. The analysis of the product is shown in FIG. 12. Assay volume 2OOOO 0217. After this, the protein was tested in an activity assay and stored preparing glycerol stocks (1:1 dilution with 50% 0207. The enzymatic assay was used to define the enzyme glycerol). activity. The results are shown in FIG. 11. The enzyme activ 0218 Enzyme activity on glucarate as substrate of the ity was 318 U/ml. purified enzyme was measured by semicarbazide assay at 250 US 2014/0171683 A1 Jun. 19, 2014

nm at 25°C. The assay was done in 96-well UV-microtiter DNase Stock-Solution (10 mg/ml DNase) per 10 ml and 25ul plates containing the following components: of 1 M MgSO were added and incubated for 20 min at room temperature for DNA cleavage. After centrifugation 45 minat 40.000 g at 4°C. to clarify the cell extract, the supernatant was loaded on a 5 ml HisTrap FF column (GE Health Care Stock Stock Solution Solution pro Europe) using a Akta Purifier 100 (GE Health Care Europe). Component Assay conc. Unit conc. Unit well (ul) After sample loading, the column was washed with 5 volumes of Lysis/Wash Buffer and the protein was eluted with the HEPES (pH 7.5), 43.50 mM SO.O mM 174.00 100 mM same buffer containing 500 mM imidazole. In the final step NaCl, 10% the buffer exchange was done using a HiPrep26/10 desalting Glycerol column (GE Health Care Europe) with a mobile phase com MgCl2 S.O mM 1000.O mM 1.00 posed of 50 mM TRIS, pH 8.0, 50 mM. NaCl, 10% glycerol Glucarate 1O.OO mM 100.00 nM 2O.OO and 1 mM dithiothreitol. After this the protein was tested in an 195.00 activity assay and stored preparing glycerol stocks (1:1 dilu Purified enzyme S.OO tion with 50% glycerol). The analysis of the product is shown in FIG. 14. Assay volume 2OOOO 0229. Enzyme activity on keto-deoxy-glucarate as sub strate of the purified enzyme was measured by coupled assay 0219. The enzymatic assay was used to define the enzyme using an aldehyde dehydrogenase to generate O-keto-glut activity. The results are shown in FIG. 13. The enzyme activ arate at 340 nm at 25° C. The assay was done in 96-well ity was 289 U/ml. microtiter plates containing the following components: 0220 2.5 Keto-deoxy-Glucarate-Dehydratase from Acinetobacter baylvi ADP 1 (KdgD) 0221) The genekdgD for the enzyme keto-deoxy-glucar ate-dehydratase from Acinetobacter baylvi ADP1 was a syn Stock Stock thetic gene codon-optimized for expression in Escherichia Assay Solution Solution pro coli. It was cloned into a derivative of plT28a with an alter Component conc. Unit conc. Unit well (ul) native MCS carrying additionally the recognition sites for the HEPES (pH 7.5), 32.12 mM SO.O mM 129.25 two restriction-endonucleases Bsal and BfuAl. 100 mM NaCl, 10% 0222. The enzyme expression was done with a modified Glycerol terrific broth medium containing additionally 1M sorbitol and MgCl2 3.75 nM 1000.0 mM 0.75 5 mM betaine. NAD 1.O mM 2000 nM 10.00 Keto-deoxy-Glucarate 10.00 mM 40.OO mM SO.OO Aldehyde S.O Terrific-Broth dehydrogenase

0223 195.00 Purified enzyme S.OO Assay volume 200.00 Component 1 liter Casein 12g Yeast extract 24g 0230. The enzymatic assay was used to define the enzyme Sorbitol 182g activity. The results are shown in FIG. 15. The enzyme activ KHPO 12.5g KHPO. 2.3g ity was 32 U/ml. 0231 2.6 Alcohol-Dehydrogenase from Escherichia coli 0224 5 M Betaine Stock solution K-12 (YigB) 0225 58.575g 0232. The gene yigB for the enzyme alcohol-dehydroge 0226 ad. 100 ml HO nase from Escherichia coli K-12 was cloned from genomic 0227. The plasmid pCBR-NH-KdgD-A.b. carrying the DNA for expression in Escherichia coli. It was cloned into a keto-deoxy-glucarate-dehydratase from Acinetobacter bay pET28a using the two restriction endonucleases Ncol and lvi ADP1 was used to transform E. coli BL21 (DE3) for use XhoI. for protein expression. The recombinant E. coli BL21 strain 0233. The enzyme expression was done with an autoin was cultivated in terrific broth medium containing 1 M sor duction-media developed by F. W. Studier and colleagues. bitol, 5 mM betaine and 90 ug/ml kanamycin. The bacteria The method is based upon a buffered medium that contains a culture was cultivated at 37° C. at 150 rpm until reaching an mixture of carbon sources, including lactose. The medium Asoo of 1. Isopropyl B-D-thiogalactopyranoside was added at allows the recombinant protein expression without any addi a concentration of 250 uM to induce protein production, and tional inducer Substances. In the following section the the culture was transferred to 16°C. at 150 rpm for addition reagents and stock Solutions are described: ally 16 h. 0228. After centrifugation, cells were frozen or directly 0234 ZY used and suspended in Lysis/Wash Buffer (50 mM phosphate, 0235 10 g tryptone pH 8.0, 500 mMNaCl, 10% glycerol, 20 mMimidazole) and for that 1 g of cells were resuspended in 10 ml Buffer. Forcell 0236 5 g yeast extract disruption a cell-disrupter was used. After this step 10 ul 0237 925 ml water US 2014/0171683 A1 Jun. 19, 2014 14

0238 20xNPS of Lysis/Wash Buffer and the protein was eluted with the same buffer containing 500 mM imidazole. In the final step the buffer exchange was done using a HiPrep26/10 desalting column (GE Health Care Europe) with a mobile phase com Component 1 liter molliter posed of 50 mM TRIS, pH 8.0. After this the protein was did H2O tested in an activity assay and stored at -20°C. The analysis (NH4)2SO 66 g O.SM of the product is shown in FIG. 16. KH2PO 136 g 1M 0251 Enzyme activity on 2,5-dioxo-pentanoate as sub NaHPO. 142g 1M strate of the purified enzyme was measured with a coupled assay using keto-deoxy-glucarate-dehydratase to produce the 0239 50x5052 substrate for YigE from keto-deoxy-glucarate at 340 nm at 25°C. The assay was done in 96-well microtiter plates con taining the following components: Component 1 liter Glycerol (weigh in beaker) 250 g HO 730 in Stock Stock Glucose 25 g Assay Solution Solution pro C-Lactose 100 g Component conc. Unit conc. Unit well (ul) HEPES (pH 7.5), 31.06 mM SO.O mM 124.25 100 mM NaCl, 10% 0240 1 M MgSO Glycerol 0241. 24.65 g MgSO.7HO MgCl2 3.75 nM 1000.0 mM 0.75 0242 Water to make 100 ml NADPH O.3 mM 4.00 mM 1S.OO 0243 100 mM ZnSO Keto-deoxy-Glucarate 10.00 mM 40.OO mM SO.OO 0244 0.287 g ZnSO.7HO Keto-deoxy-Glucarate S.O 0245 Water to make 10 ml dehydratase 0246 ZYP-5052 Rich Medium for Auto-Induction 195.00 0247. Add 1 MMgSO before adding 20xNPS to avoid Purified enzyme S.OO precipitate 0248 Kanamycin is used at significantly higher con Assay volume 2OO.OO centrations (100 ug/ml) than is normally (25-40 ug/ml). Studier has found that in the T7 expression strains in 0252. The enzymatic assay was used to define the enzyme these rich media, it does not provide adequate selection activity. The results are shown in FIG. 17. The enzyme activ at the lower concentration ity was 1.7 U/ml. 0253 2.7 KdcA from Lactococcus lactis IL1403 0254 For the decarboxylation of 5-hydroxy-2-oxo-pen Component 200 ml tanoate a branched-chain decarboxylase from Lactococcus lactis IL1403 can be used. The enzyme was prepared as ZY 186 ml 1MMgSO 0.2 ml described in Adv. Synth. Catal. 2007, 349, 1425-1435. SOX SOS2 4 ml 0255 2.8 Alcohol-dehydrogenase from Escherichia coli 2Ox NPS 10 ml K-12 (YohD) 100 mM ZnSO 0.2 ml Kanamycin (30 mg/ml) 0.667 ml 0256 The geneychD for the enzyme alcohol-dehydroge nase from Escherichia coli K-12 was cloned from genomic DNA for expression in Escherichia coli. It was cloned into a 0249. The plasmid plT28a-NH-yigB-E.c. carrying the pET28a using the two restriction endonucleases Ncol and alcohol-dehydrogenase from Escherichia coli was used to XhoI. transform E. coli BL21 (DE3) for use for protein expression. 0257 The enzyme expression was done with a modified The recombinant E. coli BL21 strain was cultivated in auto terrific broth medium containing additionally 1 M sorbitol induction media described above with the following proce and 5 mM betaine. dure. First the bacteria culture was cultivated at 37° C. and 150 rpm for 3 h, after that the culture was transferred to 16°C. at 150 rpm for additionally 21 h. Terrific-Broth 0250. After centrifugation, cells were frozen or directly used and suspended in Lysis/Wash Buffer (50 mM phosphate, 0258 pH 8.0, 500 mMNaCl, 10% glycerol, 20 mMimidazole) and for that 1 g of cells were resuspended in 10 ml Buffer. Forcell disruption a cell-disrupter was used. After this step 10 ul Component 1 liter DNase Stock-Solution (10 mg/ml DNase) per 10 ml and 25ul Casein 12g of 1 MMgSO were added and incubated for 20 min at room Yeast extract 24g temperature for DNA cleavage. After centrifugation 45 minat Sorbitol 182g 40.000 g at 4°C. to clarify the cell extract, the supernatant was KHPO 12.5g loaded on a 5 ml HisTrap FF column (GE Health Care KH2PO 2.3g Europe) using a Akta Purifier 100 (GE Health Care Europe). After sample loading, the column was washed with 5 volumes US 2014/0171683 A1 Jun. 19, 2014 15

0259 5 M Betaine Stock Solution monitored. The analysis showed the presence of glucuronic 0260 58.575g acid (FIG. 19), glucaric acid (FIG. 20), 5-keto-4-deoxy-glu 0261 Water to make 100 ml HO caric acid (FIG. 21), 2,5-dioxopentanoic acid (FIG. 22) and 0262 100 mM ZnSO 5-hydroxy-2-oxo-pentanoic acid (FIG. 23) supporting the 0263 0.287 g ZnSO.7HO invented pathway. 0264. Water to make 10 ml 0265. The plasmid pET28a-CH-ygB-E.c. carrying the REFERENCES alcohol-dehydrogenase from Escherichia coli K-12 was used 0268 1. Farrell, A. E., et al., Ethanol Can Contribute to to transform E. coli BL21 (DE3) for use for protein expres Energy and Environmental Goals. Science, 2006. sion. The recombinant E. coli BL21 strain was cultivated in 3.11(5760): p. 506-508. terrific broth medium containing 1 M sorbitol, 5 mM betaine, 0269 2. Morris, D., The next economy: from dead carbon 0.1 mMZnSO and 90 ug/mlkanamycin. The bacteria culture to living carbon. Journal of the Science of Food and Agri was cultivated at 37° C. at 150 rpm until reaching an Act of culture, 2006. 86(12): p. 1743-1746. 1. Isopropyl B-D-thiogalactopyranoside was added at a con (0270. 3. Hempel, M., Novel Process Windows-A Contri centration of 250 uM to induce protein production, and the bution to More Sustainable Chemistry? Chemical Engi culture was transferred to 16° C. at 150 rpm for additionally neering & Technology, 2009. 32(11): p. 1651-1654. 16 h. 0271. 4. Bechthold, I., et al., Succinic Acid. A New Plat 0266. After centrifugation, cells were frozen or directly form Chemical for Biobased Polymers from Renewable used and suspended in Lysis/Wash Buffer (50 mM phosphate, Resources. Chemical Engineering & Technology, 2008. pH 8.0, 500 mMNaCl, 10% glycerol, 20 mMimidazole) and 31(5): p. 647-654. for that 1 g of cells were resuspended in 10 ml Buffer. Forcell 0272 5. Anastas, P. T. and J. B. Zimmerman, Peer disruption a cell-disrupter was used. After this step 10 ul Reviewed: Design Through the 12 Principles of Green DNase Stock-Solution (10 mg/ml DNase) per 10 ml and 25ul Engineering. Environmental Science & Technology, 2003. of 1 MMgSO were added and incubated for 20 min at room 37(5): p. 94A-101A. temperature for DNA cleavage. After centrifugation 45 minat (0273 6. Werpy, T. and G. Petersen, Top Value Added 40.000 g at 4°C. to clarify the cell extract, the supernatant was Chemicals from Biomass: Volume 1-Results of Screening loaded on a 5 ml HisTrap FF column (GE Health Care for Potential Candidates from Sugars and Synthesis Gas, in Europe) using a Akta Purifier 100 (GE Health Care Europe). Other Information: PBD. 1 August 2004. 2004. p. After sample loading, the column was washed with 5 volumes Medium: ED: Size: 76 pp. pages. of Lysis/Wash Buffer and the protein was eluted with the 0274 7. Patel, M., et al., Medium and Long-Term Oppor same buffer containing 500 mM imidazole. In the final step tunities and Risks of the Biotechnological Production of the buffer exchange was done using a HiPrep26/10 desalting Bulk Chemicals from Renewable Resources—The Poten column (GE Health Care Europe) with a mobile phase com tial of White Biotechnology. The Brew Project, European posed of 50 mM TRIS, pH 8.0. After this the protein was Commission's GROWTH Programme (DG Reserach). tested in an activity assay and stored preparing glycerol 2006: Utrecht. stocks (1:1 dilution with 50% glycerol). The analysis of the 0275 8. Haas, T., et al., New diol processes. 1,3-pro product is shown in FIG. 18. panediol and 1,4-butanediol. Applied Catalysis A: Gen eral, 2005. 280(1): p. 83-88. Example 3 0276 9. Schmid, A., et al., Industrial biocatalysis today and tomorrow. Nature, 2001. 4.09(6817): p. 258-68. Analysis of Intermediates (0277 10. Schoemaker, H. E., D. Mink, and M. G. Wub 0267 To identify different intermediates of the reaction bolts, Dispelling the Myths—Biocatalysis in Industrial described in Example 1 those carrying a carboxylic acid Synthesis. Science, 2003. 299(5613): p. 1694-1697. group were analysed by HPLC-MS. 150 ul of the sample of 0278 11. Boysen, Mike M. K. Carbohydrates as Syn Example 1 were mixed with 200 uL of 8 mM 4-APEBA and thetic Tools in Organic Chemistry. Chemistry—A Euro 150 uL 125 mM EDC and incubated for 1 h at 20° C. 12. pean Journal, 2007. 13(31): p. 8648-8659. Chromatographic separation was done with 0.1% formic acid 0279 12. Eggink, M., et al., Targeted LC-MS derivatiza as eluent and a gradient of acetonitrile. A Triart column tion for aldehydes and carboxylic acids with a new deriva (100x2 mm, 2 um) was used. Detection was done by mass tization agent 4-APEBA. Analytical and Bioanalytical spectrometry. The appearance of specific mass values was Chemistry, 2010, 397: 665-675

SEQUENCES Uronate-Dehydrogenase from Agrobacterium tumefaciens C 58 MKRLLWTGAA GOLGRVMRER LAPMAEILRL ADLSPLDPAG PNEECVOCDL ADANAVNAMW

AGCDGIWHLG GISVEKPFEO ILOGNIIGLY NLYEAARAHG OPRIVFASSN HTIGYYPOTE

RLGPDWPARP DGLYGWSKCF GENLARMYFD KFGOETALVR IGSCTPEPNN YRMLSTWFSH

DDFWSLIEAW FRAPWLGCPW WWGASANDAG WWDNSHLGFL GWKPKDNAEA FRRHITETTP

PPDPNDALVR FOGGTFWDNP IFKOS

US 2014/0171683 A1 Jun. 19, 2014 17

- Continued

SEQUENCES

RANVMWAATO ALNGLIGAGV PODWATHMLG HELTAMHGLD HAQTLAIVLP ALWNEKRDTK

RAKLLOYAER WWNITEGSDD ERIDAAIAAT RNFFEOLGVP HLSDYGLDG SSIPALLKKL

EEHGMTOLGE INHDITLDVSR RIYEAAR

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS: 6

<210s, SEQ ID NO 1 &211s LENGTH: 265 212. TYPE: PRT <213> ORGANISM: Agrobacterium tumefaciens

<4 OOs, SEQUENCE: 1 Met Lys Arg Lieu. Lieu Val Thr Gly Ala Ala Gly Glin Lieu. Gly Arg Val 1. 5 1O 15 Met Arg Glu Arg Lieu Ala Pro Met Ala Glu Ile Lieu. Arg Lieu Ala Asp 2O 25 3O Lieu. Ser Pro Lieu. Asp Pro Ala Gly Pro Asn. Glu Glu. Cys Val Glin Cys 35 4 O 45 Asp Lieu Ala Asp Ala Asn Ala Val Asn Ala Met Val Ala Gly Cys Asp SO 55 6 O Gly Ile Val His Leu Gly Gly Ile Ser Val Glu Lys Pro Phe Glu Gln 65 70 7s 8O Ile Lieu. Glin Gly Asn. Ile Ile Gly Lieu. Tyr Asn Lieu. Tyr Glu Ala Ala 85 90 95 Arg Ala His Gly Glin Pro Arg Ile Val Phe Ala Ser Ser Asn His Thr 1OO 105 11 O Ile Gly Tyr Tyr Pro Gln Thr Glu Arg Lieu. Gly Pro Asp Val Pro Ala 115 12 O 125 Arg Pro Asp Gly Lieu. Tyr Gly Val Ser Lys Cys Phe Gly Glu Asn Lieu 13 O 135 14 O Ala Arg Met Tyr Phe Asp Llys Phe Gly Glin Glu Thir Ala Lieu Val Arg 145 150 155 160 Ile Gly Ser Cys Thr Pro Glu Pro Asn Asn Tyr Arg Met Leu Ser Thr 1.65 17O 17s Trp Phe Ser His Asp Asp Phe Val Ser Lieu. Ile Glu Ala Val Phe Arg 18O 185 19 O Ala Pro Val Lieu. Gly Cys Pro Val Val Trp Gly Ala Ser Ala Asn Asp 195 2OO 2O5 Ala Gly Trp Trp Asp Asn. Ser His Lieu. Gly Phe Lieu. Gly Trp Llys Pro 21 O 215 22O Lys Asp Asn Ala Glu Ala Phe Arg Arg His Ile Thr Glu Thir Thr Pro 225 23 O 235 24 O Pro Pro Asp Pro Asn Asp Ala Leu Val Arg Phe Glin Gly Gly Thr Phe 245 250 255 Val Asp Asn Pro Ile Phe Lys Glin Ser 26 O 265 US 2014/0171683 A1 Jun. 19, 2014 18

- Continued

<210s, SEQ ID NO 2 &211s LENGTH: 442 212. TYPE: PRT <213> ORGANISM: Actinobacillus succinogenes

<4 OOs, SEQUENCE: 2 Met Ser Thr Pro Ile Ile Thr Glu Met Glin Val Ile Pro Val Ala Gly 1. 5 1O 15 His Asp Ser Met Leu Lleu. Asn Lieu Ser Gly Ala His Ser Pro Tyr Phe 2O 25 3O Thir Arg Asn. Ile Val Ile Lieu Lys Asp Asn. Ser Gly Asn Thr Gly Val 35 4 O 45 Gly Glu Val Pro Gly Gly Glu Lys Ile Arg Glin Thr Lieu. Glu Asp Ala SO 55 6 O Llys Pro Lieu Val Ile Gly Llys Thr Lieu. Gly Glu Tyr Lys Asn. Wal Met 65 70 7s 8O Asn Thr Val Arg Glin Thr Phe Asn Asp His Asp Ala Gly Gly Arg Gly 85 90 95 Lieu Gln Thr Phe Asp Lieu. Arg Thr Thr Ile His Val Val Thr Ala Ile 1OO 105 11 O Glu Ala Ala Met Lieu. Asp Lieu. Lieu. Gly Glin Phe Lieu. Gly Val Thr Val 115 12 O 125 Ala Ser Lieu. Lieu. Gly Asp Gly Glin Glin Arg Asp Ala Val Glu Met Lieu 13 O 135 14 O Gly Tyr Lieu. Phe Phe Ile Gly Asp Arg Llys Llys Thir Thr Lieu Ala Tyr 145 150 155 160 Glin Asn Glin Glu Asn Asp Pro Cys Asp Trp Tyr Arg Val Arg His Glu 1.65 17O 17s Glu Ala Met Thr Pro Glu Ser Val Val Arg Lieu Ala Glu Ala Ala Tyr 18O 185 19 O Glu Lys Tyr Gly Phe Asn Asp Phe Llys Lieu Lys Gly Gly Val Lieu. Asp 195 2OO 2O5 Gly Phe Glu Glu Ala Glu Ala Val Thir Ala Lieu Ala Lys Arg Phe Pro 21 O 215 22O Asp Ala Arg Ile Thr Lieu. Asp Pro Asn Gly Ala Trp Ser Lieu. Asp Glu 225 23 O 235 24 O Ala Wall Lys Ile Gly Lys Glin Lieu Lys Gly Val Lieu Ala Tyr Ala Glu 245 250 255 Asp Pro Cys Gly Ala Glu Glin Gly Tyr Ser Gly Arg Glu Ile Met Ala 26 O 265 27 O Glu Phe Arg Arg Ala Thr Gly Lieu Pro Thr Ala Thr Asn Met Ile Ala 27s 28O 285 Thr Asp Trp Arg Gln Met Gly His Thr Ile Ser Leu Gln Ser Val Asp 29 O 295 3 OO Ile Pro Leu Ala Asp Pro His Phe Trp Thr Met Glin Gly Ser Ile Arg 3. OS 310 315 32O Val Ala Gln Met Cys His Glu Trp Gly Lieu. Thir Trp Gly Ser His Ser 3.25 330 335 Asn Asn His Phe Asp Ile Ser Lieu Ala Met Phe Thr His Val Ala Ala 34 O 345 35. O Ala Ala Pro Gly Asp Ile Thr Ala Ile Asp Thr His Trp Ile Trp Glin 355 360 365 US 2014/0171683 A1 Jun. 19, 2014 19

- Continued Glu Gly Asn Glin Arg Lieu. Thir Lys Glu Pro Phe Glin Ile Lys Gly Gly 37 O 375 38O Lieu Val Glu Val Pro Llys Llys Pro Gly Lieu. Gly Val Glu Lieu. Asp Met 385 390 395 4 OO Asp Glin Val Met Lys Ala Asn. Glu Lieu. Tyr Lys Ser Met Gly Lieu. Gly 4 OS 41O 415 Ala Arg Asp Asp Ala Met Ala Met Glin Phe Lieu. Ile Pro Gly Trp Llys 42O 425 43 O Phe Asp Asn Llys Llys Pro Cys Lieu Val Arg 435 44 O

<210s, SEQ ID NO 3 &211s LENGTH: 3O3 212. TYPE: PRT <213> ORGANISM: Acinetobacter baylyi <4 OOs, SEQUENCE: 3 Met Asp Ala Lieu. Glu Lieu Lys Asn. Ile Val Ser Asp Gly Lieu. Lieu. Ser 1. 5 1O 15 Phe Pro Val Thir Asp Phe Asp Glin Asn Gly Asp Phe Asn Ala Ala Ser 2O 25 3O Tyr Ala Lys Arg Lieu. Glu Trp Lieu Ala Pro Tyr Gly Ala Ser Ala Lieu. 35 4 O 45 Phe Ala Ala Gly Gly Thr Gly Glu Phe Phe Ser Lieu. Thr Gly Asp Glu SO 55 6 O Tyr Ser Asp Val Ile Llys Thr Ala Val Asp Ala Cys Lys Gly Ser Val 65 70 7s 8O Pro Ile Ile Ala Gly Ala Gly Gly Pro Thr Arg Glin Ala Ile Lieu. Glin 85 90 95 Ala Glin Glu Ala Glu Arg Lieu. Gly Ala His Gly Ile Lieu. Lieu Met Pro 1OO 105 11 O His Tyr Lieu. Thr Glu Ala Ser Glin Glu Gly Lieu Val Glu. His Val Lys 115 12 O 125 Glin Val Cys Asn Ala Val Asn Phe Gly Val Ile Phe Tyr Asn Arg Ser 13 O 135 14 O Val Ser Lys Lieu. Asn Val Asp Ser Lieu. Glin Glin Lieu Val Glu Ser Cys 145 150 155 160 Pro Asn Lieu. Ile Gly Phe Lys Asp Ser Ser Gly Glin Ile Asp Met Met 1.65 17O 17s Thr Glu Val Val Glin Thr Lieu. Gly Asp Arg Lieu. Ser Tyr Lieu. Gly Gly 18O 185 19 O Lieu Pro Thr Ala Glu Ile Phe Ala Ala Pro Tyr Lys Ala Lieu. Gly Ser 195 2OO 2O5 Pro Val Tyr Ser Ser Ala Val Phe Asin Phe Ile Pro Llys Thr Ala Met 21 O 215 22O Glu Phe Tyr Asn Ala Lieu. Arg Asn Asp Asp Phe Ala Thir Thr Glin Arg 225 23 O 235 24 O Lieu. Ile Arg Asp Phe Phe Lieu Pro Lieu. Ile Lys Ile Arg Asn Arg Llys 245 250 255 Ser Gly Tyr Ala Val Ser Met Val Lys Ala Gly Ala Lys Ile Val Gly 26 O 265 27 O His Asp Ala Gly Pro Val Arg Pro Pro Lieu. Ser Asp Lieu. Thr Pro Glin 27s 28O 285 US 2014/0171683 A1 Jun. 19, 2014 20

- Continued Asp Tyr Glu Asp Lieu Ala Ala Lieu. Ile Ala Thr Lieu. Gly Pro Glin 29 O 295 3 OO

<210s, SEQ ID NO 4 &211s LENGTH: 353 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 4 Met Leu Tyr Thr Ser Glin Thir Thr Pro Glu Lys Asp Gln Lys Met Ser 1. 5 1O 15 Met Ile Llys Ser Tyr Ala Ala Lys Glu Ala Gly Gly Glu Lieu. Glu Val 2O 25 3O Tyr Glu Tyr Asp Pro Gly Glu Lieu. Arg Pro Glin Asp Val Glu Val Glin 35 4 O 45 Val Asp Tyr Cys Gly Ile Cys His Ser Asp Lieu. Ser Met Ile Asp Asn SO 55 6 O Glu Trp Gly Phe Ser Glin Tyr Pro Leu Val Ala Gly His Glu Val Ile 65 70 7s 8O Gly Arg Val Val Ala Lieu. Gly Ser Ala Ala Glin Asp Llys Gly Lieu. Glin 85 90 95 Val Gly Glin Arg Val Gly Ile Gly Trp Thir Ala Arg Ser Cys Gly His 1OO 105 11 O Cys Asp Ala Cys Ile Ser Gly Asin Glin Ile Asn. Cys Glu Glin Gly Ala 115 12 O 125 Val Pro Thir Ile Met Asn Arg Gly Gly Phe Ala Glu Lys Lieu. Arg Ala 13 O 135 14 O Asp Trp Gln Trp Val Ile Pro Lieu Pro Glu Asn. Ile Asp Ile Glu Ser 145 150 155 160 Ala Gly Pro Lieu. Lieu. Cys Gly Gly Ile Thr Val Phe Llys Pro Lieu. Lieu 1.65 17O 17s Met His His Ile Thr Ala Thir Ser Arg Val Gly Val Ile Gly Ile Gly 18O 185 19 O Gly Lieu. Gly His Ile Ala Ile Llys Lieu. Lieu. His Ala Met Gly Cys Glu 195 2OO 2O5 Val Thir Ala Phe Ser Ser Asn Pro Ala Lys Glu Glin Glu Val Lieu Ala 21 O 215 22O Met Gly Ala Asp Llys Val Val Asn. Ser Arg Asp Pro Glin Ala Lieu Lys 225 23 O 235 24 O Ala Lieu Ala Gly Glin Phe Asp Lieu. Ile Ile Asn Thr Val Asn Val Ser 245 250 255 Lieu. Asp Trp Gln Pro Tyr Phe Glu Ala Lieu. Thr Tyr Gly Gly Asn Phe 26 O 265 27 O His Thr Val Gly Ala Val Lieu. Thr Pro Leu Ser Val Pro Ala Phe Thr 27s 28O 285 Lieu. Ile Ala Gly Asp Arg Ser Val Ser Gly Ser Ala Thr Gly Thr Pro 29 O 295 3 OO Tyr Glu Lieu. Arg Llys Lieu Met Arg Phe Ala Ala Arg Ser Llys Val Ala 3. OS 310 315 32O Pro Thir Thr Glu Lieu. Phe Pro Met Ser Lys Ile Asn Asp Ala Ile Glin 3.25 330 335 His Val Arg Asp Gly Lys Ala Arg Tyr Arg Val Val Lieu Lys Ala Asp 34 O 345 35. O US 2014/0171683 A1 Jun. 19, 2014 21

- Continued

Phe

<210s, SEQ ID NO 5 &211s LENGTH: 547 212. TYPE: PRT <213> ORGANISM; Lactococcus lactis

<4 OOs, SEQUENCE: 5 Met Tyr Thr Val Gly Asp Tyr Lieu. Lieu. Asp Arg Lieu. His Glu Lieu. Gly 1. 5 1O 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Lieu. Glin Phe Leu 2O 25 3O Asp Glin Ile Ile Ser Arg Glu Asp Met Lys Trp Ile Gly Asn Ala Asn 35 4 O 45 Glu Lieu. Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Llys SO 55 6 O Ala Ala Ala Phe Lieu. Thir Thr Phe Gly Val Gly Glu Lieu. Ser Ala Ile 65 70 7s 8O Asn Gly Lieu Ala Gly Ser Tyr Ala Glu Asn Lieu Pro Val Val Glu Ile 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Glin Asn Asp Gly Llys Phe Val His 1OO 105 11 O His Thr Lieu Ala Asp Gly Asp Phe Llys His Phe Met Lys Met His Glu 115 12 O 125 Pro Val Thir Ala Ala Arg Thr Lieu. Lieu. Thir Ala Glu Asn Ala Thr Tyr 13 O 135 14 O Glu Ile Asp Arg Val Lieu. Ser Glin Lieu. Lieu Lys Glu Arg Llys Pro Val 145 150 155 160 Tyr Ile Asn Lieu Pro Val Asp Wall Ala Ala Ala Lys Ala Glu Lys Pro 1.65 17O 17s Ala Leu Ser Lieu. Glu Lys Glu Ser Ser Thr Thr Asn Thr Thr Glu Gln 18O 185 19 O Val Ile Lieu. Ser Lys Ile Glu Glu Ser Lieu Lys Asn Ala Glin Llys Pro 195 2OO 2O5 Val Val Ile Ala Gly His Glu Val Ile Ser Phe Gly Lieu. Glu Lys Thr 21 O 215 22O Val Thr Glin Phe Val Ser Glu Thir Lys Lieu Pro Ile Thr Thr Lieu. Asn 225 23 O 235 24 O Phe Gly Lys Ser Ala Val Asp Glu Ser Lieu Pro Ser Phe Lieu. Gly Ile 245 250 255 Tyr Asn Gly Lys Lieu. Ser Glu Ile Ser Lieu Lys Asn. Phe Val Glu Ser 26 O 265 27 O Ala Asp Phe Ile Lieu Met Lieu. Gly Val Lys Lieu. Thir Asp Ser Ser Thr 27s 28O 285 Gly Ala Phe Thr His His Lieu. Asp Glu Asn Llys Met Ile Ser Lieu. Asn 29 O 295 3 OO Ile Asp Glu Gly Ile Ile Phe Asn Llys Val Val Glu Asp Phe Asp Phe 3. OS 310 315 32O Arg Ala Val Val Ser Ser Lieu. Ser Glu Lieu Lys Gly Ile Glu Tyr Glu 3.25 330 335 Gly Glin Tyr Ile Asp Lys Glin Tyr Glu Glu Phe Ile Pro Ser Ser Ala 34 O 345 35. O

Pro Lieu. Ser Glin Asp Arg Lieu. Trp Glin Ala Val Glu Ser Lieu. Thr Glin US 2014/0171683 A1 Jun. 19, 2014 22

- Continued

355 360 365 Ser Asn Glu Thir Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 37 O 375 38O Ser Thir Ile Phe Lieu Lys Ser Asn Ser Arg Phe Ile Gly Glin Pro Leu 385 390 395 4 OO Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Lieu. Gly Ser Glin Ile 4 OS 41O 415 Ala Asp Llys Glu Ser Arg His Lieu. Lieu. Phe Ile Gly Asp Gly Ser Lieu 42O 425 43 O Glin Lieu. Thr Val Glin Glu Lieu. Gly Lieu. Ser Ile Arg Glu Lys Lieu. Asn 435 44 O 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 45.5 460 Ile His Gly Pro Thr Glin Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 47s 48O Ser Lys Lieu Pro Glu Thir Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 Lys Ile Val Arg Thr Glu Asn. Glu Phe Val Ser Val Met Lys Glu Ala SOO 505 51O Glin Ala Asp Val Asn Arg Met Tyr Trp Ile Glu Lieu Val Lieu. Glu Lys 515 52O 525 Glu Asp Ala Pro Llys Lieu Lleu Lys Llys Met Gly Llys Lieu. Phe Ala Glu 53 O 535 54 O Glin Asn Lys 5.45

<210s, SEQ ID NO 6 &211s LENGTH: 386 212. TYPE: PRT <213> ORGANISM: Escherichia coli

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

Glin Ala Phe His Ser Ala His Val Glin Pro Val Phe Ala Val Lieu. Asp US 2014/0171683 A1 Jun. 19, 2014 23

- Continued

1.65 17O 17s

Pro Wall Tyr Thr Tyr Thir Lell Pro Pro Arg Glin Wall Ala Asn Gly Val 18O 185 19 O

Wall Asp Ala Phe Wall His Thir Wall Glu Glin Tyr Wall Thr Llys Pro Val 195 2O5

Asp Ala Ile Glin Asp Arg Phe Ala Glu Gly Ile Lieu. Lieu. Thir Lieu 21 O 215

Ile Glu Asp Gly Pro Lys Ala Luell Glu Pro Glu Asn Tyr Asp Val 225 23 O 235 24 O

Arg Ala Asn. Wall Met Trp Ala Ala Thir Glin Ala Lell Asn Gly Lieu. Ile 245 250 255

Gly Ala Gly Val Pro Glin Asp Trp Ala Thir His Met Lieu. Gly. His Glu 26 O 265 27 O

Lell Thir Ala Met His Gly Lell Asp His Ala Glin Thir Lieu Ala Ile Wall 285

Lell Pro Ala Lieu Trp Asn Glu Lys Arg Asp Thir Lys Arg Ala Lys Lieu. 29 O 295 3 OO

Lell Glin Tyr Ala Glu Arg Wall Trp Asn Ile Thir Glu Gly Ser Asp Asp 3. OS 310 315 32O

Glu Arg Ile Asp Ala Ala Ile Ala Ala Thir Arg Asn Phe Phe Glu Glin 3.25 330 335

Lell Gly Wall Pro His Lell Ser Asp Tyr Gly Luell Asp Gly Ser Ser Ile 34 O 345 35. O

Pro Ala Lieu. Luell Lell Glu Glu His Gly Met Thr Gln Leu Gly 355 360 365

Glu Asn His Asp Ile Thir Lell Asp Wall Ser Arg Arg Ile Tyr Glu Ala 37 O 375 38O

Ala Arg 385

1. Process for producing an alcohol of formula (I) or an -continued (IV) amine of formula (II): OH OH

(I) R r OH OH (II) wherein the process comprises the following reactions: (a) oxidation of at least one terminal C-atom r (b) dehydration NH2 (c) decarboxylation (d) reduction in case of the product of the formula (I) and from a substrate of the formula (III) and/or (IV) amination in the case of the product of the formula (II) and wherein at least reaction b is enzyme-catalyzed and R is a Co alkyl, wherein the alkyl may be substituted with one or more hydroxyl groups. (III) OH 2. The process of claim 1, wherein R is —CH2OH. —CH (OH) CH(OH) or CH(OH) CH(OH) CH(OH). 3. The process of claim 1, wherein the substrate is a Co-polyol, C-Sugar or C-Sugar acid. OH 4. The process of claim 1, wherein the alcohol of formula (I) is 1.4 butanediol or wherein the amine of formula (II) is 1,4-diaminobutane. US 2014/0171683 A1 Jun. 19, 2014 24

5. The process of claim 1, wherein each of the steps a-d is 11. The process of claim 1, wherein the process is carried enzyme-catalyzed. out in a cell-free in vitro system. 6. The process of claim 1, wherein reaction a is catalyzed 12. The process of claim 11, wherein the process is per by an oxidoreductase, reaction b is catalyzed by a dehy formed in a single reaction vessel, in more than one reaction dratase, reaction c is catalyzed by a decarboxylase and reac vessel, or in a bioreactor. tion d is catalyzed by an aminotransferase or an oxidoreduc 13. The process of claim 5, wherein the process is carried tase. out in a microbial cell, which recombinantly expresses the enzymes. 7. The process of claim 6, wherein the oxidoreductase is an 14. An alcohol of formula (I) or an amine of formula (II) alcoholdehydrogenase, aldehyde dehydrogenase, amino acid formed by the process of any one of claim 1. dehydrogenase, alcohol oxidase and/or aldehyde oxidase. 15. (canceled) 8. The process of claim 1, wherein the process is catalyzed 16. A method of converting 2,5-dioxopentanoate into 5-hy by less than 10 enzymes. droxy-2-oxo-pentanoate comprising treating 2.5 dioxopen 9. The process of claim 1, wherein the process is performed tanoate with alcohol dehydrogenase YigB from E. coli. in the presence of one or more cofactors for transfer of reduc 17. Enzyme mixture comprising less than 10 enzymes tion equivalents. wherein the enzymes are selected from oxidoreductases, 10. The process of claim 9, wherein one cofactor is NAD/ dehydratases, decarboxylases, and aminotransferases. NADH. k k k k k