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(12) Patent Application Publication (10) Pub. No.: US 2014/0171683 A1 Sieber Et Al 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 substrate of formula (III) and/or (IV) into a product 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 enzyme-catalyzed. In a preferred embodiment, all (2), (4) Date: Dec. 9, 2013 reactions are enzymatically catalyzed. The enzymes cata lyzing the reactions are selected from oxidoreductases, 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 Alcohol dehydrogenase 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/Glucose oxidase 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 i?,is 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.
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