US2023159902A1PendingUtilityA1
Alcohol dehydrogenase variants
Est. expiryOct 4, 2033(~7.2 yrs left)· nominal 20-yr term from priority
C12Y 101/01001C12Y 101/01244C12N 9/0006Y02P20/10C12P 7/24Y02E50/10C12P 7/18
72
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Claims
Abstract
Described herein are non-natural NAD+-dependent alcohol dehydrogenases (ADHs) capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to its unmodified counterpart. Nucleic acids encoding the non-natural alcohol dehydrogenases, as well as expression constructs including the nucleic acids, and engineered cells comprising the nucleic acids or expression constructs are described. Also described are engineered cells expressing a non-natural NAD+-dependent alcohol dehydrogenase, optionally include one or more additional metabolic pathway transgene(s), methanol metabolic pathway genes, target product pathway genes, cell culture compositions including the cells, methods for promoting production of the target product or intermediate thereof from the cells, compositions including the target product or intermediate, and products made from the target product or intermediate.
Claims
exact text as granted — not AI-modified1 - 24 . (canceled)
25 . A NAD + -dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase having a catalytic efficiency (k cat /K m ) for the conversion of methanol to formaldehyde of 8.6×10 −4 or greater.
26 . (canceled)
27 . A method of producing a target product or an intermediate of the target product, the method comprising
culturing an engineered cell in a culture medium comprising methanol or ethanol to produce the target product (TP) or its intermediate (INT), wherein the engineered cell expresses a non-natural NAD + -dependent alcohol dehydrogenase, and during culturing the cell has increased conversion of methanol or ethanol to formaldehyde or acetaldehyde, as compared to an engineered that does not express the non-natural NAD + -dependent alcohol dehydrogenase, wherein the target product or intermediate is formed from the formaldehyde or acetaldehyde.
28 . The method of claim 27 further comprising a step of isolating or purifying target product (TP) or its intermediate (INT).
29 . The method of claim 28 wherein the step of isolating or purifying comprises one or more of continuous liquid-liquid extraction, pervaporation, evaporation, filtration, membrane filtration (including reverse osmosis, nanofiltration, ultrafiltration, and microfiltration), membrane filtration with diafiltration, membrane separation, reverse osmosis, electrodialysis, distillation, extractive distillation, reactive distillation, azeotropic distillation, crystallization and recrystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, carbon adsorption, hydrogenation, and ultrafiltration.
30 . The method of claim 29 wherein the target product and isolating or purifying is selected from the group consisting of: (a) target product: 1,4-butanediol, purification: distillation; (b) target product: 1,3-butanediol, purification:distillation; (c) target product:Butadiene, purification:distillation; (d) target product:6-AminoCaproic Acid, purification:crystallization, (a) target product: caprolactam, purification:distillation as a final step; (e) target product: hexamethylenediame (HMDA), purification:crystallization; (f) target product: Adipic acid, purification: crystallization (adipic acid crystals); (g) target product: Crotyl alcohol, purification:distillation (h) target product: methyl vinyl carbinol, purification:distillation; (i) target product: succinic acid-crystallization (succinic acid crystals); (j) target product:n-propanol, purification:distillation; (k) target product: isopropanol, purification:distillation; (l) target product:propylene, purification:distillation; (m) target product: methacrylic acid, purification:crystallization, distillation, or extraction (n) target product: methylmethacrylate (MMA) or another ester, purification: distillation or crystallization.
31 . The method of claim 28 wherein the step of isolating or purifying further comprises distillation.
32 . The method of claim 28 wherein the target product is a diol.
33 . The method of claim 28 wherein the target product is a diol is 1,4-butanediol or 1,3-butanediol.
34 . The method of claim 27 wherein the engineered cell further comprises one or more alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a), a formate dehydrogenase (EM8), a formaldehyde activating enzyme (EM10), a formaldehyde dehydrogenase (EM11), a S-(hydroxymethyl)glutathione synthase (EM12), a glutathione-dependent formaldehyde dehydrogenase (EM13), a S-formylglutathione hydrolase (EM14), a formate hydrogen lyase (EM15), and a hydrogenase (EM16).
35 . The method of claim 27 wherein the engineered cell further comprises one or more alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a succinyl-CoA reductase (aldehyde forming) (EB3), a 4-hydroxybutyrate (4-HB) dehydrogenase (EB4), a 4-HB kinase (EB5), a phosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7), a 1,4-butanediol dehydrogenase (EB8), a succinate reductase (EB9), a succinyl-CoA reductase (alcohol forming) (EB10), 4-hydroxybutyryl-CoA transferase (EB11), a 4-hydroxybutyryl-CoA synthetase (EB12), a 4-HB reductase (EB13), and a 4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15), a succinyl-CoA transferase (EB1), and a succinyl-CoA synthetase (EB2A).
36 - 52 . (canceled)
53 . The method of claim 27 , wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to an NAD + -dependent alcohol dehydrogenase template selected from the group consisting of SEQ ID NO:1 (MDH MGA3_17392), EIJ77596.1, AAA22593.1, EIJ77618.1, EIJ78790.1, EIJ80770.1, EIJ78397.1, EIJ83020.1, EFI69743.1, YP_004860127.1, YP_001699778.1, ZP_11313277.1, ZP_05587334.1, YP_004681552.1, AGF87161, YP_002138168.1, YP_359772.1, YP_001343716.1, ZP_16224338.1, AAC45651.1, YP_007491369.1, YP_002434746, YP_005052855, NP_561852.1, YP_001447544, YP_001113612.1, YP_011618, ZP_01220157.1, YP_003990729.1, ZP_07335453.1, NP_717107, YP_003310546.1, ZP_10241531.1, YP_001337153.1, YP_026233.1, YP_694908, YP_725376.1, YP_001663549, EKC54576, YP_001126968.1 or a fragment of said template having said dehydrogenase activity with an amino-terminal deletion, carboxy-terminal deletion, or both, the fragment having a sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to the template.
54 - 58 . (canceled)
59 . The method of claim 53 wherein the non-natural alcohol dehydrogenase has a sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of claim 53 , and wherein the dehydrogenase comprises one or more amino acid substitutions based on formula: R 1 XR 2 , where R 1 is an original amino acid at position X of the template, and R 2 is the variant amino acid that replaces R 1 at a position on the template corresponding to X, wherein XR 2 is selected from the group consisting of (a) 11T, 38N, 42Q, 48D, 53I, 56K, 60E, 61A, 63F, 65Q, 70N, 71I, 71T, 71V, 74S, 81G, 84R, 86K, 87K, 94V, 99P, 99T, 103V, 106L, 107S, 108V, 108W, 109Y, 112K, 112R, 115H, 116F, 117D, 117Q, 117Y, 120H, 120R, 121A, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122A, 122P, 123D, 123I, 123L, 123R, 123Y, 124I, 124L, 124R, 125C, 125G, 125W, 126G, 126V, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 130F, 130I, 130Y, 134T, 143T, 145M, 146N, 147R, 148A, 148F, 148G, 148I, 148T, 148V, 148W, 149L, 149M, 149T, 149V, 150A, 150I, 152M, 155V, 157N, 158E, 158H, 158K, 158W, 161A, 161G, 161Q, 161S, 161V, 163F, 163N, 163Q, 163T, 164G, 164N, 165G, 181R, 184T, 186M, 190A, 190S, 199V, 217K, 226M, 256C, 267H, 269S, 270M, 270S, 270Y, 296S, 298H, 300T, 302V, 312V, 316V, 323M, 333L, 336L, 337C, 343D, 344A, 344G, 345E, 350K, 354M, 355D, 355I, 355K, 358G, 360A, 360G, 360K, 360R, 360S, 361N, 361R, 363K, and 379M or group consisting of (b) 38N, 60E, 71I, 71V, 87K, 99T, 103V, 107S, 108V, 108W, 109Y, 115H, 116F, 117D, 117Q, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122P, 123D, 123I, 123L, 123R, 123Y, 124I, 124L, 125C, 125G, 125V, 125W, 126G, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 129V, 130F, 130I, 130Y, 134T, 143T, 146N, 149L, 149M, 149T, 149V, 150A, 157N, 158E, 158H, 158K, 158W, 163Q, 164N, 267H, 270M, 270S, 270Y, 345E, 355D, 360G, 360K, 360R, 360S, and 361R.
60 . (canceled)
61 . (canceled)
62 . The method of claim 53 wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of claim 53 , and comprises original amino acids at all positions of group (a) 11, 38, 42, 48, 53, 56, 60, 61, 63, 65, 70, 71, 74, 81, 84, 86, 87, 94, 99, 103, 106, 107, 108, 109, 112, 115, 116, 117, 117, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 145, 146, 147, 148, 149, 150, 152, 155, 157, 158, 161, 163, 164, 165, 181, 184, 186, 190, 199, 217, 226, 256, 267, 269, 270, 296, 298, 300, 302, 312, 316, 323, 333, 336, 337, 343, 344, 345, 350, 354, 355, 358, 360, 361, 363 and 379; or of group (b) 38, 60, 71, 87, 99, 103, 107, 108, 109, 115, 116, 117, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 146, 149, 150, 157, 158, 163, 164, 267, 270, 345, 355, 360, and 361.
63 - 81 . (canceled)
82 . A non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to an NAD + -dependent alcohol dehydrogenase template selected from the group consisting of SEQ ID NO:1 (MDH MGA3_17392), EIJ77596.1, AAA22593.1, EIJ77618.1, EIJ78790.1, EIJ80770.1, EIJ78397.1, EIJ83020.1, EFI69743.1, YP_004860127.1, YP_001699778.1, ZP_11313277.1, ZP_05587334.1, YP_004681552.1, AGF87161, YP_002138168.1, YP_359772.1, YP_001343716.1, ZP_16224338.1, AAC45651.1, YP_007491369.1, YP_002434746, YP_005052855, NP_561852.1, YP_001447544, YP_001113612.1, YP_011618, ZP_01220157.1, YP_003990729.1, ZP_07335453.1, NP_717107, YP_003310546.1, ZP_10241531.1, YP_001337153.1, YP_026233.1, YP_694908, YP_725376.1, YP_001663549, EKC54576, YP_001126968.1 or a fragment of said template having said dehydrogenase activity with an amino-terminal deletion, carboxy-terminal deletion, or both, the fragment having a sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to the template.
83 . (canceled)
84 . The non-natural alcohol dehydrogenase of claim 82 wherein the non-natural alcohol dehydrogenase has a sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of claim 53 , and wherein the dehydrogenase comprises one or more amino acid substitutions based on formula: R 1 XR 2 , where R 1 is a original amino acid at position X of the template, and R 2 is the variant amino acid that replaces R 1 at a position on the template corresponding to X, wherein XR 2 is selected from the group consisting of (a) 11T, 38N, 42Q, 48D, 53I, 56K, 60E, 61A, 63F, 65Q, 70N, 71I, 71T, 71V, 74S, 81G, 84R, 86K, 87K, 94V, 99P, 99T, 103V, 106L, 107S, 108V, 108W, 109Y, 112K, 112R, 115H, 116F, 117D, 117Q, 117Y, 120H, 120R, 121A, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122A, 122P, 123D, 123I, 123L, 123R, 123Y, 124I, 124L, 124R, 125C, 125G, 125W, 126G, 126V, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 130F, 130I, 130Y, 134T, 143T, 145M, 146N, 147R, 148A, 148F, 148G, 148I, 148T, 148V, 148W, 149L, 149M, 149T, 149V, 150A, 150I, 152M, 155V, 157N, 158E, 158H, 158K, 158W, 161A, 161G, 161Q, 161S, 161V, 163F, 163N, 163Q, 163T, 164G, 164N, 165G, 181R, 184T, 186M, 190A, 190S, 199V, 217K, 226M, 256C, 267H, 269S, 270M, 270S, 270Y, 296S, 298H, 300T, 302V, 312V, 316V, 323M, 333L, 336L, 337C, 343D, 344A, 344G, 345E, 350K, 354M, 355D, 355I, 355K, 358G, 360A, 360G, 360K, 360R, 360S, 361N, 361R, 363K, and 379M or the group consisting of
(b) 38N, 60E, 71I, 71V, 87K, 99T, 103V, 107S, 108V, 108W, 109Y, 115H, 116F, 117D, 117Q, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122P, 123D, 123I, 123L, 123R, 123Y, 124I, 124L, 125C, 125G, 125V, 125W, 126G, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 129V, 130F, 130I, 130Y, 134T, 143T, 146N, 149L, 149M, 149T, 149V, 150A, 157N, 158E, 158H, 158K, 158W, 163Q, 164N, 267H, 270M, 270S, 270Y, 345E, 355D, 360G, 360K, 360R, 360S, and 361R.
85 . (canceled)
86 . (canceled)
87 . The non-natural alcohol dehydrogenase of claim 82 wherein the non-natural alcohol dehydrogenase has sequence identity of 45% or greater, 55% or greater, 65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater to any one of the templates of claim 53 , and comprises original amino acids at all positions of group (a) 11, 38, 42, 48, 53, 56, 60, 61, 63, 65, 70, 71, 74, 81, 84, 86, 87, 94, 99, 103, 106, 107, 108, 109, 112, 115, 116, 117, 117, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 145, 146, 147, 148, 149, 150, 152, 155, 157, 158, 161, 163, 164, 165, 181, 184, 186, 190, 199, 217, 226, 256, 267, 269, 270, 296, 298, 300, 302, 312, 316, 323, 333, 336, 337, 343, 344, 345, 350, 354, 355, 358, 360, 361, 363 and 379; or of group (b) 38, 60, 71, 87, 99, 103, 107, 108, 109, 115, 116, 117, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 146, 149, 150, 157, 158, 163, 164, 267, 270, 345, 355, 360, and 361.
88 - 90 . (canceled)
91 . The non-natural alcohol dehydrogenase of claim 82 comprising a sequence motif selected from one of groups (i)-(xv) as follows:
(i)
(SEQ ID NO: 79)
TNA and VTNAF;
(ii)
(SEQ ID NO: 80)
VEV and GVEVA;
(iii)
(SEQ ID NO: 81)
DIA, PDIAD.
(SEQ ID NO: 82)
DVA, and PDVAD;
(iv)
(SEQ ID NO: 83)
EKC and QEKCD;
(v)
(SEQ ID NO: 84)
STH and GSTHD;
(vi)
(SEQ ID NO: 85)
TVK and DTVKA;
(vii)
(SEQ ID NO: 86)
SLV, GW, GWV, GLY, ISLVA,
(SEQ ID NO: 87)
IGWA,
(SEQ ID NO: 88)
IGWVA,
and
(SEQ ID NO: 89)
IGLYA;
(viii)
(SEQ ID NO: 90)
HIN, RFN, RID, RIP, GHIND,
(SEQ ID NO: 91)
GRFND,
(SEQ ID NO: 92)
GRIDD,
(SEQ ID NO: 93)
and GRIQD;
(ix)
(SEQ ID NO: 94)
DVNSVEKPVV,
(SEQ ID NO: 95)
EVNSVEKPVV,
(SEQ ID NO: 96)
LVNSVEKPVV,
(SEQ ID NO: 97)
MVNSVEKPVV,
(SEQ ID NO: 98)
RVNSVEKPVV,
(SEQ ID NO: 99)
SVNSVEKPVV,
(SEQ ID NO: 100)
TVNSVEKPVV,
(SEQ ID NO: 101)
VVNSVEKPVV,
(SEQ ID NO: 102)
WVNSVEKPVV,
(SEQ ID NO: 103)
YVNSVEKPVV,
(SEQ ID NO: 104)
GPNSVEKPVV,
(SEQ ID NO:105)
GVDSVEKPVV,
(SEQ ID NO: 106)
GVISVEKPVV,
(SEQ ID NO: 107)
GVLSVEKPVV,
(SEQ ID NO: 108)
GVRSVEKPVV,
(SEQ ID NO: 109)
GVYSVEKPVV,
(SEQ ID NO: 110)
GVNIVEKPVV,
(SEQ ID NO: 111)
GVNLVEKPVV,
(SEQ ID NO:112)
GVNSCEKPVV,
(SEQ ID NO: 113)
GVNSGEKPVV,
(SEQ ID NO: 114)
GVNSWEKPVV,
(SEQ ID NO: 115)
GVNSVGKPVV,
(SEQ ID NO: 116)
GVNSVECPVV,
(SEQ ID NO: 117)
GVNSVERPVV,
(SEQ ID NO: 118)
GVNSVEKAVV,
(SEQ ID NO:119)
GVNSVEKRVV,
(SEQ ID NO: 120)
GVNSVEKSVV,
(SEQ ID NO: 121)
GVNSVEKPAV.
(SEQ ID NO: 122)
GVNSVEKPMV.
(SEQ ID NO: 123)
GVNSVEKPPV.
(SEQ ID NO: 124)
GVNSVEKPSV.
(SEQ ID NO: 125)
GVNSVEKPVF.
(SEQ ID NO: 126)
GVNSVEKPVI,
and
(SEQ ID NO: 127)
GVNSVEKPVY;
(x)
(SEQ ID NO: 128)
TETT,
(SEQ ID NO: 129)
SETN,
(SEQ ID NO:130)
GTETTS,
(SEQ ID NO: 131)
and
GSETNS;
(xi)
(SEQ ID NO: 132)
LLVI,
(SEQ ID NO: 133)
LMVI,
(SEQ ID NO: 134)
LTVI,
(SEQ ID NO: 135)
LWI,
(SEQ ID NO: 136)
and
LAAI;
(xii)
(SEQ ID NO: 137)
NVKMPVID,
(SEQ ID NO: 138)
KEKMPVID,
(SEQ ID NO: 139)
KHKMPVID,
(SEQ ID NO: 140)
KKKMPVID,
(SEQ IDNO: 141)
KWKMPVID,
(SEQ ID NO: 142)
KVKMPVQD,
(SEQ ID NO: 143)
and
KVKMPVIN;
(xiii)
(SEQ ID NO: 144)
HVGG,
(SEQ ID NO: 145)
QVGM,
(SEQ ID NO:146)
PVGS,
(SEQ ID NO: 147)
and
PVGY;
(xiv)
(SEQ ID NO: 148)
VEE
and
GVEEE;
(xV)
(SEQ ID NO: 149)
DAYEDVC,
(SEQ ID NO: 150)
NAYEDGC,
(SEQ ID NO: 151)
NAYEDKC,
and
(SEQ ID NO: 152)
NAYEDRC,
and
(SEQ ID NO: 153)
NAYEDSC,
and
(SEQ ID NO: 154)
NAYEDVR.
92 - 119 . (canceled)Join the waitlist — get patent alerts
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