# US 12,215,359 B2 · Engineered glucose dehydrogenases and methods for the reductive amination of ketone and amine compounds > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,215,359 B2 - Document ID: US12215359B2 - Status: Granted - Kind: B2 - Application number: 17/600,357 - Filed: 2020-04-23 - Published: 2022-05-12 - Granted: 2025-02-04 - Earliest claimed: 2019-05-01 - Assignees: Codexis, Inc. - Applicants: Codexis, Inc. - Inventors: Xiang Yi; Oscar Alvizo; Ravi David Garcia; David Entwistle; Charlene Ching; James Nicholas Riggins; Nandhitha Subramanian - Web page: https://patentgrep.com/patent/US12215359B2 ## CPC - C12N 9/0006: {acting on CH-OH groups as donors (1.1)} - C12N 1/20: Bacteria; Culture media therefor - C12N 15/72: Expression systems using regulatory sequences derived from the lac-operon - C12Y 101/01047: Glucose 1-dehydrogenase (1.1.1.47) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## Abstract The present application provides engineered glucose dehydrogenase polypeptides having imine reductase activity, polynucleotides encoding the engineered polypeptides, host cells capable of expressing the engineered polypeptides, and methods of using these engineered polypeptides with a range of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds. ## Claims ### Claim 1 An engineered glucose dehydrogenase polypeptide comprising a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4, wherein said engineered glucose dehydrogenase polypeptide comprises a substitution at amino acid position 147 in said polypeptide sequence selected from the group consisting of H147A/S/I/T/Q/R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:4, and wherein said engineered glucose dehydrogenase polypeptide exhibits improved production of compound (1) compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:4. ### Claim 2 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said polypeptide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:4, wherein said engineered glucose dehydrogenase polypeptide further comprises at least one substitution or substitution set in said polypeptide sequence at one or more amino acid positions selected from 96, 96/118,155, 155/253, 195, 200, and 256, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 4. ### Claim 3 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said polypeptide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:14, wherein said engineered glucose dehydrogenase polypeptide comprises at least one substitution or substitution set in said polypeptide sequence at one or more amino acid positions selected from 17, 95/96, 95/96/118, 95/96/118/156/200/253, 95/96/147, 95/96/147/200, 95/96/147/200/253, 95/96/155/156, 95/96/155/195/200, 95/96/159/195/253, 95/155/156, 95/155/159/200, 95/155/200, 96, 96/147/195/200/253, 96/155, 96/155/159, 96/155/159/195, 96/155/159/200, 96/156/159/195, 96/156/159/195/200, 96/195,147/155, 147/155/156, 147/155/156/200, 147/155/159, 147/195/200/253, 155, 155/156, 155/156/195/200/253, 155/159/200, and 253, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 14. ### Claim 4 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said polypeptide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:34, wherein said engineered glucose dehydrogenase polypeptide comprises at least one substitution or substitution set in said polypeptide sequence at one or more amino acid positions selected from 17, 17/21, 17/21/42, 17/21/42/155/260, 17/21/49, 17/21/49/51, 17/21/49/51/96, 17/21/49/51/260, 17/21/49/96/155/260, 17/21/49/260, 17/21/51/96, 17/21/51/195, 17/21/96, 17/21/96/155, 17/21/96/195/260, 17/21/96/253, 17/21/147/155/253, 17/21/155/253, 17/21/195, 17/21/253, 17/42, 17/42/49/195/260, 17/42/96, 17/44, 17/44/47/51/96, 17/44/47/51/96/114/147/195/199/253, 17/44/47/51/96/114/147/199, 17/44/47/51/96/114/195/199, 17/44/47/51/96/147/253, 17/44/47/51/178/195, 17/44/47/195/199, 17/44/51/114/195/253, 17/46/47/51/96/147, 17/46/47/51/96/195/253, 17/46/47/51/155, 17/46/47/51/155/195/199, 17/46/47/96/195, 17/46/47/178, 17/46/96/253, 17/47, 17/47/51, 17/47/51/96/114/147/195, 17/47/51/96/114/155/178/253, 17/47/51/96/195, 17/47/51/96/195/199, 17/47/96/114/155/253, 17/47/96/147/155/195/199, 17/47/96/147/195/199/253, 17/47/96/155, 17/47/96/195/199, 17/47/114, 17/47/114/155/195/199/253, 17/47/147/195/199/253, 17/49, 17/49/51, 17/49/51/96/195/253, 17/49/51/155/253, 17/49/51/155/253/260, 17/49/51/155/260, 17/49/51/253, 17/49/96, 17/49/96/155/253, 17/49/96/195, 17/49/260, 17/51, 17/51/96/114, 17/51/96/155/178, 17/51/96/155/195/199/253, 17/51/96/155/199, 17/51/96/155/253, 17/51/96/178/195/199, 17/51/96/195, 17/51/96/260, 17/51/114, 17/51/114/253, 17/51/147, 17/51/155/195, 17/51/178/195/253, 17/51/253, 17/96, 17/96/114/147/155, 17/96/114/147/155/178/195/199, 17/96/147, 17/96/147/155, 17/96/155/178, 17/96/155/178/253, 17/96/155/195, 17/96/155/195/199, 17/96/178, 17/96/178/195, 17/96/195, 17/96/195/199, 17/96/195/199/253, 17/96/253, 17/114, 17/114/253, 17/147/155/253, 17/147/178,17/147/199, 17/155, 17/155/178, 17/155/178/195, 17/155/195, 17/155/195/253, 17/155/199, 17/155/253, 17/178, 17/178/195/253, 17/195, 17/195/199, 17/195/199/253, 17/195/253, 17/199, 17/253, 17/260, 21/49/51, 21/49/96/195, 21/49/96/253, 21/49/195, 21/51/96/155, 21/96, 21/96/195, 21/96/253, 44/46/47/51/114/178/253, 44/46/47/51/155/178/253, 44/46/47/96/155/195, 44/46/51/96/147/178/195/199, 44/46/51/96/155/195/199/253, 44/46/51/96/195, 44/46/51/147/155/178/195/253, 44/47/51/96/114, 44/47/51/96/177/178/199/253, 44/47/51/114/178/253, 44/47/96/114, 44/47/96/195/253, 44/47/147/155, 44/47/147/155/199, 44/96/155/178, 46/47/51/96/114/178, 46/47/51/114/147/195/199, 46/47/51/155/195/199, 46/47/96/114/195/199, 46/47/96/155/178/195/253, 46/47/155/195/199, 46/114/147/155/178/195/199, 46/114/195/199/253, 47/51/96, 47/51/96/114, 47/51/96/147/155/195/199, 47/51/96/147/195/199, 47/51/96/178/195/199/253, 47/51/96/195, 47/51/96/195/199/253, 47/51/114/253, 47/51/155/178/195, 47/51/155/195/253, 47/96/114, 47/96/114/253, 47/96/155/178/195, 47/96/155/195, 47/96/155/195/199, 47/96/178/195/253, 47/96/178/253, 47/96/195/199/253, 47/96/195/253, 47/114, 47/114/155/195/199, 47/114/178, 47/147/195, 47/155/253, 47/178/195, 47/178/195/199, 47/195/199, 49/51/96, 49/96, 49/96/253, 51/96, 51/96/114/155/195/199/253, 51/96/155/178, 51/96/155/178/195/253, 51/96/155/195, 51/96/155/195/253, 51/96/195/253, 51/96/199, 51/96/253, 51/114/155/195/199, 51/114/195/199, 51/147/253, 51/155/195/199, 51/195, 51/253, 96, 96/114/147/155/195/199, 96/114/155/178/195/199/253, 96/114/155/195/253, 96/114/199, 96/147, 96/147/155/178/195/253, 96/147/178/195/199, 96/147/195, 96/147/195/199, 96/147/253, 96/155/178/199, 96/155/195, 96/155/199, 96/155/253, 96/178/195, 96/178/195/199, 96/195, 96/195/199, 96/195/253, 96/199, 96/199/253, 96/253,114, 114/147/155/253, 114/178/195/199/253, 114/195, 114/195/199/253, 132, 147/155, 149, 154, 155/178, 155/178/195/199, 155/195, 155/195/199, 155/195/199/253, 155/195/253, 155/253, 157, 167, 170, 178, 178/195, 178/199, 195, 196, 198, 214, 218, 251, 252, 256, 257, 259, and 260, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 34. ### Claim 5 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said polypeptide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 112, wherein said engineered glucose dehydrogenase polypeptide comprises at least one substitution or substitution set in said polypeptide sequence at one or more amino acid positions selected from 21/42/96/178/195/253, 21/42/96/195, 21/96, 21/96/147/195, 21/96/149, 21/96/155/195/253, 21/96/167, 21/96/167/195, 21/96/178, 21/96/179/195/251/253, 21/96/195, 21/147/149, 21/147/253, 21/149/178/195, 21/155/178/253/256, 21/178/195/256, 42/96/149/253/256, 94, 96, 96/114/155/198/218/253/256/257, 96/114/155/252/253/257, 96/114/155/253, 96/114/196/214/253/256, 96/114/214/253/256, 96/147, 96/147/149, 96/147/149/155, 96/147/167/195, 96/149/155/167/178/253, 96/149/155/195,96/149/178/179/251/256, 96/149/178/253, 96/149/198/252/253/257, 96/155, 96/155/167, 96/155/167/178, 96/155/195, 96/155/196/218/253/256/257, 96/155/196/252/253, 96/155/198, 96/155/252/253, 96/167, 96/178, 96/178/179/195, 96/195, 96/196, 96/196/198, 96/196/201/256, 96/251/253/256, 96/253/257, 96/256/257, 98, 103, 114/253/256/257, 147/149, 149/167, 149/195, 149/218, 149/253, 149/257, 155/167, 155/178/195, 155/178/195/251/253, 155/195/251/253/256, 155/195/253/256, 155/196/198/253, 167, 167/195, 167/195/251/253, 196, 196/253/256/257, 198/253/256, 198/256, 205, 214/253/256, 251, and 257, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 112. ### Claim 6 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said polypeptide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 736, wherein said engineered glucose dehydrogenase polypeptide comprises at least one substitution or substitution set in said polypeptide sequence at one or more amino acid positions selected from 21/47/96/114/155/251, 21/47/149/257, 21/47/155/178/257, 21/47/178, 21/47/178/251/256/257, 21/96/114/155/178, 21/114/155/251/257, 21/114/178/179, 21/155/178/209/256, 25/155, 25/178/256, 42/47/155/178, 47/114/149/178/251, 47/114/178/257, 47/149, 47/178, 47/178/179/251/257, 47/178/251/257, 96/114/155/198, 96/114/155/253/257, 96/253,114/149/155/251/256, 114/149/178/256/257, 114/155/195/198/256, 114/209/256/257, 149, 149/256/257, 155, 155/178/179, 155/178/198/252/253, 155/195/198, 155/195/198/253/256/257, 155/256/257, 178/253, 178/256, 179/256/257, 251, and 253, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 736. ### Claim 7 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said polypeptide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 908, wherein said engineered glucose dehydrogenase polypeptide comprises at least one substitution or substitution set in said polypeptide sequence at one or more amino acid positions selected from 21/42/149, 21/42/149/214/253, 21/42/179/214/244/253, 21/42/253/260/261, 21/149, 21/149/179, 21/149/179/253, 21/149/179/253/260, 21/149/179/253/260/261, 21/149/179/261, 21/149/260/261, 21/149/261, 21/179/214/253, 21/179/253, 21/179/261, 21/214/253, 21/253, 40, 42/149/179/253/260, 42/149/179/260/261, 42/149/214/253, 42/179/253/261, 42/253,104, 111, 149, 149/179, 149/179/253, 149/179/253/260/261, 149/179/260, 149/179/260/261, 149/179/261, 149/253, 149/261, 160, 178, 179/214/253, 179/253, 179/253/260, 179/253/260/261, 179/253/261, 179/260/261, 179/261, 253,253/260/261, 253/261, and 255, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 908. ### Claim 8 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said polypeptide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 956 wherein said engineered glucose dehydrogenase polypeptide comprises at least one substitution or substitution set in said polypeptide sequence at one or more amino acid positions selected from 3, 21/104/160/178/179, 21/104/160/255/258, 21/104/178/179/255, 21/104/178/255/258, 21/104/255, 21/104/255/258, 21/178/179/255/260, 21/255/260, 31, 32, 40, 40/104/178/179, 40/160/178, 46/82/259, 103, 104/149/255/258/260, 104/149/258, 104/160, 104/160/178,104/160/178/179/255, 104/160/178/179/260, 104/160/178/255/260, 104/160/179, 104/178/179/255/258, 104/178/255/258/260, 104/178/255/260,104/179,104/255/260,104/258/260, 111/149/255/258,111/149/255/260,111/160/178/179/255/258/260, 111/160/255/260,154,156,160, 160/178/179, 174, 194, 197, 205, 205/229, 222, 253, 257, 258/260, and 260, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 956. ### Claim 9 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said polypeptide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1062, wherein said engineered glucose dehydrogenase polypeptide comprises at least one substitution or substitution set in said polypeptide sequence at one or more amino acid positions selected from 3/31/154/205/257, 3/31/205, 3/154/205, 31/55/154/205, 31/55/205, 31/154/205, 31/154/205/257, 31/205, 31/205/222,40/55/205,40/154/205, 40/154/205/257, 40/174/205/229, 40/205/229, 54/205/253, 63, 63/149/154/174/178/253, 63/149/154/174/205/253, 63/149/156/178/205/253, 63/149/205,63/149/205/229/253, 63/149/205/253,63/154, 63/154/178/205/253, 63/154/205,63/154/205/229,63/154/205/229/253, 63/154/205/253,63/156, 63/156/205, 63/205, 63/205/253, 63/253, 67, 98,149/154, 149/154/205, 149/156/253, 149/205, 149/205/253, 154, 154/160, 154/160/205, 154/160/205/222/257, 154/160/253, 154/174, 154/174/178/205, 154/174/205/229, 154/205, 154/205/222, 154/205/222/257, 154/205/229, 154/205/253, 154/205/257, 154/229,154/253, 156/205, 156/205/253, 156/229/253, 160,160/205, 160/205/229, 194, 205, 205/222, 205/229, 205/229/253, 205/253, 208, 226, and 253, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1062. ### Claim 10 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said engineered glucose dehydrogenase polypeptide comprises a polypeptide sequence that is at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4. ### Claim 11 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said engineered glucose dehydrogenase polypeptide comprises SEQ ID NO: 14. ### Claim 12 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said engineered glucose dehydrogenase polypeptide comprises an amino acid sequence with at least 92% sequence identity to any even-numbered sequence set forth in SEQ ID NO:6, 8, 14, and 32-1384. ### Claim 13 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said engineered glucose dehydrogenase polypeptide comprises a polypeptide sequence set forth in the even numbered sequences of SEQ ID NOs: 6-1384. ### Claim 14 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said engineered glucose dehydrogenase polypeptide further exhibits improved production of a compound of structural Formula (IV) wherein R1 is selected from a hydrogen atom, or optionally substituted alkyl, alkenyl, alkynyl, alkoxy, arylalkoxy, hydroxyalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; and wherein R2 is independently selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, alkylamino, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; and wherein R3 is independently selected from methyl, D3-methyl and ethyl. ### Claim 15 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said engineered glucose dehydrogenase polypeptide further exhibits improved utilization of compound (2) ### Claim 16 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said engineered glucose dehydrogenase polypeptide further exhibits improved utilization of compound (3) ### Claim 17 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said engineered glucose dehydrogenase polypeptide exhibits improved production of compound (1) (1) from compound (2) and compound (3) ### Claim 18 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said engineered glucose dehydrogenase polypeptide further exhibits improved enantioselectivity. ### Claim 19 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said engineered glucose dehydrogenase polypeptide further exhibits improved stability. ### Claim 20 (depends on 1) The engineered glucose dehydrogenase polypeptide of claim 1, wherein said engineered glucose dehydrogenase polypeptide is purified. ### Claim 21 (depends on 1) A composition comprising at least one glucose dehydrogenase polypeptide provided in claim 1.