# US 10,370,688 B2 · Methods for hydroxylyation of chemical compounds

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- Number: US 10,370,688 B2
- Document ID: US10370688B2
- Status: Granted
- Kind: B2
- Application number: 15/357,668
- Filed: 2016-11-21
- Published: 2017-05-04
- Granted: 2019-08-06
- Earliest claimed: 2012-05-08
- Assignees: Codexis, Inc.
- Applicants: Codexis, Inc.
- Inventors: Haibin Chen; Yong Koy Bong; Fabien L. Cabirol; Anupam Gohel Prafulchandra; Tao Li; Jeffrey C. Moore; Martina Quintanar-Audelo; Yang Hong; Steven J. Collier; Derek Smith
- Web page: https://patentgrep.com/patent/US10370688B2

## CPC

- C12P 17/12: containing a six-membered hetero ring
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- C12P 13/24: Proline; Hydroxyproline; Histidine
- C12P 17/188: {Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms (ergot-alcaloids C12P17/183)}
- C12Y 114/11002: Procollagen-proline dioxygenase (1.14.11.2), i.e. proline-hydroxylase
- Y02P 20/52: using catalysts, e.g. selective catalysts
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The present disclosure provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.

## Claims

### Claim 1

A process for preparing a product compound of formula (Ia),
the process comprising:
i. contacting a substrate compound of formula (IIa)
with an engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2,
ii. in the presence of α-ketoglutaric acid at a concentration equimolar or higher than that of the substrate compound concentration, under suitable reaction conditions to prepare a product compound of formula (Ia);
wherein for the compound of formula (Ia) and the compound of formula (IIa),
Q is (C1-C5)alkylene;
L is (C1-C4)alkylene;
R6 is halo;
and
wherein the sum of ring carbon atoms of said Q +L is an integer from 2 to 5;
with the provisos that
(i) when the sum of ring carbon atoms of said Q +L is 2, then L is a methylene; and
(ii) when the sum of ring carbon atoms of said Q +L is 3, then L is either a bond or ethylene; and
wherein the engineered polypeptide comprises a mutation of X166T/Q/L.

### Claim 2 (depends on 1)

The process of claim 1, in which the compound of formula (Ia) comprises the compound of formula (Ib),
the process comprising:
i. contacting substrate compound of formula (IIb)
with an engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2,
ii. in the presence of α-ketoglutaric acid at a concentration equimolar or higher than that of the substrate compound concentration, under suitable reaction conditions to prepare a product compound of formula (Ib);
wherein for the compound of formula (Ib) and the compound of formula (IIb),
R6 is halo;
k is an integer from 1 to 5;
r is an integer from 0 to 4; and
wherein k +r is 3, 4 or 5;
with the proviso that when k +r is 3, then k is 1 or 3; and
wherein the engineered polypeptide further comprises one or more residue differences at residue positions selected from the group consisting of: X2, X3, X4, X5, X9, X13, X25, X26, X29, X30, X36, X42, X52, X57, X58, X59, X66, X86, X92, X95, X103, X112, X113, X115, X116, X121, X131, X150, X151, X225, X230, X270, and X271.

### Claim 3 (depends on 1)

The process of claim 1, in which the compound of formula (Ia) comprises the compound of formula (Ic),
the process comprising:
i. contacting substrate compound of formula (IIc)
with an engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2,
ii. in the presence of α-ketoglutaric acid at a concentration equimolar or higher than that of the substrate compound concentration, under suitable reaction conditions to prepare a product compound of formula (Ic);
wherein for the compound of formula (Ic) and the compound of formula (IIc),
R6 is halo; and
wherein the engineered polypeptide further comprises one or more residue differences at residue positions selected from the group consisting of: X2, X3, X4, X5, X9, X13, X25, X26, X29, X30, X36, X42, X52, X57, X58, X59, X66, X86, X92, X95, X103, X112, X113, X115, X116, X121, X131, X150, X151, X225, X230, X270, and X271.

### Claim 4 (depends on 1)

The process of claim 1, in which the compound of formula (Ia) comprises the compound of formula (Ie),
the process comprising:
i. contacting substrate compound of formula (IIe)
with an engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2,
ii. in the presence of α-ketoglutaric acid at a concentration equimolar or higher than that of the substrate compound concentration, under suitable reaction conditions to prepare a product compound of formula (Ie);
wherein for the compound of formula (Ie) and the compound of formula (IIe),
R6 is halo; and
wherein the engineered polypeptide further comprises one or more residue differences at residue positions selected from the group consisting of: X2, X3, X4, X5, X9, X13, X25, X26, X29, X30, X36, X42, X52, X57, X58, X59, X66, X86, X92, X95, X103, X112, X113, X115, X116, X121, X131, X150, X151, X225, X230, X270, and X271.

### Claim 5 (depends on 1)

The process of claim 1, in which the suitable reaction conditions comprise Fe+2, optionally in which the Fe+2 is in the form of (NH4)2(FeSO4)2.

### Claim 6 (depends on 1)

The process of claim 1, in which the suitable reaction conditions comprise a reductant that reduces Fe+3 to Fe+2, optionally in which the reductant comprises ascorbic acid present in at least about 0.1 fold, 0.2 fold, 0.3 fold, 0.5 fold, 0.75 fold, 1 fold, 1.5 fold, or at least 2 fold the molar concentration of substrate compound.

### Claim 7 (depends on 1)

The process of claim 1, in which the suitable reaction conditions comprise O2, optionally in which the O2 is provided by forced aeration.

### Claim 8 (depends on 1)

The process of claim 1, in which the suitable reaction conditions comprise (a) substrate loading at about 10 g/L to 100 g/L; (b) about 1 g/L to about 50 g/L of the engineered polypeptide; (c) α-ketoglutarate at a concentration of about 1 to 2 molar equivalents of the substrate compound; (d) ascorbic acid at a concentration of about 0.25 to 0.75 molar equivalents of the substrate compound; (e) about 0.5 mM to about 12 mM of FeSO4; (f) a pH of about 6 to 8; (g) a temperature of about 20° to 40° C.; and (h) a reaction time of 6 to 120 h.

### Claim 9 (depends on 1)

The process of claim 1, wherein said engineered polypeptide further comprises at least one mutation at a residue selected from the group consisting of 2K, 2T, 3S, 4Q, 4L, 4E, 4S, 5I, 5L, 5M, 91, 13T, 25R, 26T, 29A, 30V, 30P, 36T, 42E, 52P, 57T, 57A, 58A, 59G, 66Q, 86S, 92V, 95M, 103L, 103Q, 112T, 112V, 113E, 115E, 115H, 115D, 115G, 115S, 115A, 116L, 121F, 131Y, 131F, 150S, 1515, 225L, 225Y, 225W, 230V, 270E, 271K, and 271, wherein said residue positions are numbered relative to SEQ ID NO:2.

### Claim 10 (depends on 1)

The process of claim 1, wherein said engineered polypeptide further comprises a mutation or combination of mutations, as compared to the sequence of SEQ ID NO: 2, selected from the group consisting of:
(a) 103L;
(b) 52P and 255Y;
(c) 4E/L/S and 115A;
(d) 25R and 58A;
(e) 29A;
(f) 115H/D/G and 121F;
(g) 3S and 103L;
(h) 103L and 131Y/F;
(i) 26T and 103L;
(j) 25R, 66Q, 92V and 115E;
(k) 25R, 66Q, 92V, 103L, and 115E; and
(l) 3S, 25R, 66Q, 92V, 103L, and 115E.

### Claim 11 (depends on 1)

The process of claim 1, wherein said engineered polypeptide further comprises one or more differences in at least one residue position, as compared to the sequence of SEQ ID NO: 2 at residue positions selected from the group consisting of: 17, 24, 26, 62, 88, 98, 114, 140, 151, 186, 188, and 205.

### Claim 12 (depends on 1)

The process of claim 1, wherein said engineered polypeptide further comprises at least one difference in at least one residue position, as compared to the sequence of SEQ ID NO: 2, wherein said residue position is selected from the group consisting of: 17V, 24R, 24S, 26R, 26W, 62Q, 88R, 98F, 98T, 114N, 140L, 151A, 151H, 186G, 188G, and 205V.