Granted Patent B2

US 12,480,100 · App. 18/671,324 · Granted Nov 25, 2025

Inventors: Oscar Alvizo (Fremont, CA); Steven J. Collier (Concord, MA); Hans-Georg Joerg Hennemann (Bedburg, DE); Seong Ho Oh (Singapore, SG); Wenjuan Zha (Singapore, SG)

Assignee: Codexis, Inc.

C12N9/0006A61K31/137C12P13/001C12Y101/01184

Abstract

Claims (25)

1 . An engineered polypeptide capable of converting 1-(3-hydroxyphenyl)-2-(methylamino) ethanone to (R)-phenylephrine, wherein the amino acid sequence of the polypeptide has at least 90% identity to SEQ ID NO: 2 and comprises a residue difference E145A relative to SEQ ID NO: 2.

2 . The engineered polypeptide of claim 1 , wherein the polypeptide further comprises at least one residue difference relative to SEQ ID NO: 2 selected from I11L, A64V, T76I, V95M, S96L, V99L, F147L, F147I, V148I, T152A, L153M, S159T, Y190C, Y190G, D197A, E200P, A202F, M206C, and Y249F.

3 . The engineered polypeptide of claim 1 , wherein the polypeptide is capable of stereospecifically converting 1-(3-hydroxyphenyl)-2-(methylamino) ethanone to (R)-phenylephrine in an enantiomeric excess of at least 99%.

4 . An engineered polynucleotide encoding the engineered polypeptide of claim 1 .

5 . A method for producing a compound of Formula I from a compound of Formula II:

wherein R 2 is a group selected from: —H, —Cl, —Br, —I, —F, —CH 3 , —OH, —OCH 3 , —SH, —SCH 3 , —NH 2 , —NHCH 3 , or a long chain alkyl;

wherein R 3 is a group selected from: —H, —Cl, —Br, —I, —F, —CH 3 , —OH, —OCH 3 , —SH, —SCH 3 , —S(O)CH 3 , —NH 2 , —NHCH 3 , —N(CH 3 ) 2 , —OR, —SR, —NR 2 , —SO 2 NR 2 (wherein R=—H, —CH 3 , or alkyl), ethyl, propyl, isopropyl, cyclopropyl, or a long chain alkyl;

wherein R 4 is a group selected from: —H, —Cl, —Br, —I, —F, —CH 3 , —OH, —OCH 3 , —SH, —SCH 3 , —S(O)CH 3 , —SO 2 CH 3 , —NH 2 , —NHCH 3 , —N(CH 3 ) 2 , SO 2 NR 2 (wherein R=—H, —CH 3 );

wherein R 5 is a group selected from: —H, —Cl, —Br, —I, —F, —CH 3 , —OH, —OCH 3 , —SH, —SCH 3 , —S(O)CH 3 , —SO 2 CH 3 , —NH 2 , —NHCH 3 , —N(CH 3 ) 2 , —OR, —SR, —NR 2 , —SO 2 NR 2 (wherein R=—H, —CH 3 , or alkyl), ethyl, propyl, isopropyl, or cyclopropyl;

wherein R 6 is a group selected from: —H, —Cl, —Br, —I, —F, —CH 3 , —OH, —SH, or —NH 2 ;

wherein R 2 and R 3 , R 3 and R 4 , or R 4 and R 5 can optionally be connected as part of a 5 or 6 membered ring;

wherein R α is a group selected from: —H, —CH 3 , ethyl, propyl, isopropyl, cyclopropyl, or a long chain alkyl; wherein R β is a group selected from: —H, —CH 3 , ethyl, propyl, isopropyl, or cyclopropyl;

wherein R α and R β can form a ring, or wherein the R α -R β unit is a carbonyl or imino functional group;

wherein R N1 and R N2 can be independently a group selected from: —H, —CH 3 , —OH, —OCH 3 , —OR, —C(O)R (wherein R=—H, —CH 3 , or alkyl), ethyl, propyl, isopropyl, cyclopropyl, long chain alkyl, carbonyl, or carboxy;

the method comprising contacting a mixture comprising the compound of Formula II with a polypeptide of claim 1 , under reaction conditions suitable to convert the compound of Formula II to the compound of Formula I.

6 . The method of claim 5 , comprising contacting a polypeptide of claim 1 with a mixture comprising a 1-(3-hydroxyphenyl)-2-(methylamino) ethanone substrate and a buffer under reaction conditions suitable to convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone to (R)-phenylephrine.

7 . The method of claim 5 , wherein the reaction conditions comprise a temperature of about 25° C. to about 35° C.

8 . The method of claim 5 , wherein the reaction conditions comprise a temperature of about 30° C.

9 . The method of claim 5 , wherein the reaction conditions comprise a pH of about 6.5 to about 7.0.

10 . The method of claim 5 , wherein the reaction conditions comprise an initial pH of about 7.0 and then adjusting the initial pH to about 6.75 after about 2 hours.

11 . The method of claim 5 , wherein the mixture comprises 50% isopropyl alcohol.

12 . The method of claim 5 , wherein the buffer is selected from 0.1 M triethanolamine and 0.05 M potassium phosphate.

13 . The method of claim 5 , wherein the mixture comprises about 0.05 g/L NADP.

14 . The method of claim 5 , wherein the reaction conditions comprise an inert atmosphere.

15 . The method of claim 5 , wherein the reaction conditions comprise a pH of about 6.75-7.0, a temperature of about 30° C., about 50% isopropyl alcohol, about 0.05 g/L NADP, about 100 g/L 1-(3-hydroxyphenyl)-2-(methylamino) ethanone, and about 0.9-1.1 g/L of the polypeptide, wherein the reaction is carried out under N 2 for about 19-24 hours.

Continuity (9)

References Cited (156)