Biocatalysts and methods for the synthesis of armodafinil

Patent metadata

Field	Value
Publication number	US-9938509-B2
Application number	US-201715698319-A
Country	US
Kind code	B2
Filing date	Sep 7, 2017
Priority date	Dec 8, 2010
Publication date	Apr 10, 2018
Grant date	Apr 10, 2018

Abstract

The present invention relates to non-naturally occurring polypeptides useful for preparing armodafinil, polynucleotides encoding the polypeptides, and methods of using the polypeptides. The non-naturally occurring polypeptides of the present invention are effective in carrying out biocatalytic conversion of the (i) 2-(benzhydrylsulfinyl)acetamide to (−)-2-[(R)-(diphenylmethyl)sulfinyl]acetamide (armodafinil), or (ii) benzhydryl-thioacetic acid to (R)-2-(benzhydrylsulfinyl)acetic acid, which is a pivotal intermediate in the synthesis of armodafinil, in enantiomeric excess.

First claim

What is claimed is:

1. A non-naturally occurring polypeptide having cyclohexanone monooxygenase (CHMO) activity, wherein the amino acid sequence of the polypeptide has a least 90% sequence identity to SEQ ID NO: 136, and one or more amino acid substitutions at one or more positions corresponding to positions in SEQ ID NO: 136 selected from the group consisting of 37, 277, 278, 280, 281, 326, 432, 433, 435, and 490.

2. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide is further capable of converting the acid substrate compound (1b) to compound (2b) (R-enantiomer) or its opposite enantiomer compound (S-enantiomer)

with at least 2-fold improved activity relative to the wild-type polypeptide of SEQ ID NO: 2.

3. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid substitutions relative to SEQ ID NO: 136, wherein the polypeptide comprises an alanine, glutamic acid, glycine, isoleucine, lysine, proline, serine, threonine, or valine at a position corresponding to position 246 of SEQ ID NO:136.

4. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring further is capable of converting the acid substrate of compound (1b) to the R-enantiomer compound (2b) in at least 50% enantiomeric excess.

5. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid differences relative to SEQ ID NO: 136, wherein said polypeptide further comprises one or more substitutions selected from the group consisting of a glycine at position 143, glycine at position 278, and lysine at position 490.

6. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises lysine at a position corresponding to position 490 of SEQ ID NO: 136.

7. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide is further capable of converting compound (1a) to compound (2a)

in at least 75% enantiomeric excess under suitable reaction conditions.

8. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide is further capable of converting compound (1a) to compound (2a) with an activity increased at least 2-fold relative to the activity of the polypeptide of SEQ ID NO: 136, under suitable reaction conditions.

9. The non-naturally occurring polypeptide of claim 6 , wherein said non-naturally occurring polypeptide further comprises a combination of amino acid substitutions relative to SEQ ID NO: 136 selected from the following group consisting of:
(a) the amino acid at position 143 is glycine;
(b) the amino acid at position 143 is glycine, and the amino acid at position 484 is cysteine; and
(c) the amino acid at position 62 is valine, the amino acid at position 143 is glycine, the amino acid at position 275 is serine, the amino acid at position 329 is valine, and the amino acid at position 484 is cysteine.

10. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide is further capable of converting compound (1b) to compound (2b)

in enantiomeric excess under suitable reaction conditions.

11. The non-naturally occurring polypeptide of claim 6 , wherein said non-naturally occurring polypeptide is further capable of converting compound (1b) to compound (2b) in at least 75% enantiomeric excess under suitable reaction conditions.

12. The non-naturally occurring polypeptide of claim 6 , wherein said non-naturally occurring polypeptide is further capable of converting compound (1b) to compound (2b) with an activity increased at least 2-fold relative to the activity of the polypeptide of SEQ ID NO: 136, under suitable reaction conditions.

13. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide is capable of at least 90% or greater conversion of compound (1b) to compound (2b) in 24 h with a substrate loading of about 50 g/L.

14. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide further comprises amino acid substitutions at positions corresponding to positions in SEQ ID NO: 136 selected from the group consisting of 32, 40, 42, 54, 62, 74, 123, 135, 163, 171, 176, 182, 192, 227, 246, 288, 290, 313, 314, 322, 329, 336, 348, 364, 373, 382, 430, 472, 478, 489, 538, and 539.

15. The non-naturally occurring polypeptide of claim 14 , wherein said polypeptide comprises one or more substitutions corresponding to substitutions in SEQ ID NO: 136, selected from the group consisting of: the amino acid at position 32 is glutamic acid, the amino acid at position 40 is glycine, the amino acid at position 42 is isoleucine, the amino acid at position 54 is valine, the amino acid at position 62 is valine, the amino acid at position 74 is glutamic acid, the amino acid at position 123 is alanine, the amino acid at position 135 is lysine, the amino acid at position 163 is leucine or tyrosine, the amino acid at position 171 is glycine, the amino acid at position 176 is serine, the amino acid at position 182 is valine, the amino acid at position 192 is valine, the amino acid at position 227 is aspartic acid or glutamic acid, the amino acid at position 264 is tyrosine, the amino acid at position 288 is leucine or valine, the amino acid at position 290 id aspartic acid, the amino acid at position 313 is glutamic acid, the amino acid at position 314 is leucine or threonine, the amino acid at position 322 is glycine or methionine, the amino acid at position 329 is valine, the amino acid at position 336 is serine, the amino acid at position 348 is alanine, the amino acid at position 364 is lysine, the amino acid at position 373 is valine, the amino acid at position 382 is arginine, the amino acid at position 430 is arginine, the amino acid at position 489 is glycine, the amino acid at position 538 is glutamic acid, and the amino acid at position 539 is glutamic acid.

16. A expression vector comprising a non-naturally occurring polynucleotide encoding the non-naturally occurring polypeptide of claim 1 .

17. A host cell comprising the expression vector of claim 16 .

Assignees

• Codexis Inc

Inventors

• Ang Ee Lui
• Alvizo Oscar
• Behrouzian Behnaz
• Clay Michael D
• Collier Steven J
• Eberhard Ellen D
• Fan Fu
• Song Shiwei
• Smith Derek J
• Widegren Magnus
• Wilson Robert
• Xu Junye
• Zhu Jun

Classifications

• C12P11/00

  Preparation of sulfur-containing organic compounds · CPC title

• C12P13/02

  Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)} · CPC title

• C12P41/002

  by oxidation/reduction reactions · CPC title

• C12Y114/13022

  Cyclohexanone monooxygenase (1.14.13.22) · CPC title

• C12N9/0073 Primary

  with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13 · CPC title