Granted Patent
B2
US 12,480,111 · App. 18/189,091 · Granted Nov 25, 2025
Penicillin-G acylases
Inventors:
Rama Voladri (Pleasanton, CA); Christopher Michael Micklitsch (Philadelphia, PA); Oscar Alvizo (Fremont, CA); Jovana Nazor (Milpitas, CA); Da Duan (Foster City, CA); Judy Victoria Antonio Viduya (Greenbrae, CA); Stephan Jenne (Foster City, CA); Chihui An (Scotch Plains, NJ); Keith Allen Canada (Freehold, NJ); Paul N. Devine (Tinton Falls, NJ); Iman Farasat (Rahway, NJ); Anna Fryszkowska (New York, NY); Katrina W. Lexa (Santa Rosa, CA); Robert Kevin Orr (Cranford, NJ)
Assignee:
Codexis, Inc.
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Abstract
The present invention provides engineered penicillin G acylase (PGA) enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, and methods of using the engineered PGA enzymes.
Claims (13)
1 . An engineered polynucleotide encoding an engineered penicillin G acylase variant having at least 95% sequence identity to SEQ ID NO:6, and substitutions at positions 254, 255, and 370 and at least one additional substitution selected from 54C, 62G, 115A/P, 125L/T, 127S/V, 185V, 253K/V, 256Q, 257I, 257V, 260A/P, 268S/V, 322P, 325G, 348C, 348Q, 369L, 369P, 369V, 369W, 372A/H/L, 373F/M, 377P 378H, 384A, 384F/513Q/536M, 384G/L, 388T, 389L, 391P/S, 435R, 461A, 517L/P, 530C/Y, 554A/E/P/V, 556G, 557G/S, 559P/S, 560I, 600T/623V, 623A/G/R/W, 624A, 626G, 627G/H, 705G/P, 706G, 707S, 723A/G, 740L, 748G, and 752E, and wherein said engineered penicillin G acylase variant is capable of deacylating a di or tetra-protected insulin dimer, wherein said positions are numbered with reference to SEQ ID NO:6.
2 . The engineered polynucleotide encoding an engineered penicillin G acylase variant of claim 1 comprising the substitutions 254W, 255G, and a substitution set selected from 103V/370F/444S/706G/766G, 103V/369W/370F/442I/444S/536M/556G/766G, 103V/369W/370F/444S, 103V/369W/370F/444S/556G/706G/766G, 103V/369W/370F/444S/765P/766G, 103V/369W/370F/765P/766G, 257V/362V/370I/384A/451R, 257V/362V/370I/384L/451R/723L, 362V/370I/451R/705D, 369P/370F, 369P/370F/444S/706G/766G, 369P/370F/556G/766G, 369V/370F/388T/444S/556G/766G, 369V/370F/444S, 369V/370F/444S/556G/766G, 369V/370F/556G, 369V/370F/556G/765P, 369V/370F/556G/766G, 369V/370F/766G, 369W/370F/444S/556G, 369W/370F/444S/556G/612A/766G, 369W/370F/444S/556G/706G/765P, 369W/370F/444S/706G/765P/766G, 370I/372A/373M/384L/513Q/560G, 370I/372A/384L/451R/705D, 370I/372A/384L/560G/705D, 370I/384A/451R/560G/705D/723L, 370I/384L/451R/705D/723L, 370I/451R/560G/705D/723L, and 370I/451R/705D/723L.
3 . The engineered polynucleotide encoding an engineered penicillin G acylase variant of claim 2 comprising the substitutions 370I, 384L, 513Q, 560G and a substitution set is selected from 103V/254W/255G/372S/373F/557G, 253H/254W/255G/322T/369W/372A/373M/623G, 253H/254Q/255G/322T/369W/372A/373M/623G, 253H/254Q/255G/369W/372A/373M/391A/623G/723A, 253H/254Q/255G/369W/372A/373M/619R/623G/723A, 253H/254Q/255G/369W/372A/373M/623G/723A, 253H/254Q/255G/372A/373L/623G/723A, 253H/254S/255V/369W/372A/373M/623S/723A, 253H/254S/255G/369W/372A/373M, 253H/254W/255G/322T/369W/372A/373W/723A, 253H/254W/255G/369W/372A/373M/623G/723A, 253H/254W/255G/372A/373L/623S, 253S/254S/255V/322T/369W/372A/373M/619R/723A, 254W/255G/260S/372S/373F/556G, 254W/255G/260S/372S/373F/556G/557V/559S, 254W/255G/322T/369W/372A/373M, 254W/255G/322T/369W/372A/373W/723A, 254W/255G/322T/369W/372A/373M/623G/723A, and 254W/255G/369W/372A/373F/556G.
4 . The engineered polynucleotide encoding an engineered penicillin G acylase variant of claim 1 , wherein said engineered penicillin G acylase variant comprises a histidine tag.
5 . The engineered polynucleotide encoding an engineered penicillin G acylase variant of claim 4 , wherein said histidine tag is present at the C-terminus of said engineered penicillin G acylase variant.
6 . The engineered polynucleotide encoding an engineered penicillin G acylase variant of claim 1 , wherein said engineered penicillin G acylase variant comprises a polypeptide sequence set forth in variant numbers 1-308.
7 . The engineered polynucleotide encoding an engineered penicillin G acylase variant of claim 1 , wherein said engineered penicillin G acylase variant comprises a polypeptide sequence selected from the even-numbered sequences between SEQ ID NO:4 and SEQ ID NO:90.
8 . The engineered polynucleotide sequence of claim 1 , said wherein said sequence comprises a polynucleotide sequence that is at least 95% identical to a sequence selected from the odd-numbered sequences between SEQ ID NO:5 and SEQ ID NO:89.
9 . A vector comprising the engineered polynucleotide sequence of claim 1 .
10 . The vector of claim 9 , further comprising at least one control sequence.
11 . A host cell comprising the vector of claim 10 .
12 . A method for producing an engineered penicillin G acylase variant, comprising culturing said host cell of claim 11 under conditions that said engineered penicillin G acylase variant is produced by said host cell.
13 . The method of claim 12 , further comprising the step of recovering said engineered penicillin G acylase variant produced by said host cell.
Assignments (4)
SECURITY INTEREST
Recorded Feb 15, 2024
From: CODEXIS, INC.
To: INNOVATUS LIFE SCIENCES LENDING FUND I, LP, AS COLLATERAL AGENT
Reel/Frame 066600/0650 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Feb 9, 2024
From: VOLADRI, RAMA; MICKLITSCH, CHRISTOPHER MICHAEL; ALVIZO, OSCAR; NAZOR, JOVANA; DUAN, DA; VIDUYA, JUDY VICTORIA ANTONIO; JENNE, STEPHAN
To: CODEXIS, INC.
Reel/Frame 066433/0001 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Feb 9, 2024
From: ORR, ROBERT KEVIN; LEXA, KATRINA W.; FRYSZKOWSKA, ANNA; FARASAT, IMAN; DEVINE, PAUL N.; CANADA, KEITH ALLEN; AN, CHIHUI
To: MERCK SHARP & DOHME CORP.
Reel/Frame 066433/0254 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Feb 8, 2024
From: VOLADRI, RAMA; MICKLITSCH, CHRISTOPHER MICHAEL; ALVIZO, OSCAR; NAZOR, JOVANA; DUAN, DA; VIDUYA, JUDY VICTORIA ANTONIO; JENNE, STEPHANE; MERCK SHARP & DOHME; MERCK SHARP & DOHME
To: CODEXIS, INC.
Reel/Frame 066529/0054 →
Continuity (4)
Continuation
16616041
Provisional Application
62527199
· Jun 30, 2017
Provisional Application
62525404
· Jun 27, 2017
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