US2023416789A1PendingUtilityA1

Olivetolic acid cyclase variants with improved activity for use in production of phytocannabinoids

Assignee: HYASYNTH BIOLOGICALS INCPriority: Nov 20, 2020Filed: Nov 17, 2021Published: Dec 28, 2023

Est. expiryNov 20, 2040(~14.3 yrs left)· nominal 20-yr term from priority

Inventors:Timothy S. Liao Letian Song Louis Hom Curtis Walton Daniel Furlong Mindy Melgar Devanshi Bhargava

C12P 7/42C12N 9/88C12Y 404/01026C12P 7/22C12P 17/06

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References

Claims

Abstract

The present disclosure relates generally to methods, isolated polypeptides and polynucleotides, expression vectors, and host cells for the production of olivetolic acid and phytocannabinoids. A method of producing olivetolic acid (OVLa) and/or a phytocannabinoid in a heterologous host cell having OVLa-producing or phytocannabinoid-producing capacity comprises transforming the host cell with a nucleotide encoding a variant olivetolic acid cyclase (OAC) protein having at least 6 amino acid mutations relative to the wild type OAC protein, and culturing the transformed host cell to produce OVLa and/or phytocannabinoids therefrom. The variant OAC protein (SEQ ID NO:92) has at least 85% sequence identity with the wild type OAC protein (SEQ ID NO:91). Exemplary variants having improved OVLa or phytocannabinoid production capacity are described.

Claims

exact text as granted — not AI-modified

1 . A method of producing olivetolic acid (OVLa) or a phytocannabinoid produced therefrom in a heterologous host cell comprising OVLa-producing or phytocannabinoid-producing capacity, said method comprising:
 transforming said host cell with a nucleotide encoding a variant olivetolic acid cyclase (OAC) protein having at least 6 amino acid mutations relative to the wild type OAC protein, and   culturing said transformed host cell to produce olivetolic acid and/or phytocannabinoids therefrom,   wherein said variant OAC protein comprises at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the wild type OAC protein sequence according to SEQ ID NO:91.   
     
     
         2 . The method of  claim 1 , wherein at least 4 of the at least 6 amino acid mutations are in residues selected from the group consisting of: residues 28, 31, 41, 43, 44, 68, 74, 84, 100 and 102 of SEQ ID NO:91. 
     
     
         3 . The method according to  claim 2 , wherein at least one of the at least 4 mutations in amino acid residue 28, 31, 41, 43, 44, 68, 74, 84, 100 or 102 is a conservative amino acid substitution. 
     
     
         4 . The method according to  claim 2 , wherein at least one of the at least 4 mutations in amino acid residue 28, 31, 41, 43, 44, 68, 74, 84, 100 or 102 is a non-conservative amino acid substitution. 
     
     
         5 . The method of  claim 1 , wherein said variant OAC protein has amino acid substitutions in at least 10, at least 9, at least 8, at least 7, at least 6, or at least 5 amino acid residues at positions 28, 31, 41, 43, 44, 68, 74, 84, 100 or 102. 
     
     
         6 . The method according to  claim 1 , wherein any amino acid mutation at a residue other than 28, 31, 41, 43, 44, 68, 74, 84, 100 or 102 is a conservative amino acid substitution. 
     
     
         7 . The method according to  claim 1 , wherein the nucleotide encoding the variant olivetolic acid cyclase (OAC) protein has a sequence comprising:
 (a) a nucleotide sequence according to SEQ ID NO:5, SEQ ID NO: 3, SEQ ID NO:4, or SEQ ID NO:6-SEQ ID NO:39;   (b) a nucleotide sequence having at least 85%, at least 90%, at least 95%, or at least 99%, identity with the sequence of (a); or   (c) a nucleotide sequence that hybridizes with the complementary strand of the nucleotide having the sequence of (a).   
     
     
         8 . The method according to  claim 1 , wherein the variant OAC protein comprises a sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:43-SEQ ID NO:76, and sequences having, at least 90%, at least 95%, or at least 99% sequence identity therewith. 
     
     
         9 . The method according to  claim 1 , wherein at least 4 of the at least 6 amino acid mutations relative to the wild type OAC protein are selected from the group consisting of:
 V28A;   V31G;   Y41T, Y41S or Y41V;   K44V;   T68L or T68R;   174E, 174R, 174D or 174G;   V84R;   R100M or R100E; and   G102R, G102S, or G102STOP.   
     
     
         10 . The method of  claim 1 , wherein the host cell is transformed with a nucleotide encoding variant olivetolic acid cyclase (OAC) protein with at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity of any one of the following sequences with the indicated substitutions from OAC wild type (SEQ ID NO:91) being present:
 V28A/V31G/Y41S/G43SILENT(=GGG)/K44V/T68L/174R/V84R/R100E/G102R (SEQ ID NO:42),   V28A/Y41T/G43SILENT(=GGG)/T68L/174E/V84R/R100M/G102R (SEQ ID NO:40),   Y41S/G43SILENT(=GGG)/K44V/T68R/174R/V84R (SEQ ID NO:41),   V28A/Y41T/G43SILENT(=GGG)/T68L/174G/V84R/R100E (SEQ ID NO:43),   V28A/Y41T/G43SILENT(=GGG)/K44V/T68L/174D/V84R/R100M/G102R (SEQ ID NO:44),   V28A/Y41T/G43SILENT(=GGG)/T68L/174D/V84R/G102R (SEQ ID NO:45),   V28A/Y41T/G43SILENT(=GGG)/K44V/T68L/174R/V84R/R100E/G102R (SEQ ID NO:46),   Y41T/G43SILENT(=GGG)/T68R/174R/V84R/R100M/G102STOP (SEQ ID NO:47),   V28A/Y41V/G43SILENT(=GGG)/K44V/T68L/174G/V84R/R100E/G102R (SEQ ID NO:48),   V28A/Y41T/G43SILENT(=GGG)/K44V/T68R/174G/V84R/G102STOP (SEQ ID NO:49),   V28A/Y41T/G43SILENT(=GGG)/K44V/T68R/174G/V84R/G102STOP (SEQ ID NO:50),   V28A/V31G/Y41T/G43SILENT(=GGG)/K44V/T68R/174E/V84R/R100E (SEQ ID NO:51),   V28A/Y41S/G43SILENT(=GGG)/T68R/174R/V84R/R100M/G102STOP (SEQ ID NO:52),   Y41T/G43SILENT(=GGG)/K44V/T68L/174G/V84R/G102R (SEQ ID NO:53),   V28A/Y41T/G43SILENT(=GGG)/K44V/T68R/174R/V84R/R100E/G102R (SEQ ID NO:54),   V28A/V31G/Y41S/G4351LENT(=GGG)/K44V/T68R/174R/V84R (SEQ ID NO:55),   V28A/G43SILENT(=GGG)/K44V/174D/V84R/R100E/G102R(=CGC) (SEQ ID NO:56),   V28A/Y41V/G43SILENT(=GGG)/K44V/T68L/174G/V84R/G102R (SEQ ID NO:57),   Y41T/G43SILENT(=GGG)/T68L/174G/V84R/R100M/G102R (SEQ ID NO:58),   V28A/V31G/Y41T/G43SILENT(=GGG)/K44V/T68R/174R/V84R/R100E/G102R (SEQ ID NO:59),   V31G/Y41S/G43SILENT(=GGG)/K44V/T68R/V84R/R100E (SEQ ID NO:60),   V28A/Y41V/G43SILENT(=GGG)/T68L/174D/R100E/G102STOP (SEQ ID NO:61)   V28A/Y41V/G43SILENT(=GGG)/T68R/174G/V84R/R100M/G102R (SEQ ID NO:62),   V31G/G43SILENT(=GGG)/174G/V84R/R100E (SEQ ID NO:63),   V28A/Y41S/G43SILENT(=GGG)/K44V/T68R/174R/V84R/R100M/G102R (SEQ ID NO:64),   V28A/V31G/Y41V/G43SILENT(=GGG)/K44V/T68L/174G/V84R (SEQ ID NO:65),   V28A/Y41V/G43SILENT(=GGG)/K44V/T68L/174G/V84R/R100M/G102R (SEQ ID NO:66),   V28A/V31G/G43SILENT(=GGG)/T68L/174R/V84R/R100E/G102R (SEQ ID NO:67),   V31G/Y41V/G43SILENT(=GGG)/K44V/T68L/174R/V84R/R100E/G102STOP (SEQ ID NO:68),   V31G/Y41T/G43SILENT(=GGG)/K44V/T68R/174D/V84R/G102R (SEQ ID NO:69),   V31G/Y41T/G43SILENT(=GGG)/K44V/T68R/174D/V84R/R100E/G102R (SEQ ID NO:70),   V28A/Y41S/G43SILENT(=GGG)/K44V/T68R/174G/V84R/R100M/G102R (SEQ ID NO:71),   V28A/Y41V/G43SILENT(=GGG)/K44V/174R/R100E/G102STOP (SEQ ID NO:72),   V28A/V31G/Y41T/G43SILENT(=GGG)/K44V/174E/V84R/R100M/G102R (SEQ ID NO:73),   V28A/G43SILENT(=GGG)/K44V/T68R/174E/V84R/R100E/G102STOP (SEQ ID NO:74),   V31G/Y41T/G43SILENT(=GGG)/K44V/T68L/174D/V84R/G102R (SEQ ID NO:75), or   Y41V/G43SILENT(=GGG)/K44V/T68L/174R/V84R/G102R (SEQ ID NO:76).   
     
     
         11 . The method of  claim 1 , wherein said phytocannabinoid is cannabigerol (CBG), cannabigerolic acid (CBGa), cannabigerovarin (CBGV), cannabigerovarinic acid (CBGVa), cannabigerocin (CBGO), cannabigerocinic acid (CBGOa), a cannabivarin, tetrahydrocannabinol (THC), or tetrahydrocannabinolic acid (THCa). 
     
     
         12 . The method of  claim 1 , wherein the host cell additionally comprises a divarinic acid synthase and produces divarinic acid. 
     
     
         13 . The method of  claim 1 , wherein said host cell is a yeast cell, a bacterial cell, a fungal cell, a protist cell, or a plant cell said host cell is  S. cerevisiae, E. coli, Yarrowia lipolytica , or  Komagataella phaffii.    
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein said transformed host cell additionally comprises a polynucleotide encoding a polyketide synthase enzyme and/or a polynucleotide encoding a prenyltransferase enzyme. 
     
     
         16 . An isolated polypeptide having olivetolic acid cyclase activity comprising an amino acid sequence of at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 92, wherein 6 or more amino acid residues comprise mutations relative to SEQ ID NO: 91, said mutations being located at positions selected from the group consisting of: residues 28, 31, 41, 43, 44, 68, 74, 84, 100 and 102 of SEQ ID NO:91. 
     
     
         17 . The isolated polypeptide of  claim 16 , wherein the isolated polypeptide comprises an amino acid sequence according to SEQ ID NO:42, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:43-SEQ ID NO:76. 
     
     
         18 . An isolated polynucleotide comprising:
 (a) a nucleotide sequence according to SEQ ID NO:5, SEQ ID NO: 3, SEQ ID NO:4, or SEQ ID NO:6-SEQ ID NO:39;   (b) a nucleotide sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence of (a), or   (c) a nucleotide sequence that hybridizes with the complementary strand of the nucleotide having the sequence of (a).   
     
     
         19 . An expression vector comprising the polynucleotide according to  claim 18 , encoding a variant olivetolic acid cyclase (OAC) protein with a sequence according to SEQ ID NO:92 having 6 or more amino acid mutations relative to the wild type OAC protein. 
     
     
         20 . The expression vector of  claim 19 , wherein the polynucleotide encoding the variant OAC protein comprises the nucleotide sequence according to SEQ ID NO:5, SEQ ID NO: 3, SEQ ID NO:4, or SEQ ID NO:6-SEQ ID NO:39. 
     
     
         21 . A host cell transformed with the expression vector of  claim 19 , optionally wherein the host cell additionally comprises a polynucleotide encoding a polyketide synthase enzyme and/or a polynucleotide encoding a prenyltransferase enzyme. 
     
     
         22 . (canceled)

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