Diterpene production in Yarrowia

Patent metadata

Field	Value
Publication number	US-10273519-B2
Application number	US-201414906497-A
Country	US
Kind code	B2
Filing date	Jul 23, 2014
Priority date	Jul 23, 2013
Publication date	Apr 30, 2019
Grant date	Apr 30, 2019

Abstract

The present invention relates to a method for the production of a diterpene or a glycosylated diterpene, which method comprises: a. fermenting a recombinant microorganism of the genus Yarrowia in a suitable fermentation medium at a temperature of about 29° C. or higher, wherein the microorganism comprises one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; and a polypeptide having kaurenoic acid 13-hydroxylase activity and whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol; and b. recovering the diterpene or glycosylated diterpene.

First claim

The invention claimed is:

1. A method for the production of a steviol glycoside, which method comprises:
fermenting a recombinant microorganism of the genus Yarrowia in a suitable fermentation medium at a temperature of about 29° C. or higher and below 45° C., wherein the temperature of fermentation in such method is from 32° C. to 34° C. during the steviol glycoside production phase,
wherein the microorganism comprises one or more nucleotide sequence(s) encoding:
a. a polypeptide having ent-copalyl pyrophosphate synthase activity, wherein said nucleotide sequence comprises:
i. a nucleotide sequence encoding a polypeptide having ent-copalyl pyrophosphate synthase activity, said polypeptide comprising an amino acid sequence that has at least about 70% sequence identity with the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 18, 20, 60 or 62;
ii. a nucleotide sequence that has at least about 70% sequence identity with the nucleotide sequence of SEQ ID NOs: 141, 142, 151, 152, 153, 154, 159, 160, 182 or 184;
iii. a nucleotide sequence, the complementary strand of which will hybridize to a nucleic acid molecule of sequence (i) or (ii), under stringent conditions, wherein said hybridization is conducted in a 1 M salt solution, for at least 10 hours, with washing at 65° C. for at least one hour, with at least two changes of the washing solution, wherein the washing solution comprises a 0.1 M salt solution; or
iv. a nucleotide sequence which differs from the nucleotide sequence of (i), (ii) or (iii) due to the degeneracy of the genetic code;

b. a polypeptide having ent-Kaurene synthase activity, wherein said nucleotide sequence comprises:
i. a nucleotide sequence encoding a polypeptide having ent-Kaurene synthase activity, said polypeptide comprising an amino acid sequence that has at least about 70% sequence identity with the amino acid sequence of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 64 or 66;
ii. a nucleotide sequence that has at least about 70% sequence identity with the nucleotide sequence of SEQ ID NOs: 143, 144, 155, 156, 157, 158, 159, 160, 183 or 184;
iii. a nucleotide sequence, the complementary strand of which will hybridize to a nucleic acid molecule of sequence (i) or (ii), under stringent conditions, wherein said hybridization is conducted in a 1 M salt solution, for at least 10 hours, with washing at 65° C. for at least one hour, with at least two changes of the washing solution, wherein the washing solution comprises a 0.1 M salt solution; or
iv. a nucleotide sequence which differs from the nucleotide sequence of (i), (ii) or (iii) due to the degeneracy of the genetic code;

c. a polypeptide having ent-Kaurene oxidase activity, wherein said nucleotide sequence comprises:
i. a nucleotide sequence encoding a polypeptide having ent-Kaurene oxidase activity, said polypeptide comprising an amino acid sequence that has at least about 70% sequence identity with the amino acid sequence of SEQ ID NOs: 22, 24, 26, 68 or 86;
ii. a nucleotide sequence that has at least about 70% sequence identity with the nucleotide sequence of SEQ ID NOs: 145, 161, 162, 163, 180 or 186;
iii. a nucleotide sequence, the complementary strand of which will hybridize to a nucleic acid molecule of sequence (i) or (ii), under stringent conditions, wherein said hybridization is conducted in a 1 M salt solution, for at least 10 hours, with washing at 65° C. for at least one hour, with at least two changes of the washing solution, wherein the washing solution comprises a 0.1 M salt solution; or
iv. a nucleotide sequence which differs from the nucleotide sequence of (i), (ii) or (iii) due to the degeneracy of the genetic code; and

d. a polypeptide having kaurenoic acid 13-hydroxylase activity, wherein said nucleotide sequence comprises:
i. a nucleotide sequence encoding a polypeptide having kaurenoic acid 13-hydroxylase activity, said polypeptide comprising an amino acid sequence that has at least about 70% sequence identity with the amino acid sequence of SEQ ID NOs: 28, 30, 32, 34, 70, 90, 92, 94, 96 or 98;
ii. a nucleotide sequence that has at least about 70% sequence identity with the nucleotide sequence of SEQ ID NOs: 146, 164, 165, 166, 167 or 185;
iii. a nucleotide sequence, the complementary strand of which will hybridize to a nucleic acid molecule of sequence (i) or (ii), under stringent conditions, wherein said hybridization is conducted in a 1 M salt solution, for at least 10 hours, with washing at 65° C. for at least one hour, with at least two changes of the washing solution, wherein the washing solution comprises a 0.1 M salt solution; or
iv. a nucleotide sequence which differs from the nucleotide sequence of (i), (ii) or (iii) due to the degeneracy of the genetic code;
whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol, and
wherein the recombinant microorganism further comprises one or more nucleotide sequences encoding a polypeptide having UDP-glucosyltransferase activity, whereby expression of the further nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least one of steviolmonoside, steviolbioside, stevioside or rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rubusoside, and dulcoside A at a concentration of above 5 mg/L in the fermentation medium; and

recovering the steviol glycoside.

2. The process according to claim 1 , wherein the recombinant microorganism comprises a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of glucose to the C-13 position of steviol,
whereby expression of the nucleotide sequence confers on the microorganism the ability to produce at least steviolmonoside.

3. The process according to claim 1 , wherein the recombinant microorganism comprises a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a glucose at C-13 position of steviol or steviolmonoside,
whereby expression of the nucleotide sequence confers on the microorganism the ability to produce at least steviolbioside.

4. The process according to claim 1 , wherein the recombinant microorganism comprises a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of glucose to the C-19 position of steviolbioside,
whereby expression of the nucleotide sequence confers on the microorganism the ability to produce at least stevioside.

5. The process according to claim 1 , wherein the recombinant microorganism comprises a nucleotide sequence encoding a polypeptide capable of catalyzing glucosylation of the C-3′ of the glucose at the C-13 position of stevioside,
whereby expression of the nucleotide sequence confers on the microorganism the ability to produce at least rebaudioside A.

6. The process according to claim 1 , wherein the recombinant microorganism comprises a nucleotide sequence encoding a polypeptide capable of catalyzing the glucosylation of stevioside or rebaudioside A,
whereby expression of the nucleotide sequence confers on the microorganism the ability to produce at least rebaudioside D.

7. The process according to claim 1 , wherein the recombinant microorganism comprises a nucleotide sequence encoding a polypeptide capable of catalyzing the glucosylation of stevioside,
whereby expression of the nucleotide sequence confers on the microorganism the ability to produce at least rebaudioside E.

8. The process according to claim 1 , wherein the recombinant microorganism comprises a nucleotide sequence encoding a polypeptide capable of catalyzing the glucosylation of rebaudioside E, whereby expression of the nucleotide sequence confers on the microorganism the ability to produce at least rebaudioside D.

Assignees

• Dsm Ip Assets Bv

Inventors

• Broers Nicolette Jasmijn
• Boer Viktor Marius
• Lawrence Adam G

Classifications

• C12N9/88

  Lyases (4.) · CPC title

• C12N9/0073

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

• C07C62/32

  containing hydroxy or O-metal groups · CPC title

• C12N9/90

  Isomerases (5.) · CPC title

• C07C2603/86

  containing four rings · CPC title