Diterpene production in Yarrowia

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.