Microbial production of triterpenoids including mogrosides
The present invention provides host cells and methods for making mogrol glycosides, including Mogroside V (Mog. V), Mogroside VI (Mog. VI), Iso-Mogroside V (Isomog. V), and glycosylation products that are minor products in Siraitia grosvenorii . The invention provides engineered enzymes and engineered host cells for producing mogrol glycosylation products, such as Mog. V, Mog. VI, and Isomog. V, at high purity and/or yield. The present technology further provides methods of making products containing mogrol glycosides, such as Mog. V, Mog. VI, and Isomog. V, including food products, beverages, oral care products, sweeteners, and flavoring products.
1. A method for making a triterpenoid, comprising:
providing a recombinant microbial host cell expressing a heterologous enzyme pathway catalyzing the conversion of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to one or more triterpenoids, the pathway comprising:
a farnesyl diphosphate synthase (FPPS),
a squalene synthase (SQS), wherein the SQS comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 11;
a squalene epoxidase (SQE), wherein the squalene epoxidase comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 39; and
culturing the host cell at a temperature within the range of 28° C. to 37° C., and under conditions for producing the triterpenoid;
wherein the microbial host cell is a bacterium or yeast.
2. The method of claim 1 , wherein the SQS comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 11.
3. The method of claim 2 , wherein the SQS comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 11.
4. The method of claim 2 , wherein the SQS comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 11, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
5. The method of claim 1 , wherein the microbial host cell is a bacterium.
6. The method of claim 5 , wherein the microbial host cell is E. coli.
7. The method of claim 6 , wherein the E. coli produces increased MEP pathway products, and has an overexpression of one or more MEP pathway enzymes.
8. The method of claim 1 , wherein the squalene epoxidase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 39.
9. The method of claim 1 , wherein the host cell is E. coli that coexpresses an SQS enzyme comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11, and a squalene epoxidase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 39.
10. The method of claim 1 , wherein the heterologous enzyme pathway further comprises a triterpene cyclase.
11. The method of claim 10 , wherein the heterologous enzyme pathway further comprises an epoxide hydrolase (EPH).
12. The method of claim 11 , wherein the heterologous pathway further comprises one or more oxidases.
13. The method of claim 12 , wherein at least one oxidase is a cytochrome P450 enzyme.
14. The method of claim 12 , wherein the heterologous enzyme pathway produces mogrol.
15. The method of claim 14 , wherein the heterologous enzyme pathway further comprises one or more uridine diphosphate-dependent glycosyltransferase (UGT) enzymes, thereby producing one or more mogrol glycosides.
16. The method of claim 15 , wherein the one or more mogrol glycosides are selected from Mog. II-E, Mog. III-A-2, Mog. III-E, Mog. IIIx, Mog. IV-A, Mog. IV-E, Siamenoside, Isomog. IV, and Mog. V.
17. The method of claim 9 , wherein the E. coli produces increased MEP pathway products, and has an overexpression of one or more MEP pathway enzymes.