Monoterpene-producing genetically modified host cells and methods of use of same
The present disclosure provides a genetically modified host cell capable of producing linalool (or 3,7-dimethylocta-1,6-dien-3-ol).
1. A genetically modified host cell, wherein the host cell is a diploid or tetraploid yeast cell and is genetically modified with a heterologous nucleic acid comprising a nucleotide sequence encoding linalool synthase, wherein the linalool synthase lacks a plastid targeting sequence and wherein the linalool synthase comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:13.
2. The genetically modified host cell of claim 1 , wherein the linalool synthase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:13.
3. The genetically modified host cell of claim 1 , wherein the genetically modified host cell produces linalool in an amount of at least 0.05 mg per liter of medium or in an amount of at least 1 mg per dry cell weight.
4. The genetically modified host cell of claim 1 , wherein the genetically modified host cell is genetically modified with a nucleic acid comprising a nucleotide sequence encoding a truncated 3′-hydroxy-3-methylglutaryl-coenzyme A reductase (tHMGR).
5. The genetically modified host cell of claim 1 , wherein the genetically modified host cell is genetically modified such that the endogenous farnesyl diphosphate synthase (FPPS) is modified to comprise one or more amino acid substitutions at positions selected from F96, N127, and K197, relative to the amino acid sequence set forth in SEQ ID NO:17.
6. The genetically modified host cell of claim 5 , wherein the modified FPPS comprises an amino acid sequence having at least 85% amino acid sequence identity to:
a) the FPPS amino acid sequence set forth in SEQ ID NO:18, and comprises an F96W substitution and an N127W substitution; or
b) the FPPS amino acid sequence set forth in SEQ ID NO:19, and comprises a K197E substitution.
7. The genetically modified host cell of claim 5 , wherein the genetically modified host cell is genetically modified with a heterologous nucleic acid comprising a nucleotide sequence encoding a geraniol synthase.
8. The genetically modified host cell of claim 7 , wherein the geraniol synthase comprises an amino acid sequence having at least 95% amino acid sequence identity to the geraniol synthase set forth in any one of SEQ ID NOs:20-23 and 28-31.
9. The genetically modified host cell of claim 7 , wherein the genetically modified host cell produces linalool and geraniol.
10. The genetically modified host cell of claim 9 , wherein the linalool synthase has a length of from 530 amino acids to 550 amino acids.
11. The genetically modified host cell of claim 7 , wherein the geraniol synthase does not comprise a plastid targeting sequence.
12. The genetically modified host cell of claim 1 , wherein the genetically modified host cell is genetically modified with a heterologous nucleic acid comprising: a) a promoter; and b) a nucleotide sequence encoding a geraniol reductase, wherein the nucleotide sequence encoding the geraniol reductase is operably linked to the promoter, and wherein the promoter is heterologous to the nucleotide sequence encoding the geraniol reductase.
13. The genetically modified host cell of claim 12 , wherein the geraniol reductase comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:32.
14. The genetically modified host cell of claim 1 , wherein the linalool synthase comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:10.
15. The genetically modified host cell of claim 14 , wherein the linalool synthase comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:10.
16. A composition comprising:
a) the genetically modified host cell of claim 1 ; and
b) a culture medium.
17. A method of producing a fermented beverage, the method comprising culturing the composition of claim 16 for a time period and under conditions suitable for fermentation.