Heterologous production of 10-methylstearic acid
Nucleic acids and cells comprising a methyltransferase gene and/or a reductase gene are disclosed. These nucleic acids and cells may be used to produce branched (methyl)lipids, such as 10-methylstearate.
1. A method of producing a branched (methyl)lipid or an exomethylene-substituted lipid comprising contacting a yeast cell with oleic acid, methionine, or both oleic acid and methionine,
wherein the branched (methyl)lipid or the exomethylene-substituted lipid is a carboxylic acid, carboxylate, ester, thioester, or amide,
wherein (a) the branched (methyl)lipid comprises (i) a saturated branched aliphatic chain comprising a branching methyl group or (ii) an unsaturated branched aliphatic chain comprising a branching methyl group, or (b) the exomethylene-substituted lipid comprises a branched aliphatic chain wherein the branched aliphatic chain is substituted with an exomethylene group,
wherein the yeast cell comprises a methyltransferase gene encoding a Thermomonospora curvata tmsB enzyme, and
wherein the yeast cell produces the branched (methyl)lipid or the exomethylene-substituted lipid.
2. The method of claim 1 , wherein the branched (methyl)lipid or the exomethylene-substituted lipid comprises a linear lipid with a chain length of 14-20 carbons and a methyl branch at the Δ9 position, the Δ10 position, or the Δ11 position.
3. The method of claim 2 , wherein the branched (methyl)lipid or the exomethylene-substituted lipid is a diacylglycerol, a triacylglycerol, or a phospholipid, and wherein the diacylglycerol, triacylglycerol, or phospholipid comprises an ester of 10-methylstearate or an ester of 10-methylenestearate.
4. The method of claim 1 , wherein at least 1% by weight of fatty acids of the yeast cell are one or more linear fatty acids with a chain length of 14-20 carbons and a methyl branch at the Δ9 position, the Δ10 position, or the Δ11 position.
5. The method of claim 1 , wherein the yeast cell comprises at least 1% lipid as measured by % dry cell weight.
6. The method of claim 1 , wherein the yeast cell further comprises a recombinant reductase gene.
7. The method of claim 6 , wherein the recombinant reductase gene encodes tmsA from Thermomonospora curvata.
8. The method of claim 6 , wherein the yeast cell encodes a fusion protein comprising (A) a reductase protein encoded by the recombinant reductase gene and (B) the tmsB enzyme.
9. The method of claim 1 , wherein the methyltransferase gene is codon-optimized for the yeast cell, or wherein the yeast cell further comprises a reductase gene and the reductase gene is codon-optimized for the yeast cell.
10. The method of claim 1 , wherein the yeast cell is Arxula, Saccharomyces , or Yarrowia.
11. The method of claim 10 , wherein the yeast cell is Arxula adeninivorans, Saccharomyces cerevisiae , or Yarrowia lipolytica.
12. The method of claim 1 , wherein the yeast cell comprises a methyltransferase protein encoded by the methyltransferase gene, wherein the methyltransferase protein comprises an amino acid sequence with at least 95% sequence identity with the amino acid sequence of SEQ ID NO:76.
13. The method of claim 12 , wherein the yeast cell comprises a methyltransferase protein encoded by the methyltransferase gene, wherein the methyltransferase protein comprises the amino acid sequence of SEQ ID NO:76.
14. The method of claim 1 , wherein the methyltransferase gene comprises a nucleotide sequence with at least 95% sequence identity with the nucleotide sequence of SEQ ID NO:75.
15. The method of claim 14 , wherein the methyltransferase gene comprises the nucleotide sequence of SEQ ID NO:75.