Enzymes, cells, and methods for producing lactones
AssigneeManus Bio Inc.
InventorsArthur J. SHAW, Stephen SARRIA, Liwei LI, Christine Nicole S. SANTOS, Ajikumar PARAYIL KUMARAN
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Abstract
The present disclosure relates to microbial hosts, enzymes, and methods for biosynthesis of lactones.
Claims36
A microbial cell for producing one or more lactones, the microbial cell expressing an enzyme having fatty acid hydroxylase activity, wherein the microbial cell is engineered relative to a parent strain to have one or more modifications selected from:
- (a) a decrease in expression and/or activity of one or more enzymes involved in fatty acid activation and degradation,
- (b) a decrease in expression and/or activity of one or more β-oxidation and/or peroxisome enzymes;
- (c) a modification in expression and/or activity of one or more lipases;
- (d) a decrease in expression and/or activity of ω-oxidation enzymes;
- and
- (e) a decrease in expression and/or activity of one or more acyl-CoA synthetase enzymes;
- (f) an increase in metabolic NADPH supply;
- (g) a decrease in expression and/or activity of one or more neutral lipid biosynthesis enzymes;
- and/or
- (h) a decrease in expression and/or activity of one or more of citric acid cytoplasmic exporter, and one or more NADPH dependent aldehyde reductases.
The microbial cell of claim 2, wherein the enzyme is a cytochrome P450 enzyme selected from CYP505, CYP116, CYP703, and CYP102, or a derivative thereof.
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A microbial cell for producing one or more lactones, the microbial cell expressing a P450 enzyme having fatty acid hydroxylase activity,
- wherein the P450 enzyme comprises an amino acid sequence that has at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or 100% sequence identity to amino acids 1 to 461 of SEQ ID NO:
- 1, and comprises a CPR domain having at least 70% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity or 100% sequence identity to a CPR domain selected from SEQ ID NOS:
- 37 to 60.
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A P450 enzyme having fatty acid hydroxylase activity, the P450 enzyme comprising an amino acid sequence that has at least 80% sequence identity to amino acids 1 to 461 of SEQ ID NO:
- 1, and comprises a CPR domain having at least 70% sequence identity to a CPR domain selected from SEQ ID NOS:
- 37 to 60.
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A P450 enzyme having fatty acid hydroxylase activity, the P450 enzyme comprising an amino acid sequence that has at least 80% sequence identity to amino acids 1 to 461 of SEQ ID NO:
- 1, and comprises a CPR domain having at least 70% sequence identity to a CPR domain selected from SEQ ID NOs:
- 61 to 64, wherein the P450 enzyme comprises a substitution at one or more positions selected from Q53, T332, P363 and E443 with respect to SEQ ID NO:
- 29, wherein the substitutions are optionally selected from:
- (i) Q53 is substituted with an amino acid selected from Arg, Ile, Leu, His, Lys, Gly, Ala, and Val;
- (ii) T332 is substituted with Ser, Gly, Ala, Pro, Cys, Ile, Leu, and Val;
- (iii) P363 is substituted with an amino acid selected from Ala, Leu, Ile, and Val;
- and
- (iv) E443 is substituted with Gln or Asn.
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Dependencies are inferred from the claim text.