Car enzymes and improved production of fatty alcohols
The disclosure relates to variant carboxylic acid reductase (CAR) enzymes for the improved production of fatty alcohols in recombinant host cells.
1. A variant carboxylic acid reductase (CAR) polypeptide comprising an amino acid sequence having at least 85% sequence identity to the wild-type CAR polypeptide of SEQ ID NO: 7, wherein the variant CAR polypeptide is genetically engineered to have at least one mutation at an amino acid position selected from the group consisting of E636K; L1128S; M259I; T396S; A574T; T90M; E20F; E20L; E20Y; V191A; Q473A; Q473F; Q473K; Q473M; Q473R; Q473V; V926G; S927I; S927V; L1128A; L1128G; L1128K; L1128M; L1128P; L1128R; L1128T; L1128V; L1128Y; D18V and D292N; E20K and V191A; P809L and M1062V; D143E and A612T; M1062T and R1080H; E936K, and P1134R; I870V, S927I, S985I, and I1164F; L799M, V810F, S927R, M1062L, A1158V, and F1170I; and L80Q, T231M, F288L, A418T, V530M, A541V, G677D, and P712A, wherein expression of the variant CAR polypeptide in a recombinant host cell results in a higher titer of fatty alcohol compositions compared to a recombinant host cell expressing the wild type CAR polypeptide of SEQ ID NO:7.
2. The variant CAR polypeptide of claim 1 , wherein said variant polypeptide comprises:
(a) the mutations P809L and M1062V;
(b) the mutations D143E and A612T;
(c) the mutations M1062T and R1080H;
(d) the mutations E936K, and P1134R;
(e) the mutations I870V, S927I, S985I, and I1164F;
(f) the mutations L799M, V810F, S927R, M1062L, A1158V, and F1170I; or
(g) the mutations L80Q, T231M, F288L, A418T, V530M, A541V, G677D, and P712A.
3. A recombinant host cell comprising a polynucleotide sequence encoding a variant carboxylic acid reductase (CAR) polypeptide having at least 85% sequence identity to the wild-type CAR polypeptide of SEQ ID NO: 7 and having at least one mutation at an amino acid position selected from the group consisting of E636K; L1128S; M259I; T396S; A574T; T90M; E20F; E20L; E20Y; V191A; Q473A; Q473F; Q473K; Q473M; Q473R; Q473V; V926G; S927I; S927V; L1128A; L1128G; L1128K; L1128M; L1128P; L1128R; L1128T; L1128V; L1128Y; D18V and D292N; E20K and V191A; P809L and M1062V; D143E and A612T; M1062T and R1080H; E936K, and P1134R; I870V, S927I, S985I, and I1164F; L799M, V810F, S927R, M1062L, A1158V, and F1170I; and L80Q, T231M, F288L, A418T, V530M, A541V, G677D, and P712A, wherein the recombinant host cell produces a fatty alcohol composition at a higher titer or yield than a host cell expressing the wild type CAR polypeptide of SEQ ID NO: 7 when cultured in a medium containing a carbon source under conditions effective to express said variant CAR polypeptide.
4. The recombinant host cell of claim 3 , further comprising a polynucleotide encoding a thioesterase polypeptide.
5. The recombinant host cell of claim 4 , further comprising a polynucleotide encoding
(i) a FabB polypeptide and a FadR polypeptide, or
(ii) a fatty aldehyde reductase.
6. The recombinant host cell according to claim 3 , wherein said recombinant host cell has a titer or a yield that is at least 3 times greater than the titer or the yield of a host cell expressing the corresponding wild type CAR polypeptide when cultured under the same conditions as the recombinant host cell.
7. The recombinant host cell of claim 6 , wherein said recombinant host cell has a titer of from 30 g/L to 250 g/L, or a titer of from 90 g/L to 120 g/L.
8. The recombinant host cell of claim 6 , wherein said recombinant host cell has a yield from 10% to 40%.
9. A cell culture comprising the recombinant host cell of claim 3 .
10. The cell culture of claim 9 , wherein said cell culture has productivity that is at least 3 times greater than the productivity of a cell culture that expresses the corresponding wild type CAR polypeptide.
11. The cell culture of claim 10 , wherein said productivity ranges from 0.7 mg/L/hr to 3 g/L/hr.
12. The cell culture of claim 11 , wherein the culture medium comprises a fatty alcohol composition.
13. The recombinant host cell according to claim 3 , wherein the fatty alcohol composition collects in an organic phase extracellularly.
14. The recombinant host cell of claim 13 , wherein the fatty alcohol composition comprises one or more of a C6, C8, C10, C12, C13, C14, C15, C16, C17, or C18 fatty alcohol.
15. The recombinant host cell of claim 13 , wherein the fatty alcohol composition comprises a C10:1, C12:1, C14:1, C16:1, or a C18:1 unsaturated fatty alcohol.
16. The recombinant host cell of claim 13 , wherein the fatty alcohol composition comprises C12 and C14 fatty alcohols.
17. The recombinant host cell of claim 16 , wherein the fatty alcohol composition comprises C12 and C14 fatty alcohols at a ratio of 3:1.
18. The recombinant host cell of claim 13 , wherein the fatty alcohol composition comprises unsaturated fatty alcohols or saturated fatty alcohols.
19. The recombinant host cell of claim 18 , wherein the fatty alcohol composition comprises an unsaturated fatty alcohol having a double bond at position 7 in the carbon chain between C7 and C8 from the reduced end of the fatty alcohol.
20. The recombinant host cell of claim 13 , wherein the fatty alcohol composition comprises branched chain fatty alcohols.
21. A method of making a fatty alcohol composition at a high titer, yield or productivity, comprising the steps of:
(a) engineering a recombinant host cell according to claim 3 ;
(b) culturing said recombinant host cell in a medium comprising a carbon source; and
(c) isolating said fatty alcohol composition from said medium.