ACYL-ACP Reductase With Improved Properties

Abstract

The disclosure relates to acyl-ACP reductase (AAR) enzyme variants that result in improved fatty aldehyde and fatty alcohol production when expressed in recombinant host cells. The disclosure further relates to methods of making and using such AAR variants for the production of fatty alcohol compositions having particular characteristics.

1 .- 82 . (canceled) 
     
     
         83 . A recombinant host cell expressing a variant acyl-ACP reductase (AAR) polypeptide comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 80, wherein said variant AAR polypeptide comprises a mutation at an amino acid position selected from the group consisting of Q40V, G52V, G273E, K303G, H340P, L344A, L344D, L344S, L344T, A345R, V346P, V346G, and A345*, and wherein said AAR polypeptide catalyzes the conversion of an acyl-ACP to a fatty aldehyde. 
     
     
         84 . The recombinant host cell of  claim 83 , wherein the recombinant host cell produces a fatty aldehyde or fatty alcohol composition with a titer that is at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, or at least 30% greater than the titer of a fatty aldehyde or alcohol composition produced by a host cell expressing a corresponding wild type AAR polypeptide, when cultured in medium containing a carbon source under conditions effective to express the variant AAR polypeptide. 
     
     
         85 . The recombinant host cell of  claim 84 , wherein the fatty alcohol composition is produced at a titer of about 30 g/L to about 250 g/L. 
     
     
         86 . The recombinant host cell of  claim 84 , wherein the fatty alcohol composition is released from the cell. 
     
     
         87 . A cell culture comprising a recombinant host cell that expresses a variant acyl-ACP reductase (AAR) polypeptide comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 80, wherein said variant AAR polypeptide comprises a mutation at an amino acid position selected from the group consisting of Q40V, G52V, G273E, K303G, H340P, L344A, L344D, L344S, L344T, A345R, V346P, V346G, and A345*, and wherein said AAR polypeptide catalyzes the conversion of an acyl-ACP to a fatty aldehyde. 
     
     
         88 . The cell culture of  claim 87 , wherein the recombinant host cell produces a fatty alcohol composition that comprises one or more of a C6, C8, C10, C12, C13, C14, C15, C16, C17, and a C18 fatty alcohol. 
     
     
         89 . The cell culture of  claim 88 , wherein the fatty alcohol composition comprises one or more of a C10:1, C12:1, C14:1, C16:1, and a C18:1 unsaturated fatty alcohol. 
     
     
         90 . The cell culture of  claim 88 , wherein the fatty alcohol composition comprises an unsaturated fatty alcohol. 
     
     
         91 . The cell culture of  claim 88 , wherein the fatty alcohol composition comprises a 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. 
     
     
         92 . The cell culture of  claim 88 , wherein the fatty alcohol composition comprises a saturated fatty alcohol. 
     
     
         93 . A method of producing a fatty alcohol composition having an increase in titer, comprising:
 i. culturing with a carbon source, a recombinant host cell that expresses a variant acyl-ACP reductase (AAR) polypeptide comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 80, wherein said variant AAR polypeptide comprises a mutation at an amino acid position selected from the group consisting of Q40V, G52V, G273E, K303G, H340P, L344A, L344D, L344S, L344T, A345R, V346P, V346G, and A345*, and wherein said AAR polypeptide catalyzes the conversion of an acyl-ACP to a fatty aldehyde, and   ii. harvesting a fatty alcohol composition.   
     
     
         94 . The method of  claim 93 , wherein the fatty alcohol composition has a fatty alcohol titer that is at least 20% to 30% greater than a fatty alcohol titer of a fatty alcohol composition produced by a wild-type AAR-expressing host cell. 
     
     
         95 . The method of  claim 93 , wherein the fatty alcohol composition has an increased fraction of C14 fatty alcohols.