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Fatty alcohol forming acyl reductase (FAR) variants and methods of use

a technology of acyl reductase and fatty alcohol, which is applied in the field of fatty alcohol forming acyl reductase (far) variants, can solve the problem that the process requires a significant amount of energy

Inactive Publication Date: 2014-12-04
CODEXIS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this process requires a significant amount of energy and involves the use of a non-renewable energy source.

Method used

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  • Fatty alcohol forming acyl reductase (FAR) variants and methods of use
  • Fatty alcohol forming acyl reductase (FAR) variants and methods of use
  • Fatty alcohol forming acyl reductase (FAR) variants and methods of use

Examples

Experimental program
Comparison scheme
Effect test

example 1

Wild-type M. algicola DG893 FAR Gene Acquisition and Vector Construction

[0254]Gene acquisition of wild-type M. algicola DG893 FAR (“FAR Maa”) is described in the published application WO / 2011 / 008535. The genomic sequence of M. algicola DG893 can also be found at GenBank Accession No. NZ_ABCP01000001.1. The amino acid sequence of the encoded FAR polypeptide is designated SEQ ID NO:2. A codon optimized polynucleotide sequence encoding the FAR polypeptide of SEQ ID NO: 2 is designated SEQ ID NO:1. The M. algicola DG893 FAR gene and genes encoding variants of the M. algicola DG893 FAR were cloned into the vector pCK110900 (depicted as FIG. 3 in U.S. Patent Appln. Pub. 2006 / 0195947) under the control of a lac promoter, as described in WO 2011 / 008535. The resulting plasmids were introduced into E. coli BW25113, BW25113 ΔfadE, or BW25113 Ltor R (Baba et al., Molecular Systems Biology, 2006 doi:10,1038 / msb4100050 Article No. 2006.0008), W3110-ΔfhuA and MG1655-7740 by routine transformation ...

example 2

Evaluation of FAR Variants Using Microtiter Plate Assays

[0255]FAR variants were screened under several slightly differing conditions. Variant Nos. 1-144 were grown in 96-well shallow plates containing 180 μL Luria Bertani (LB) or M9YE medium supplemented with 1% glucose and 30 μg / mL chloramphenicol (CAM), for approximately 16-18 hours (overnight) in a shaker-incubator at 30° C., 200 rpm. A 5% inoculum was used in 96-deep-well plates to initiate fresh 380 μL culture containing 2×YT broth medium supplemented with 30 μg / mL CAM and 0.4% glucose. Some variants from later rounds of screening were grown in 96-well shallow plates containing 180 μL M9YE medium supplemented with 1% glucose and 30 μg / mL chloramphenicol (CAM), for approximately 16-18 hours (overnight) in a shaker-incubator at 30° C. or 37° C., 200 rpm. A 5% inoculum was used in 96-deep-well plates to initiate fresh 380 μL culture containing M9YE broth medium supplemented with 30 μg / mL CAM and 5% glucose. Other variants from lat...

example 3

Chain Length Profile of Fatty Alcohols Exhibited by Representative FAR Variants

[0260]The chain length profile of a subset of FAR variants was evaluated. Table 3 provides the relative chain length distribution of fatty alcohols exhibited by recombinant E. coli strains expressing wild-type FAR Maa or FAR variants when cultured at 30° C. As described above for Example 2, the total fatty alcohol titer was determined by adding the titers of each fatty alcohol measured (C10:0-OH, C12:1-OH, C12:0-OH, C13:0-OH, C14:1-OH, C14:0-OH, C15:-OH, C16:1-OH, C16:0-OH, C18:1-OH, and C18:0-OH). The percentage of each fatty alcohol species was then calculated as a percentage of the total fatty alcohols measured.

TABLE 3Relative chain length distribution of fatty alcoholsFARVariantRelative Chain Length Distribution of fatty alcoholsaNo.C12:0C14:0C14:1C16:1C16:0C18:1Wild-type030103651(SEQ IDNO: 2)Variant04023314226Variant09037282685Variant09028382592Variant0190403110118Variant129452123129aThe relative cha...

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Abstract

The present disclosure provides methods useful for producing fatty alcohol compositions from recombinant host cells. The disclosure further provides fatty acyl-CoA reductase (FAR) variant enzymes, polynucleotides encoding the FAR variant enzymes, and vectors and host cells comprising polynucleotides encoding the FAR variant enzymes.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS[0001]This application claims the benefit of priority of U.S. Provisional Application No. 61 / 577,756, filed Dec. 20, 2011; of U.S. Provisional Application No. 61 / 578,673, filed Dec. 21, 2011; of U.S. Provisional Application No. 61 / 636,044, filed Apr. 20, 2012; and of U.S. Provisional Application No. 61 / 674,053, filed Jul. 20, 2012; the entire content of each of which is incorporated herein by reference.REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII FILE[0002]The Sequence Listing written in file 90834-853678_ST25.TXT, created on Dec. 13, 2012, 121,968 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference.FIELD OF THE INVENTION[0003]This invention relates to fatty alcohol forming acyl reductases (FAR) variants, recombinant bacterial microorganisms incorporating polynucleotides encoding the FAR variants, and production of fatty alcohols by the engin...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P7/04C12N9/02A61Q5/00A61K8/34A61Q19/00
CPCC12P7/04A61K8/342A61Q19/00C12Y102/01C12N9/0008A61K2800/10A61Q5/00C07C31/125C12Y102/0105C12Y102/0108Y02P20/52
Inventor CLARK, LOUIS A.KARLSHOEJ, KRISTIANCHOUDHARY, PATRICIA
Owner CODEXIS INC
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