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
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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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