Knockout Of FadD Gene And Influence Of ω_-1-hydroxy Fatty Acid Yield Of Bacillus Pumilus

ω_-1,ω_-2,ω_-3-hydroxy fatty acids of C18 long-chain hydroxy fatty acids due to its hydroxyl group close to the first C-terminal and reaction nature live, can be applied to the surface-active agent, cosmetic ingredients, lubricants and anti-rust agent in machinery industry,and anti-bacterial agents in the food industry. They also work as a monomer in the synthesis of polymers to provide a maximum carbon chain which is no single-molecule side-chain and make the polymers'performance superior.ω-1-hydroxy fatty acid is one of the best monomers in research and development of green polymers. Bacillus pumilus M-F641 is a high-yielding strain forω-1-hydroxy fatty acids production obtained by mutagenesis, and it is easy to incubate. In order to improve the conversion rate of hydroxy fatty acids (the ratio of hydroxy fatty acids and total adding fatty acids) and the hydroxy fatty acid production rate (the ratio of hydroxy fatty acids and fatty acid consumption), in this paper, a key enzyme gene of fadD involved in long-chain fatty acid activation, transmembrane transport and encoding acyl-CoA synthetase in fatty acidβ-oxidation metabolic process was studied. The main research contents are as follows:Firstly, B. pumilus M-F641's fadD gene was amplified by PCR method and the primer was designed according to published fadD genes of Bacillus pumilus and other Bacillus strains. Full-length sequences of fadD gene about 1720 bp were obtained. The sequences waere analyzed by using the MEGA 3.1 and DNAStar software;Secondly, the recombinant expression plasmid pET32M-fadD was transformed into E. coli BL21 (DE3).after IPTG inducing , SDS-PAGE electrophoresis analysis showed a clear expression of pET32M-fadD. The recombinant expression acyl-CoA synthetase activity recached to 79.2U/mg;Finally, the suicide vector pMD18-T-fadD::Tetr was succeessfully constructed,adopted deletion-insertion to disrupt fadD gene of B.pumilus M-F641 and got fadD knockout mutant B. pumilus M-F641A.After 24h simple fermentation validation, theω-1 hydroxy fatty acid production rate of B. pumilus M-F641A was 16.95%, but it can not prove that the yield has increased an subject to further fermentation to verify.