| Mortierella alpina is an oleaginous filamentous fungi that could produce high-yield PUFAs and has been used in arachidonic acid industrial production.After nearly20 years research,the PUFAs anabolic network has been gradually analyzed in Mortierella alpina,but the mechanism of fatty acid accumulation needs to be further studied.In order to explore the p5cdh gene in glutamate metabolism pathway on fatty acid accumulation mechanism,Mortierella alpina was used for the research object to construct recombinant strains by homologous overexpression method in order to verify the effect of the p5cdh gene on fatty acid synthesis in the study.Then the fermentation medium of the recombinant strain was optimized based on the response surface methodology to obtain the maximum arachidonic acid(ARA)production of the recombinant strain.The main research results are as follows:1.In the process of fatty acid synthesis in Mortierella alpina,p5cdh gene was homologous overexpressed and three recombinant strains,Ma-p5cdh-1,Ma-p5cdh-2and Ma-p5cdh-3,were constructed.Compared with the wild-type Mortierella alpina,the NADPH level and total fatty acid of the recombinant strains were significantly increased,which demonstrated that p5cdh gene in the glutamate metabolic pathway provided the NADPH for Mortierella alpina to synthesize fatty acid.2.The fermentation medium of recombinant strain Ma-p5cdh-1 was optimized by response surface methodology(RSM).The response surface methodology was used to determine the optimal concentrations of producing ARA by the recombinant strain:92.69 g/L glucose,15.41 g/L yeast extract,2.95 g/L KH2PO4 and 2.97 g/L Na NO3.Under that condition,the average ARA yield was 9.02 g/L by verification experiment.The results suggested that an average 27.04%increase in ARA production compared with preliminary experiment was achieved after medium optimization and this indicated that the optimization of MA-p5cdh-1 fermentation medium had been realized successfully. |
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