Comparative Study On Major NADPH Supplying Genes In Fatty Acid Synthesis Of Mortierella Alpina

Fatty acids play an important role in many fields,especially for human health.At present,its main sources are plant extraction,deep sea fish oil and microbial extraction.Plant extraction has been gradually eliminated due to its long growth cycle,high cost and only producing short chain fatty acids.Deep sea fish are facing a series of problems such as environmental protection and marine pollution.Mortierella alpina has become the target strain of microbial oil production due to its high safety,rich fatty acid production and long chain fatty acid production.Up to now,the research on Mortierella alpina at home and abroad mainly focused on the screening of strains and the optimization of fermentation conditions,but few studies on the regulation mechanism of key enzyme genes in fatty acid synthesis process.In this study,the role of GDH in fatty acid synthesis was investigated by constructing a homologous overexpression strain encoding glutamate dehydrogenase gene(GDH)and a glutamine synthetase(GS)gene silencing strain.At the same time,the research results of NADPH(reduced nicotinamide adenine dinucleotide)gene and GDH gene yield reduction ability analysis were compared.The conclusions are as follows:(1)The homologous overexpression strains Mortierella alpina-GDH1 and Mortierella alpina-GDH2 were successfully constructed.The main results were as follows: compared with the control strain,the results showed that the expression of GDH1 and GDH2 was 13.67 and 9.7 times higher than that of control group;the content of NADPH and linolenic acid in GDH1 and GDH2 overexpression strains increased by 28.5% and 44.6%,respectively;the average contents of total fatty acids in GDH1 and GDH2 overexpression strains increased by 10.4% and 21.5%,respectively;oleic acid and linolenic acid in GDH1 overexpression strains increased by 28.7% and 16.7%,respectively.In GDH2 overexpression strain,oleic acid and linolenic acid increased by 29.4% and 17.0%,respectively.These results indicate that overexpression of GDH promotes the production of NADPH,which is required in fatty acid synthesis,and increases the fatty acid content and unsaturation.(2)The recombinant Mortierella alpina-i GS was successfully constructed by RNAi.The results are as follows: compared with the control strain,the expression of GS gene in M.alpina-i GS was reduced by about 60%;the content of NADPH in M.alpina-i GS increased by 35.7%;the content of total fatty acid increased by 19.9%;the content of oleic acid and linolenic acid was increased to a certain extent.In conclusion,GS gene can regulate fatty acid synthesis of Mortierella alpina,which is consistent with the result of overexpression of GDH gene.(3)In this paper,we compared the ability of producing NADPH and fatty acid of main NADPH source genes.When the expression levels of NADPH main source genes were increased by 50%,the fatty acid content and NADPH content of different gene overexpression strains were significantly different.Among them,GDH2 has a great influence on NADPH content,next to G6PD2.On the other hand,GDH gene may be one of the key speed limiting enzymes for fatty acid synthesis and NADPH production,which has a very important impact on fatty acid content.