Optimization Of Culture Medium For Arachidonic Acid By Response Surface Method And Pilot Study On Construction Of High-Yield EPA Genetically Engineered Strain

Polyunsaturated fatty acids(PUFAs)are long-chain fatty acids containing more than one double bond in their carbon skeleton,and are mainly classified into two series of ?-6 and ?-3.Arachidonic acid(ARA)and Eicosapentaenoic acid(EPA)are typical representatives.ARA and EPA,as essential fatty acids in human body,play an important role in promoting brain and nerve development,preventing cardiovascular diseases,and regulating inflammatory reactions.They have been widely used in food,medicine and health care products and cosmetics.ARA and EPA are traditionally derived from animal liver and deep-sea fish oil.Due to high extraction cost and insufficient yield,it is difficult to meet market demand.Large-scale production of ARA and EPA by liquid fermentation of Mortierella alpina has become a research hotspot.At present,Mortierella alpina has become the designated strain for industrial production of ARA.Due to its low content,EPA has not yet been industrialized,but it still has good application prospects.In this paper,the ARA yield was improved by the response surface optimization medium,and the EPA production was increased by exogenous expression of 17des dehydrogenase.The CRISPR/Cpfl knockout plasmid was constructed and a gene editing system was established in M alpina G12.In this paper,the strain M.alpina G12 was selected from the laboratory,and the ARA fermentation medium of Mortierella alpina was optimized by response surface methodology.Based on the pre-laboratory study and related literature research,the Plackett-Burman method was used to screen three main factors affecting ARA yield from the initial medium:yeast powder,glucose,corn syrup dry powder;steepest climbing method The maximum response value of ARA production was approximated.Finally,the three-factor and three-level experiments were designed by Box-Behnken method.The optimal medium was obtained by the result analysis and subsequent experiments.The composition was:yeast powder 12.6g/L,glucose 75.6g/L,corn syrup dry powder 7.1g/L,MgSO4 0.5g/L,KH2PO4 1g/L,CaCl20.5g/L,pH 6.5.After 7 days of fermentation,the ARA yield reached 5.12 g/L,which was 27.3%higher than that before optimization.It can be seen from the fatty acid anabolic pathway that ARA dehydrogenates to form EPA under the action of omega-3 fatty acid dehydrogenase,whereas Mortierella alpina omega-3 dehydrogenase induces expression only at low temperature(18?)and the expression amount is limited.In this paper,Agrobacterium tumefaciens-mediated genetic transformation system of Mortierella alpina was established.A binary vector pBIG-CBXB-17des containing the omega-3 type ?17 dehydrogenase and the rust-resistant resistance gene of Saprolegnia diclina expressed at room temperature was constructed and successfully transformed into M.alpina spores and fermentation verified that the EPA content is improved compared to the control.CRISPR/Cpfl is the third generation gene editing technology after ZFNs and TALENs.It has the advantages of simple operation,high editing efficiency and wide versatility.In order to study the function of key enzyme genes in fatty acid metabolism pathway,this paper attempts to establish the CRISPR/Cpfl gene editing system in M.alpina G12.The sgRNA was designed with ura5 as the target gene,and the binary vector pBIG-sgRNA-nlsCpfl containing the Cpfl gene and sgRNA was constructed.An attempt was made to transform the M.alpina G12 by Agrobacterium tumefaciens,and the ura5 partial gene fragment was knocked out to obtain a ua5-auxotrophic strain.No transformants have been screened yet,and this part need to be further studied.