Metabolic Engineering Of Pichia Pastoris To Improve α-Farnesene Production

α-Farnesene is the simplest acyclic sesquiterpenoid and has potential applications in medicine and bioenergy.Enzymes that catalyze the production of terpenoids are usually complex and require complicated protein modifications for activity.Pichia pastoris,however,has strong protein modification abilities,making it a promising method for producing terpenoids.In this study,P.pastoris X33 was used as the starting strain,and various genetic engineering methods were used to increase the supply of the important precursor acetyl-CoA,increase the carbon flux in theα-farnesene synthesis pathway,and weaken the branch pathways of its important precursors.Ultimately,a highly efficient P.pastoris strain for producingα-farnesene was obtained,providing reference for the efficient production of acyclic sesquiterpenoids by microorganisms.The main research results are as follows:（1）Determining the optimal promoter for expressingα-farnesene synthase:α-farnesene synthase（aFS）was regulated using the promoters P_GAP,P_TEF1,P_CGW14,P_HXI11,P_SER1 and P_G1,and theα-farnesene production of each recombinant strain was measured.According to the results,P_GAP was determined to be the most suitable promoter for expressing aFS.And the recombinant strain XF1,which was regulated by P_GAP to express aFS,had a shake-flask fermentation yield ofα-farnesene of 0.463±0.03 g·L^-1.In addition,the expression strength of the different promoters was estimated,revealing that P_SER1was the weakest promoter.（2）Overexpression of key genes in theα-farnesene synthesis pathway:First,the rate-limiting enzyme genes t HMG1,IDI,and AFSLERG20 in the pathway were overexpressed,and the copy number of IDI and AFSLERG20 genes was increased to strengthen the mevalonic acid（MVA）pathway.The results showed that the production ofα-farnesene was further improved.Then,acetyl-CoA acetyltransferase from different sources,ERG10 from P.pastoris and Ato B from Escherichia coli MG1655,were separately overexpressed.The results showed that Ato B had higher catalytic activity and was more capable of promotingα-farnesene synthesis.Finally,the t HMG1 gene copy number was increased to further increaseα-farnesene production,and it was found that the recombinant strain XF13,carrying three copies of the t HMG1 gene,was most conducive toα-farnesene synthesis with a shake-flask fermentation yield of 1.32±0.03 g·L^-1.（3）Reducing pathway metabolism:To further increaseα-farnesene production,the squalene synthase（ERG9）promoter was replaced by the serine-inhibitory promoter P_SER1 in the XF13 strain to reduce squalene production.The diacylglycerol diphosphate phosphatase（DPP1）gene was knocked out to reduce the production of farnesol,allowing the precursor farnesene pyrophosphate（FPP）to further accumulate,resulting in an increase inα-farnesene production.In addition,theα-farnesene synthase（aFS）copy number was optimized to further increase its yield.The results showed that the recombinant strain XF19 carrying three copies of aFS had a highα-farnesene production yield,with a shake-flask fermentation yield of1.76±0.03 g·L^-1.（4）Increasing the supply of acetyl-CoA:Acetyl-CoA is an important precursor for the synthesis of terpenes,thus increasing acetyl-CoA flux will promote the synthesis ofα-farnesene.Then,pyruvate dehydrogenase（PDH）and acetyl-CoA synthase（ACS）from E.coli MG1655 were heterologously expressed using glucose inhibition inducible promoter P_G1 in strain XF19 to increase the acetyl-CoA supply in the cytoplasm of P.pastoris.The resultant strain XF22 produced 17.92±1.35 g·L^-1 ofα-farnesene with a productivity of 1.07×10^-1 g·L^-1·h^-1 and a yield of 7.80×10^-2 g·g^-1·glucose after 168 h in fed-batch fermentation during carrying out in a 7-L fermentor.（5）Increasing the intracellular NADPH supply:6 mol of NADPH are need to produce 1mol ofα-farnesene using acetyl-CoA as precursor.Based on strain XF22,overexpression of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase effectively improves intracellular NADPH supply,resulting in the strains XF22Z and XF22S.During 72hours of shaking flask fermentation,XF22Z and XF22S respectively showed an increase of approximately 6.1%and 11.2%inα-farnesene production compared to XF22,with a yield of2.27±0.09 g·L^-1 and 2.39±0.07 g·L^-1,respectively.Finally,the highest yield strain XF22S accumulated 19.35±1.05 g·L^-1 ofα-farnesene in a 7-L fed-batch fermentation.