Excavation Of Metabolic Pathways For Increasing The Production Of Isoprene In Saccharomyces Cerevisiae

Isoprene is a terpenoid hydrocarbon molecule with volatile and hydrophobic properties.It is the simplest of isoprene compounds and is the composition skeleton of terpenes.Isoprene has a wide range of industrial applications in the fields of rubber production,perfume and pesticide manufacturing.Nowadays,petroleum is mainly used as a raw material for isoprene synthesis.However,with the depletion of petroleum resources and the pollution caused by using,the synthesis of isoprene is restricted.In recent years,with the development of synthetic biology and metabolic engineering,microorganisms as cell factories,biosynthesis of isoprene has become a focus of current research.At present,isoprenoid compounds are mainly synthesized by Saccharomyces cerevisiae and E.coli as host bacteria.Among them,Saccharomyces cerevisiae,as a eukaryotic model strain,not only has the advantages of clear genetic background,high glucose metabolism rate,and strong ability to adapt to industrial process conditions,but also has a natural mevalonate(MVA)pathway,which is a production good host for isoprene products.In this study,Saccharomyces cerevisiae was used as the chassis cell to explore new pathways to increase the production of isoprene by increasing the acetyl-CoA(Acetyl-CoA)flux in the cytoplasm and screening for key enzymes in the mevalonate(MVA)pathway.The results are as follows:(1)By knocking out the genes that lead to the mitochondrial tributary of Acetyl-CoA(Acetyl-CoA),the flux of acetyl-CoA in the cytoplasm is increased.Knockout of the mitochondrial pyruvate carrier gene(MPC2),mitochondrial porin gene(POR2),and the E1αsubunit gene of the pyruvate dehydrogenase complex(PDA1)to cut off the tributary of Acetyl-CoA to the mitochondria to excavate and improve S.cerevisiae.Metabolic pathways for pentadiene production.The expression vector containing the isoprene synthase gene ispS of Populus alba was transferred into the above three knockouts and subjected to shake flask fermentation.The results showed that the effect of knocking out the PDA1 gene was the best and the yield of isoprene reached 35 μg·L-1.Compared with the original strain,the yield increased by 0.3 folds.Then,on the basis of knocking out the PDA1 gene,the genes MPC2 and POR2 were further knocked out respectively.The fermentation results showed that the production of isoprene was not further improved.(2)Based on the single knock-out PDA1 gene strain,overexpression of xylulose 5-pho sphate-specific phosphotransketase(LmPK)and Clostridium kluyveris phosphotransacetylase gene(CkPTA)from Leuconostoc mesenteroide in the cytoplasm was further increased Acetyl-CoA flux in the cytoplasm.The fermentation results show that the production of isoprene has not been further improved.(3)On the basis of the above,the key enzyme genes of the MVA pathway are screened and the expression is enhanced to increase the downstream metabolic flux.In this study,the IDI1 genes(IPP isomerase)from E.coli,Saccharomyces cerevisiae and Solanum lycopersicum were screened separately,and the results showed that the IDI1 gene from E.coli had the best effect and the yield of isoprene reached 54 μg·L-1,which is about 1 folds higher.In addition,the introduction of HMGR genes from Saccharomyces cerevisiae and Ruegeria pomeroyi,respectively,through flask fermentation,the results showed that the yeast HMGR gene is better,and the production of isoprene reached 103 μg·L-1,compared with The original strain increased by 2 folds.In this study,we combined genetic engineering and metabolic engineering to strengthen acetyl-CoA in the cytoplasm of Saccharomyces cerevisiae,and then regulated the key enzymes in the MVA pathway,and explored the metabolic pathways to improve the production of Saccharomyces cerevisiae isoprene.The further research provided reference,and also contributed to the early industrialization of isoprene.