Engineering Acetyl-CoA Synthesis Pathway And Farnesyl Diphosphate Synthase To Improve The Production Of Geraniol In Saccharomyces Cerevisiae

In our previous work,we obtained high geraniol production yeast strain by genetic engineering such as the introduction of geraniol synthetase(GES)gene,the overexpression of key metabolic and regulatory genes in mevalonate acid(MVA)pathway,dynamic control of geraniol metabolism,and the elimination of geraniol metabolism downstream pathway.The final strain achieved geraniol production up to 1.69 g9L,which 1s the highest geraniol production in engineered yeast reported so far.However,compared with the sesquiterpenes and diterpenoid titers reported,the monoterpenes titer is still relative low.It is mainly due to the lack of key precursor geranyl pyrophosphate(GPP)in yeast and the toxicity of monoterpene.This study aims to improve the synthesis of monoterpene using geraniol as a representative product through enhancing the precursor GPP supply.In Saccharomyces cerevisiae,there is no specific GPP synthase and GPP is produced by a farnesyl diphosphate synthase(FPPS,Erg20p).Therefore,we first did point mutations of Erg20p to improve its GPP synthase activity.We built a model of the yeast FPPS,and chose three amino acid sites close to FPPS enzyme active center including Argl09,Gln 168 and Tyr201.We found out that compared with the control strain,the change of Gln168 to Arg improved the geraniol production by 53.03%.In addition,the geraniol production improved about 41%by changing Tyr201 to R or H.We also increase the supply of GPP indirectly by enhancing the supply of cytoplasmic Acetyl-CoA.Phosphoketolase(PHK)pathway was introduced and the pathway for Acetyl-CoA synthesis was optimized.We then compared the different combinations of different genes,which included xylulose 5-phosphate phosphoketolase(xpk)/phosphotransacetylase(pta),xylulose 5-phosphate phosphoketolase(xpk)/acetokinase(ack),and fructose 6-phosphate phosphoketolase(fpk)/phosphotransacetylase(pta)pathway,and confirmed the positive roles of xpk/pta pathway.In addition,we overexpressed four key enzymes of the oxidation stage in pentose phosphate pathway which included ribulose-5-phosphate epimerase encoded by RPEI,ribulose-5-phosphate isomerase encoded by RKI1,transketolase encoded by TKL1,and transaldolase encoded by TAL1.Additionally,we also overexpressed of the key gene ZWF1 encoding the glucose 6-phosphate dehydrogenase in the non-oxidation stage of pentose phosphate pathway.Engineerred strains of SYJZ9 and SYJZ10 were constructed and the geraniol production was improved by 47%through these genetic modification,whch carried xpkA and pta genes as well as the pentose phosphate pathway genes.