Metabolic Engineering Of Saccharomyces Cerevisiae Cell Factory For Efficient Synthesis Of Polyketides

Polyketides are a class of small molecular natural products that have gained significant attention as clinical drugs.Saccharomyces cerevisiae（S.cerevisiae）has been widely used as a heterologous chassis to reconstitute the polyketide biosynthetic pathway.However,due to the low efficiency of many polyketide synthases and the limited availability of precursors such as acetyl-Co A and malonyl-Co A,the yield of polyketide synthesis is generally poor.To address these issues,this study broadened the substrate supply of acetyl-Co A and malonyl-Co A by successfully using peroxisome acetyl-Co A to synthesize polyketides.In addition,we employed metabolic engineering to optimize plasmid copy number of two pairs of silent polyketide synthase（PKS）in genome mining.By doing so,we achieved a 10-fold increase in the potential yield of polyketides and successfully isolated and resolved new polyketide analogs.Lastly,we applied plasmid copy number engineering to the combinatorial biosynthesis of the newly discovered polyketides and PKS subunits from previously determined structures.This approach yielded ten additional novel polyketides.The main research results are as follows:（1）The aim of this study was to expand the substrate utilization range of acetyl-Co A by effectively utilizing them in different compartments in S.cerevisiae.We accomplished this by introducing acetyl-Co A carboxylase(Acc1^{S1157A,S659A,S686A},Acc1***)and two PKSs into yeast peroxisomes,allowing for the synthesis of two polyketides,flaviolin and triacetic acid lactone（TAL）.We also discovered that the peroxisome localization tag p PTS1 is more efficient in mediating protein peroxisome localization than previously studied e PTS1.Additionally,the transcription factors Adr1c,Oaf1c,and Pip2c were found to promote peroxisome proliferation and enhance polyketide biosynthesis through peroxisomes.Our findings demonstrate that the simultaneous use of the cytoplasmic and peroxisome pathways resulted in a significantly higher production of 1.13 m M TAL(0.14 g·L^-1),a 49.9%increase compared to using the cytoplasmic pathway alone and a 90.9%increase compared to using the peroxisome pathway alone.（2）In this study,we examined the efficacy of progressively truncated promoters to drive antibiotic selection marker genes as a means of increasing plasmid copy number.By attenuating selection marker gene expression,we observed a strong correlation between the degree of promoter truncation,plasmid copy number,and the relevant gene’s expression level.Our findings indicate that different degrees of promoter truncation can increase the plasmid copy number up to 15 or 19 times that of the wild type when using Kan R or Hyg R as selection markers,respectively.We subsequently applied this approach to optimize PKS At Cur S1-At Cur S2 expression and realized a remarkable 5-fold increase in the yield of the antitumor compound 10,11-dehydrocurvularin.In addition,we extended our findings to two groups of potential polyketide biosynthesis gene clusters,namely Hs Hyp S1-Ga BDLS2（compounds 1 and 2）and Gc BDLS1-Gc BDLS2（compounds 3-5）,with very low product yields.Our approach increased the yield of these compounds by 5.2,3.2 times,and 5.5,6.3,and 10.1 times,respectively,facilitating product separation and structure determination.（3）In the subunit exchange method,we replaced the PKS pair Ga BDLS1-Ga BDLS2 and Hs Hyp S1-Gc BDLS2 with the previously reported PKS pair.Results indicated that three new combinations,namely At Cur S1-Ga BDLS2,Gc BDLS1-At Cur S2,and Gc BDLS1-Hs Hyp S2,produced new products 6-15;however,the yields were insufficient for purification and product structure determination.Through the implementation of plasmid copy number engineering,we achieved a significant enhancement in the production of unnatural polyketides.As a result of these efforts,we have successfully identified and characterized ten novel unnatural polyketides.