Regulation Of Growth Performance In Yeast Cells Under Multidimensional Environment

Due to the concerns on climate change and sustainability,converting the renewable resource into chemicals by microbial cell factories is an increasing interest,which is more environmentally friendly,comparing with a chemical process.Yeast cells have the advantages of well-studied genetic background,ample genetic engineering tools,and high tolerance against the harsh industrial environment.Based on these,the yeast cell factories have been applied to produce organic acids,biofuels,biomaterials,pharmaceuticals,and natural products.During the production process of the chemical,the cell growth of yeast cells determines the titer,yield,and productivity.In this dissertation,the growth performance of yeast cells was improved in normal and harsh environments by engineering physiological functions,including engineering the mediator complex subunit Med3 to tune the cell cycle progress,optimizing the membrane asymmetry to improve membrane integrity,shortening the actin cable length to strengthen the budding process,and increasing actin patch density to stabilize intracellular pH.By applying the methods of microbiology and synthetic biology,the above strategies were performed and optimized,leading to the improved growth performance of Candida glabrata and Saccharomyces cerevisiae and enhanced butanol and medium-chain fatty acids production.The results are shown as follow:1.Engineering the mediator complex subunit Med3 to tune the cell cycle progress:Firstly,based on the transcriptomic data,it was found that Med3 could activate the transcriptional process of acetyl-Co A synthetase encoding gene acs2 by coordinating Ino4,and then regulate the intracellular level of acetyl-Co A.After that,homologous alignment and phenotypic analysis were applied to identify gene CAGL0M11990 g as Cg Cln3,a cyclin that regulates the transition of cell cycle progression.Moreover,the transcriptional process of Cg Cln3 was dependent on the level of acetyl-Co A,and then Cg Cln3 could regulate the cell size and budding index of C.glabrata.Finally,overexpression of Med3 could shorten the lag phase of cell growth and the specific growth rate and final biomass were increased by 29.9%and 12.1%,respectively,comparing with that of the control.2.Optimizing the membrane asymmetry to enhance membrane integrity: the membrane asymmetry regulators Lem3 and Sfk1 were identified by transcriptomic sequencing from an adaptive laboratory evolution(ALE)mutant strain.Then,Co-overexpression of LEM3 and SFK1 [strain Lem3(M)-Sfk1(H)] through promoter engineering remodeled the membrane phospholipid distribution,leading to an increased accumulation of phosphatidylethanolamine in the inner leaflet of the plasma membrane.s a result,membrane potential and membrane integrity were increased by 131.5% and 29.2%,respectively;while the final OD600 in the presence of hexanoic acid,octanoic acid,and decanoic acid was improved by 57.7%,79.6%,and 73.4%,respectively.3.Shortening actin cytoskeleton cable length to improve budding capacity for butanol production: The engineering targets SPA2 and CDC42 to shorten actin cable length and improve budding capacity were identified,based on literature mining,genetic engineering,and phenotypic characterization.Then,the butanol responsive promoter-based autonomous bidirectional signal conditioner was constructed to temporally tune the expression of SPA2 and CDC42.And the ABSC-butanol system was introduced into butanol producing strain to construct LH002-B5.Finally,after the 120 h fermentation process,the budding index of LH002-B5 was increased by 13.0%,leading to enhancement in titer(1674.3 mg/L),yield(120.3 mg/g),and productivity(14.0 mg/L/h),respectively.4.Improving actin cytoskeleton patch density to stabilize intracellular pH for medium-chain fatty acids production: Firstly,the analysis based on the pH-sensitive fluorescent protein pHluorin,lipophilic dye FM4-64,and fluorescent-labeled ?-factor revealed that decanoic acid could damage the endocytosis process,then lead to low pHi,and finally caused growth performance defect.Then,the engineering targets CLC1 and SLA2 to improve actin patch density and stabilize intracellular pH were identified,basing on literature mining,genetic engineering,and phenotypic characterization.After that,the medium-chain fatty acid responsive promoter-based autonomous bidirectional signal conditioner was constructed to regulate the expression of CLC1 and SLA2 in medium-chain fatty acids producing strain,resulting in strain LH003-M3.Finally,after the 60 h fermentation process,the intracellular pH of strain LH003-M5 was stabilized at 6.6.As a result,the titer,yield,and productivity of strain LH003-M5 were increased to 692.3 mg/L,19.3 mg/g,and 11.5 mg/L/h,respectively.