Engineering Modification Of Saccharomyces Cerevisiae And Escherichia Coli For The Utilization Study Of Formate And Acetate

The excessive development and use of non-renewable fossil resources have led to global climate change and food shortages.Therefore,the development of sustainable green biotechnology is of great significance in alleviating this serious situation.Formic acid and acetic acid,as a new type of green resource,can be produced by catalytic reduction of CO_2,and their reduction and utilization contribute to reducing carbon emissions and achieving carbon neutrality goals.Compared to traditional sugar-based fermentation carbon sources,formic acid has the advantages of low cost and direct production from CO₂ fixation;acetic acid has the advantages of abundant sources and can be directly utilized by microorganisms.However,the editing tools for natural methylotrophic microorganisms are not mature yet,and the utilization efficiency of formic acid is low,which is not conducive to large-scale application.Escherichia coli and Saccharomyces cerevisiae are commonly used model microorganisms with well-studied gene functions and metabolic pathways,and mature gene-editing tools,as well as simple cultivation methods.In addition,these two model microorganisms are often applied in fermentation industries and have industrial application potentials.Therefore,the development of pathways for utilizing formic acid and acetic acid as substrates in model microorganisms such as Escherichia coli and Saccharomyces cerevisiae has significant importance.The main content of this study is as follows:First,a formic acid-formaldehyde-ribulose-5-phosphate pathway was constructed in brewing yeast by expressing exogenous MDH,ACS-ACDH,HPS,and PHI genes,successfully creating a strain capable of growing on a single carbon source of formic acid.Further optimization of pathway gene expression and peroxisome targeting strategy improved the growth ability of the strain under formic acid conditions,with 2.5 g/L of formic acid consumed in 200 hours.Subsequently,the peroxisomes were further modified by knocking out the PEX31,PEX32,LPL1,IZH3 genes and overexpressing the SFA1 and PEX5 genes to further enhance the growth of the strain under formic acid conditions.To verify the strain’s ability to produce chemicals using formic acid,a strain of brewing yeast capable of accumulating FFA was constructed by knocking out the POX1,FAA1,and FAA4 genes,using FFA（free fatty acid）synthesis as an example.Finally,based on the electrochemical basis of CO₂ fixation to formic acid,the target of catalyzing CO₂ to FFA through whole-cell"one-pot"was achieved by adjusting the balance between electrocatalysis and microbial fermentation.Under glucose conditions of 20 g/L,12.5 mg/L of FFA could be accumulated after being electrified.Second,in E.coli,acetate was used as an auxiliary carbon source and glucose as the main carbon source for the production of pyruvate.By knocking out the pox B,pfl B,and ace EF genes,preliminary accumulation of pyruvate from glucose was achieved.The addition of acetate as an auxiliary carbon source restored the strain’s growth on glucose.The accumulation of pyruvate was 2.62 g/L,with a byproduct of lactate accumulating at 2.02 g/L.Further knockout of the ldh A and mgs A genes resulted in zero lactate production.Finally,by optimizing the ratio of glucose and acetate,pyruvate production reached 8.6 g/L,with yields of0.91 g pyruvate/g glucose and 0.73 g pyruvate/g mixed carbon source.To further achieve the production of pyruvate using acetate as the sole carbon source,overexpression of genes involved in the glyoxylate cycle and acetate utilization was carried out,resulting in the generation of 0.6 g/L pyruvate from 8 g/L acetate.These results successfully demonstrated the conversion of acetate to pyruvate.Third,the acetate utilization pathway in brewing yeast was strengthened by expressing the ACS1 gene in the cytoplasm and introducing a point mutation into the HAA1 gene on the genome,enhancing the conversion of acetate to acetyl-Co A and shortening the strain’s lag phase.Subsequently,the expression of exogenous Ca MCR gene enabled the conversion of acetate to 3-HP,with 0.8 g/L of 3-HP produced from 12g/L of acetate.By regulating the TCA cycle and glyoxylate cycle,the rate of acetate utilization was further improved.Fermentation conditions were optimized to achieve 2.2 g/L of 3-HP production.Finally,using fermentation broth containing 18 g/L of acetate obtained from Clostridium ljungdahlii through synthesis gas（CO₂/H₂）fermentation,2.05 g/L of 3-HP was accumulated.This experiment used a"two-step"method to fix CO₂using Clostridium and produce acetate coupled with brewing yeast fermentation,successfully generating the high-value compound 3-HP.Fourth,adaptive evolution was used to enhance the acetate tolerance and utilization ability of brewing yeast.After 280 generations of continuous domestication in an acetate medium,a domesticated strain that could tolerate 24 g/L of acetate was successfully obtained.Under high concentration acetate conditions,the lag phase of the strain was shortened.Transcriptome analysis revealed that the improved tolerance of the evolved strain was mainly related to cell protein synthesis,phospholipid metabolism,and cell transport functions.In addition,there were changes in the downregulation of sugar metabolism genes,upregulation of ribosome synthesis pathway genes,and upregulation of ion channel protein expression,among others.By reducing the from-scratch synthesis flux of secondary metabolites and many intracellular compounds,the efficiency of intracellular material utilization was improved.The strength of the cell membrane structure was increased to enhance the strain’s resistance to acetate and acetaldehyde toxicity.Transcriptome analysis results provide a research foundation and direction for further analysis of the mechanism of acetate tolerance.In summary,this thesis established a microbial platform for the utilization of formic acid and acetate,achieved catalytic conversion of CO₂driven by renewable energy,and constructed pathways for brewing yeast to utilize formic acid and acetate.This provides research potential for the synthesis of high-value compounds using formic acid or acetate as the sole carbon source and provides a research foundation for microbial electrocatalytic CO₂ fixation.In addition,by using adaptive evolution and systems biology analysis,some potential targets for acetate tolerance in brewing yeast were identified.The results of this thesis provide a research case for CO₂-driven biological fermentation.