Construction And Screening Of Stress-resistant Escherichia Coli For High-yield Isobutanol Production

Energy,resources and environmental issues have become increasingly critical with the continuous development of modern industry,and there is an urgent need to innovate the traditional manufacturing industry.Green bio-manufacturing is a new industry that uses microbial cells or enzyme proteins to catalyze low-cost and renewable raw materials for highefficiency and large-scale material processing and material conversion to produce high valueadded products.It has good application potentials in the production of biofuels,pharmaceutical intermediates and chemical products with excellent social and economic benefits.Isobutanol is a new generation of biofuels that outperforms ethanol.High-yielding,stable and resistant cell factories are important to achieve isobutanol green bio-manufacturing,and their construction relies on synthetic biology techniques.CRISPR/ Cas9,a novel and efficient gene editing technology that has been successfully applied in a number of species,was used for the construction of isobutanol-producing Escherichia coli(E.coli).In this study,an E.coli CRISPR/Cas9 gene editing system was constructed and was used to accomplish the homologous recombination dependent deletion and replacement of a large fragment over 50 kb for the first time in the world.In this study,I constructed the target gene sg RNA plasmid,repair template donors(containing purple protein gene for screening).The overexpressed Cas9 protein in E.coli digested the chromosome to remove the target fragment,and the E.coli homologous recombination repair system was used to place the purple protein gene to the corresponding position in the genome.In this study,colony color(purple)can be used as an indicator for rapid selection of positive clones.According to the system established in this work,our laboratory deleted 28 large fragments(over 50 kb each)in the genome of E.coli and obtained a series of mutant strains.In this paper,the above-mentioned mutants were cultured under various natural stress conditions,such as high temperature,Na Cl,Na AC,furfural,isobutanol and so on.The growth rates of each mutant strain under stress and non-stress environments were measured to determine the beneficial and harmful genes for the microbial resistance to the stresses.Finally,a number of mutant strains that can effectively resist the specific harmful conditions were screened out.In this study,the genotypes and the anti-stress target genes were further analyzed to clarify the specific gene functions and to provide new information for following strain engineering.Furthermore,two stress conditions,e.g.acid stress and nutrient deficiency,were taken as examples to optimize the gene expression,culture conditions and process control of isobutanol-producing strains.The fermentation level of isobutanol was improved to 30 g/L.Taken together,this study aimed to investigate the stress problem faced by the host cells in the industrial fermentation process.CRISPR/Cas9 technology was used in E.coli to highthroughput screen stress-resistant genes.The production of isobutanol under the acid stress was used as an example to study how to maintain the production of metabolites under harmful conditions.The results of this paper will help to improve the industrial strains and optimize the fermentation process.