Studies On Continuous Genome Evolution Of Saccharomyces Cerevisiae

Saccharomyces cerevisiae is one of the preferred chassis cells for metabolic engineering.Due to its complex metabolic and regulatory networks,many complex physiological phenotypes cannot be clearly understood.Genome-wide evolution is one of the most effective synthetic biology methods to overcome our limited knowledge.In this study,by repression of the expression of genes related to genome replication and stability,genome mutagenesis and selection could be coupled into a continuous process in S.cerevisiae.The established continuous genome evolution strategy could be used for the construction of yeast cell factories efficiently and rapidly.CRISPR interference(CRISPRi)efficiency could be affected by using different gRNAs targeting the same mutator gene,accordingly regulating the genome mutation rate.In this thesis,we designed different gRNAs targeting the same mutator gene to accurately regulate its expression level to create programmable mutators:MTRC,M-190,T-190,R-190 and C-190.The mutation rate of each mutators was evaluated by using the canavanine-resistance test method.It was confirmed that different mutators resulted in different mutation rates and mutation types.With BY4741-iAID6 as the parent strain,various high-performance traits were selected for genome evolution studies,with an aim to construct efficient microbial cell factories.Under iso-butanol stress,the mutator strain BY4741-iAID6-MTRC grew faster than WT and was able to tolerate 6.5%or higher concentrations of iso-butanol.When ?-carotene production was chosen as the target phenotype,the mutator strain BY4741-iAID6-CrtIEYB-MTRC produced a maximum titer of ?-carotene as high as 257.21 mg/L,which is 1.5 to 3-fold higher than the control strain.To further determine the optimal mutation rate and mutation type of iso-butanol genome continuous evolution,the MTRC strain in 6.5%iso-butanol was subjected to high-throughput sequencing.The second-generation sequencing results showed that the accumulation of point mutations with high mutation rate caused by the repression of MSH2 gene was conducive to the evolution of iso-butanol tolerance.In summary,by inhibiting the expression of genes related to genome replication and stability in S.cerevisiae,we established a continuous genome evolution technique for the eukaryotic systems.With high-performance traits as the research object,we successfully verified the effectiveness of the programmable mutator system in continuous genome evolution of S.cerevisiae.