Application Of SCRaMbLE System To Improve Formic Acid Tolerance And Utilization In Saccharomyces Cerevisiae

With the development of biological technology,synthetic genome has become feasible.Saccharomyces cerevisiae is the first eukaryotic microorganism with whole genome sequencing and one of the model microorganisms.Redesigning the synthetic S cerevisiae genome will enable researchers to better understand and utilize it.In S.cerevisiae,whole-genome rearrangement of chromosomes can be made based on Synthetic Chromosome Rerangement and Modified by Loxp-mediated Evolution(SCRaMbLE),resulting in a large number of mutant strains with different genotypes and phenotypes.In this study,a yeast,yXZX565,that can make use of formic acid,was screened out from S cerevisiae containing different synthetic chromosomes.With yXZX565 as the background strain,strain Yy1.1 was obtained through the directional screening with formic acid as a single carbon source through the application of SCRaMbLE system.It is proved that this strategy can be used to improve the tolerance and utilization of formic acid in yeast.Then Yy1.1 was used as the starting strain for iterative rearrangement,and strains Yy2.1,Yy3.7 and Yy4.8 were screened to further improve the tolerance and utilization of formic acid.Secondly,we reported that of knockout SPT2,could enhance yeast tolerance to alkaline environment,maintain yeast activity,so that it can better use formic acid.Finally,by integrating malonyl-CoA reductase(MCR)into the yeast genome,the co-culture of glucose and formic acid was carried out.When 2.5 g/L formic acid was added 24 hours later,the growth of the screening strain Yy2.1 was comparable to that of the control strain yXZX565,the screening strain Yy2.1 formic acid used 2.32g/L,which was 120% more than that of the control strain.Moreover,the production of 3-hydroxypropionic acid of Yy2.1 was 3.55 g/L,which was66% higher than that of the control.Finally,the strain Yy2.1 was sequenced whole-genome,and the results showed that Yy2.1 had two large fragments inversion on chromosome 5 compared with yXZX565,speculating that genomic structural variation affected the genotype and phenotype of yeast.The study of this subject has proved that SCRaMbLE system can well improve yeast utilization to formic acid,which shed lights for yeast to use formic acid and fix carbon dioxide in the future.