The Effects Of Autonomously Replicating Sequence On Heterogeneous Gene Expression And DNA Replication Robustness In Saccharomyces Cerevisiae

Saccharomyces cerevisiae is an excellent production strain of exogenous products,which is widely used in synthetic biology and metabolic engineering.More and more attention has been paid to the expression of exogenous genes in the host.Using red fluorescent protein as a reporter gene,a set of methods for detecting the position effect on the expression of exogenous genes in the whole genome were established.Through the detection of 1044 integration loci,the relative fluorescence intensity of the recombinant strains varied from 0.98 to 12.98,and the maximum fold was 13.2.The discovery of extreme high and extreme low expression of exogenous gene integration sites revealed that the relative fluorescence intensity of near the centromere and telomere integration loci were low,and the conformation of 3Dstructure also affects the expression of exogenous genes.It was confirmed that the copy number was not responsible for the position effect,and the post-transcriptional translation process of the reporter gene also had a position effect in S.cerevisiae.The robustness of the position effect was confirmed by the replacement of reporter genes,promoters and carbon sources.When the carotenoid metabolic pathway was used as a reporter gene,the scope of influence of position effects was found to be limited.In the exploration of position effect,we found the activity of autonomous replication sequence(ARS)may affect the expression of exogenous genes.When the exogenous gene was integrated near the active ARS on chromosome III,the gene expression was significantly higher than near the inactive ARS.The relative fluorescence intensity of RFP ranged from 1.79 to 10.27.The integration and deletion the active ARS affect the expression of nearby genes.Whether it is integrated in the chromosome or in the plasmid,the expression of the exogenous gene nearby the active ARS is higher than that in the inactive ARS.In addition,it has been proved that the expression of genes is not only affected by the efficiency of ARS,but also affected by the 3D structure of chromosomes.ARS deficiency did not affect the growth of the strain,in order to explore how the robustness of DNA replication was maintained after ARS deficiency.Firstly,on the basis of the left arm of synthetic chromosome V,the comfirmed ARS,Likely ARS and Dubious ARS were knocked out,and the growth of the recombinant strain were not affected.The ACS model was used to predict potential ARS and the method of chromosome segmentation to test the function of ARS.Some ARSs with weak replication efficiency were found on chromosomes,and the recombinant strain could still grow after the iterative deletion of these ARS on chromosomes.But the ARS deletion strain is more sensitive to DNA replication pressure.In addition,CHIP-qpcr and plasmid loss rate experiments proved that,when comfirmed ARS was knockedout,the weak efficiency ARS would significantly fire to maintaining DNA replication and chromosome stability robust in S.cerevisiae.