Characterization And Application To Lycopene Synthesis Pathway Of Terminators In Saccharomyces Cerevisiae

The terminator is the DNA sequence of RNA polymerase transcriptional termination,which is responsible for the "output" of gene expression.Eukaryotic terminator can play an important regulatory role in the transcription process by affecting the stability of mRNA,the transcription efficiency and the location.In this paper,we cloned and characterized the native yeast terminators by analyzing the genome of Saccharomyces cerevisiae,and the lycopene biosynthesis pathway was used to explore the potential application of terminator in gene expression and fine regulation of pathway.The main results are as follows:(1)100 yeast terminators were cloned from the S.cerevisiae glycolytic pathway and TCA cycle.A terminator library was established by connecting to verification vector that contained the promoter TYSlp and green fluorescent protein gene eGFP.There are 45 strong terminators,31 moderate terminators and 24 weak terminators relative to that of PGK1 t standard terminator,range from 0.0613 to 1.8002,with a mean relative FI of 0.9945.(2)The efficiency element is found to be important element in controlling terminator strength by mutating the control elements(efficiency element,position element,and Ploy(A))of the yeast terminator.The strong terminator can lead to increase mRNA expression levels and protein production,indicating the gene expression can be directly affected by terminator selection.Then,the terminator strengths were characterized by combining different types and strengths promoters,it was found that the inducible GAL1 p and the strong FBA1 p had little effect on the terminator strength.However,the FI of the weak promoter(CDC60p)combined with the medium strength terminator is 5 times than the weak terminator.And the study shows that the same gene expression result can be achieved by pairing a strong terminator with a weak promoter replace a strong promoter.The terminator as a genetic component that is independent of the reporter gene and sequence of coding regions by studing different reporter genes.(3)With lycopene exogenous synthesis pathway to validation object,different strength promoters and terminators were used to design orthogonal experiment that used to construct gene expression modules under the control of different terminators,which used to verify the application potential of terminators in metabolic engineering.The study show when the strong terminator ATP15 t was combined with the weak terminator TDH3 t to control the expression of the lycopene synthesis pathway,the highest yield of the synthesized lycopene was 0.5681 mg/L,which was 5 times the lowest yield of lycopene.In summary,the terminator is important gene regulatory element that has great potential in the regulation of gene expression and metabolic pathway.