Design Of Terminator Of Saccharomyces Cerevisiae And Application In Pathway Engineering

Termination of RNA polymerase II transcription represents the final step in transcription cycle.Termination influences pre-mRNA processing,localization and stability.In this paper,analyzing the sequence and structural characteristics of the key core region of Saccharomyces cerevisiae terminator,cloned and characterized synthesis yeast terminators by designing synthetic terminator according to the principle of regulation and control.The fine regulation of lycopene biosynthesis pathway in S.cerevisiae was used as the verification object,and the regulation of synthesis terminators on metabolic pathway and the regulation of metabolic pathway of synthetic pathway were studied.The main results are as follows:(1)Through designing and constructing synthetic terminators library have characterized 266 of synthetic yeast terminators through measuring the expression of an enhanced green fluorescent protein(eGFP)under the control of promoter TYS1 p.The fluorescence protein expression intensity(FI)of the eGFP gene was used as a characterization index for the strength of synthetic terminator.The strength of yeast synthesis terminator ranged from 2.3648 to 3.5270,and the mean fluorescence values was 3.0719.(2)By sequencing the synthetic terminator and sequence analysis of terminator linker 1,the strength of synthetic terminator decreases as GC content increases of linker 1 sequence.The 88 strong synthetic terminators,90 medium strong synthetic terminators and 88 weak synthetic terminators in statistical library found that probability of occurrence of base G at any position of linker 1 sequence in the weak synthetic terminators are always greater than that of strong terminators.And the strength increases of synthetic terminator with base T increase in linker 1 sequence.By mutating the stem length of synthetic terminator,it found that changes in stem length of weak terminator and medium-strength terminator can result in both increased mRNA expression levels and protein production,indicating that the strength of terminator can be modulated by altering stem length of the terminator.(3)Taking the exogenous synthetic pathway of lycopene as verification object,parallel experiments were designed using synthetic terminators of different strengths to construct gene expression modules under the control of different strength synthesis terminators to verify the application potential of synthetic terminators in pathway engineering.It found that the lycopene production of the medium-strength terminator T-307 was 26.72 mg/L,which was 34.74% higher than that of the weak terminator T-414.Studies have shown that the strength of the synthetic terminator has flexible regulation and has great application potential in gene expression and fine regulation of metabolic pathways.