Design Of Yeast Core Promoter And Genome Rearrangement To Enhance Naringenin Synthesis

Basic research mode of synthetic biology is to redesign the DNA sequence of components,endow them with new coding,function,structure,and realize the regulation of life process.This study mainly focuses on two key factors that affect gene expression :(1)transcriptional regulatory sequence changes in gene promoters,and(2)gene expression unit sequence changes triggered by genome rearrangement.In order to reflect the research features of high-throughput "design-build-detect-learn" in synthetic biology,and to quickly screen the effects of two types of DNA sequence modification libraries on yeast cell metabolism,this study established a rapid screening scheme of yeast colony-color chromatography.Two kinds of color products,carotenoids and flavonoids,were selected as colony screening markers.By observing the color distribution and depth,we can quickly screen the strains that meet the target demand,and then test them by HPLC.On the first hand,the sequence of the core promoter in Yarrowia lipolytica,the sequence between the TATA box and the transcriptional starting site,were respectively replaced by the corresponding core sequence in EXP1 p and GPDp by a bottom-up DNA assembly strategy.Combined with the different expression intensities of ?-carotene synthase Crt Y will directly affect the conversion of lycopene to carotene Efficiency and yield can result in a variety of colony color spectra,thereby enhancing the promoter by high-throughput screening,and obtaining a LE7-Y1 artificial promoter with a 5.5-fold improvement in lycopene to ?-carotene conversion.It was verified that its transcription intensity was 4 times stronger than that of the natural promoter.On the other hand,in haploid yeast,by replacing 4-coumaric acid coenzyme A ligase,a gene related to naringenin synthesis,with Arabidopsis thaliana,the naringenin production can be rapidly increased to 53 mg/L in haploid strains,a 165 % improvement over the starting haploid strain(20 mg/L).Then use synthetic genomic rearrangement technology(SCRa Mb LE),combined with the predeoxyviolacein(PDV)synthetic pathway as a screen for synthetic chromosome structural variants(SV)rearrangement of Saccharomyces cerevisiae,and biosensor Fde R to screen aromatic Strains with increased ring production,which in turn increased the amount of coumaric acid and naringenin produced by the target product.The yield of naringenin was increased by46% compared with that of the original diploid strain.In this study,the relationship between DNA sequence redesign and the synthesis of cell-specific secondary metabolites were revealed,through two respectives of work:(1)de novo design of gene promoter,and(2)global gene expression sequence changes owing to genome rearrangement.The obtained conclusions offered certain reference significance to synthetic biology in designing and reconstruction of life processes.