Reverse Metabolic Engineering Of Saccharomyces Cerevisiae For The Regulation Of Tyrosine Synthesis Pathway

Tyrosine is an important aromatic amino acid,which is widely used in food and medicine.Its derivatives include flavonoids,organic acids and alkaloids.Saccharomyces cerevisiae,as a eukaryotic model of industrial microorganisms,it is often used in the industrial production of various biological products,but S.cerevisiae cannot accumulate high concentration of amino acids.S.cerevisiae efficiently expressed tyrosine downstream product betaxanthin which has obvious color.It is easier to detect than tyrosine.In this study,betaxanthin,a tyrosine derivative with yellow fluorescence,was used as a screening index,and a recombinant strain with improved tyrosine synthesis ability was obtained by mutagenesis.We determined the production of p-coumaric acid by tyrosine by shake flask fermentation,then strengthened the supply of precursors and weaken the bypass metabolic pathways to improve the synthesis of p-coumaric acid.We carried out comparative genome sequencing analysis and quantitative PCR analysis of related genes that efficiently accumulate p-coumaric acid,initially excavated partial key genes that might regulate the synthesis of p-coumaric acid.The main results of this study are as follows:(1)Heterologous expression of betaxanthin in flowering plants in LTH0 accumulates betaxanthin.After expression,the color of the colonies turned to yellow.The relative yellow fluorescence intensity reached 1 203�45 AU�OD^-1 after cultured for 72 hours.We used UV mutagenesis,ARTP mutagenesis and compound mutagenesis methods to screen high-fluorescence mutants after multi-generations,the final 9-generations compound mutant strain LTHCB had a fluorescence intensity of 33 811�873 AU�OD^-1,which was 28 times of the control LTHA.There was no significant decrease in multiple passages.We selected 26high-fluorescence mutants from the mutant library from these three mutations.(2)LTH0 extracellularly accumulated 5.3 mg�L^-1 extracellular tyrosine in shake flask fermentation,while 19 of the 26 mutant strains had increased tyrosine production.The highest tyrosine production mutant strain accumulated 34.8 mg�L^-1 tyrosine extracellularly,which was 5.56 times higher than that of the control.After integrating and expressing the tyrosine ammonia lyase Fj TAL derived from Flavobacterium johnsoniae,the recombinant strain of the control strain LTH0 was fermented to obtain 189.4 mg�L^-1 p-coumaric acid,and the mutant strain had a maximum p-coumaric acid accumulation of 341.9 mg�L^-1.The yield of p-coumaric acid was 81.0%higher than that of the control.On this basis,knockout of PDC1,PDC6,TRP2 and PHA2 had no significant effect on the accumulation of p-coumaric acid,and knocking out of TRP2 or PHA2 increased the production of p-coumaric acid by 10.6%and23.1%respectively.Knocking out ARO3 and RIC1 increased the accumulation of shikimate pathway precursors,and increased the production of p-coumaric acid by 20%and 17.7%respectively.Optimizing fermentation conditions in shake flask,using 30 g�L^-1 glucose and peptone as carbon and nitrogen sources,supplementing with sodium carbonate and controlling the initial p H to 6,the recombinant strain LTHH0 accumulated 442.7 mg�L^-1p-coumaric acid.(3)In accordance with the strategy of reverse metabolic engineering,the obtained optimal p-coumaric acid-producing recombinant strain LTHH0 and the starting strain LTH0were re-sequenced and compared to find potential regulatory sites from the mutation sites,and 217 sites were found in the analysis.There are mutations in the upstream and downstream of genes or exon regions.After GO enrichment analysis and Pathway enrichment analysis,22related genes were selected for fluorescent quantitative PCR analysis.Among them,18 genes whose transcription levels have changed significantly are selected according to gene function.It was found that knockout of VBA4,GAL2,GPP1 and overexpression of TKL2 and ESBP6increased the production of p-coumaric acid by 9.4%,23.3%,15.2%,13.8%and 20.1%respectively.Finally,we have constructed the recombinant strain LTHHH11 which accumulated 582.78 mg�L^-1 p-coumaric acid which was 2.08 times higher than LTH0.