Synthesis Of β-Carotene From Saccharomyces Cerevisiae By Metabolic Engineering

β-Carotene is a terpenoid,belonging to the carotenoid family,which exists in plants,fruits and microorganisms with important commercial value.It has an important effect on cardiovascular disease,prostate cancer,especially lung cancer,so that its market continues to grow,and is concerned by many researchers.In recent years,the production ofβ-carotene in microbial cell factories have become more and more important,and the use of microbial cells to synthesize high value-added naturalβ-carotene is of great research significance.Saccharomyces cerevisiae has a clear genetic background,and is food safety,and its products are relatively safe.In this study,Saccharomyces cerevisiae LFD18 was used as the starting strain,and key genes were modified to construct a recombinant strain of Saccharomyces cerevisiae with efficient synthesis ofβ-carotene through transcriptomics,metabolic engineering and other methods,the main research contents and results are as follows:（1）β-Carotene synthetase genes Car RA and Car B from Blakeslea trispora,Car RP and Car B from Mucor circinelloides were expressed under constitutive promoter TEF_prGPD_pr and inducible promoter GAL1_prGAL10_pr to construct expression boxes,and were transformed into Ythmg1.The results showed that theβ-carotene synthetase gene product derived from Blakeslea trispora was more productive with inductively expressed GAL1_prGAL10_prpromoter.（2）The aboveβ-carotene synthetase gene expression box was transformed into LFD18strain to obtain ZS19 strain.During plate spreading experiments one reddish mutant was found,whose colony color was significantly darker than that of its surrounding colonies.It was named ZS19 reddish,and itsβ-carotene content was significantly increased.GO,PATHWAY and q PCR analysis showed that the expression levels of 6 genes related to carbon metabolism and liposome synthesis,including lpp1,dpp1,hxk1,fdh1,hmg2 and gre2,were up-regulated.By simultaneously overexpressing lpp1,dpp1,hxk1 and fdh1 genes,the recombinant strain named ZS20,produced 194.3 mg·L^-1β-carotene,which was 1.3 times than that of the ZS19 reddish strain and 2.1 times than that of the ZS19 strain.（3）β-Carotene production by strain ZS20 was optimized in fermenters.Glucose was added at a constant rate of 20 g·h^-1in fermenter,and the yield ofβ-carotene was 226.9 mg·L^-1.The above fermentation process was further optimized by substrates selection,nitrogen source increaseing,amino acids adding and supplement rate changing.The final yield ofβ-carotene reached 526.3 mg·L^-1,232%higher than before optimization,with a unit DCW yield of 40.8mg·g^-1 DCW.