Prpduction Carotenoids By Saccharomyces Cerevisiae And Related Enzyme Research

Carotenoids are a class of natural pigment material, widely exists in the nature and has many physiological functions. It is far cannot satisfy the need by chemical synthesis and extracted from plants. As carotenoids synthetic genes have been cloned, biological synthetic methods of carotene showed strong vitality. Blakeslea trispora as commonly used in industry carotenoid production strain, the carotenoid production is very high, but there are some problems such as positive and negative bacteria separated for culture and so on.This article made carotenoids synthetic genes of Blakeslea trispora heterologous expressed in saccharomyces cerevisiae, using the host’s own MVA pathway provide precursor GGPP to produce carotenoids, and try to separate the domains of bifunctional enzyme carRA. Specific work is as follows:1. Carotenoids synthetic genes of Blakeslea trispora are built onto the vecter, then transform into the host, get through the carotenoids synthetic pathway, thereby gaining the recombinant yeast that produce carotenoids;2. Build the recombinant vecters containing speed limit enzymes’genes, then transform into the host, increase the production of carotenoids. We chose the speed limit enzymes’genes, ipi, ggps, tHMGR, build the recombinant vecters. We obtain pyAB/prG, pyAB/prGI, pyAB/prGIT recombinant yeast;3. Using the recombinant strains for fermentation, determination of β-carotene production. The highest yield we obtained is697.6μg/g DCW;4. Study the bifunctional enzyme carRA, try to separate the domains. We separate the bifunctional enzyme carRA which has two independent structure with different enzyme activity domain. Then build phytoene synthase activity gene into the recombinant vecter, transform into the host. In addition, we analyzed the hydrophobic of bifunctional enzyme carRA, made a signal peptide gene, that can anchor in saccharomyces cerevisiae on the membranes, connect to the phytoene synthase activity gene. Then build the recombinant vecter, transform into the host, observe the activity.