The Cloning And Expression Of Phaiffa Rhodozyma Carotenogenie Genes In Saccharomvces Cerevisiae To Construct β-carotene Biosynthetic Pathway

Carotenoids are a class of pigments with important biological functions and can be usedas the coloring agents for food and feed products. In addition, β-carotene is the precursor ofvitamin A, thus plays an important role in the vision process. Red yeast Phaffia rhodozymaproduces astaxanthin as the final product and also accumulates β-carotene as an intermediate.In this work, several carotenogenic genes of P. rhodozyma were cloned and expressed inSaccharomyces cerevisiae and the β-carotene biosynthesis was researched. The main contentsand results are as follows.crtE, crtYB and crtI cDNAs of P. rhodozyma CGMCC2.1557, as well as the truncatedgene encoding the catalytic domain of3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA)reductase (cHMG1) of S. cerevisiae were cloned and inserted into pFA6a-HIS3MX6toconstructe the plasmids pFA6a-cHMG1, pFA6a-crtE, pFA6a-crtI and pFA6a-crtYB, whichcontain the gene expression cassettes of crtE, crtYB, crtI and cHMG1. The obtained geneexpression cassettes were then inserted into three Escherichia coli/S. cerevisiae shuttle vectors,yielding the four gene expression plasmids, YEplac195-crtE/crtYB, YEp351-crtI,pRS200-cHMG1and pRS200-cHMG1/crtI. The four plasmids were transformed into the S.cerevisiae strain INVSc1, yielding the recombinant strains Sc-EYBI, Sc-EYBIH andSc-EYBIH+I.Cell growth of the recombinant S. cerevisiae strains and control strain Sc-vector wasmeasured. Compare to the control strain Sc-vector, the recombinant S. cerevisiae strainsSc-EYBI, Sc-EYBIH and Sc-EYBIH+I exhibited the enhanced cell growth. On the one hand,β-carotene productions in the recombinant S. cerevisiae were found to be quite different at20°C and30°C. The recombinant S. cerevisiae strains accumulated much more β-carotene at20°C than30°C. Furthermore, additional expression of the catalytic domain of HMG-CoAreductase and crtI increased the β-carotene level to528μg/g and1136μg/g,2-fold and4.4-fold higher than the control strain Sc-EYBI.In order to realize the thermal control and β-carotene accumulation difference among therecombinant S. cerevisiae strains, transcription level of these genes at different cultivationtemperatures was analysed. The recombinant cells was found to accumulate much moreβ-carotene at20°C than30°C, which might be partially due to a little higher transcriptionlevel of crt genes and cHMG1gene at20°C. In Sc-EYBIH and Sc-EYBIH+I, the transcriptionlevel of crtI genes and cHMG1were higher than that in Sc-EYBI, which might be the mainreason of different β-carotene accumulation levels in these strains. Furthermore, the death rateof the recombinant strain Sc-EYBIH and Sc-EYBIH+I was much lower than the control strainSc-vector. The research here establishes the foundation for the further study of astaxanthin.