The Study Of High Production Of 3S, 3’S Astaxanthin By Transgenic Xanthophyllomyces Dendrorhous

Astaxanthin is a xanthophyll of great interest in animal nutrition and human health.The market prospect in the nutraceutics industries for this health-protective molecule is very promising. Astaxanthin is synthesized by several bacteria , algae and plants fromβ-carotene by aequential action of two enzymes: aβ-carotene 3,3’-hydroxylase that introduces an hydroxyl group at the 3 (and 3’) positions of each of the twoβ-ionone rings ofβ-carotene, and aβ-carotene ketolase that introduces keto groups at carbons 4 and 4’ of theβ-ionone rings. Astaxanthin is also produced by the yeast-like basidiomycete Xanthophyllomyces dendrorhous. A gene asy involved in the conversionofβ-carotene to astaxanthin has been cloned. The configuration of astaxanthin produced by X. dendrorhous is 3R, 3’R, and thus made it difficult to be utilized. In this thesis, the heterologus genesβ-carotene 3, 3’-hydroxylase andβ-carotene ketolase were co-transformed to X. dendrorhous mutant CBS6938 pr-1-104 which produceβ-carotene, and the 3S, 3’S astaxanthin was produced by the transformants.Firstly, some X. dendrorhous expression vectors were constructed: pPRcDNA1-bkt, pPR2TNH-bkt, pPRcDNA1-crtZ, pPRCDNA1crtZ-bkt, pPR2TNHasy.β-carotene ketolase bkt,β-carotene hydroxylase crtZ and astaxanthin synthase asy are originated from Haematococcus plucialis, Erwinia uredovora and X. dendrorhous respectively. Secondly, the electrotransformation method for X. dendrorhous is optimized: the X. dendrorhous cells in exponential growth phase were treated with 50 mmol/ L DTT, and the plasmid concentration must reach to 0.7μg/μL, the electric plus was controlled at 5KV/cm, under that condition, 2.9×103 transformants /4μg DNA were got in one transformation.Then, the bkt and crtZ genes were successfully transformed to X. dendrorhous mutant CBS6938 pr-1-104 respectively. With antibiotic gradient dish selection and thin layer chromatography selection, the high carotenoids production transformants were obtained. After optimizing the fermentation medium, the X. dendrorhous transformants with bkt gene can produce total carotenoids 420μg/g (DW), and the canthaxanthin concentration can reach to 45%; meantime, the X. dendrorhous transformants with crtZ gene can produce total carotenoids 402μg/g (DW), and the canthaxanthin concentration can also reach to 45%. To get the 3S, 3’S astaxanthin, theβ-carotene ketolase bkt andβ-carotene hydroxylase crtZ were successfully co-transformed to X. dendrorhous mutant CBS6938 pr-1-104. With high concentration antibiotic selection, the high carotenoids production transformants were selected. After the mediun and fermentation were optimized, the X. dendrorhous transformants with crtZ and bkt can produce total carotenoids 580μg/g (DW), and the canthaxanthin concentration can also reach to 17.22%.