The Combination Of β-Carotene Biosynthesis

Saccharomyces cerevisiae as a model organism in eukaryotes, with characteristics of genetic information clear and fermentation conditions mature. Carotenoids are high value added four terpene compounds with antioxidant, anticancer and many other kinds of physiological functions. Based on synthetic β-carotene genes the of different sources, we use different methods to construct efficient β-carotene biosynthetic pathway in saccharomyces cerevisiae and the fermentation conditions were optimized. Specific work is as follows:1、Different recombinant bacteria InvSC1-1 (pYES2-carRA-carB/pRS42-ggps-idi)、InvSC1-2 (pYES2-carRA-carB/pRS425-ggps) and InvSC1-3 (pYES2-carRA-carB-ggps) were used for fermentation respectively. The production of β-carotene were 1.495 mg·L-1、2.053 mg·L-1、2.137 mg·L-1 respectively. Using different carbon sources on fermentation of InvSC1-3, β-carotene yield was the highest,3.188 mg·L-1 when the carbon source was for 1% glucose plus 2% galactose. Different effectors were added to the medium for fermentation on InvSC1-1. When the concentration of isoniazid was for 0.8 g·L-1, the β-carotene production of the unit of biomass increased by 4.57 times,164.56 μg·g-1; when ketoconazole concentration was 10 mg·L-the β-carotene production of the unit of biomass increased by 2.74 times, 98.77 μg·g-2·We amplificated carotenoid biosynthesis genes crtI·crtY·crtB·crtE from Erwinia with plasmid PACCAR 16AcrtX as templates to build β-carotene biosynthetic pathway in saccharomyces cerevisiae. We compared the ability to produce β-carotene with the same function enzyme genes between Erwinia and Blakeslea trispora in different strains (InvSC1·BY4741). We made sure that genes from different sources in InvSC1 was better.3·Overexpression of a single enzyme or two enzymes involved in β-carotene biosynthetic pathway and then screened the rate limiting enzyme genes. Ultimately, curB was determined the rate limiting enzyme. Overexpression of carB, the β-carotene production increased by 1.22 times, 40.13 mg·L-1.4·We use 2A system to build β-carotene biosynthetic pathway (pYES2-crtY-crtl-crtB-crtE, pYES2-carR4-curB-ggps) from different sources in saccharomyces cerevisiae. The β-carotene production was 16.51 mg·L-1 and 24.50 mg·L-1 respectively.