Optimization Of Lycopene Synthesis Pathway By Saccharomyces Cerevisia

Lycopene(C₄₀H₅₆)has many physiological functions such as quenching singlet oxygen,scavenging free radicals and inhibiting lipid peroxidation.It is used in the field of health care widely.Similar to most terpenoids,lycopene has low content in plants and low yield by chemical synthesis.The rapid development of synthetic biotechnology has achieved the production of some terpenoids by microbial hosts,and has been applied commercially.In this study,combining genetic engineering and fermentation engineering methods,the exogenous lycopene synthesis module was introduced into Saccharomyces cerevisiae,and the synthesis of lycopene in S.cerevisiae was realized by optimizing the metabolic pathway and fermentation process.The main research results as follows:The key enzymes involved in the synthesis of lycopene were searched through the literature and screened in the NCBI database.The CrtB derived from Erwinia and the CrtI derived from Xanthophyllomyces dendrorhous.According to the preference of the codon of S.cerevisiae,the codon of CrtB and CrtI were optimized to be expressed in single copy plasmid pRS41H,and high performance liquid chromatography?HPLC?analysis showed that lycopene was successfully synthesized in engineered yeast,with a yield of 0.038 mg/L.The adaptation between lycopene biosynthesis pathway and metabolism of the chassis was optimized through investigating the specie of chassis host,the copy number of functional gene and the type of promoter.The results proved that lycopene can be efficiently produced when using S.cerevisiae CEN.PK2-1C as chassis,coupling with multi-copy plasmid pRS42K,and employing constitutive promoter to control the expression of lycopene biosynthesis pathway.Comparing with the initial engineered strain,the yield of lycopene was increased by about 6 times,reaching 0.152 mg/L.In order to optimize the supply of precursors in lycopene biosynthetic engineering strain and use S.cerevisiae CEN.PK2-1C as the host,the synthesis level of lycopene by expressing the isoamyl pyrophosphate isomerase from Escherichia coli and GGPP synthetase derived from Sulfolobus acidocaldarius and strengthening the MVA synthesis pathway by genome integration method.The yield increased by 945.6 times than that of the initial strain,reaching23.64 mg/L.The optimum conditions for the fermentation conditions of engineered strain showed that the optimum conditions for the synthesis of lycopene were:yeast extract powder 10 g/L,sucrose 20 g/L,?NH₄?₂SO₄ 20 g/L,MgSO4 0.05 g/L,K₂HPO₄·3H₂O 0.02 g/L,A₆₀₀ 0.05,loading amount 24%,rocking speed 200 r min^-1,pH 6,fermentation temperature 28?.The yield of lycopene reached 79.42 mg/L by fed batch fermentation with citric acid,which was about 3.36 times higher than that before fermentation.The results showed that introducing the exogenous lycopene synthesis module into Saccharomyces cerevisiae,and the metabolic pathway and fermentation process were optimized,which the synthesis of lycopene was improved greatly.