高效合成番茄红素酿酒酵母菌株的构建

Construction of a highly efficient synthetic lycopene engineered Saccharomyces cerevisiae

摘要:

番茄红素作为一种高附加价值的萜类化合物已受到国内外研究者的广泛关注。首先对酿酒酵母Saccharomyces cerevisiae模式菌株S288c和YPH499合成番茄红素的能力进行分析比较，结果表明YPH499更适合作为底盘细胞用于番茄红素的合成。随后比较组成型启动子GPDpr、TEF1pr和诱导型启动子GAL1pr、GAL10pr对番茄红素合成的影响，结果发现以GPDpr、TEF1pr作为番茄红素合成途径基因crtE、crtB和crtI的启动子，摇瓶发酵60 h后，番茄红素产量为15.31 mg/L；以GAL1pr和GAL10pr为启动子时，其产量为123.89 mg/L，提高8.09倍。继续改造甲羟戊酸 (MVA) 途径，过量表达N-末端截短的关键酶基因tHMG1 (3-羟基-3-甲基戊二酸单酰辅酶A还原酶)，番茄红素产量为265.68 mg/L，单位菌体产量72.79 mg/g。文中所设计构建的异源表达番茄红素合成途径的酿酒酵母菌株单位细胞产量高，可以进一步改造和优化后用于番茄红素的工业化生产。

Abstract:

Lycopene, as a high value-added terpene compound, has been widely concerned by researchers at home and abroad. Firstly, the ability of lycopene synthesis of Saccharomyces cerevisiae model strains S288c and YPH499 was analyzed and compared. The results showed that YPH499 was more suitable for lycopene synthesis as yeast chassis. Subsequently, the effects of constitutive promoters GPDpr, TEF1pr and inducible promoters GAL1pr, GAL10pr on Lycopene synthesis were compared. The results showed that when GPDpr and TEF1pr were used as promoters of crtE, crtB and crtI in lycopene synthesis pathway, the production of lycopene was 15.31 mg/L after 60 h fermentation in shaking flask. When GAL1pr and GAL10pr were used as promoters, the production was 123.89 mg/L, which was 8.09 times higher. In addition, the methylvaleric acid (MVA) pathway was further modified to overexpress the key enzyme gene of N-terminal truncation, tHMG1 (3-hydroxy-3-methylglutaryl coenzyme A reductase). The lycopene production was 265.68 mg/L, and the yield per cell was 72.79 mg/g. The Saccharomyces cerevisiae strain designed and constructed in this study can express lycopene in high yield per cell, thus could be used in the industrial production of lycopene after further construction and optimization.