重组酿酒酵母发酵生产瓦伦西亚烯及其衍生物

Production of (+)-valencene and related sesquiterpenoids by engineered Saccharomyces cerevisiae

以酿酒酵母为宿主菌株,重构其体内代谢途径,生物合成瓦伦西亚烯及其衍生物。该研究在酿酒酵母菌株中引入来源于纯天仙子的细胞色素P450单加氧酶(cytochrome P450 monooxygenase,HPO)、来源于黄扁柏的瓦伦西亚烯氧化酶(valencene oxidase,CnVO),并构建了它们对应的4个突变体HPOM、CnVO-3、CnVO-4、CnVO-34,与来源于拟南芥的细胞色素还原酶(cytochrome P450 reductase,AtCPR)组合表达,通过静息细胞试验,筛选得到表达HPO和HPOM的菌株催化瓦伦西亚烯的效果最优,选取HPOM作为后续试验的基础。进一步引入来自黄扁柏的瓦伦西亚烯合成酶(valencene synthase,CnVS),过表达醇脱氢酶(alcohol dehydrogenase,ADH1),截短的3-羟基-3-甲基戊二酰辅酶A还原酶(truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase,tHMG1)。最终获得1株原位生产瓦伦西亚烯及其衍生物的重组酵母菌株PK2-24,进行3 L发酵罐的发酵试验,158 h后总萜产量达310.94 mg/L,较原始菌株提高了111倍。该研究为利用酵母规模化生产瓦伦西亚烯及其衍生物奠定了重要基础。

The metabolic pathway of Saccharomyces cerevisiae was modified to produce(+)-valencene and related sesquiterpenoids.The genes of cytochrome P450 monooxygenase HPO derived from Hyoscyamus muticus,valencene oxidase CnVO derived from Callitropsis nootkatensis and four of their mutants(HPOM,CnVO-3,CnVO-4 and CnVO-34)were introduced into S.cerevisiae.Their effect on(+)-valencene hydroxylation were investigated by co-expressed with cytochrome reductase AtCPR derived from Arabidopsis thaliana.Through resting cell assays,the catalytic efficiencies of strains expressing HPO and HPOM showed the best results,and HPOM was selected for future study.Strain S.cerevisiae PK2-24 was constructed by expressing(+)-valencene synthase CnVS from Callitropsis nootkatensis and overexpressing ADH1(alcohol dehydrogenase)and tHMG1(truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase).After 158 h fermentation in a 3 L bioreactor,total terpenoid yielded was 310.94 mg/L,which was 111 times higher than that of the original strain.In conclusion,this study presents a new candidate for industrial scale production of(+)-valencene and its related sesquiterpenoids.