产玉米黄质的人工酵母细胞的构建

Engineered Yeast Cell for Producing Zeaxanthin

为了实现微生物异源合成天然类胡萝卜素玉米黄质,以一株产β-胡萝卜素的酿酒酵母为底盘细胞,利用合成生物学技术构建人工酵母细胞。通过在染色体整合玉米黄质生物合成关键酶-β-胡萝卜素羟化酶(Crt Z),并对其9种来源进行筛选,发现整合欧文氏菌来源的β-胡萝卜素羟化酶的菌株获得玉米黄质的最高产量。法尼基焦磷酸(FPP)作为合成萜烯类天然产物的重要前体,通过敲除Lpp1和Dpp1基因,削减法尼基焦磷酸向法呢醇的转化,为玉米黄质的合成提供更多的前体,使玉米黄质的产量提高了1.27倍(从29 mg/L提高到36.8 mg/L)。在此基础上,通过增加欧文氏菌来源Crt Z的基因拷贝数及调节其启动子的强弱来增强β-胡萝卜素羟化酶的表达强度,使得玉米黄质的摇瓶产量达到96.2 mg/L,是目前公开报道中产量最高的。

For heterologous synthesize natural carotenoid zeaxanthin in microorganism, a β-carotene producing Saccharomyces cerevisiae was chozen as the host cell to construct engineered yeast with synthetic biology method. The key enzymes β-carotene hydroxylase encoding gene CrtZ from nine sources were integrated in the chromosomal, separately. As the result, the strain carrying CrtZ from Erwinia uredovora achieved the highest titer of zeaxanthin. Moreover, the conversion from farnesyl pyrophosphate （an important precursor for terpenoid natural products） to farnesol was reduced by knocking out gene Lppl and Dppl, providing more precursors for zeaxanthin synthesis. The zeaxanthin yield increased 1.27 fold （from 29 mg/L to 36.8 mg/L） accordingly. Furthermore, a titer of zeaxanthin was achieved as 96.2 mg/L in shake-flask through increasing the CrtZ gene copy number and regulating its promoter＇ s activity,which is the highest reported microbial titer known.