酿酒酵母产脂肪酸乙酯代谢途径改造

Metabolic Pathway Modification of Saccharomyces cerevisiae for Overproducing Fatty Acid Ethyl Ester

脂肪酸乙酯(fatty acid ethyl ester,FAEE)是目前最具有潜力的新型能源,微生物细胞工厂生产FAEE比目前普遍的化学合成方法具有诸多优势,例如对环境污染小、生产成本低等。酿酒酵母具备良好的产乙醇和脂肪酰辅酶A的能力,我们试图通过改造酿酒酵母代谢途径来提高FAEE的前体——脂肪酰辅酶A的产量。通过引入根据酿酒酵母密码子优化的外源基因WS2(蜡酯合成酶基因)构建一条可以利用葡萄糖为底物、经过一系列反应生成FAEE的代谢通路。富集脂肪酰辅酶A需要进一步改造分支途径,例如阻断甾醇酯途径(ΔARE1、ΔARE2),三酰基甘油途径(ΔDGA1,ΔLRO1)和β氧化途径(ΔPXA2)来减少脂肪酰辅酶A的分流,从而相应的富集脂肪酰辅酶A。结果表明:3个途径的敲除大大增加了FAEE的产量,相较于在原始出发菌株BY4741中表达WS2即菌株BYW2增加了9倍,摇瓶产量达到11.72 mg/L。由于目的产物FAEE产量较低,在摇瓶发酵阶段进行了发酵优化,对是否添加乙醇和菜籽油、发酵时间以及乙醇添加模式这3个因素进行优化。结果表明:添加乙醇和菜籽油明显提升了FAEE产量,最佳发酵时间为60 h,乙醇添加模式为从20 h开始每间隔6 h补加发酵体积的2%,此时FAEE产量达到最高,为144.4 mg/L,是未优化前FAEE产量的12.3倍,是出发菌株BY4741中FAEE产量的480倍。为进一步提升FAEE产量,采用发酵罐发酵并对比了尿嘧啶缺陷菌株BYD5W2和尿嘧啶未缺陷菌株BYD5W2*的FAEE产量的差异,结果显示尿嘧啶缺陷菌株BYD5W2的FAEE最高产量为0.618 g/L,尿嘧啶未缺陷菌株BYD5W2*的FAEE最高产量为1.35 g/L,是目前报道的利用酿酒酵母产FAEE产量中较高的。

Fatty acid ethyl ester(FAEE)is the most potential new energy source in the new century.Compared with the current common chemical synthesis methods,the production of FAEE in microbial cell factories has many advantages,such as low environmental pollution and low production cost.Saccharomyces cerevisiae has the talent to produce ethanol and fatty acyl-CoA.The production of fatty acyl-CoA,i.e.,the precursor of FAEE,was thus improved by modifying the metabolic pathways of S.cerevisiae.By introducing codon-optimized heterogeneous gene WS2(wax ester synthase),a metabolic pathway was constructed and it could use glucose as a substrate to generate FAEE through a series of reactions.To enrich the fatty acyl-CoA,it was necessary to further reform its branches,such as blocking sterol ester way(ΔARE1,ΔARE2),triacylglycerol pathway(ΔDGA1,ΔLRO1)andβ-oxidation pathway(ΔPXA2)to reduce fatty acyl-CoAutilization.The results showed that the knockout of three pathways greatly increased the yield of FAEE.Strain BYW2 expressing WS2 gene had a 9-fold increase in FAEE yield compared to the original strain BY4741,and the shake flask yield reached 11.72 mg/L.Due to the low yield of the target product FAEE,the fermentation was optimized in the shaking flask.Three factors including the addition of ethanol and rapeseed oil addition,fermentation time and ethanol addition mode were optimized.The results showed that the addition of ethanol and rapeseed oil significantly increased the yield of FAEE.The optimal fermentation time was 60 h,and the ethanol addition mode was to add 2%of the fermentation volume every 6 h after 20 h.Under the optimal conditions,the highest yield of FAEE reached144.4 mg/L,which was 12.3 times higher than that before optimization,and 480 times higher than that in the original strain BY4741.In order to further increase the yield of FAEE,the difference of FAEE yield between uracil deficient strain BYD5 W2 and uracil non-deficient strain BYD5 W2*was compared by fermentation tank.The results showed that the highest FAEE yield of uracil deficient strain BYD5 W2 was 0.618 g/L,and that of uracil non-deficient strain BYD5 W2*was 1.35 g/L,which was the highest reported FAEE yield by S.cerevisiae currently.