α-法尼烯在巴斯德毕赤酵母中的生物合成

Metabolic engineering of Pichia pastoris forα-farnesene production

由可再生原料微生物生产α-法尼烯是一种有前途的替代传统石油基工艺的方法。尽管已经报道α-法尼烯可以通过常规模型菌株如大肠杆菌和酿酒酵母异源产生,但是其发酵规模不易扩大。巴斯德毕赤酵母是生产类异戊二烯的良好平台且可高密度培养,具有大规模生产α-法尼烯的潜力。在该项研究中,首先确定tHmg1,IDI1,ERG19,AFSLERG20是甲羟戊酸途径和α-法尼烯合成途径的限速酶基因。然后对限速酶基因进行组合过表达并优化基因拷贝数以平衡代谢路径增大流向α-法尼烯合成的代谢通量,最终获得菌株F16,其α-法尼烯产量为(1.09±0.02)g/L。最后,通过外源添加不饱和脂肪酸促进α-法尼烯分泌到细胞外,当培养基添加20 mmol/L的油酸,在摇瓶中获得最高的α-法尼烯产量约1.40 g/L[0.32 g/g细胞干重(dry cell weight,DCW)]。这是出发菌株F1产量的3.1倍。该研究是首次以巴斯德毕赤酵母作为底盘微生物细胞来生产α-法尼烯,并为其他倍半萜的异源生物合成提供了新的思路。

Microbial production ofα-farnesene from renewable feedstock is a promising alternative to traditional petroleum-based processes.Although it has been reported thatα-farnesene can be heterologously produced by conventional model strains such as Escherichia coli and Saccharomyces cerevisiae,its fermentation scale is not easy to expand.Pichia pastoris is a good platform for the production of isoprene and has the potential for large-scale production ofα-farnesene at high density.tHmg1,IDI1,ERG19,and AFSLERG20 were confirmed as the rate-limiting enzymes of the mevalonate pathway and theα-farnesene synthesis pathway.Then,the metabolic flux toα-farnesene synthesis was increased by combining overexpression rate limiting enzyme genes and optimizing the copy number of genes to balance the metabolic pathway.Strain F16 was obtained,and itsα-farnesene yield was(1.09±0.02)g/L.Furthermore,the secretion ofα-farnesene into extracellular space was promoted by adding unsaturated fatty acids,when 20 mmol/L of oleic acid was added to the medium,the highestα-farnesene titer in the shake flask was about 1.40 g/L[0.32 g/g(dry cell weight,DCW)],which was 3.1 times of that in the starting strain F1.For the first time,P.pastoris was used as a chassis microbial cell to produceα-farnesene and the results provide a new idea for the heterologous biosynthesis of other sesquiterpenes.