State Key Laboratory of Microbial Technology, Shandong University, No. 72 Binhai Road, Qingdao, 266237, People's Republic of China.
Environment Research Institute, Shandong University, Qingdao, 266237, People's Republic of China.
Microb Cell Fact. 2021 Jan 7;20(1):7. doi: 10.1186/s12934-020-01500-3.
The sesquiterpene germacrene A is a direct precursor of ß-elemene that is a major component of the Chinese medicinal herb Curcuma wenyujin with prominent antitumor activity. The microbial platform for germacrene A production was previously established in Saccharomyces cerevisiae using the germacrene A synthase (LTC2) of Lactuca sativa.
We evaluated the performance of LTC2 (LsGAS) as well as nine other identified or putative germacrene A synthases from different sources for the production of germacrene A. AvGAS, a synthase of Anabaena variabilis, was found to be the most efficient in germacrene A production in yeast. AvGAS expression alone in S. cerevisiae CEN.PK2-1D already resulted in a substantial production of germacrene A while LTC2 expression did not. Further metabolic engineering the yeast using known strategies including overexpression of tHMGR1 and repression of squalene synthesis pathway led to an 11-fold increase in germacrene A production. Site-directed mutagenesis of AvGAS revealed that while changes of several residues located within the active site cavity severely compromised germacrene A production, substitution of Phe23 located on the lateral surface with tryptophan or valine led to a 35.2% and 21.8% increase in germacrene A production, respectively. Finally, the highest production titer of germacrene A reached 309.8 mg/L in shake-flask batch culture.
Our study highlights the potential of applying bacterial sesquiterpene synthases with improved performance by mutagenesis engineering in producing germacrene A.
倍半萜 germacrene A 是 β-榄香烯的直接前体，β-榄香烯是具有显著抗肿瘤活性的中国药用植物莪术的主要成分。先前已在酿酒酵母中建立了 germacrene A 生产的微生物平台，该平台使用的是莴苣（Lactuca sativa）的 germacrene A 合酶（LTC2）。
我们评估了 LTC2（LsGAS）以及其他来自不同来源的 9 种已鉴定或假定的 germacrene A 合酶在生产 germacrene A 方面的性能。发现鱼腥藻（Anabaena variabilis）的合酶 AvGAS 在酵母中生产 germacrene A 的效率最高。单独在酿酒酵母 CEN.PK2-1D 中表达 AvGAS 已经导致大量 germacrene A 的产生，而表达 LTC2 则没有。通过已知策略（包括过表达 tHMGR1 和抑制角鲨烯合成途径）进一步对酵母进行代谢工程改造，导致 germacrene A 的产量增加了 11 倍。AvGAS 的定点突变表明，尽管位于活性腔内部的几个残基的变化严重损害了 germacrene A 的产生，但将位于侧表面的苯丙氨酸 23 替换为色氨酸或缬氨酸分别导致 germacrene A 的产量增加了 35.2%和 21.8%。最后，在摇瓶分批培养中， germacrene A 的最高产量达到了 309.8mg/L。
我们的研究强调了通过诱变工程应用具有改进性能的细菌倍半萜合酶在生产 germacrene A 方面的潜力。
