摘要
To facilitate enzyme and pathway engineering, a selection was developed for improved sesquiterpene titers in Saccharomyces cerevisiae. α-Bisabolene, a candidate advanced biofuel, was found to protect yeast against the disruptive action of nonionic surfactants such as Tween 20 (T20). An experiment employing competition between two strains of yeast, one of which makes twice as much bisabolene as the other, demonstrated that growth in the presence of T20 provided sufficient selective pressure to enrich the high-titer strain to form 97% of the population. Following this, various methods were used to mutagenize the bisabolene synthase (BIS) coding sequence, coupled with selection by subculturing in the presence of T20. Mutagenesis targeting the BIS active site did not yield an improvement in bisabolene titers, although mutants were found which made a mixture of α-bisabolene and β-farnesene, another candidate biofuel. Based on evidence that the 3′ end of the BIS mRNA may be unstable in yeast, we randomly recoded the last 20 amino acids of the enzyme and, following selection in T20, found a variant which increased specific production of bisabolene by more than 30%. Since T20 could enrich a mixed population, efficiently removing strains that produced little or no bisabolene, we investigated whether it could also be applied to sustain high product titers in a monoculture for an extended period. Cultures grown in the presence of T20 for 14 days produced bisabolene at titers up to 4-fold higher than cultures grown with an overlay of dodecane, used to sequester the terpene product, and 20-fold higher than cultures grown without dodecane.
摘要译文
为了促进酶和途径工程，开发了用于改善酿酒酵母中倍半萜滴度的选择。发现候选先进生物燃料α-双酚是保护酵母免受非离子表面活性剂如吐温20（T20）的破坏性作用。在两种酵母菌株之间进行竞争的实验，其中一种酵母菌株产生两倍于另一种的赤霉素，证明在T20存在下的生长提供了足够的选择压力以富集高滴度菌株以形成97％的群体。在此之后，使用各种方法诱变bisabolene合酶（BIS）编码序列，并在T20存在下通过传代培养进行选择。针对BIS活性位点的诱变没有产生白藜芦醇滴度的改善，尽管发现了制备α-二硼烯和β-法呢烯（另一种候选生物燃料）的混合物的突变体。基于证据表明BIS mRNA的3'末端在酵母中可能不稳定，我们随机记录了酶的最后20个氨基酸，并且在T20中选择后，发现了一种变体，其使赤桉烯的特定产量增加了30％以上。由于T20可以富集混合种群，有效去除产生很少或不产生赤霉烯的菌株，我们研究了它是否也可以用于长期单一栽培中维持高产量滴度。在T20存在下培养14天的培养物产生的白藜芦醇的滴度比用十二烷覆盖物生长的培养物高4倍，用于螯合萜烯产物，比没有十二烷的培养物高20倍。
James Kirby, Minobu Nishimoto, Ruthie W. N. Chow, Venkata N. Pasumarthi, Rossana Chan, Leanne Jade G. Chan, Christopher J. Petzold, Jay D. Keasling. Use of Nonionic Surfactants for Improvement of Terpene Production in Saccharomyces cerevisiae[J]. Applied Microbiology, 2014,80(21): 6685-6693