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
以促进酶和途径工程，一个选择是针对在酿酒酵母改进的倍半萜滴度的发展。结果发现，以保护对酵母非离子表面活性剂如吐温20（T20）的破坏性行动。两株酵母的实验采用的竞争，其中之一使得两倍甜没药烯作为其他，表明，生长在T20的存在提供了足够的选择压力，以丰富的高滴度应变以形成人口的97％。在此之后，各种方法被用于通过传代培养在T20的存在诱变的甜没药烯合酶（BIS）的编码序列，加上选择。诱变针对BIS活动现场并没有屈服在红没药烯效价的提高，虽然被发现突变体制作的混合物另一名候选人生物燃料。基于证据表明3发现了一个变体，它由超过30％增加特定的生产红没药烯的。由于T20能够丰富一个混合人群，有效地去除产生很少或没有红没药烯菌，我们调查是否它也可以被施加到维持高产物滴度在一个单一较长时间。在滴度高达4倍比生长用十二烷的叠加，用于螯合萜产物培养更高生长于T20的存在下14天产生的甜没药烯文化，和20倍比没有十二烷生长的培养物高。
James Kirby[a];[b]; Minobu Nishimoto[a];[b]; Ruthie W. N. Chow[a]; Venkata N. Pasumarthi[a]; Rossana Chan[a]; Leanne Jade G. Chan[b]; Christopher J. Petzold[b] ; Jay D. Keasling[a];[b];[c];[d]. Use of Nonionic Surfactants for Improvement of Terpene Production in Saccharomyces cerevisiae[J]. Applied and Environmental Microbiology, 2014,80(21): 6685-6693