PDF(Pubmed)
Abstract：
Enzyme stability is often a limiting factor in the microbial production of high-value-added chemicals and commercial enzymes. A previous study by our research group revealed that the unstable isoprene synthase from Ipomoea batatas (IspSib) critically limits isoprene production in engineered Escherichia coli. Directed evolution was, therefore, performed in the present study to improve the thermostability of IspSib. First, a tripartite protein folding system designated as lac\'-IspSib-\'lac, which could couple the stability of IspSib to antibiotic ampicillin resistance, was successfully constructed for the high-throughput screening of variants. Directed evolution of IspSib was then performed through two rounds of random mutation and site-saturation mutation, which produced three variants with higher stability: IspSibN397V A476V, IspSibN397V A476T, and IspSibN397V A476C. The subsequent in vitro thermostability test confirmed the increased protein stability. The melting temperatures of the screened variants IspSibN397V A476V, IspSibN397V A476T, and IspSibN397V A476C were 45.1 ± 0.9°C, 46.1 ± 0.7°C, and 47.2 ± 0.3°C, respectively, each of which was higher than the melting temperature of wild-type IspSib (41.5 ± 0.4°C). The production of isoprene at the shake-flask fermentation level was increased by 1.94-folds, to 1,335 mg/L, when using IspSibN397V A476T. These findings provide insights into the optimization of the thermostability of terpene synthases, which are key enzymes for isoprenoid production in engineered microorganisms. In addition, the present study would serve as a successful example of improving enzyme stability without requiring detailed structural information or catalytic reaction mechanisms.IMPORTANCEThe poor thermostability of IspSib critically limits isoprene production in engineered Escherichia coli. A tripartite protein folding system designated as lac\'-IspSib-\'lac, which could couple the stability of IspSib to antibiotic ampicillin resistance, was successfully constructed for the first time. In order to improve the enzyme stability of IspSib, the directed evolution of IspSib was performed through error-PCR, and high-throughput screening was realized using the lac\'-IspSib-\'lac system. Three positive variants with increased thermostability were obtained. The thermostability test and the melting temperature analysis confirmed the increased stability of the enzyme. The production of isoprene was increased by 1.94-folds, to 1,335 mg/L, using IspSibN397V A476T. The directed evolution process reported here is also applicable to other terpene synthases key to isoprenoid production.
摘要：
酶稳定性通常是微生物生产高附加值化学品和商业酶的限制因素。我们的研究小组先前的研究表明，来自Ipomoeabatatas（IspSib）的不稳定异戊二烯合酶严重限制了工程大肠杆菌中的异戊二烯生产。定向进化是，因此,在本研究中进行，以提高IspSib的热稳定性。首先,称为lac\'-IspSib-\'lac的三方蛋白折叠系统，这可以将IspSib的稳定性与抗生素氨苄青霉素抗性结合起来，已成功构建用于高通量筛选变体。然后通过两轮随机突变和位点饱和突变进行IspSib的定向进化，产生了三个具有更高稳定性的变体：IspSibN397VA476V，IspSibN397VA476T,和IspSibN397VA476C。随后的体外热稳定性测试证实了增加的蛋白质稳定性。筛选的变体IspSibN397VA476V的解链温度，IspSibN397VA476T,和IspSibN397VA476C为45.1±0.9°C，46.1±0.7°C,和47.2±0.3°C，分别,均高于野生型IspSib的解链温度（41.5±0.4°C）。摇瓶发酵水平的异戊二烯产量增加了1.94倍，至1,335毫克/升，使用IspSibN397VA476T时。这些发现为优化萜烯合酶的热稳定性提供了见解，是工程微生物中生产类异戊二烯的关键酶。此外,本研究将作为一个成功的例子，提高酶的稳定性，而不需要详细的结构信息或催化反应机制。IspSib的不良热稳定性严重限制了工程大肠杆菌中异戊二烯的生产。称为lac\'-IspSib-\'lac的三方蛋白折叠系统，这可以将IspSib的稳定性与抗生素氨苄青霉素抗性结合起来，首次成功建造。为了提高IspSib的酶稳定性,IspSib的定向进化是通过错误PCR进行的，使用lac\'-IspSib-\'lac系统实现了高通量筛选。获得了具有增加的热稳定性的三个阳性变体。热稳定性测试和解链温度分析证实了酶的稳定性增加。异戊二烯的产量增加了1.94倍，至1,335毫克/升，采用IspSibN397VA476T。此处报道的定向进化过程也适用于对类异戊二烯生产至关重要的其他萜烯合酶。
