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首页> 外文期刊>RSC Advances >Directed evolution of mevalonate kinase in Escherichia coli by random mutagenesis for improved lycopene
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Directed evolution of mevalonate kinase in Escherichia coli by random mutagenesis for improved lycopene

机译：通过改进的番茄红素的随机诱变指导甲羟戊酸激酶在大肠杆菌中的进化

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摘要

Lycopene is a terpenoid pigment that has diverse applications in the fields of food and medicine. Metabolic engineering in microbial hosts has shown that mevalonate kinase (MK, EC2.7.1.366) is one of the rate-limiting enzymes in the lycopene synthetic pathway. In this study, a directed evolution strategy in Escherichia coli was used to optimize the activity of Saccharomyces cerevisiae MK. Using three rounds of error-prone PCR; screening the development of a lycopene-dependent color reaction; and combinatorial site-specific saturation mutagenesis, three activity-enhancing mutations were identified: V13D, S148I, and V301E. V13D was near the MK catalytic center, in the β-sheet that forms a salt-bridge with nearby Arg-248. S148I was located in the α-helix lid and improved the stability of the α-helix. V301E may increase MK folding by influencing its secondary structure. The K _(m ( RS )-mevalonate) of purified mutant MK decreased by 74% compared with the K _(m ( RS )-mevalonate) of the wild-type MK, and the K _(cat ( RS )-mevalonate) was improved by 26% compared with wild type. Fermentation experiments revealed that lycopene production of the mutant MK increased 2.4-fold compared with wild-type MK.
机译：番茄红素是一种萜烯类颜料，在食品和医药领域具有多种应用。微生物宿主中的代谢工程表明，甲羟戊酸激酶（MK，EC2.7.1.366）是番茄红素合成途径中的限速酶之一。在这项研究中，在大肠杆菌中的定向进化策略被用来优化酿酒酵母MK的活性。使用三轮易错PCR;筛选番茄红素依赖性显色反应的发展;结合位点特异性饱和诱变，鉴定出三个增强活性的突变：V13D，S148I和V301E。 V13D在MK催化中心附近，在β-折叠中与附近的Arg-248形成盐桥。 S148I位于α螺旋盖中，提高了α螺旋的稳定性。 V301E可能会通过影响其二级结构来增加MK折叠。纯化的突变体MK的K _（m（RS）-甲羟戊酸）与野生型MK的K _（m（RS）-甲羟戊酸）和K _（cat（RS）-甲羟戊酸）相比降低了74％ ）比野生型提高了26％。发酵实验表明，与野生型MK相比，突变型MK的番茄红素产量增加了2.4倍。

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