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.

中文翻译：

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通过随机诱变在大肠杆菌中定向进化甲羟戊酸激酶以改善番茄红素†

番茄红素是一种萜类色素，在食品和医药领域有多种应用。微生物宿主的代谢工程表明，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，使用大肠杆菌中的定向进化策略来优化酿酒酵母的活性MK。使用三轮易错PCR；筛选依赖番茄红素的颜色反应的发展；和组合位点特异性饱和诱变，确定了三种活性增强突变：V13D、S148I 和 V301E。V13D 在 MK 催化中心附近，在与附近的 Arg-248 形成盐桥的 β 折叠中。S148I位于α-螺旋盖中，提高了α-螺旋的稳定性。V301E 可能通过影响其二级结构来增加 MK 折叠。与野生型MK的K m ( RS )-甲羟戊酸和K cat _{( RS )} -_甲羟戊酸相比，纯​​化突变MK的K _{m ( RS )-甲羟戊酸}降低了74%与野生型相比提高了 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。