Directed evolution of mevalonate kinase in Escherichia coli by random mutagenesis for improved lycopene.
Directed evolution of mevalonate kinase in Escherichia coli by random mutagenesis for improved lycopene.
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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
DOI：
10.1039/c8ra01783b
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发表时间：
2018-04-18
期刊：
影响因子：
3.9
通讯作者：
Liu, Huizhou
中科院分区：
文献类型：
--
作者：
Chen, Hailin;Liu, Changqing;Li, Meijie;Zhang, Haibo;Xian, Mo;Liu, Huizhou
番茄红素是一种萜类色素，在食品、医药等领域有着广泛的应用。微生物代谢工程研究表明，甲氧戊酸激酶(MK，EC2.7.1.366)是番茄红素合成途径中的限速酶之一。本研究采用定向进化策略对酿酒酵母MK的活性进行了优化。通过三轮容易出错的聚合酶链式反应、番茄红素依赖的显色反应的筛选和结合位点特异性饱和突变，鉴定出三个活性增强突变：V13D、S148I和V301E。V13D位于MK催化中心附近的β-Sheet中，与附近的Arg-248形成盐桥。S148I位于α-螺旋的盖子上，提高了α-螺旋的稳定性。V301E可能通过影响其二级结构而增加MK折叠。纯化突变体MK的Km(RS)-甲戊酸比野生型MK的Km(RS)-甲戊酸降低了74%，Kcat(RS)-甲戊酸比野生型提高了26%。发酵实验表明，突变株MK的番茄红素产量是野生型MK的2.4倍。
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 Km (RS)-mevalonate of purified mutant MK decreased by 74% compared with the Km (RS)-mevalonate of the wild-type MK, and the Kcat (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.
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影响因子：
11.4
作者：
Choudhari, Sheetal M.;Ananthanarayan, Laxmi;Singhal, Rekha S.
通讯作者：
Singhal, Rekha S.
影响因子：
5.1
作者：
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15.3
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通讯作者：
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