Directed evolution of mevalonate kinase inEscherichia coliby random mutagenesis for improved lycopene

Abstract

a 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 ratelimiting 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 b-sheet that forms a salt-bridge with nearby Arg-248. S148I was located in the a-helix lid and improved the stability of the a-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.

Modular engineering for microbial production of carotenoids

“…The expression of mevalonate kinase (MK) can be strongly feedback inhibited by GPP and FPP (Dorsey and Porter, 1968; Hinson et al., 1997). Directed evolution (Anthony et al., 2009) and overexpression (Chen et al., 2018b) of MK has been applied to increase yields of IPP downstream products. In addition, several feedback-resistant MKs have been identified and characterized (Kazieva et al., 2017; Primak et al., 2011), which can be expressed in other microorganisms for carotenoid overproduction.…”

Alternative metabolic pathways and strategies to high-titre terpenoid production inEscherichia coli

“…An E. coli strain harbouring the S. cerevisiae M5K V13D/S148I/V301E variant produced 1.43 g L À1 lycopene, a titre that is 2.4-fold higher compared to the strain containing the wild-type homolog. 77 Protein engineering has also been used to generate a catalytically superior S. cerevisiae IDI variant (L141H/Y195F/W256C), which improves lycopene production 1.8-fold, reaching 1.2 g L À1 . 78 Moreover, type II IDIs recently identied in some archaea, [79][80][81] Gram-positive bacteria 82,83 and cyanobacteria 84 might also be benecial in terpenoid production using the MVA pathway when compared to type I IDIs.…”

“…This might involve the engineering of enzymes that are more resistant to feedback control, or enzymes with improved catalytic efficiency, or new specicities towards desired target activities/products. 77,137,168,173,186,198,257,285,286 The use of alternative and articial pathways with this ever-expanding enzyme resource and pathway engineering tools should extend current capabilities, thereby allowing high-titre production of a wide range of terpenoids in E. coli and other microbial species.…”

Enhanced Lycopene Production in Escherichia coli by Expression of Two MEP Pathway Enzymes from Vibrio sp. Dhg

“…Some carotenoids are known to function as membrane protective anti-oxidants [2,3], which has led to a high demand for carotenoids for medical and pharmaceutical applications [4,5]. Lycopene is one of the most highly valuable carotenoids owing to its potent antioxidant [6] and disease prevention properties [7,8]. The lycopene market is growing at 3.5 percent annually and is expected to exceed 133 million dollars in 2023, thus supporting high demand and value [9,10].…”