Enhancement of the catalytic activity of Isopentenyl diphosphate isomerase (IDI) from Saccharomyces cerevisiae through random and site-directed mutagenesis

Abstract

BackgroundLycopene is a terpenoid pigment that has diverse applications in the food and medicine industries. A prospective approach for lycopene production is by metabolic engineering in microbial hosts, such as Escherichia coli. Isopentenyl diphosphate isomerase (IDI, E.C. 5.3.3.2) is one of the rate-limiting enzymes in the lycopene biosynthetic pathway and one major target during metabolic engineering. The properties of IDIs differ depending on the sources, but under physiological conditions, IDIs are limited by low enzyme activity, short half-life and weak substrate affinity. Therefore, it is important to prepare an excellent IDI by protein engineering.ResultsDirected evolution strategy (error-prone PCR) was utilized to optimize the activity of Saccharomyces cerevisiae IDI. 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: L141H, Y195F, and W256C. L141H, located near the active pocket inside the tertiary structure of IDI, formed a hydrogen bond with nearby β-phosphates of isopentenylpyrophosphate (IPP). Phe-195 and Cys-256 were nonpolar amino acids and located near the hydrophobic group of IPP, enlarging the hydrophobic scope, and the active pocket indirectly. Purified IDI was characterized and the result showed that the Km of mutant IDI decreased by 10% compared with Km of the parent IDI, and Kcat was 28% fold improved compared to that of the original IDI. Results of a fermentation experiment revealed that mutant IDI had a 1.8-fold increased lycopene production and a 2.1-fold increased yield capacity compared to wild-type IDI.ConclusionWe prepared an engineered variant of IDI with improved catalytic activity by combining random and site directed mutagenesis. The best mutants produced by this approach enhanced catalytic activity while also displaying improved stability in pH, enhanced thermostability and longer half-life. Importantly, the mutant IDI could play an important role in fed-batch fermentation, being an effective and attractive biocatalyst for the production of biochemicals.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0913-z) contains supplementary material, which is available to authorized users.

Microbial proteases: ubiquitous enzymes with innumerable uses

“…(root-knot nematode) Actinomycetes Saccharomonospora viridis strain Hw G550 (Darwesh et al 2019 ) Alkaline protease ↓ nematode reproduction, no. of females, eggs, galls, egg-masses, larvae, and developmental stages Meloidogyne incognita (root-knot nematode) Fungus Lecanicillium psalliotae (Yang et al 2019 ) Serine protease Ver112 Degradation of cuticular constituents and egg shells Radopholus similis (burrowing nematode) Genetically modified Pseudomonas fluorescens pf36 (p4MCS & p4Tn5) (Chen et al 2018a , b ) Protease PASE4 Nematostatic (↓ growth) and nematicidal Meloidogyne incognita (root-knot nematode) Root bacteria in tomato plants Enterobacter asburiae HK169 (Oh et al 2018 ) Collagenases Chitinases Serine proteases Nematicidal ↓ gall formation Meloidogyne ethiopica and Xiphinema index Seven Rhizobacteria strains (Aballay et al 2017 ) Exoenzymes: Collagenases Chitinases Serine proteases Lipases Nematicidal ↓ egg hatching Caenorhabditis elegans (soil nematode) Soil bacterium Bacillus thuringiensis (Peng et al 2016 ) Collagenase metalloproteases Degradation of intestine of host nematode C. elegans and Meloidogyne incognita Alcaligenes faecalis ZD02 (Ju et al 2016 ) Extracellular serine protease Nematostatic (↓ growth) and nematicidal …”

“…Site-directed mutagenesis is done generally to impart the proteins with desirable properties like improved catalysis or activity, stability like thermostability, substrate specificity, solubility, expression, etc. while also being utilized for immobilization of enzymes for the targeted industrial application (Baweja et al 2016a , b ; Chen et al 2018a , b ).…”

Modular engineering for microbial production of carotenoids

“…Type 2 Idi (mainly exists in plants, some gram-positive eubacteria and archaea) tends to perform better than the type 1 (has a conserved cysteine in the NxxCxHP consensus sequence and a conserved glutamate in the ExE consensus sequence that encode for the active site and mainly exists in plants, mammals, some Gram-positive eubacteria and most Gram-negative eubacteria) for isoprenoid production in E. coli (Rad et al., 2012). In addition, directed evolution of idi from S. cerevisiae increased its activity and affinity, leading to a 1.8-fold increase of lycopene production in E. coli (Chen et al., 2018a).…”

Fermentation Strategies for Production of Pharmaceutical Terpenoids in Engineered Yeast

“…In another study, alkaline medium with pH ranging from 7 to 8 enhanced farnesol accumulation by S. cerevisiae [ 100 ]. It was also reported that the optimum pH for the wild type S. cerevisiae , and mutant strain in isopentenyl diphosphate isomerase activity (one of the important rate-limiting enzymes in terpenoid production), was 7.5 [ 101 ]. Alkaline pH might enhance the synthesis and solubility of terpenoids in the cultivation medium, but growth and biomass accumulation might be low, not allowing high cell density cultivation as desirable in industrial terpenoids production.…”