2,5-Diketo-gluconic acid reductase from Corynebacterium glutamicum: Characterization of stability, catalytic properties and inhibition mechanism for use in vitamin C synthesis
Highlights
► 2,5-Diketo-gluconic acid (2,5-DKG) reductase activity was found in 9 bacterial strains. ► 2,5-DKG reductase synthesizes the immediate precursor of vitamin C. ► Corynebacterium glutamicum 2,5-DKG reductase was expressed in high yield and characterized. ► The catalytic mechanism and inhibition kinetics were elucidated to guide enzyme and process engineering.
Introduction
The bacterial enzyme 2,5-diketo-d-gluconic acid reductase (2,5-DKG reductase; 2,5-didehydrogluconate reductase; EC 1.1.1.274) is one of more than 140 members of the aldo-keto reductases (AKRs), an enzyme superfamily of NAD(P)(H)-dependent oxidoreductases [1], [2]. This enzyme catalyses the stereo specific reduction of 2,5-diketo-d-gluconic acid (2,5-DKG) at position C-5 to 2-keto-l-gulonic acid (2-KLG) [3], which is an intermediate that can be transformed into l-ascorbic acid (vitamin C) by a single chemical step [4].
The first microorganisms available for conversion of 2,5-DKG to 2-KLG were isolated from soil and sewage by Sonoyama and colleagues in the 1970s [5], [6]. These 2-KLG producing strains belong to the Brevibacterium, Arthrobacter, Micrococcus, Staphylococcus, Pseudomonas, Bacillus and Corynebacterium genera. In 1987, the conversion of 2,5-DKG to 2-KLG by Corynebacterium sp. was identified as a single catalytic step of 2,5-DKG reductase in the cytosol [7]. To date, only a few enzymes with 2,5-DKG reductase activity have been biochemically characterized. Those include two native DKGRs from a species of Corynebacterium (2,5-DKG reductase A; AKR5C and 2,5-DKG reductase B; AKR5D) [7], [8], two homologous expressed 2,5-DKG reductase from Escherichia coli (YqhE and YafB) [9], [10] and two heterologous expressed 2,5-DKG reductases from uncultured microbes. Two heterologous expressed 2,5-DKG reductases from uncultured microbes have been found by screening environmental DNA expression libraries [11]. Structurally, only 2,5-DKG reductase A from Corynebacterium sp. [12], [13], [14] and its quadruple mutant [15] have been studied. Native 2,5-DKG reductase A from Corynebacterium sp. is a monomeric enzyme (about 34 kDa) composed of eight α-helices and eight parallel β-strands (TIM barrel; (α/β)8), similar to most microbial AKRs [1], [12]. The reduced form of pyridine nucleotide NADP(H) is bound to the C-terminal face of the barrel. The absence of a canonical Rossman fold in active site set AKRs apart from numerous dehydrogenases [16], [17]. Mutation studies of residues in the coenzyme binding site and substrate binding pocket in the apo and coenzyme-bound form of 2,5-DKG reductase show that binding of NADPH causes communicated and coordinated structural changes into these regions [14].
2,5-DKG reductase is of high interest for the biocatalytic production of the key intermediate 2-KLG by the 2,5-diketo-d-gluconic acid pathway from d-glucose via d-gluconate, 2-keto-d-gluconate and 2,5-diketo-d-gluconate. Vitamin C can be obtained through transformation and refining of 2-KLG [18]. Sonoyama et al. [19] invented a two-stage fermentation process for 2-KLG production where glucose is oxidized to 2,5-DKG by a mutated Erwinia sp. and is then reduced to 2-KLG by a mutant strain of Corynebacterium sp. The second step reaction is catalyzed by NADPH dependent 2,5-DKG reductase. A tandem fermentation process to produce 2-KLG from gluconic acid by using co-immobilized cells of Gluconobacter oxydans and Corynebacterium sp. has also been suggested [20]. Also the genetically engineered Erwinia strains; Erwinia herbicola [3] and E. citreus [21], which naturally accumulate 2,5-DKG from d-glucose have been employed. The gene encoding for 2,5-DKG reductase was cloned from Corynebacterium sp. into the above mentioned Erwinia strains, allowing an elegant one-organism fermentation of 2-KLG directly from d-glucose. According to Powers [22], the transport of 2,5-DKG into, and the diffusion of 2-KLG out of the 2-KLG synthesizing cells, appear to be the rate-limiting steps. Based on this, Genencor established an in vitro biocatalytic four steps method to produce 2-KLG from d-glucose [23], [24]. Four enzymes: NADP+ dependent glucose dehydrogenase (GDH) from Thermoplasm acidophilum, NADPH dependent 2,5-DKG reductase from Corynebacterium sp., gluconate dehydrogenase and 2-keto-d-gluconate dehydrogenase (both from permeabilized, modified Pantoea citrea cells with glucose dehydrogenase activity) are involved in the continuous conversion of d-glucose. The first two, soluble enzymes are exogenously added and regenerate the coenzyme in situ.
Nowadays a remarkable part of vitamin C industrial production is performed in a two-step fermentation process [4], [25], [26], but the traditional Reichstein process [27] which involves several environmentally hazardous chemical and energy consuming steps is still utilized for vitamin C synthesis (Fig. 1, process route A). In our previous published work the synthesis of 2-KLG from d-glucose was established (process route B) [28]. First, d-glucose is converted by Pectobacter cypripedii strain HEPO1 (DSMZ 12393) into 2,5-DKG, which is then enzymatically converted with NADPH-dependent 2,5-DKG reductase from Corynebacterium glutamicum to 2-KLG. NADPH is regenerated in situ by GDH from Bacillus sp. and d-glucose in the second biocatalytic step. Using this bi-enzymatic system, 2,5-DKG is completely reduced to 2-KLG. Here, we describe the screening and cloning of a 2,5-DKG reductase from C. glutamicum DSM 20301, its expression in E. coli and biochemical characterization in regard to process relevant properties of the recombinant enzyme. A detailed kinetic study of the catalytic mechanism of 2,5-DKG reductase and its inhibition by cations and anions provides mechanistic insights for further enzyme and process engineering.
Section snippets
Materials
Salts, acids and bases for enzyme assays and media preparation were purchased from commercial suppliers at the highest level of purity possible. Media components were obtained from Sigma–Aldrich, Roth and Merck. 2,5-Diketo-d-gluconic acid (2,5-DKG) was produced by fermentation of P. cypripedii as previously described [28] and isolated from the culture broth by methanol precipitation. It was further purified by liquid chromatography, using isocratic elution with ultrapure water on Amberlite
Screening
Twenty-two bacterial strains were screened in shaking flask cultures for the ability to reduce 2,5-DKG to 2-KLG (Table 1). Different optimal growth conditions (22–37 °C, 6 different media, pH 6.0–7.3, for detailed information see: Supplementary Tables S1 and S2) resulted in different specific growth rates. Therefore, the cultivation time to reach maximum cell densities was determined in preliminary experiments (20–50 h) and three classes of organisms were defined, which were harvested after 24,
Conclusions
2,5-DKG reductase from C. glutamicum can be recombinantly produced by E. coli in high yields and easily purified by affinity chromatography. Its physical and catalytic properties make the enzyme ideally suited for the application in the synthesis of 2-KLG, a precursor of l-ascorbic acid production. The investigated inhibition mechanism of 2,5-DKG reductase by anions and cations is of high relevancy for further process engineering. Having the recombinant enzyme in hands, powerful protein
Acknowledgements
The authors thank Mr. Dominik Jeschek for superb DSC measurements and MSc. Shima Khazaneh for careful reading and discussions.
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2018, Biotechnology AdvancesCitation Excerpt :In the NTFR, D-glucose is first converted into 2,5-diketo-gluonic acid (2,5-DKG) by glucose dehydrogenase, gluconate dehydrogenase and 2-keto-D-gluconate dehydrogenase by Erwinia sp., (e.g. Erwinia herbicola ATCC 21988) in the first fermentation process (Anderson et al., 1985; Grindley et al., 1988). In the second fermentation step, 2,5-DKG is converted into 2-KLG by 2,5-DKG reductase in Corynebacterium sp. (Kaswurm et al., 2013a, 2012; Miller et al., 1987; Sanli et al., 2004) (Fig. 4). However, 2,5-DKG can be completely destroyed during sterilisation prior to the second fermentation step because it is highly thermally unstable.
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