Abstract
2,5-diketo-D-gluconic acid reductase (2,5-DKG reductase) catalyses the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-keto-L-gulonic acid (2-KLG), a direct precursor (lactone) of L-ascorbic acid (vitamin C). This reaction is an essential step in the biocatalytic production of the food supplement vitamin C from D-glucose or D-gluconic acid. As 2,5-DKG reductase is usually produced recombinantly, it is of interest to establish an efficient process for 2,5-DKG reductase production that also satisfies food safety requirements. In the present study, three recently described food grade variants of the Lactobacillales based expression systems pSIP (Lactobacillus plantarum) and NICE (Lactococcus lactis) were evaluated with regard to their effictiveness to produce 2,5-DKG reductase from Corynebacterium glutamicum. Our results indicate that both systems are suitable for 2,5-DKG reductase expression. Maximum production yields were obtained with Lb. plantarum/pSIP609 by pH control at 6.5. With 262 U per litre of broth, this represents the highest heterologous expression level so far reported for 2,5-DKG reductase from C. glutamicum. Accordingly, Lb. plantarum/pSIP609 might be an interesting alternative to Escherichia coli expression systems for industrial 2,5-DKG reductase production.
Highlights
The bacterial enzyme 2,5-diketo-D-gluconic acid reductase (2,5-didehydrogluconate reductase; 2,5-DKG reductase; EC 1.1.1.274) is an NAD(P)(H)-dependent oxidoreductase assigned to the aldo-keto reductase (AKR) family (Ellis 2002). 2,5-DKG reductase 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), a key intermediate in the production of L-ascorbic acid (Anderson et al 1985)
As judged by SDSPAGE of the crude extracts (Figure 2), 2,5-DKG reductase could successfully be expressed with the three food–grade expression systems Nisin controlled gene expression (NICE), pSIP603 and pSIP609
The majority of the so far published studies concerned with the heterologous expression of dkr genes (Corynebacterium sp.) were focussed on 2,5-DKG reductase optimization by site-directed mutagenesis and the kinetic characterisation of the obtained mutants after expression in E. coli, rather than the optimization of expression yields (Banta et al 2002a, b, Powers 1996, Sanli et al 2004, Banta and Anderson 2002)
Summary
The bacterial enzyme 2,5-diketo-D-gluconic acid reductase (2,5-didehydrogluconate reductase; 2,5-DKG reductase; EC 1.1.1.274) is an NAD(P)(H)-dependent oxidoreductase assigned to the aldo-keto reductase (AKR) family (Ellis 2002). 2,5-DKG reductase 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), a key intermediate in the production of L-ascorbic acid (Anderson et al 1985). An efficient hybrid process for the production of 2-KLG comprising the conversion of D-glucose or D-gluconic acid into 2,5-DKG by Pectobacter cypripedii HEPO1 (DSM 12939) and the subsequent reduction of 2,5-DKG to 2-KLG using 2,5-DKG reductase from Corynebacterium glutamicum was previously developed in our laboratory (Pacher et al 2008). Examples of true food grade host/ vector combinations have been presented and applied using the expression systems Lactobacillus plantarum / pSIP (Nguyen et al 2011a) and Lactococcus lactis / NICE (Maischberger et al 2010) In both systems, antibiotic resistance marker genes have been replaced by selection markers (pSIP: alr, alanine racemase gene; NICE: lacF, gene encoding the soluble carrier enzyme IIA of the lactose specific phosphotransferase system) complementing corresponding gene deletions in the host chromosomes
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