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  • Two key enzymes play important roles in the bioconversion of

    2019-11-05

    Two key enzymes play important roles in the bioconversion of xylitol from d-arabitol, namely d-arabitol dehydrogenase (ArDH) and NADH-dependent xylitol dehydrogenase (XDH), respectively (Qi et al., 2014). In an earlier study, we developed a high xylitol yield strain by expressing xdh in E. coli BL21 from Gluconobacter oxydans CGMCC1.49, where the mixed culture of recombinant and G. oxydans strains produced 0.83 g/g of xylitol from d-arabitol in the presence of exogenous NADH (Qi et al., 2016). In another study, we have constructed two recombinant strains, namely BL21-xdh and BL21-ardh that contained novel xdh and ardh, respectively, from Gluconobacter sp. JX-05 and produced 26.1 g/L of xylitol with a yield of 0.87 g/g in a mixed culture of both strains using exogenous NADH (Qi et al., 2017). However, the recombinant strains developed in the above studies were devoid of system of regenerating NADH by the PPM-18 mg themselves even though sufficient supply of NADH is required for the activity of XDH during bioconversion of xylulose to xylitol. It has resulted the requirement of adding a good quantity of exogenous NADH, which does not offer a cost-effective bioconversion system. Therefore, a cofactor regeneration system in the recombinant cell factory is very important for enhanced xylitol yield from d-arabitol and co-factor self-sufficiency of whole cell biocatalysis (Jo et al., 2015, Wang et al., 2017a). One of the most convenient ways to produce indigenous NADH by the cells is the introduction of adh in the cell factory along with the addition of a co-substrate, such as ethanol (Weckbecker et al., 2010, Zhou et al., 2012). Therefore, a recombinant E. coli BL21 (DE3) was developed in this study by cloning and expressing a novel adh from G. thailandicus CGMCC1.3748 that generated the recombinant BL21-adh strain. Likewise, a novel xdh was also cloned from G. thailandicus to construct the recombinant BL21-xdh. Both adh and xdh strains were studied for whole cell co-biotransformation of d-arabitol along with the original G. thailandicus strain in single and mixed cultures for xylitol production.
    Materials and methods
    Results and discussion
    Conclusion In this study, the novel xdh and adh genes were successfully cloned and expressed to develop recombinant strains. The whole cell co-biotransformation of d-arabitol using the mixed cultures of recombinant and original strains produced enhanced amount of xylitol without using any exogenous NADH supply. The concentration and yield of xylitol produced by the recombinant strains in this study are nicely comparable with those reported in the previous studies using the strains that required external NADH for converting d-arabitol. Therefore, strains with self-regenerating system of NADH could be considered as the promising biological agent for biotransformation of d-arabitol into xylitol.
    Introduction Escherichia coli is a facultative heterotroph microorganism that grows well under both aerobic and anaerobic conditions. The mitochondrial pyruvate dehydrogenase (PDH) complex regulates the critical step in carbohydrate utilization, namely the conversion of pyruvate to acetyl-CoA and NADH [1,2]. Genes encoding the PDH complex have been characterized in E. coli. The three components of the complex are encoded by a single operon that includes a regulatory gene (pdhR) and the structural genes, aceE (pyruvate dehydrogenase, E1), aceF (dihydrolipoamide acetyltransferase, E2), and lpd (dihydrolipoamide dehydrogenase, E3) [3]. Although PDH is significant for aerobic growth of the bacteria, this activity can also be detected in cell extracts of E. coli when grown under anaerobic conditions. While, PDH activity is normally deficient in vivo of E. coli under anaerobic conditions. During aerobic growth, the NADH generated in glycolysis is ultimately oxidized by oxygen whereas the organic compounds generated from glycolysis act as electron acceptors to keep redox balance and maintain bacterial growth under anaerobic conditions. Therefore the intracellular [NADH]/[NAD+] ratio (∼ 0.75) of an anaerobic cell is much higher than that (∼0.03) of an aerobic cell because of differences in electron acceptors between the two growth patterns [4]. In anaerobic E. coli cultures, PDH activity is either very low or undetectable due to inhibition by high level of NADH [5,6]. Further study has verified that NADH sensitivity of the PDH complex resides in the dihydrolipoamide dehydrogenase (LPD) component as only this enzyme interacts with NAD+ as a substrate [7].