Production of 5-ketogluconic acid using recombinant strains of Gluconobacter oxydans

Abstract: 5-Keto-D-gluconic acid (5-KGA) is a valuable compound that has a wide range of applications in the chemical and food industries and is commonly used as a precursor for manufacturing tartaric acid (TA). Industrial production of 5-KGA through chemical approaches was practiced in the early 1920’s and was dropped immediately due to several drawbacks because of its complicated process that resulted in lower 5-KGA yield and higher accumulation of impure gluconic acid. As a promising solution for manufacturing 5-KGA, the biological approach through the mechanism of microbial fermentation was found to be cost-efficient with higher productivity of 5-KGA. Gluconobacter oxydans (G. oxydans) belonging to the Acetobacteraceae family was commonly preferred due to their ability to perform incomplete oxidation suitable for industrial production. Compared to wild-type, over-expression of polyol dehydrogenase in recombinant strains of G. oxydans has proven to be successful in enhancing the production of 5-KGA and eliminating the co-production of other undesired products during fermentation. The aim of this study was to increase the production of 5-KGA in G. oxydans strain DSM 50049 carrying plasmid pBBR1MCS-5 integrated with the gene encoding alcohol dehydrogenase (XADH) from Xanthomonas campestris. The recombinant G. oxydans strain was cultivated at different culture parameters for determining optimal conditions for maximal activity and expression of XADH. During cultivation in glucose medium, the recombinant G. oxydans generated a maximum yield of ~ 0.7 g 5-KGA / g glucose at 25°C, pH-6.0 along with co-production of 2-KGA. The results obtained from SDS-PAGE, activity assay and resting cell reaction were found inconclusive to prove the expression of XADH using low-copy number plasmid, pBBR1MCS-5. Furthermore, the formation of target protein bands from the insoluble cell fractions of BL21 (DE3) strain carrying pET30a vector was found satisfying to continue further studies on the expression of XADH. Results have shown that cultivation parameters affect the performance of the enzymes during oxidation which lends support the idea of developing different stages of pH during fermentation for increasing the productivity of 5-KGA. If the yields of 5-KGA were improved after optimizing the strain and culture conditions for maximal XADH activity, further studies will be focused on large-scale cultivation and developing framework for developing strategies to improve 5-KGA productivity.