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

English. 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.

English. From the beginning of the 21st century, the production and consumption of industrially manufactured chemicals tend to rise yearly, contributing billions of dollars to this industry. With a focus on manufacturing chemicals in a sustainable and eco-friendly way, industries began to explore the possibilities of utilizing microbial fermentation as a replacement for existing traditional methods, mostly constitutes of using harmful chemicals and harsh manufacturing processes. 5-Keto-D-gluconic acid (5-KGA) is an unique chemical compound used as a key ingredient in the manufacturing of manufacturing L (+) tartaric acid, Rochelle salt, insecticides, wool dyes, etc. Manufacturing of 5-KGA was initially performed using chemical synthesis and dropped mainly due to lower yield and high operational costs. Biological routes for producing 5-KGA were originally achieved through microbial fermentation in the late 1950s using Acetobacter species, which was later replaced by the Gluconobacter species primarily for its wide industrial applications. Gluconobacter oxydans (G. oxydans) is a gram-negative, non-motile bacterium belonging to the family Acetobacteraceae having an optimum growth temperature of 25-30°C and pH 5.5-6.0. These obligate aerobes require oxygen to survive and can cause incomplete oxidation of alcohols, sugars, and acids which causes a significant production of gluconic acid from D-glucose, L-sorbose from D-sorbitol, acetaldehyde from ethanol, and D-galactonic acid from D-galactose. During fermentation process, the wild strains of G. oxydans oxidize D-glucose to form 5-KGA and other undesirable co-products including 2-keto-D-gluconic acid (2-KGA) and 2,5-diketo-D-gluconic acid (2,5-DKGA). To prevent the formation of undesirable products and to enhance the productivity of 5-KGA, several studies were performed with a focus on developing recombinant strains of G. oxydans. Using gene technology, recombinant plasmids were designed to knock out genes responsible to produce 2-KGA and 2,5-DKGA. For improving the 5-KGA yield in G. oxydans, membrane-bound major polyol dehydrogenase enzymes are cloned and expressed into host organisms during transformation. This study was focused on expressing alcohol dehydrogenase enzymes from Xanthomonas campestris in the recombinant strains of G. oxydans and Escherichia coli. As part of gene expression, G. oxydans cells were cultivated at different pH and temperatures to understand the activity and expression rate of alcohol dehydrogenase. During cultivation, the concentration of D-glucose, gluconic acid and 5-KGA present in the fermentation broth were constantly monitored to assess the activity of membrane-bound enzymes.