(620bl) Conversion of Glucose to (2R, 3R)-Tartaric Acid By Sequential Whole-Cell Catalyzed Oxidation and Chemical Catalysis

Abstract Development of bio-refineries has recently attracted increasing attention as a means to provide sustainable alternative solutions to reducing the dependence on petroleum resources and preventing environmental deterioration. (2R, 3R)-tartaric acid, an important natural existing hydroxyl carboxylic acid, is mainly used in pharmacy, food and dye industry. Currently, (2R, 3R)-tartaric acid on the market is produced by traditional technology from the cream of tartar or stereospecific hydrolysis of the cis-epoxysuccinic acid, derived from maleic anhydride, by cis-epoxysuccinate hydrolase. However, the former method is limited by the availibity of raw material and the latter is dependent on a relatively expensive petrochemical material. Therefore, new processes for the economical preparation of (2R, 3R)-tartaric acid from carbohydrate or renewable resource would be much more attractive. It is reported that glucose can be partially oxidized to 5-keto-gluconic acid (5-KGA) by several microbial cells, and then 5-KGA can be further transferred to (2R, 3R)-tartaric acid by chemical catalysis. But the efficiency of this process is still unsatisfactory. To obtain a robust whole-cell biocatalyst, Gluconobacter oxydans DSM2343 was metabolically engineered to improve the production of 5-KGA. First, the GOX1231 and GOX1081 genes, encoding gluconate-2 dehydrogenase and pyruvate decarboxylase, respectively, were eliminated via a clean deletion system to obtain the recombiant strain G. oxydans ZJU2. Then, the sorbitol dehydrogenase gene (GOX0855-0856, sldAB) was overexpressed in G. oxydans ZJU2 under a carefully selected promoter. The resulting strain, G. oxydans ZJU2/pB-P₀₁₆₉-sldAB, produced 673 mM (about 131 g/L) of 5-KGA with a productivity of 2.08 g/(L·h) in 15 L fermentor. Further investigation to the metabolic pathway reveals that the electron transferring chain and its related coenzyme could be the crucial factors that hindered the conversion of gluconic acid to 5-KGA in this oxidation fermentation process. Hence, the gene cluster for the biosynthesis of coenzyme PQQ, pqqABCDE (GOX0983-0987), as well as tldD (GOX1104) gene, related to PQQ synthesis, and cyoBACD (GOX1911-1914) gene for terminal ubiquinol cytochrome bo₃ oxidase were fused into pUCpr plasmid and transferred into G. oxydans ZJU2/pB-P₀₁₆₉-sldAB. The resultant recombinant strain G. oxydans ZJU2/pB-P₀₁₆₉-sldAB/pUCpr-P₀₁₆₉-pqqABCDE-tldD-P₀₁₆₉-cyoBACD could produce 162 g/L 5-KGA within 64 h by using fed-batch fermentation and the productivity was 2.53 g/(L·h). At last, the transformation of 5-KGA to (2R, 3R)-tartaric acid was investigated with various transition metal catalysts in alkaline solution buffer in shaking flasks. After 72 h reaction, the molar conversion ratio and the selectivity with Cu²⁺ as catalyst were 74.58% and 76.18%, respectively. The reaction mechanism of 5-KGA to (2R, 3R)-tartaric acid by Cu²⁺ was also discussed.