Manufacture Of L-(+)-tartaric Acid By Sequential Whole-cell Oxidation And Chemical Catalysis

L-(+)-tartaric acid (L-TA), an important hydroxyl-carboxylic acid chelating agent, is widely used in the pharmaceutical, food, dyes and other industries. Currently, the L-TA 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 availability of raw material and the latter depends on the petrochemical material. In the 21st century, with the depletion of the petrochemical resources and the unstable crude oil price, great attentions have been paid to produce chemicals from renewable biomass feedstock. Therefore, substitute processes for the economical preparation of L-TA from carbohydrate or renewable resource would be much more attractive. Based on comprehensive overview of L-TA, such as properties, usages, market demands, production methods, and bio-manufacturing progresses, the construction of the efficient Gluconobacter oxydans cell factories for 5-KGA production and chemical catalysis for L-TA were mainly studied in this thesis, aiming to settle the industrial issues in the biological production route of L-TA from the renewable resource.First, on the basis of analyzing the 5-KGA production pathway of G. oxydans DSM2343, the gluconate-2-dehydrogenase and pyruvate decarboxylase genes were markerless deleted, cutting off the 2-KGA and acetic acid metabolic pathway. The 5-KGA production of the engineered G. oxydans ZJU2 was 20.89 g/L on the flask, which was 2.65 fold high than the compared strain. Then, the kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (qs) and specific 5-KGA production rate (qp) were analyzed and a simple and easy-to-operate DOT control strategy was proposed, aimed at achieving high yield and high productivity of 5-KGA. The batch fermentation on the 15-L fermenter was carried out. The achieved 5-KGA production and molar conversion rate was 75.66 g/L and 70.21%, respectively with the productivity of 1.72 g/L·h, and the final DCW was 3.8 g/L. However, the residual gluconate was about 23.19 g/L, which was about 21.29% against the initial glucose.High accumulation of the gluconate showed the conversion of gluconate to 5-KGA was the rate-limiting step, which could be released by overexpression of the key enzyme and helped to improve the 5-KGA level. The PQQ dependent SLDH from G. oxydans and the secondary alcohol dehydrogenase (GCD) from Xanthomonas campestris DSM3586 were expressed in G. oxydans ZJU2, respectively under the selective P0169 promoter. It was showed that the 5-KGA productions by G. oxydans ZJU2/pBB5-P0169-gcd and G. oxydans ZJU2/pBB5-P0169-sldAB were 119.14 g/L and 123.78 g/L respectively with the productivity of 1.86 g/L·h and 1.93 g/L·h after 64 h, meanwhile, the 5-KGA molar conversion rate were 73.71% and 76.58%, respectively. This results indicated that the strain G. oxydans ZJU2/pBB5-P0169-sldAB(G. oxydans ZJU3) was more suite for 5-KGA production.Overexpression of the PQQ-dependent SLDH resulted in the imbalance between the PQQ level and the amount of the enzyme, affecting the effect of SLDH expression. Hence, the PQQ synthesis and respiratory models were enhanced in the recombinant strain G. oxydans ZJU3, generating G. oxydans ZJU7 strain. The PQQ level and the H+/O were 757.83±2.43μg/L and 2.01±0.16, respectively. While the quinol oxidase activity was 0.80±0.06μmol/min·mg protein, with the 5-KGA production of 144.52 g/L. The molar conversion rate and productivity were 84.07% and 2.26 g/L·h. Under the optimized fermentation process parameters, the glucose fed-batch fermentation process was established. The final 5-KGA production and productivity were 162 g/L and 2.53 g/L·h after 64 h, and the molar conversion reached 83.31%.At last, the chemical catalysis of 5-KGA to L-TA in alkaline solution was investigated. The CuSO4·5H2O was screened as the catalyst, and reaction conditions were also optimized.100 g/L of 5-KGA-K and 0.5 g/L of CUSO4·5H2O were added into the alkaline solution. Subsequently, the reaction system was incubated (220 rpm,30℃) for a span of 72 h, and 5-KGA molar conversion rate could be up to 73%-75%, with a selectivity of about 76.18%. Compared with the NH4VO3, Pd-C etc., the CuSO4·5H2O catalyst was low cost, non-toxic, and high efficiency. Based on an analysis of the reaction process, a mechanism for the metal chelate catalytic oxidation of 5-KGA to L-TA is proposed, which was completely different from that associated with catalysis by vanadate.Manufacture of L-TA by sequential whole-cell oxidation and chemical catalysis from renewable resources can get rid of the dependence on petrochemical material, achieving the industrial raw materials shift. This also has the important significant on the L-TA industry and its international competitiveness.