| Malonic acid is a valuable C3 platform compound which is widely used in food,agriculture,medical and chemical synthesis.Due to the active methylene and carboxyl groups in its structure,malonic acid can participate in many types of chemical reactions.Therefore,malonic acid is an important organic intermediate which can be used to synthesize dyes,herbicides,additives and pharmaceutical intermediates.Due to the need for sustainable green development,the biosynthesis of malonic acid using renewable resources has become a hot research topic.Two non-natural metabolic pathways of malonic acid have been developed,using semialdehyde malonate and malonyl coenzyme A as a precursor,respectively.However,the production efficiency of biological synthesis of malonic acid is not enough to compete with chemical synthesis.To further improve the yield of malonic acid,the following three studies were performed using Escherichia coli BL21（DE3）as a chassis microorganism in this study:（1）Construction of malonic acid biosynthesis pathway in E.coli BL21（DE3）.To produce malonic acid in Escherichia coli,the malonic acid biosynthesis pathway was introduced and the related genesβ-pyruvate transaminase gene（pa0132）,succinate semialdehyde dehydrogenase gene（yne I）,aspartate ammonialyase gene（asp A）and aspartateα-decarboxylase gene（pan D）were overexpressed.Then,the expression of succinate dehydrogenase gene（sdh C）and phosphoenolpyruvate carboxylase gene（ppc）was up-regulated to increase the supply of precursors,and the engineering strain BL21（PPP）was constructed which could produce 0.24 g·L-1 malonic acid in LB medium in shake flask fermentation.（2）Optimization of the malonic acid fermentation process.The fermentation process was optimized by single factor,including the type of culture medium,initial glucose concentration,inducer concentration,as well as the strategies of adding exogenous biotin and fumaric acid to promote the synthesis of malonic acid.The optical basic fermentation conditions were:SOB medium supplemented with 4 g·L-1 glucose was used as fermentation medium,and 0.05 m M IPTG was added to induce protein expression.In addition,the addition of 75μg·L-1 biotin contributed to malonic acid production,which was increased by 32.59%compared with that of the control group without biotin.The yield of malonic acid was increased to 1.23 g·L-1 by adding 8 g·L-1 fumaric acid,and the conversion rate was 0.171 mol·mol-1（malonic acid/fumaric acid）.In fed-batch fermentation,we found that intermittent feeding fermentation was more conducive to malonic acid production,with a2.59 g·L-1 yield in 5 L fermentor.Then,the fermentation experiment was carried out by adding fumaric acid in 5 L fermentor,and the optimization strategies such as gradient cooling（37℃→34℃→30℃→25℃）and the addition of inducer IPTG in 36 h were adopted.Finally,the yield of malonic acid reached to 12.42 g·L-1 and the conversion rate was 0.346 mol·mol-1（malonic acid/fumaric acid）.（3）Establishment of a whole cell catalytic system for malonic acid production.The whole cell catalysis experiment was carried out by using the recombinant strain BL21（PPP）,and the whole cell incubation conditions and catalytic conditions were optimized by single factor.The optimum whole cell incubation conditions were determined as follows:LB medium was used for cell culture at 37℃and when cells grew to OD600=1.2,IPTG with a final concentration of 0.75 m M was added and cells was induced for 32 hours.The optimum catalytic conditions were as follows:the reaction temperature was 25℃,the p H of reaction system was 8.0,the concentration of fumaric acid was 16 g·L-1,and addition of metal ion Ni2+.In the final single batch whole cell catalysis experiment,the yield of malonic acid was 6.70 g·L-1 and the conversion rate was 0.467 mol·mol-1（malonic acid/fumaric acid）,which was 1.59 times higher than that before the optimization of the whole cell catalysis experiment and 1.34 times higher than that of biological fermentation. |
