关键词: 琥珀酸, 大肠杆菌, 乳酸脱氢酶, Red同源重组, 发酵优化

Abstract:

Succinic acid, as one of the most promising chemical intermediates, has received extensive attention from the society. The production of succinic acid by microbial methods has the advantages of environmental friendliness and low cost, but it also has the problems of low fermentation yield and complex by-products. In this paper, by engineering Escherichia coli (E. coli SUC37), the yield of succinic acid was effectively increased. Firstly, using E. coli SUC37 as the starting strain, a lactate dehydrogenase gene (ldhA) inactivation mutant strain was constructed by Red homologous recombination to block the major redundant metabolic branches, reduce the accumulation of by-products and thus increase the yield of succinate. The activities of mitochondrial isocitrate dehydrogenase (NAD-IDH), NAD-malic enzyme (NAD-ME), malate synthase (MS), isocitrate cleavage enzyme (ICL), coenzyme Ⅰ NAD(H), and NAD-malate dehydrogenase (NAD-MDH) were analyzed against those of the starting and engineered strains during the fermentation process. Metabolic differences of the key enzymes during the fermentation process of the high-yielding strains were further explored. The optimization of the fermentation conditions for succinic acid production using corn starch industrial waste corn syrup as the nitrogen source yielded the following results: under the conditions of the initial glucose content of 9%, the initial corn syrup content of 2.5%, the initial magnesium carbonate content of 6.8%, the inoculum amount of 3%, and the fermentation temperature of 37℃, the conversion rate of succinic acid for 60h of fermentation was 0.658g/g glucose, which was improved by 30.2%, and the yield reached 54.9g/L, an increase of 20.7%; the by-product lactic acid accumulated 19.16g/L, a decrease of 46.5%; laying the foundation for the industrialized production of succinic acid.

Key words: succinic acid, Escherichia coli, lactate dehydrogenase, Red homologous recombination, fermentation optimization