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

摘要：

琥珀酸作为最具有前途的化学中间体之一，受到社会广泛的关注。利用微生物法生产琥珀酸具有环境友好、成本低廉等优点，但是也存在发酵得率低、副产物复杂等问题。本文通过对大肠杆菌（E. coli SUC37）的工程改造，有效提高了琥珀酸的产量。首先以E. coli SUC37为出发菌株，通过Red同源重组技术，构建乳酸脱氢酶基因（ldhA）失活突变菌株，阻断主要冗余代谢支路，减少副产物的积累进而增加琥珀酸的产量。对照分析了发酵过程中出发菌株和工程菌株中线粒体异柠檬酸脱氢酶（NAD-IDH）、NAD-苹果酸酶（NAD-ME）、苹果酸合酶（MS）、异柠檬酸裂合酶（ICL）、辅酶ⅠNAD(H)、NAD-苹果酸脱氢酶（NAD-MDH）的活性，进一步探索高产菌株发酵过程中关键酶的代谢差异，发现ldhA的敲除主要通过增强TCA循环的还原支路以及乙醛酸途径，从而促进琥珀酸的积累。优化以玉米淀粉工业废弃物玉米浆为氮源产琥珀酸的发酵条件得到，初始葡萄糖含量为9%、初始玉米浆含量为2.5%、初始碳酸镁含量为6.8%、接种量为3%、发酵温度为37℃的条件下，发酵60h琥珀酸的转化率为0.658g/g葡萄糖，提高了30.2%；产量达到54.9g/L，提高了20.7%；副产物乳酸积累了19.16g/L，降低了46.5%，为琥珀酸的工业化生产奠定了基础。

关键词: 琥珀酸, 大肠杆菌, 乳酸脱氢酶, Red同源重组, 发酵优化