柠檬酸是全球产量和需求量最大的有机酸,作为食品饮料酸味剂、医药和化工原料应用广泛。目前我国产柠檬酸黑曲霉的选育及其深层发酵技术居世界领先水平。该研究旨在探究过表达黑曲霉柠檬酸胞外转运蛋白和丙酮酸脱氢酶,提高其柠檬酸胞外转运速度,增加柠檬酸产量。以黑曲霉CGMCC 10142(Aspergillus niger CGMCC 10142)为出发菌株,运用基因工程技术,构建过表达柠檬酸转运蛋白基因cexA和丙酮酸脱氢酶E1基因pdhA的重组菌株,通过摇瓶发酵筛选,获得一株高产柠檬酸重组菌株94-18,其柠檬酸产量达138.9 g/L,较出发菌株提高了7.7%。经30 L罐发酵,94-18菌株比出发菌株CGMCC 10142的发酵周期缩短4 h,60 h时产柠檬酸酸量189.3 g/L,发酵强度3.15 g/(L·h),显著高于出发菌株。对94-18菌株cexA和pdhA表达量进行检测,发现24 h时94-18菌株的cexA和pdhA表达量较出发菌株分别上调了25倍和13倍;在48 h时,分别上调了63倍和37倍。进一步分析该时间点三羧酸循环中关键有机酸的积累量,发现同时过表达cexA和pdhA有效加速糖酵解途径代谢流并增加丙酮酸的分解速度,使柠檬酸合成和转运速度加快,减少苹果酸和富马酸积累量,最终实现缩短柠檬酸发酵周期,增加柠檬酸产量。该基因改造策略及其所构建的菌株具有较好的工业生产价值。
Citric acid is the most widely produced and demanded organic acid globally, with extensive applications as an acidulant in food and beverages, and as a raw material in pharmaceuticals and chemical industries.Currently, China's selection and breeding of Aspergillus niger for citric acid production and its fermentation technology are at the world's leading level.This study aims to explore the co-overexpression of the citric acid extracellular transporter and pyruvate dehydrogenase in A.niger to enhance the extracellular transport rate and increase yield of citric acid.Using A.niger CGMCC 10142 as the initial strain, genetic engineering techniques were used to construct a recombinant strain that co-overexpresses the citric acid transporter gene cexA and the pyruvate dehydrogenase E1 gene pdhA.Through shake flask fermentation screening, a high-yielding citric acid recombinant strain 94-18 was obtained.The citric acid production reached 138.9 g/L, an increase of 7.7% compared to the CGMCC 10142 strain.In 30 L bioreactor fermentation, the fermentation cycle of strain 94-18 was shortened by 4 hours compared to the CGMCC 10142 strain.At 60 hours, the citric acid production was 189.3 g/L, with a fermentation intensity of 3.15 g/(L·h), significantly higher than that of the initial strain.The expression levels of cexA and pdhA in strain 94-18 were detected and found to be upregulated 25 and 13 times, respectively, compared to the CGMCC 10142 strain at 24 hours;at 48 hours, they were upregulated 63 and 37 times, respectively.Further analysis of the accumulation of important organic acids in the TCA cycle at these time points revealed that the co-overexpression of cexA and pdhA effectively accelerated the EMP metabolic flux and increased the breakdown rate of pyruvate, speeding up citric acid synthesis and transport, reducing the accumulation of malic and fumaric acids, and ultimately achieving efficient citric acid synthesis.This also accelerated the extracellular transport rate of citric acid, shortened the fermentation cycle, and increased citric acid production.The genetic modification strategy and the constructed strain have significant industrial production value.
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