Liu Jingjing, Xie Zhipeng, Shin Hyun-Dong, Li Jianghua, Du Guocheng, Chen Jian, Liu Long
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
Hangzhou Bioking Biochemical Engineering Co. Ltd, Hangzhou 311106, China.
J Biotechnol. 2017 Jul 10;253:1-9. doi: 10.1016/j.jbiotec.2017.05.011. Epub 2017 May 12.
Aspergillus oryzae finds wide application in the food, feed, and wine industries, and is an excellent cell factory platform for production of organic acids. In this work, we achieved the overproduction of L-malate by rewiring the reductive tricarboxylic acid (rTCA) pathway and L-malate transport pathway of A. oryzae NRRL 3488. First, overexpression of native pyruvate carboxylase and malate dehydrogenase in the rTCA pathway improved the L-malate titer from 26.1gL to 42.3gL in shake flask culture. Then, the oxaloacetate anaplerotic reaction was constructed by heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase from Escherichia coli, increasing the L-malate titer to 58.5gL. Next, the export of L-malate from the cytoplasm to the external medium was strengthened by overexpression of a C4-dicarboxylate transporter gene from A. oryzae and an L-malate permease gene from Schizosaccharomyces pombe, improving the L-malate titer from 58.5gL to 89.5gL. Lastly, guided by transcription analysis of the expression profile of key genes related to L-malate synthesis, the 6-phosphofructokinase encoded by the pfk gene was identified as a potential limiting step for L-malate synthesis. Overexpression of pfk with the strong sodM promoter increased the L-malate titer to 93.2gL. The final engineered A. oryzae strain produced 165gL L-malate with a productivity of 1.38gLh in 3-L fed-batch culture. Overall, we constructed an efficient L-malate producer by rewiring the rTCA pathway and L-malate transport pathway of A. oryzae NRRL 3488, and the engineering strategy adopted here may be useful for the construction of A. oryzae cell factories to produce other organic acids.
米曲霉在食品、饲料和酿酒行业有着广泛应用，并且是生产有机酸的优良细胞工厂平台。在本研究中，我们通过改造米曲霉NRRL 3488的还原性三羧酸（rTCA）途径和L-苹果酸转运途径实现了L-苹果酸的过量生产。首先，在摇瓶培养中，rTCA途径中天然丙酮酸羧化酶和苹果酸脱氢酶的过表达使L-苹果酸产量从26.1 g/L提高到42.3 g/L。然后，通过异源表达来自大肠杆菌的磷酸烯醇式丙酮酸羧激酶和磷酸烯醇式丙酮酸羧化酶构建了草酰乙酸回补反应，使L-苹果酸产量提高到58.5 g/L。接下来，通过过表达来自米曲霉的C4-二羧酸转运基因和来自粟酒裂殖酵母的L-苹果酸通透酶基因，加强了L-苹果酸从细胞质向胞外培养基的输出，使L-苹果酸产量从58.5 g/L提高到89.5 g/L。最后，在与L-苹果酸合成相关的关键基因表达谱转录分析的指导下，鉴定出由pfk基因编码的6-磷酸果糖激酶是L-苹果酸合成的潜在限制步骤。用强sodM启动子过表达pfk使L-苹果酸产量提高到93.2 g/L。最终构建的工程米曲霉菌株在3-L补料分批培养中产生了165 g/L的L-苹果酸，生产效率为1.38 g/L·h。总体而言，我们通过改造米曲霉NRRL 3488的rTCA途径和L-苹果酸转运途径构建了高效的L-苹果酸生产者，这里采用的工程策略可能有助于构建用于生产其他有机酸的米曲霉细胞工厂。
