代谢工程改造黑曲霉生产葡萄糖二酸  

Metabolic Engineering Modification of Aspergillus niger for the Production of D-glucaric Acid

作　　者：郭宇飞 闫荣媚 张小茹 曹威 刘浩 GUO Yu-fei;YAN Rong-mei;ZHANG Xiao-ru;CAO Wei;LIU Hao(Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,Tianjin Key Laboratory of Industrial Microbiology,College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457)

机构地区：[1]天津科技大学生物工程学院天津市工业微生物重点实验室工业发酵微生物教育部重点实验室,天津300457

出　　处：《生物技术通报》2022年第11期227-237,共11页Biotechnology Bulletin

基　　金：国家重点研发计划(2021YFC2100700)。

摘　　要：葡萄糖二酸是葡萄糖的一种二元羧酸衍生物,是重要的平台化合物,被应用于医药、化工等领域。本研究以黑曲霉为底盘细胞,通过表达来自恶臭假单胞菌Pseudomonas putida KT2440的糖醛酸脱氢酶基因ppudh,成功在黑曲霉中实现了葡萄糖二酸的合成,产量为18.74 mg/L;在此基础上通过共过表达黑曲霉自身来源的肌醇加氧酶(anmioxA)和肌醇-1-磷酸合酶(aninoA)、酿酒酵母Saccharomyces cerevisiae S288C来源的羧酸转运蛋白(scJEN1),强化了合成通路和外泌途径,将产量提高至102.10 mg/L;通过表达来自乳酸乳球菌Lactococcus lactis subsp.cremoris MG1363的NADH氧化酶(llnox),建立NAD~+辅因子循环系统,使产量进一步提高至115.65 mg/L;利用RNA干扰技术对竞争支路中的关键酶磷酸果糖激酶(pfkA)和葡萄糖-6-磷酸脱氢酶(zwf)进行弱化表达,葡萄糖二酸最终的产量达到313.65 mg/L。本研究为微生物高效生产葡萄糖二酸和下游相关产品生产奠定基础。D-glucaric acid,a dicarboxylic acid derivative of glucose,is an important platform compound and is used in medicine,chemical industry and other fields.Using Aspergillus niger as the chassis cell for D-glucaric acid production,expressing the uronate dehydrogenase gene ppudh from Pseudomonas putida KT2440 in A.niger,D-glucaric acid biosynthesis with a titer of 18.74 mg/L was successfully achieved.Strengthening D-glucaric acid biosynthesis secretion,by overexpressing the endogenous inositol oxygenase(anmioxA)and inositol-1-phosphate synthase(aninoA)as well as the carboxylate transporter(scJEN1)from Saccharomyces cerevisiae S288C,D-glucaric acid production was improved significantly with a titer of 102.10 mg/L.Further establishing a NADcofactor recycling system by expressing a NADH oxidase(llnox)from Lactococcus lactis subsp.cremoris MG1363 D-glucaric acid yield was improved to 115.65 mg/L.Finally reducing the carbon flux to glycolysis and pentose phosphate pathway enabled the highest D-glucaric acid production of 313.65 mg/L,which lays the foundation for efficient production of glucaric acid and the corresponding downstream chemicals by microorganisms.

关 键 词：葡萄糖二酸 黑曲霉 辅因子循环系统 RNA干扰 

分 类 号：TQ216[化学工程—有机化工]