代谢改造重组谷氨酸棒杆菌C4途径高效合成5-氨基乙酰丙酸  

Engineering the C4 pathway of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid

作　　者：王丽君[1] 闫思翰 杨套伟[1] 徐美娟[1] 张显[1] 邵明龙[1] 李华钟[1] 饶志明[1] Lijun Wang;Sihan Yan;Taowei Yang;Meijuan Xu;Xian Zhang;Minglong Shao;Huazhong Li;Zhiming Rao(Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《生物工程学报》2021年第12期4314-4328,共15页Chinese Journal of Biotechnology

基　　金：国家重点研发计划项目(No.2018YFA0900300);国家自然科学基金(No.31770058);宁夏回族自治区重点研发计划(No.2019BCH01002);国家轻工技术与工程一流学科自主课题(No.LITE2018-06)资助。

摘　　要：5-氨基乙酰丙酸(5-aminolevulinic acid,5-ALA)在医药和农业等领域有着广泛作用,目前主要采用大肠杆菌或谷氨酸棒杆菌以微生物发酵法合成。为了进一步提高谷氨酸棒杆菌合成5-ALA的能力,对其C4代谢途径进行了系统代谢改造。首先分别在谷氨酸棒杆菌中异源表达荚膜红杆菌和沼泽红假单胞菌的5-氨基乙酰丙酸合成酶ALAS,选择酶活相对较高的沼泽红假单胞菌的RphemA基因作为关键合成酶基因,并筛选到能显著增强RphemA的酶活性的核糖体结合位点RBS5。重组菌株ALAS的比酶活可达(221.87±3.10)U/mg,且5-ALA产量提高了14.3%;随后通过敲除α-酮戊二酸脱氢酶抑制蛋白基因(odhI)和琥珀酸脱氢酶基因(sdhA),促进了前体琥珀酰CoA向5-ALA途径的流动;通过sRNA抑制hemB表达减少了5-ALA的降解;并且过表达半胱氨酸/O-乙酰丝氨酸转运蛋白eamA提高了5-ALA的输出效率;使用重组菌株C.glutamicum13032/?odhI/?sdhA-sRNAhemB-RBS5RphemA-eamA摇瓶发酵,5-ALA最高产量达11.90g/L,较出发菌株提高了57%。最后,在5L发酵罐中进行补料分批发酵,48h内5-ALA的产量达25.05g/L,为目前以葡萄糖为碳源发酵的最高产量。本研究构建了高产5-ALA重组谷氨酸棒杆菌,具有良好的工业应用前景。5-aminolevulinic acid(5-ALA)plays an important role in the fields of medicine and agriculture.5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum.We systematically engineered the C4 metabolic pathway of C.glutamicum to further improve its ability to produce 5-ALA.Firstly,the hemA gene encoding 5-ALA synthase(ALAS)from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C.glutamicum,respectively.The RphemA gene of R.palustris which showed relatively high enzyme activity was selected.Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA.The ALAS activity of the recombinant strain reached(221.87±3.10)U/mg and 5-ALA production increased by 14.3%.Subsequently,knocking out genes encodingα-ketoglutarate dehydrogenase inhibitor protein(odhI)and succinate dehydrogenase(sdhA)increased the flux of succinyl CoA towards the production of 5-ALA.Moreover,inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA,while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA.Shake flask fermentation using the engineered strain C.glutamicum 13032/?odhI/?sdhA-sRNAhemBRBS5 RphemA-eamA resulted in a yield of 11.90 g/L,which was 57%higher than that of the original strain.Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h,which is the highest reported-to-date yield of 5-ALA from glucose.

关 键 词：谷氨酸棒杆菌 5-氨基乙酰丙酸 代谢改造 C4途径 

分 类 号：TQ226.36[化学工程—有机化工]