Wang Lijun, Yan Sihan, Yang Taowei, Xu Meijuan, Zhang Xian, Shao Minglong, Li Huazhong, Rao Zhiming
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2021 Dec 25;37(12):4314-4328. doi: 10.13345/j.cjb.210057.
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 encoding α-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-sRNAhemB- RBS5RphemA-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.
5-氨基乙酰丙酸（5-ALA）在医学和农业领域发挥着重要作用。5-ALA可由工程化的大肠杆菌和谷氨酸棒杆菌生产。我们系统地改造了谷氨酸棒杆菌的C4代谢途径，以进一步提高其生产5-ALA的能力。首先，分别将来自荚膜红细菌和沼泽红假单胞菌的编码5-ALA合酶（ALAS）的hemA基因在谷氨酸棒杆菌中进行异源表达。选择了具有相对较高酶活性的沼泽红假单胞菌的RphemA基因。筛选最佳核糖体结合位点序列RBS5显著提高了RphemA的活性。重组菌株的ALAS活性达到（221.87±3.10）U/mg，5-ALA产量提高了14.3%。随后，敲除编码α-酮戊二酸脱氢酶抑制剂蛋白（odhI）和琥珀酸脱氢酶（sdhA）的基因增加了琥珀酰辅酶A向5-ALA生产的通量。此外，通过sRNA抑制hemB的表达减少了5-ALA的降解，而过表达半胱氨酸/O-乙酰丝氨酸转运蛋白eamA提高了细胞内5-ALA的输出效率。使用工程菌株谷氨酸棒杆菌13032/∆odhI/∆sdhA-sRNAhemB-RBS5RphemA-eamA进行摇瓶发酵，产量为11.90 g/L，比原始菌株高57%。使用该工程菌株在5 L发酵罐中进行补料分批发酵，在48小时内产生了25.05 g/L的5-ALA，这是迄今为止报道的从葡萄糖生产5-ALA的最高产量。
