Zhang Zihan, Hu Baodong, Zhou Jingwen, Li Jianghua, Chen Jian, Du Guocheng, Zhao Xinrui
Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China.
Metabolites. 2025 Feb 23;15(3):151. doi: 10.3390/metabo15030151.
Heme is an important cofactor and plays crucial roles in the correct folding of hemoproteins. The synthesis of heme can be enhanced by the plasmid-based expression of heme biosynthetic genes. However, plasmid-based expression is genetically unstable and requires the utilization of antibiotics to maintain high copy numbers of plasmids.
The rate-limiting steps in heme biosynthesis were first analyzed based on previous studies and the accumulation of heme intermediates was achieved by adding heme precursor (5-aminolevulinic acid, ALA). Next, the intracellular accumulation of porphyrin was increased by deleting the porphyrin transporter TolC. Finally, the heme synthetic genes were modified by integrating the and genes into the and locus, assembling the rate-limiting enzymes HemC and HemD with RIAD-RIDD tags, replacing the promoters of genes with the constitutive promoter P, and deleting the heme degradation gene .
An enhanced heme supply HEME2 strain was obtained with a heme titer of 0.14 mg/L, which was 4.60-fold higher than that of the C41(DE3) strain. The HEME2 strain was applied to produce human hemoglobin and leghemoglobin. The titer and peroxidase activity of human hemoglobin were 1.29-fold and 42.4% higher in the HEME2-hHb strain than the values in the control strain C41-hHb. In addition, the peroxidase activity and heme content of leghemoglobin were increased by 39.2% and 53.4% in the HEME2-sHb strain compared to the values in the control strain C41-sHb.
A plasmid-free C41(DE3) strain capable of efficient and stable heme supply was constructed and can be used for the production of high-active hemoglobins.
血红素是一种重要的辅因子，在血红蛋白的正确折叠中起关键作用。基于质粒的血红素生物合成基因表达可增强血红素的合成。然而，基于质粒的表达在遗传上不稳定，需要使用抗生素来维持高拷贝数的质粒。
首先根据先前的研究分析血红素生物合成中的限速步骤，并通过添加血红素前体（5-氨基乙酰丙酸，ALA）来实现血红素中间体的积累。接下来，通过删除卟啉转运蛋白TolC来增加细胞内卟啉的积累。最后，通过将 和 基因整合到 和 位点，将限速酶HemC和HemD与RIAD-RIDD标签组装，用组成型启动子P替换 基因的启动子，并删除血红素降解基因 ，对血红素合成基因进行修饰。
获得了血红素供应增强的HEME2菌株，血红素滴度为0.14 mg/L，比C41(DE3)菌株高4.60倍。HEME2菌株用于生产人血红蛋白和豆血红蛋白。与对照菌株C41-hHb相比，HEME2-hHb菌株中人血红蛋白的滴度和过氧化物酶活性分别高1.29倍和42.4%。此外，与对照菌株C41-sHb相比，HEME2-sHb菌株中豆血红蛋白的过氧化物酶活性和血红素含量分别提高了39.2%和53.4%。
构建了一种能够高效稳定供应血红素的无质粒C41(DE3)菌株，可用于生产高活性血红蛋白。
