Liu Jiameng, Liu Yexue, Zhao Chenxu, Wang Wenhang, Li Qinggang, Lu Fuping, Li Yu
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Bioengineering, Tianjin University of Science & Technology, Tianjin 300457, China.
School of Food Science and Engineering, Tianjin University of Science & Technology, Tianjin 300457, China.
Sheng Wu Gong Cheng Xue Bao. 2023 Mar 25;39(3):1119-1130. doi: 10.13345/j.cjb.220504.
Heme, which exists widely in living organisms, is a porphyrin compound with a variety of physiological functions. is an important industrial strain with the characteristics of easy cultivation and strong ability for expression and secretion of proteins. In order to screen the optimal starting strain for heme synthesis, the laboratory preserved strains were screened with and without addition of 5-aminolevulinic acid (ALA). There was no significant difference in the heme production of strains BA, BAΔ6 and BAΔ6Δ. However, upon addition of ALA, the heme titer and specific heme production of strain BAΔ6Δ were the highest, reaching 200.77 μmol/L and 615.70 μmol/(L·g DCW), respectively. Subsequently, the gene (encoding the cytochrome assembly protein HemX) of strain BAΔ6Δ was knocked out to explore its role in heme synthesis. It was found that the fermentation broth of the knockout strain turned red, while the growth was not significantly affected. The highest ALA concentration in flask fermentation reached 82.13 mg/L at 12 h, which was slightly higher than that of the control 75.11 mg/L. When ALA was not added, the heme titer and specific heme production were 1.99 times and 1.45 times that of the control, respectively. After adding ALA, the heme titer and specific heme production were 2.08 times and 1.72 times higher than that of the control, respectively. Real-time quantitative fluorescent PCR showed that the expressions of , , , , , and genes at transcription level were up-regulated. We demonstrated that deletion of gene can improve the production of heme, which may facilitate future development of heme-producing strain.
血红素广泛存在于生物体内，是一种具有多种生理功能的卟啉化合物。是一种重要的工业菌株，具有易于培养、蛋白质表达和分泌能力强的特点。为了筛选血红素合成的最佳出发菌株，对实验室保藏菌株进行了添加和不添加5-氨基乙酰丙酸（ALA）的筛选。菌株BA、BAΔ6和BAΔ6Δ的血红素产量没有显著差异。然而，添加ALA后，菌株BAΔ6Δ的血红素滴度和比血红素产量最高，分别达到200.77 μmol/L和615.70 μmol/(L·g DCW)。随后，敲除菌株BAΔ6Δ的基因（编码细胞色素组装蛋白HemX）以探索其在血红素合成中的作用。发现敲除菌株的发酵液变红，而生长没有受到显著影响。摇瓶发酵中12 h时ALA的最高浓度达到82.13 mg/L，略高于对照的75.11 mg/L。不添加ALA时，血红素滴度和比血红素产量分别是对照的1.99倍和1.45倍。添加ALA后，血红素滴度和比血红素产量分别比对照高2.08倍和1.72倍。实时定量荧光PCR显示，、、、、、和基因在转录水平的表达上调。我们证明，基因的缺失可以提高血红素的产量，这可能有助于未来血红素生产菌株的开发。
