Li Lingfeng, Liu Pei, Luo Wen, Wang Qin, Wang Zhi, Chen Xiaobin, Li Junhui, Cai Dongbo, Ma Xin, Chen Shouwen
Environmental Microbial Technology Center of Hubei Province, State Key Laboratory of Biocatalysis and Enzyme Engineering, College of Life Sciences, Hubei University, Wuhan 430062, Hubei, China.
Hubei Provincial Key Laboratory of Industrial Microbiology, Key Laboratory of Fermentation Engineering (Ministry of Education), School of Food and Biological Engineering, Hubei University of Technology, Wuhan 430068, Hubei, China.
Sheng Wu Gong Cheng Xue Bao. 2021 Aug 25;37(8):2803-2812. doi: 10.13345/j.cjb.200623.
Bacitracin is a broad-spectrum antibiotics mainly produced by Bacillus, and is used as veterinary medicine in the fields of livestock and poultry breeding. Insufficient supply of precursor amino acids might be an important factor that hinders high-level microbial production of bacitracin. We investigated the effect of strengthening L-cysteine supply on bacitracin production by an industrial bacitracin producer, Bacillus licheniformis DW2. Overexpression of cysK encoding L-cysteine synthase led to a 9.17% increase of the bacitracin titer. Moreover, overexpression of cysE encoding L-serine acetyltransferase and cysP encoding thiosulfate/sulfate intracellular transporter increased the bacitracin titers by 7.23% and 8.52%, respectively. Moreover, overexpression of a putative cystine importer TcyP led to a 29.19% increase of intracellular L-cysteine, and bacitracin titer was increased by 7.79%. Subsequently, the strong promoter PbacA was used to replace the promoters of genes cysP, cysE and tcyP in strain DW2::ysK, respectively. The resulted strain CYS4 (DW2::cysK-PbacA-(cysP)-PbacA(cysE)- PbacA(tcyP) produced 910.02 U/mL bacitracin, which was 21.10% higher than that of the original strain DW2 (747.71 U/mL). Together with the experiments in 3 L fermenters, this research demonstrated that enhancing intracellular L-cysteine supply is an effective strategy to increase bacitracin production of B. licheniformis.
杆菌肽是一种主要由芽孢杆菌产生的广谱抗生素，在畜禽养殖领域用作兽药。前体氨基酸供应不足可能是阻碍杆菌肽高水平微生物生产的一个重要因素。我们研究了强化L-半胱氨酸供应对工业杆菌肽生产菌株地衣芽孢杆菌DW2生产杆菌肽的影响。编码L-半胱氨酸合酶的cysK的过表达导致杆菌肽效价提高了9.17%。此外，编码L-丝氨酸乙酰转移酶的cysE和编码硫代硫酸盐/硫酸盐细胞内转运蛋白的cysP的过表达分别使杆菌肽效价提高了7.23%和8.52%。此外，推定的胱氨酸转运体TcyP的过表达导致细胞内L-半胱氨酸增加了29.19%，杆菌肽效价提高了7.79%。随后，分别使用强启动子PbacA替换菌株DW2::ysK中基因cysP、cysE和tcyP的启动子。所得菌株CYS4（DW2::cysK-PbacA-(cysP)-PbacA(cysE)-PbacA(tcyP)）产生910.02 U/mL的杆菌肽，比原始菌株DW2（747.71 U/mL）高21.10%。结合在3 L发酵罐中的实验，本研究表明增强细胞内L-半胱氨酸供应是提高地衣芽孢杆菌杆菌肽产量的有效策略。
