代谢工程改造L-半胱氨酸供给模块促地衣芽胞杆菌 高效合成杆菌肽

Metabolic engineering of L-cysteine supply modules for enhanced production of bacitracin in Bacillus licheniformis

摘要:

杆菌肽是一种主要由芽胞杆菌产生的广谱性抗生素，目前作为兽药广泛应用于畜禽养殖领域。前体氨基酸供应不足可能是限制微生物发酵高产杆菌肽的重要因素。文中以杆菌肽工业生产菌株——地衣芽胞杆菌Bacillus licheniformis DW2为出发菌株，研究l-半胱氨酸供给模块强化对杆菌肽合成的影响。首先，构建了l-半胱氨酸合成酶基因cysK强化表达菌株，杆菌肽效价相比于对照菌株提高了9.47%。接着，为提高l-半胱氨酸合成前体供给，对l-丝氨酸乙酰转移酶基因cysE和硫代硫酸盐/硫酸盐胞内转运蛋白基因cysP进行强化，杆菌肽产量分别提高了7.23%和8.52%。随后，结果表明转运蛋白TcyP负责从胞外向胞内转运胱氨酸，强化表达TcyP后胞内l-半胱氨酸浓度和杆菌肽效价分别提高了29.19%和7.79%。通过组合代谢工程育种，在整合表达了基因cysK基础上，利用强启动子PbacA分别替换基因cysP、cysE和tcyP原始启动子，得到工程菌株CYS4 (DW2::cysK-PbacA(cysP)- PbacA(cysE)-PbacA(tcyP))，杆菌肽效价达到910.02 U/mL，相比于出发菌株DW2 (747.71 U/mL) 提高了21.10%。最后，通过3 L发酵罐小试实验，进一步证实了强化l-半胱氨酸有利于杆菌肽合成。研究表明，强化胞内l-半胱氨酸供给水平是提高地衣芽胞杆菌中杆菌肽产量的有效策略，为杆菌肽工业生产提供了一株具有良好应用前景的菌株。

Abstract:

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.