莫西霉素高产菌株的构建

Construction of high-producing strains of kirromycin

摘要:

【背景】 莫西霉素(kirromycin)是由山丘链霉菌(Streptomyces collinus) Tü 365产生的一种复杂线性聚酮-非核糖体肽类化合物，可与细菌延伸因子EF-Tu相互作用，阻碍蛋白质合成，具有窄谱抑菌活性。目前莫西霉素作为实验试剂用于核糖体结构和功能研究。近年来有研究报道，莫西霉素及其类似物抗沃尔巴克氏体(Wolbachia)活性优于注册药物多西环素，有望成为治疗丝状线虫感染的新先导化合物。【目的】 运用组合代谢工程方法，构建莫西霉素高产菌株，实现莫西霉素产量的提升。【方法】 基于莫西霉素生物合成基因簇以及生物合成途径分析，利用组成型启动子kasOp^*过表达或组合过表达莫西霉素生物合成基因簇内的巴豆酰辅酶A还原酶编码基因kirN、天冬氨酸- 1-脱羧酶编码基因kirD、磷酸泛酰巯基乙胺转移酶编码基因kirP、转运蛋白超家族外排泵编码基因kirTI和kirTII、天蓝色链霉菌(Streptomyces coelicolor) A3(2)中乙酰辅酶A羧化酶编码基因acc等，实现莫西霉素产量的提升。【结果】 过表达/组合过表达上述基因对莫西霉素产量均有一定提升效果，其中，单独过表达acc及组合过表达kirD、kirN、kirP、kirTI和kirTII的效果更好，莫西霉素产量较野生型菌株分别提高了57.8%和65.6%，达到198.3 mg/L和208.1 mg/L。此外，通过在公开的细菌基因组数据库中搜索莫西霉素生物合成酶的同源蛋白编码基因，发现31个预测可以合成莫西霉素类天然产物的生物合成基因簇。【结论】 本研究运用代谢工程方法有效提升了莫西霉素的产量，为进一步推动莫西霉素的应用开发奠定了基础。

Abstract:

[Background] Kirromycin is a complex linear polyketide-nonribosomal peptide compound produced by Streptomyces collinus Tü 365, which interacts with the bacterial elongation factor EF-Tu, thereby inhibiting protein synthesis and exhibiting narrow-spectrum antibacterial activity. Currently, kirromycin is widely used as an experimental reagent in studying ribosomal structure and function. Recent studies have reported that kirromycin and its analogs exhibit superior activity against Wolbachia compared to the registered drug doxycycline, making them potential lead compounds for treating filarial worm infections. [Objective] Combinatorial metabolic engineering methods were used to construct high-production strains of kirromycin to enhance kirromycin production and lay the foundation for further applications. [Methods] Based on the kirromycin biosynthetic gene cluster and biosynthetic pathways analysis, overexpression or co-overexpression of the genes encoding crotonyl CoA reductase (kirN), aspartate-1-decarboxylase (kirD), phosphopantetheinyl transferase (kirP), efflux pump proteins of the major facilitator superfamily (kirTI and kirTII), and acetyl-CoA carboxylase (acc) from Streptomyces coelicolor A3(2) under the control of constitutive promoter kasOp* were performed to increase kirromycin production in S. collinus. [Results] Overexpression or co-overexpression of the aforementioned genes had a certain effect on increasing kirromycin production. Better results were observed from the overexpression of the acc gene and co-overexpression of kirD, kirN, kirP, kirTI, and kirTII genes. Compared to the wild-type strain, kirromycin production increased by 57.8% and 65.6%, reaching 198.3 mg/L and 208.1 mg/L, respectively. A systematic search for homologous proteins-coding genes of kirromycin biosynthetic enzymes in publicly available bacterial genome databases revealed 31 biosynthetic gene clusters potentially synthesizing kirromycin-like natural products. [Conclusion] This study effectively increased kirromycin production in S. collinus using metabolic engineering methods, laying a solid foundation for further applications and development of kirromycin.