异源重构白色链霉菌土霉素生产菌株

Heterologous reconstitution of oxytetracycline producing strain in Streptomyces albus Del14

【目的】土霉素(oxytetracycline,OTC)属于第一代四环素类抗生素,对革兰氏阳性菌和阴性菌具有很好的抑菌效果,目前主要应用于畜牧业、水产养殖业和作为原料药生产二代、三代四环类抗生素。因此,在生产上具有提高产量、降低成本的迫切需求。为了解决工业菌株改造中面临着遗传操作比较困难、周期长的问题,我们通过异源重构白色链霉菌(Streptomyces albus)Del14并作为底盘生产菌株,进而评估该菌株OTC生物制造细胞工厂的潜力。【方法】通过理性工程重构,获得一系列衍生菌株Del14:Oxy、Del14:Oxy1K、Del14:Oxy1KΔotrR、Del14B:Oxy1KΔotrR。对上述菌株进行摇瓶发酵以及HPLC检测发酵产物;通过RT-qPCR检测OTC生物合成基因簇(otc cluster)相关结构基因的转录水平。【结果】S.albus Del14生长快、不结球,对OTC具有一定的耐受性。通过操纵簇内调控因子OtcR和OtrR,最终使重组菌株Del14B:Oxy1KΔotrR OTC产量在第6天达到了1.1 g/L,与原始生产菌株龟裂链霉菌(S.rimosus)M4018在第8天产量相当。【结论】本研究首次在S.albus Del14中异源表达了土霉素生物合成基因簇,初步证实了一个很有潜力的OTC生物制造底盘,为这一OTC生产菌株的进一步优化改造奠定了基础。

[Objective] Oxytetracycline(OTC) is one of the first-generation tetracycline antibiotics,with good inhibitory effects on Gram-positive and Gram-negative bacteria.It is mainly used in animal husbandry and aquaculture industry,and also served as the raw material of second-generation and third-generation tetracyclic antibiotics.Therefore,there is an urgent need to improve productivity and reduce costs in production.To simplify genetic manipulation and shorten cultivation time,we evaluated the OTC biomanufacturing capacity of Streptomyces albus Del14 by heterologous expression and cluster refactoring.[Methods] Through rational cluster refactoring,we obtained a series of derivative strains:Del14:Oxy,Del14:Oxy1K,Del14:Oxy1KΔotrR and Del14B:Oxy1KΔotrR.The above strains were fermented in shake flask and OTC was analyzed by HPLC.Additionally,the transcriptional levels of structural genes related to otc cluster were detected by RT-qPCR.[Results] S.albus Del14 showed rapid growth and sufficient dispersed mycelia as well as high resistance to OTC.By manipulation of the expression of two cluster-situated regulators OtcR and OtrR,the OTC yield of the recombinant strain Del14B:Oxy1KΔotrR reached 1.1 g/L on the 6th-day of fermentation,which was comparable to the production by the native host S.rimosus M4018 on the 8th day.[Conclusion] In this study,the otc cluster was expressed in S.albus Del14 for the first time and the potential for OTC biomanufacture as a chassis strain was preliminarily confirmed,laying the foundation for further OTC production optimization.