Heterologous reconstitution of oxytetracycline producingstrainin Streptomyces albus Del14

Abstract:

[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.