Stepwise increase of thaxtomins production in Streptomyces albidoflavus J1074 through combinatorial metabolic engineering.

Herbicide-resistance in weeds has become a serious threat to agriculture across the world. Thus, there is an urgent need for the discovery and development of herbicides with new modes of action. Thaxtomin phytotoxins are a group of nitrated diketopiperazines produced by potato common scab-causing phytopathogen Streptomyces scabies and other actinobacterial pathogens. They are generally considered to function as inhibitors of cellulose synthesis in plants, and thus have great potential to be used as natural herbicides. Generation of an overproducing strain is crucial for the scale-up production of thaxtomins and their wide use in agriculture. In the present study, we employed a stepwise strategy by combining heterologous expression, repressor deletion, activator overexpression, and optimization of fermentation media for high-level production of thaxtomins. The maximum yield of 728 mg/L thaxtomins was achieved with engineered Streptomyces albidoflavus J1074 strains in shake-flask cultures, and it was approximately 36-fold higher than S. albidoflavus J1074 carrying the unmodified cluster. Moreover, the yield of thaxtomins could reach 1973 mg/L when the engineered strain was cultivated in a small-scale stirred-tank bioreactor. This is the highest titer reported to date, representing a significant leap forward for the scale-up production of thaxtomins. Our study presents a robust, easy-to-use system that will be broadly useful for improving titers of bioactive compounds in many Streptomyces species.