Generation of Streptomyces hygroscopicus cell factories with enhanced ascomycin production by combined elicitation and pathway-engineering strategies

Ascomycin (FK520) is a macrocyclic antibiotic that also exhibits antifungal and immunosuppressive activity. However, its relatively low titer and yield have hampered commercial application. Here, we have successfully constructed an efficient ascomycin-producing strain of Streptomyces hygroscopicus with high titer and yield, using a novel combinatorial engineering approach based on the identification of targets involved in both metabolic and transcriptional regulation. First, we investigated the effects of different chemicals on ascomycin accumulation and found that dimethyl sulfoxide best stimulated ascomycin overproduction. We next compared intracellular metabolic and transcriptional profiles after dimethyl sulfoxide and control treatments and identified potential target genes (zwf and aroA, involved in metabolic precursor pathways; and luxR, iclR, fadR, and fkbN, involved in transcriptional regulation). These candidate genes were then engineered to produce strains with individual and combinatorial overexpression. Combined overexpression of aroA, fkbN, and luxR resulted in the highest yield of ascomycin (1258.30 +/- 33.49 mg/L), 4.12-fold higher than the control yield (305.60 +/- 16.90 mg/L). This integrative multilevel approach identified novel determinants involved in both metabolic and transcriptional regulation, resulting in the diversion of carbon flux towards ascomycin accumulation. This approach could be applied to boost the production of a variety of useful bacterial metabolites.