Members of the bacterial genus Streptomyces produce the majority of known microbial-origin antibiotics as well as a number of other secondary metabolites including many pharmaceutically valuable compounds. It has been proposed that the expression of antibiotic biosynthetic genes is tightly controlled through many different regulatory networks in Streptomyces species. Traditionally, random mutation has been one most widely used strategies for Streptomyces strain improvement to generate secondary metabolite over-producing industrial mutants, even though the molecular genetic basis underlying such enhanced production remains largely unknown. Recent omics-guided reverse engineering approaches including comparative transcriptomics and proteomics were successfully used to identify alterations in gene expression associated with the overproduction of secondary metabolites in industrial streptomycetes strains. Our previous results suggest that comparative transcriptome analysis using S. coelicolor microarrays with antibiotic-overproducing mutants may be an efficient approach to discover novel regulatory genes and mechanisms in Streptomyces species. Moreover, sequential targeted gene disruptions of independently-working regulatory systems could provide an efficient and rational alternative for Streptomyces strain improvement approaches. Currently, we identified wblA/SCO1712-dependent and wblA/SCO1712 independent genes using additional comparative microarray analysis of antibiotic down-regulator deleted S. coelicolorΔwblAΔSCO1712 mutant, followed by their functional expressions and RT-PCR analyses. Among wblA/SCO1712-independent genes identified, moreover, a SCO5426, an ACT precursor flux down-regulating 6-phosphofructokinase gene was additionally disrupted in a S. coelicolor ΔwblA ΔSCO1712 mutant background and further stimulated ACT production, implying that both ACT biosynthetic regulatory as well as precursor flux-controlling pathways could be synergistically optimized for antibiotic production in Streptomyces species.