More than two-third of known antibiotics are produced by actinomycetes of the genus Streptomyces. Unfortunately, the production rate from Streptomyces natural antibiotic is extremely slow and thus cannot satisfy industrial demand. In this study, the production of antibiotics by Streptomyces is enhanced by a "superplasmid" which including global regulatory factors afsR, cyclic adenosine receptor protein (CRP), RNA polymerase beta subunits (rpoB) with point mutation and acetyl coenzyme A carboxylase gene (accA(2)BE), these elements are controlled by the PermE* promoter and then transfer into Streptomyces coelicolor M145, Streptomyces mutabilis TRM45540, Streptomyces hygroscopicus XM201, and Streptomyces hygroscopicus ATCC29253 by conjugation to generate exconjugants. NMR, HPLC, and LC-MS analyses revealed that the superplasmid led to the overproduction of actinorhodin (101.90%), undecylprodigiosin (181.60%) in S. coelicolor M145:: pLQ003, of rapamycin (110%), hygrocin A (163.4%) in S. hygroscopicus ATCC29253:: pLQ003, and of actinomycin D (11.78%) in S. mutabilis TRM45540:: pLQ003, and also to the downregulation of geldanamycin in S. hygroscopicus XM201, but we found that mutant strains in mutant strains of S. hygroscopicus XM201 with regulatory factors inserted showed several peaks that were not found in wild-type strains. The results of the present work indicated that the regulator net working in Streptomyces was not uniform, the superplasmid we constructed possibly caused this overproduction and downregulation in different Streptomyces.