Genome sequencing and bioinformatics analyses indicated that Streptomyces thioluteus has great potential to produce a variety of bioactive natural products and possesses complex regulatory mechanisms of secondary metabolites. In order to investigate the biosynthetic pathways of putative secondary metabolites and regulation mechanisms in this strain, a genomic expression library of S. thioluteus was constructed by using an integrative Streptomyces plasmid pJTU2554 as the vector. Nine heterologous expression mutant strains with antibacterial activities were obtained by bioassay screening of the expression library in the heterologous host of Streptomyces lividans ZX1. Positive clones carrying the putative polyketide synthase (PKS) and noribosomal peptide synthetase (NRPS) genes were screened from the library by PCR. Results of TLC and HPLC-MS analyses showed that heterolgous expression of 6 positive cosmids containing the aureothin biosynthetic gene cluster in S. lividans ZX1 resulted in the production of the polyketide antibiotic, aureothin. It is indicated that heterologous expression and screening of the library was successful in S. thioluteus.