Regulation of secondary metabolism involves complex interactions of both pathway-specific regulators and global regulators, which may trigger or repress the expression of genes involved in antibiotic biosynthesis. Similarly, many of these global regulatory proteins belong to two-component systems. In this study, a new two-component system (TCS) AfrQ1Q2 homologous to AfsQ1Q2 of Streptomyces coelicolor was acquired from the genome sequence of Streptomyces rimosus M4018 by using bioinformatics analysis. RT-PCR results showed co-transcription of afrQ1 (RR) and afrQ2 (HK) in S. rimosus. Consequently, the significant enhancement in oxytetracycline (OTC) yield in afrQ1-disrupted mutant was observed when cultivated in the defined minimal medium (MM) with glycine as the sole nitrogen source. In order to further investigate the regulation mechanism of AfrQ1Q2 in OTC production, the transcriptional levels of five biosynthesis and regulation related genes such as oxyB, otrB, otcG, otcR and otrC were tested by qRT-PCR, which indicated a significantly up-regulatory trend in the afrQ1-disrupted mutant. Meanwhile, a down-regulatory trend of each gene was tested in the complementary mutant as compared to wild type M4018. Moreover, these selected five genes were positively correlated with OTC production. Conclusively, these findings suggested that the TCS AfrQ1Q2 could be one of the global regulators, which negatively regulates OTC production via activating pathway specific regulators in S. rimosus M4018. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.