*Article* **SspH, a Novel HATPase Family Regulator, Controls Antibiotic Biosynthesis in** *Streptomyces*

**Xue Yang 1,†, Yanyan Zhang 1,\* ,†, Shanshan Li <sup>1</sup> , Lan Ye 1,2, Xiangjing Wang <sup>2</sup> and Wensheng Xiang 1,2,\***


† These authors contributed equally to this work.

**Abstract:** *Streptomyces* can produce a wealth of pharmaceutically valuable antibiotics and other bioactive compounds. Production of most antibiotics is generally low due to the rigorously controlled regulatory networks, in which global/pleiotropic and cluster-situated regulatory proteins coordinate with various intra- and extracellular signals. Thus, mining new antibiotic regulatory proteins, particularly the ones that are widespread, is essential for understanding the regulation of antibiotic biosynthesis. Here, in the biopesticide milbemycin producing strain *Streptomyces bingchenggensis*, a novel global/pleiotropic regulatory protein, SspH, a single domain protein containing only the HATPase domain, was identified as being involved in controlling antibiotic biosynthesis. The *sspH* overexpression inhibited milbemycin production by repressing the expression of milbemycin biosynthetic genes. The *sspH* overexpression also differentially influenced the expression of various antibiotic biosynthetic core genes. Site-directed mutagenesis revealed that the HATPase domain was essential for SspH's function, and mutation of the conserved amino acid residues N54A and D84A led to the loss of SspH function. Moreover, cross-overexpression experiments showed that SspH and its orthologs, SCO1241 from *Streptomyces coelicolor* and SAVERM\_07097 from *Streptomyces avermitilis*, shared identical functionality, and all exerted a positive effect on actinorhodin production but a negative effect on avermectin production, indicating that SspH-mediated differential control of antibiotic biosynthesis may be widespread in *Streptomyces*. This study extended our understanding of the regulatory network of antibiotic biosynthesis and provided effective targets for future antibiotic discovery and overproduction.

**Keywords:** global regulator; milbemycins; *Streptomyces bingchenggensis*; SspH; actinorhodin; avermectin
