AUTHOR=Liu Ying , Khan Sabir , Wu Panpan , Li Bowen , Liu Lanlan , Ni Jingshu , Zhang Hongxia , Chen Ketao , Wu Hang , Zhang Buchang 

TITLE=Uncovering and Engineering a Mini-Regulatory Network of the TetR-Family Regulator SACE_0303 for Yield Improvement of Erythromycin in Saccharopolyspora erythraea

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=9

YEAR=2021

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2021.692901

DOI=10.3389/fbioe.2021.692901

ISSN=2296-4185

ABSTRACT=<p>Erythromycins produced by <italic>Saccharopolyspora erythraea</italic> have broad-spectrum antibacterial activities. Recently, several TetR-family transcriptional regulators (TFRs) were identified to control erythromycin production by multiplex control modes; however, their regulatory network remains poorly understood. In this study, we report a novel TFR, SACE_0303, positively correlated with erythromycin production in <italic>Sac. erythraea</italic>. It directly represses its adjacent gene <italic>SACE_0304</italic> encoding a MarR-family regulator and indirectly stimulates the erythromycin biosynthetic gene <italic>eryAI</italic> and resistance gene <italic>ermE</italic>. SACE_0304 negatively regulates erythromycin biosynthesis by directly inhibiting <italic>SACE_0303</italic> as well as <italic>eryAI</italic> and indirectly repressing <italic>ermE</italic>. Then, the SACE_0303 binding site within the <italic>SACE_0303-SACE_0304</italic> intergenic region was defined. Through genome scanning combined with <italic>in vivo</italic> and <italic>in vitro</italic> experiments, three additional SACE_0303 target genes (<italic>SACE_2467</italic> encoding cation-transporting ATPase, <italic>SACE_3156</italic> encoding a large transcriptional regulator, <italic>SACE_5222</italic> encoding α-ketoglutarate permease) were identified and proved to negatively affect erythromycin production. Finally, by coupling CRISPRi-based repression of those three targets with <italic>SACE_0304</italic> deletion and <italic>SACE_0303</italic> overexpression, we performed stepwise engineering of the SACE_0303-mediated mini-regulatory network in a high-yield strain, resulting in enhanced erythromycin production by 67%. In conclusion, the present study uncovered the regulatory network of a novel TFR for control of erythromycin production and provides a multiplex tactic to facilitate the engineering of industrial actinomycetes for yield improvement of antibiotics.</p>