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

Abstract

Erythromycins produced by Saccharopolyspora erythraea 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 Sac. erythraea. It directly represses its adjacent gene SACE_0304 encoding a MarR-family regulator and indirectly stimulates the erythromycin biosynthetic gene eryAI and resistance gene ermE. SACE_0304 negatively regulates erythromycin biosynthesis by directly inhibiting SACE_0303 as well as eryAI and indirectly repressing ermE. Then, the SACE_0303 binding site within the SACE_0303-SACE_0304 intergenic region was defined. Through genome scanning combined with in vivo and in vitro experiments, three additional SACE_0303 target genes (SACE_2467 encoding cation-transporting ATPase, SACE_3156 encoding a large transcriptional regulator, SACE_5222 encoding α-ketoglutarate permease) were identified and proved to negatively affect erythromycin production. Finally, by coupling CRISPRi-based repression of those three targets with SACE_0304 deletion and SACE_0303 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.

CRISPR/Cas9-Mediated Multi-Locus Promoter Engineering in ery Cluster to Improve Erythromycin Production in Saccharopolyspora erythraea

“…In addition to the engineering efforts targeting the biosynthetic genes, there are also many successful examples of manipulating genes outside the ery cluster to improve erythromycin biosynthesis in S. erythraea strains. For rational strain engineering, enhancing precursor supply/energy availability [ 27 , 28 , 29 ], manipulating regulators/two-component systems [ 30 , 31 , 32 ], and overexpressing heterologous non-PKS genes such as the hemoglobin gene vhb [ 33 ], are proved to be effective strategies to enhance erythromycin production in S. erythraea to various degrees. The effectiveness of these modifications collectively suggests that erythromycin biosynthesis is probably restricted by a series of complex factors, which are not only located in the ery cluster but also distributed among the bacterial genome.…”

CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria

“…CRISPRi/a tools have also been used to redirect carbon and energy flow for metabolic engineering in non-model bacteria ( Figure 1C ). CRISPRi is often used to repress a native gene(s), including essential genes, to redirect carbon flux towards a desired product ( Wang et al, 2017 ; Shabestary et al, 2018 ) or bioactive molecule ( Yu et al, 2018 ; Liu et al, 2021b ). CRISPRa can be used to activate the desired metabolic pathway to increase biosynthesis of the desired product, such as an anti-cancer drug in a weakly-expressed biosynthetic gene cluster ( Peng et al, 2018 ; Ye et al, 2019 ).…”

New Targets of TetR-type regulator SLCG_2919 for Controlling Lincomycin Biosynthesis in Streptomyces lincolnensis

“…Although 70% of the known natural antibiotics, such as lincomycin, spinosad, and avermectins are produced by actinomycetes and have important application values in the elds of medicine and agriculture (Palazzotto et al 2019), these secondary metabolites still exhibit low industrial fermentation yields. Rewiring the regulatory network of TFs in actinomycetes has manifested great potential for industrial overproduction of antibiotics (Liu et al 2021). Therefore, the de nition of these complicated regulatory mechanisms will help overcome the bottleneck of low outputs of secondary metabolites.…”