Characterization of three pathway-specific regulators for high production of monensin in Streptomyces cinnamonensis

Abstract

Monensin, a polyether ionophore antibiotic, is produced by Streptomyces cinnamonensis and worldwide used as a coccidiostat and growth-promoting agent in the field of animal feeding. The monensin biosynthetic gene cluster (mon) has been reported. In this study, the potential functions of three putatively pathway-specific regulators (MonH, MonRI, and MonRII) were clarified. The results from gene inactivation, complementation, and overexpression showed that MonH, MonRI, and MonRII positively regulate monensin production. Both MonH and MonRI are essential for monensin biosynthesis, while MonRII is non-essential and could be completely replaced by additional expression of monRI. Transcriptional analysis of the mon cluster by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and electrophoresis mobility shift assays (EMSAs) revealed a co-regulatory cascade process. MonH upregulates the transcription of monRII, and MonRII in turn enhances the transcription of monRI. MonRII is an autorepressor, while MonRI is an autoactivator. MonH activates the transcription of monCII-monE, and upregulates the transcription of monT that is repressed by MonRII. monAX and monD are activated by MonRI, and upregulated by MonRII. Co-regulation of those post-polyketide synthase (post-PKS) genes by MonH, MonRI, and MonRII would contribute to high production of monensin. These results shed new light on the transcriptional regulatory cascades of antibiotic biosynthesis in Streptomyces.

Regulatory Patterns of Crp on Monensin Biosynthesis in Streptomyces cinnamonensis

“…Then, tailoring enzyme MonCI mediates an epoxidation of the polyolefin constructed by polyketide synthases, and MonBI and MonBII cooperatively catalyze the cyclization of the resultant polyepoxide to yield the polyether skeleton of monensin. Our previous studies showed that three pathway-specific regulators (MonRI, MonRII and MonH) [26] and the global regulator DasR [27] do not regulate the transcriptions of mon PKSs genes and tailoring genes monCI, monBI and monBII. Here, our results suggested that the targets of Crp regulation would be the PKSs and monCI, monBI and monBII genes of the mon cluster.…”

“…Moreover, Crp regulations on pathway-specific regulatory genes actII-ORF4, redZ and cdaR were identified in S. coelicolor [12]. Of three pathway-specific regulators for monensin biosynthesis, MonRI and MonRII upregulated the transcription of tailoring gene monD and thioesterase gene monAX [26], while monRI was under the control of Crp, indicating that Crp possibly exerted regulatory effects on the transcription of monD and monAX through its tuning on monRI transcription.…”

“…There are twenty genes in the mon cluster, and our previous study suggested that the monensin biosynthetic gene cluster is transcribed into eight transcription units (TUs) [26], shown in Figure 5a. The RNA-seq analysis results showed that TU4, TU5, TU6, TU7 and TU8 in the mon cluster were significantly upregulated in the crp overexpression strain ST042 and correspondingly downregulated in the anti-crp strain ST043, compared to the original strain ST021 (Figure 5b).…”

“…Our laboratory focuses on the transcriptional factors for regulation of monensin biosynthesis. Our previous study showed that MonRI, MonRII and MonH are three positive pathway-specific regulators and that they cooperatively control mon genes in monensin biosynthesis [26]. The global factor DasR positively controls monensin biosynthesis via regulating both pathway-specific regulatory gene monRII and function genes such as monAIX, monE and monT [27].…”

Actinomycete-Derived Polyketides as a Source of Antibiotics and Lead Structures for the Development of New Antimicrobial Drugs

“…The additional genes found in the mon gene cluster encode enzymes involved in isomerisation, epoxidation (MonCI), hydroxylation (MonBI, MonBII, and MonD), and methylation (MonE) of the structure to the final product monensin A [98,219]. Finally, the mon gene cluster includes three regulator genes, monH, monRI, and monRII, and the gene monT, which encodes an efflux protein, believed to confer self-resistance to the host [98,217,220,221].…”