A Complex Signaling Cascade Governs Pristinamycin Biosynthesis in Streptomyces pristinaespiralis

Abstract

Pristinamycin production in Streptomyces pristinaespiralis Pr11 is tightly regulated by an interplay between different repressors and activators. A ␥-butyrolactone receptor gene (spbR), two TetR repressor genes (papR3 and papR5), three SARP (Streptomyces antibiotic regulatory protein) genes (papR1, papR2, and papR4), and a response regulator gene (papR6) are carried on the large 210-kb pristinamycin biosynthetic gene region of Streptomyces pristinaespiralis Pr11. A detailed investigation of all pristinamycin regulators revealed insight into a complex signaling cascade, which is responsible for the fine-tuned regulation of pristinamycin production in S. pristinaespiralis. Streptomycetes are filamentous, Gram-positive soil bacteria that are well known for their ability to produce varieties of bioactive secondary metabolites, including more than 70% of the commercially important antibiotics (1). The production of antibiotics is controlled by a vast array of physiological and nutritional conditions, communicated by extracellular and intracellular signaling molecules (2). The beginning of antibiotic biosynthesis is often coordinated with processes of morphological differentiation. The characteristic Streptomyces life cycle involves the formation of a feeding substrate mycelium and subsequent development of aerial hyphae, which finally septate into spores (3). Generally, antibiotic production begins as the culture enters stationary growth in liquid culture and coincidences with the onset of morphological differentiation in agar-grown cultures (reviewed in reference 4). In many Streptomyces strains, antibiotic production is regulated by low-molecular-weight compounds, called ␥-butyrolactone autoregulators (GBLs) (5, 6). GBLs are small diffusible signaling molecules that are synthesized and gradually accumulated in a growth-dependent manner, at or near the middle of the exponential phase of Streptomyces growth, when they trigger the onset of antibiotic biosynthesis and/or morphological differentiation at nanomolar concentrations (7). Often, the GBL signal is transmitted via a hierarchical signaling cascade including pleiotropic and pathway-specific regulators, which all together control the antibiotic production: when the GBL concentration reaches a critical level, the signal is transmitted into the cells by binding to specific cytoplasmic receptor proteins, the GBL receptors (7). GBL receptors belong to the TetR family of transcriptional regulators (8). In the absence of the corresponding ligand, the GBL receptor binds to conserved AT-rich, partially palindromic sequences (9), the so-called "ARE" sequences (autoregulatory element) (10), within the promoter regions of its target genes and thereby represses the transcription of these genes. By binding of the GBLs to their receptors, the latter undergo a conformational change and dissociate from the target DNA, allowing expression of the derepressed genes (11). Predominantly, targets of GBL receptors are transcriptional regulatory genes, such as TetR and SARP (Streptomyce...

Genetic engineering approaches for the fermentative production of phenylglycines

“…pristinaespiralis Pr11 wild type was grown in pristinamycin inoculum and production medium as reported previously (Mast et al 2011a). Samples were harvested after 24 h. RNA isolation and RT-PCR procedure were carried out as described before (Mast et al 2015). For RT-PCR reactions, primers RTpglfw/rv were used that anneal to overlapping regions of the pgl gene sequences.…”

“…In order to select for Phg operon containing transformants of apramycin-resistant mutants (MpglE (Mast et al 2011a) and papR5::apra (Mast et al 2015)), expression constructs were designed, which harbor a thiostrepton resistance cassette (thio R ). For this purpose, the thio R cassette was isolated as a XbaI-restricted fragment from pDrive-thio and was cloned into the XbaI restriction site of pRM4, pYM/lpg, and pYM/ dpg, resulting in the expression constructs pYMT, pYMT/lpg, and pYMT/dpg, respectively ( Fig.…”

“…As we had incident that Phg production performance in S. pristinaespiralis depends on the pristinamycin biosynthesis capability (see above for MpglE, MsnbDE samples), we aimed to further enhance Phg production by using a pristinamycin superproducer as expression host. In a previous study, we showed that the S. pristinaespiralis repressor mutant papR5::apra produces up to~300% more pristinamycin than the wild-type strain (Mast et al 2015). Due to this high pristinamycin production capability, we used papR5::apra as a host for Phg operon expression.…”

“…Within the pristinamycin biosynthetic gene region, the genes pglA, pglB, pglC, pglD, and pglE are organized in an operon-like structure (lpg) and together encode for L-Phg biosynthesis (Mast et al 2011a, Osipenkov 2016. These genes are located downstream of the NRPS genes snbC and snbDE and are under control of the pathway-specific transcriptional activator PapR2 (Mast et al 2015) (Fig. 1a).…”

Toward Systems Metabolic Engineering of Streptomycetes for Secondary Metabolites Production

“…The metabolic engineering efforts were strongly aided from previous findings of the regulatory cascade governing the biosynthesis of both PI and PII in S. pristinaespiralis. [79] For PII, the deletion of the two cluster-specific repressors PapR3 and PapR5 along with overexpression of the activators PapR4 and PapR6 resulted in a 1.5-fold higher production of PII in a mutant harboring an additional copy of the PII BGC, compared to the wild-type. Furthermore, the addition of resins to relieve both feedback inhibition and toxicity of PII resulted in the 5.26-fold higher production, compared to wild-type, when grown in 5 L bioreactors.…”