RESUMEN
Virginiamycin M (VM) of Streptomyces virginiae is a hybrid polyketide-peptide antibiotic with peptide antibiotic virginiamycin S (VS) as its synergistic counterpart. VM and VS belong to the Streptogramin family, which is characterized by strong synergistic antibacterial activity, and their water-soluble derivatives are a new therapeutic option for combating vancomycin-resistant Gram-positive bacteria. Here, the VM biosynthetic gene cluster was isolated from S. virginiae in the 62-kb region located in the vicinity of the regulatory island for virginiamycin production. Sequence analysis revealed that the region consists of 19 complete open reading frames (ORFs) and one C-terminally truncated ORF, encoding hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS), typical PKS, enzymes synthesizing precursors for VM, transporters for resistance, regulatory proteins, and auxiliary enzymes. The involvement of the cloned gene cluster in VM biosynthesis was confirmed by gene disruption of virA encoding a hybrid PKS-NRPS megasynthetase, which resulted in complete loss of VM production without any effect on VS production. To assemble the VM core structure, VirA, VirF, VirG, and VirH consisting, as a whole, of 24 domains in 8 PKS modules and 7 domains in 2 NRPS modules were predicted to act as an acyltransferase (AT)-less hybrid PKS-NRPS, whereas VirB, VirC, VirD, and VirE are likely to be essential for the incorporation of the methyl group into the VM framework by a HMG-CoA synthase-based reaction. Among several uncommon features of gene organization in the VM gene cluster, the lack of AT domain in every PKS module and the presence of a discrete AT encoded by virI are notable. AT-overexpression by an additional copy of virI driven by ermEp() resulted in 1.5-fold increase of VM production, suggesting that the amount of VirI is partly limiting VM biosynthesis.
Asunto(s)
Antibacterianos/biosíntesis , Genes Bacterianos , Familia de Multigenes/genética , Estreptograminas/biosíntesis , Streptomyces/genética , Streptomyces/metabolismo , Virginiamicina/biosíntesis , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Emparejamiento Base/genética , Secuencia de Bases , ADN Bacteriano/genética , Farmacorresistencia Bacteriana/genética , Regulación Bacteriana de la Expresión Génica , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Insercional , Péptido Sintasas/metabolismo , Filogenia , Sintasas Poliquetidas/química , Sintasas Poliquetidas/metabolismo , Estructura Terciaria de Proteína , Recombinación Genética/genética , Análisis de Secuencia de Proteína , Estreptograminas/química , Transcripción Genética , Virginiamicina/químicaRESUMEN
Heterologous higher order control modalities will be important tools for targeted multigene interventions in next-generation gene therapy, tissue engineering, and sophisticated gene-function studies. In this study, we present the design and rigorous quantitative analysis of a variety of different dual-regulated gene transcription control configurations combining streptogramin- and tetracycline-responsive expression systems in a one-vector format. Quantitative assessment of dual-regulated expression performance in various mammalian and human cell lines is based on two compatible secreted reporter genes, SEAP, the human placental secreted alkaline phosphatase, and the recently developed SAMY, the secreted alpha-amylase. Assembly of streptogramin- and tetracycline-responsive transgene control units in consecutive (--> -->), divergent (<-- -->), and convergent (--> <--) orientation showed excellent regulation characteristics in most genetic arrangements exemplified by neglectable interference and high transgene induction ratios in all four control settings (ON/ON, OFF/ON, ON/OFF, OFF/OFF). The overall regulation performance of divergent dual-regulated expression configurations could be substantially increased when placing noncoding stuffer fragments or insulator modules between the divergently oriented antibiotic-responsive promoters. Dual-regulated expression technology pioneers artificial higher order gene control networks that will likely enable new opportunities in multigene metabolic engineering and generate significant therapeutic impact.