With the development of synthetic biology in the field of (actinobacterial) specialized metabolism, new tools are needed for the design or refactoring of biosynthetic gene clusters. If libraries of synthetic parts (such as promoters or ribosome binding sites) and DNA cloning methods have been developed, to our knowledge, not many vectors designed for the flexible cloning of biosynthetic gene clusters have been constructed. We report here the construction of a set of 12 standardized and modular vectors designed to afford the construction or the refactoring of biosynthetic gene clusters in Streptomyces species, using a large panel of cloning methods. Three different resistance cassettes and four orthogonal integration systems are proposed. In addition, FLP recombination target sites were incorporated to allow the recycling of antibiotic markers and to limit the risks of unwanted homologous recombination in Streptomyces strains when several vectors are used. The functionality and proper integration of the vectors in three commonly used Streptomyces strains, as well as the functionality of the Flp-catalyzed excision, were all confirmed. To illustrate some possible uses of our vectors, we refactored the albonoursin gene cluster from Streptomyces noursei using the BioBrick assembly method. We also used the seamless ligase chain reaction cloning method to assemble a transcription unit in one of the vectors and genetically complement a mutant strain. Volume 85 Issue 16 e00485-19 aem.asm.org 2 on July 10, 2020 by guest http://aem.asm.org/ Downloaded from cloning methods in Streptomyces species, prolific producers of specialized metabolites. These vectors were designed on the model of the SEVA plasmids, although the exact architecture of these plasmids could not be used for our application. The 12 vectors were proven to be functional by the verified integration in the chromosome of three commonly used Streptomyces species. We also illustrate two possible uses of our vectors. We first refactored the albonoursin gene cluster using BioBrick assembly. Second, we genetically complemented our cgc22 mutant strain, CGCL030 (cgc22 is involved in congocidine biosynthesis [30]), by constructing a gene cassette constituted of a promoter, an RBS, cgc22, and a terminator using ligase chain reaction assembly. RESULTS AND DISCUSSIONDesign of the vectors. The vectors were designed to meet the following specifications. It should be possible to use several vectors in the same strain (orthogonality), so different antibiotic resistance cassettes and different systems of integration at specific sites in the chromosome of Streptomyces should be used for the construction of the vectors. The vectors should be E. coli/Streptomyces shuttle vectors so that genetic constructions can be prepared in E. coli before being introduced into Streptomyces strains; thus, an E. coli origin of replication has to be included. It should be possible to introduce the vectors into Streptomyces strains by E. coli/Streptomyces intergeneric conjugation, so the presence o...
Anthelvencins A and B are pyrrolamide metabolites produced by Streptomyces venezuelae ATCC 14583 and 14585. Isolated in 1965, they were reported to exhibit anthelmintic and moderate antibacterial activities. In this study, we revise the structure of anthelvencin A and identify a third anthelvencin metabolite, bearing two N-methylated pyrrole groups, which we named anthelvencin C. We sequenced the genome of S. venezuelae ATCC 14583 and identified a gene cluster predicted to direct the biosynthesis of anthelvencins. Functional analysis of this gene cluster confirmed its involvement in anthelvencin biosynthesis and allowed us to propose a biosynthetic pathway for anthelvencins. In addition to a nonribosomal peptide synthetase (NRPS), the assembly of anthelvencins involves an enzyme from the ATP-grasp ligase family, Ant23. We propose that Ant23 uses a PCP-loaded 4aminopyrrole-2-carboxylate as substrate. As observed for the biosynthesis of the other pyrrolamides congocidine (produced by Streptomyces ambofaciens ATCC 25877) and distamycin (produced by Streptomyces netropsis DSM 40846), the NRPS assembling anthelvencins is composed of stand-alone domains only. Such NRPSs, sometimes called type II NRPSs, are less studied than the classical multimodular NRPSs. Yet, they constitute an interesting model to study protein-protein interactions in NRPSs and are good candidates for combinatorial biosynthesis approaches.
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