Assembly and Heterologous Expression of the Coumermycin A₁ Gene Cluster and Production of New Derivatives by Genetic Engineering

Abstract

Many secondary metabolites of clinical importance have been isolated from different Streptomyces species. As most of the natural producers remain difficult to handle genetically, heterologous expression of an entire biosynthetic gene cluster in a well characterised host allows improved possibilities for modifications of the desired compound by manipulation of the biosynthetic genes. However, the large size of a functional gene cluster often prevents its direct cloning into a single cosmid clone. Here we describe a successful strategy to assemble the entire coumermycin A1 biosynthetic gene cluster (38.6 kb) into a single cosmid clone by lambda RED recombination technology. Heterologous expression of the reconstituted gene cluster in Streptomyces coelicolor M512 resulted in the heterologous production of coumermycin A1. Inactivation of the methyltransferase gene couO--responsible for the C-methylation at the 8-positions of the aminocoumarin moieties in coumermycin A1--and heterologous expression of the modified cluster resulted in an accumulation of a C-8-unsubstituted coumermycin A1 derivative. Subsequent expression of the halogenase gene clo-hal from the clorobiocin gene cluster in the heterologous producer strain led to the formation of two new hybrid antibiotics, containing either one or two chlorine atoms. The identities of the new compounds were verified by LC-MS, and their antibacterial activities were tested against Bacillus subtilis in an agar diffusion assay.

Biochemical and Genetic Insights into Asukamycin Biosynthesis

“…Because neither 2B9 nor 10D6 carries a full set of the predicted biosynthetic genes, we modified the conventional -Red recombination strategy (20,(23)(24)(25) and reassembled the cosmids 2B9 and 10D6 into a single clone (Fig. 3).…”

“…The biosynthetic genes involved in C 5 N moiety and CHC-CoA assembly are closely positioned and appear to be co-transcribed, but many other functionally related asu genes are not organized in any apparent order. Recently, the -Red recombination system has been extended to heterologous expression (23)(24)(25). Taking advantage of the shared vector and overlapping inserts, we simplified this system and assembled two cos-mids in a single step by skipping the PCR cloning and multiple "stitching" processes (Fig.…”

Use of a Halogenase of Hormaomycin Biosynthesis for Formation of New Clorobiocin Analogues with 5‐Chloropyrrole Moieties

“…In contrast, when we inactivated the methyltransferase genes novO and couO, which are responsible for 8'-methylation of the aminocoumarin moieties of novobiocin and clorobiocin, and expressed the corresponding 8'-halogenase from the clorobiocin cluster in the resulting mutants, more than 75 % of the aminocoumarins were accumulated as 8'-chlorinated compounds. [7,9] In these two cases, the substrates of the halogenation reactions are likely to be identical to those of the methylation reactions, [34,35] although the Clo-hal reaction has not yet been demonstrated in vitro. …”

Formation and Attachment of the Deoxysugar Moiety and Assembly of the Gene Cluster for Caprazamycin Biosynthesis

“…It has been successfully applied for functional studies and engineering of antibiotic biosynthetic machineries (17), e.g., heterologous expression (10) and combinatorial biosynthesis (11). More recently, recombineering was used for the reconstitution of large biosynthetic gene clusters from overlapping inserts (3,22,57). This "stitching" overcomes the problem that genomic library clones rarely contain an entire gene cluster due to limitations of the average insert sizes.…”