Production of the Tubulin Destabilizer Disorazol in Sorangium cellulosum: Biosynthetic Machinery and Regulatory Genes

Abstract

Myxobacteria show a high potential for the production of natural compounds that exhibit a wide variety of antibiotic, antifungal, and cytotoxic activities. The genus Sorangium is of special biotechnological interest because it produces almost half of the secondary metabolites isolated from these microorganisms. We describe a transposon-mutagenesis approach to identifying the disorazol biosynthetic gene cluster in Sorangium cellulosum So ce12, a producer of multiple natural products. In addition to the highly effective disorazol-type tubulin destabilizers, S. cellulosum So ce12 produces sorangicins, potent eubacterial RNA polymerase inhibitors, bactericidal sorangiolides, and the antifungal chivosazoles. To obtain a transposon library of sufficient size suitable for the identification of the presumed biosynthetic gene clusters, an efficient transformation method was developed. We present here the first electroporation protocol for a strain of the genus Sorangium. The transposon library was screened for disorazol-negative mutants. This approach led to the identification of the corresponding trans-acyltransferase core biosynthetic gene cluster together with a region in the chromosome that is likely to be involved in disorazol biosynthesis. A third region in the genome harbors another gene that is presumed to be involved in the regulation of disorazol production. A detailed analysis of the biosynthetic and regulatory genes is presented in this paper.

Structural and Functional Characterization of Three Polyketide Synthase Gene Clusters in Bacillus amyloliquefaciens FZB 42

“…Instead, discrete AT genes were detected upstream of the megasynthase open reading frames (Table 2). This architecture, which has been recently described as trans-AT PKS (34), is also known from a smaller number of other systems, such as the pederin and onnamide PKSs from bacterial endosymbionts of beetles (34) and sponges (36), respectively; the leinamycin (lnm) PKS from Streptomyces atroolivaceus (7); the mupirocin PKS from Pseudomonas fluorescens (11); the myxovirescin (TA) PKS from Myxococcus xanthus (30); and others (4,19,40). Experimental evidence presented for the LnmG AT suggested that the discrete ATs act iteratively by loading malonyl coenzyme A (CoA) onto all PKS modules during polyketide synthesis (7).…”

Insights into the Evolution of Macrolactam Biosynthesis through Cloning and Comparative Analysis of the Biosynthetic Gene Cluster for a Novel Macrocyclic Lactam, ML-449

“…BecA and BecC are thought to complete the synthesis of the acyl chain, which is subsequently modified into an aminoacyl. Such splitting of modules on separate proteins has been reported in several cases (7,23,32,36). The structure of ML-449 indicates incorporation of one additional acetate unit compared to what is observed in BE-14106 biosynthesis.…”

Engineering the acyltransferase substrate specificity of assembly line polyketide synthases

“…These trans-AT PKSs include those responsible for production of the disorazoles [76,77], kirromycin [78 -80] and several others [75,81,82]. A potential engineering strategy involves the selective inactivation of a cis-AT domain and complementation using a trans-AT with differing specificity [9].…”

“…ACP transacylase [77] erythromycin AT6null/ bryostatin AT1 [78] erythromycin AT6null/ bryostatin AT2 [78] MMCoA/MCoA Figure 3. Previous attempts to engineer AT domain substrate specificity.…”