Magdalena Kotowska

A cryptic type I polyketide synthase (cpk) gene cluster in Streptomyces coelicolor A3(2)

The chromosome of Streptomyces coelicolor A3(2), a model organism for the genus Streptomyces, contains a cryptic type I polyketide synthase (PKS) gene cluster which was revealed when the genome was sequenced. The ca. 54-kb cluster contains three large genes, cpkA, cpkB and cpkC, encoding the PKS subunits. Insilico analysis showed that the synthase consists of a loading module, five extension modules and a unique reductase as a terminal domain instead of a typical thioesterase. All acyltransferase domains are specific for a malonyl extender, and have a B-type ketoreductase. Tailoring and regulatory genes were also identified within the gene cluster. Surprisingly, some genes show high similarity to primary metabolite genes not commonly identified in any antibiotic biosynthesis cluster. Using western blot analysis with a PKS subunit (CpkC) antibody, CpkC was shown to be expressed in S. coelicolor at transition phase. Disruption of cpkC gave no obvious phenotype.

Type II thioesterase from Streptomyces coelicolor A3(2)

Type I polyketide synthases (PKSs) are complexes of large, multimodular enzymes that catalyse biosynthesis of polyketide compounds via repetitive reaction sequences, during which each step is catalysed by a separate enzymic domain. Many type I PKSs, and also non-ribosomal peptide synthetase clusters, contain additional thioesterase genes located adjacent to PKS genes. These are discrete proteins called type II thioesterases (TE IIs) to distinguish them from chain-terminating thioesterase (TE I) domains that are usually fused to the terminal PKS module. A gene of a new TE II, scoT, associated with the cluster of putative type I PKS genes from Streptomyces coelicolor A3(2), was found. The deduced amino acid sequence of the gene product shows extensive similarity to other authentic thioesterase enzymes, including conservation of characteristic motifs and residues involved in catalysis. When expressed in the heterologous host Streptomyces fradiae, scoT successfully complemented the resident TE II gene (tylO), and, by restoring a significant level of macrolide production, proved to be catalytically equivalent to the TylO protein. S1 nuclease mapping of scoT revealed a single potential transcription start point with expression being switched on for a short period of time during a transition phase of growth.

Roles of type II thioesterases and their application for secondary metabolite yield improvement

A large number of antibiotics and other industrially important microbial secondary metabolites are synthesized by polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). These multienzymatic complexes provide an enormous flexibility in formation of diverse chemical structures from simple substrates, such as carboxylic acids and amino acids. Modular PKSs and NRPSs, often referred to as megasynthases, have brought about a special interest due to the colinearity between enzymatic domains in the proteins working as an “assembly line” and the chain elongation and modification steps. Extensive efforts toward modified compound biosynthesis by changing organization of PKS and NRPS domains in a combinatorial manner laid good grounds for rational design of new structures and their controllable biosynthesis as proposed by the synthetic biology approach. Despite undeniable progress made in this field, the yield of such “unnatural” natural products is often not satisfactory. Here, we focus on type II thioesterases (TEIIs)—discrete hydrolytic enzymes often encoded within PKS and NRPS gene clusters which can be used to enhance product yield. We review diverse roles of TEIIs (removal of aberrant residues blocking the megasynthase, participation in substrate selection, intermediate, and product release) and discuss their application in new biosynthetic systems utilizing PKS and NRPS parts.

Streptomyces coelicolor A3(2) Produces a New Yellow Pigment Associated with the Polyketide Synthase Cpk

Streptomyces coelicolor A3(2) is an extensively studied model organism for the genetic studies of Streptomycetes – a genus known for the production of a vast number of bioactive compounds and complex regulatory networks controlling morphological differentiation and secondary metabolites production. We present the discovery of a presumptive product of the Cpk polyketide synthase. We have found that on the rich medium without glucose S. coelicolor A3(2) produces a yellow compound secreted into the medium. We have proved by complementation that production of the observed yellow pigment is dependent on cpk gene cluster previously described as cryptic type I polyketide synthase cluster. The pigment production depends on the medium composition, does not occur in the presence of glucose, and requires high density of spore suspension used for inoculation.