Recombinatorial biosynthesis of polyketides

Abstract

Modular polyketide synthases (PKSs) from Streptomyces and related genera of bacteria produce many important pharmaceuticals. A program called CompGen was developed to carry out in silico homologous recombination between gene clusters encoding PKSs and determine whether recombinants have cluster architectures compatible with the production of polyketides. The chemical structure of recombinant polyketides was also predicted. In silico recombination was carried out for 47 well-characterised clusters. The predicted recombinants would produce 11,796 different polyketide structures. The molecular weights and average degree of reduction of the chemical structures are dispersed around the parental structures indicating that they are likely to include pharmaceutically interesting compounds. The details of the recombinants and the chemical structures were entered in a database called r-CSDB. The virtual compound library is a useful resource for computer-aided drug design and chemoinformatics strategies for finding pharmaceutically relevant chemical entities. A strategy to construct recombinant Streptomyces strains to produce these polyketides is described and the critical steps of mobilizing large biosynthetic clusters and producing new linear cloning vectors are illustrated by experimental data.

Annotation of the Modular Polyketide Synthase and Nonribosomal Peptide Synthetase Gene Clusters in the Genome of Streptomyces tsukubaensis NRRL18488

“…However, the other modules did not show such similarity, suggesting that the two clusters might have diverged through recombination with another unrelated cluster. Such single-crossover events were suggested previously to be a major driving force in the evolution of clusters (24,25,31). It was possible to demonstrate that cluster St-PKS2 was transcribed by using RT-PCR (Fig.…”

Evolution and Diversity of Assembly-Line Polyketide Synthases

“…Another approach inspired by natural PKS evolution uses homologous recombination for the assembly and relies on naturally occurring regions of sequence similarity. The possibility of using homologous recombination was first assessed computationally and suggested numerous regions of sequence similarity that could potentially lead to chimeric assemblies and new molecules. , The feasibility of this approach was later shown experimentally in two different studies. First, homologous DNA recombination between DEBS and pikromycin (PIKS) clusters was shown to produce numerous functional chimeric assembly lines with splicing points located at various locations within modules, though preferentially in KS and AT domain-encoding regions .…”

Diversity oriented biosynthesis via accelerated evolution of modular gene clusters

“…This direct relationship between the sequence of PKS-encoding genes and the structure of the product molecule immediately suggested the potential for rational genetic modification to yield strains capable of producing analogues of predictable structure, and although then many groups have attempted to bioengineer these systems with varying success 5 – 7 . Several examples of targeted in vivo analogue production at reasonable titre have been described 8 – 10 , although a reduction in productivity often accompanies the generation of hybrid or modified PKSs 11 – 13 . This approach is most suited to producing focussed libraries closely related in structure to the parent molecule, limiting its industrial application to lead optimisation.…”