Heterologous expression, purification, reconstitution and kinetic analysis of an extended type II polyketide synthase.

BACKGROUND: Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products. The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase. Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain. We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC). RESULTS: The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies. Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone. Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate. CONCLUSIONS: Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces. Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS. They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems.

Domain analysis of the molecular recognition features of aromatic polyketide synthase subunits.

Bacterial aromatic polyketide synthases (PKSs) are a family of homologous multienzyme assemblies that catalyze the biosynthesis of numerous polyfunctional aromatic natural products. In the absence of direct insights into their structures, the use of gene fusions can be a powerful tool for understanding the structural basis for their properties. A series of truncated and hybrid proteins were constructed and analyzed within a family of PKS subunits, designated aromatases/cyclases (ARO/CYCs). When expressed alone, neither the N-terminal nor the C-terminal domain of the actinorhodin (act) or the griseusin (gris) ARO/CYC exhibited substantial aromatase activity. However, in the presence of each other, the half proteins were active. Furthermore, analysis of a set of hybrid proteins derived from the act and gris ARO/CYCs allowed us to localize the chain length dependence of this aromatase activity to their N-terminal domains. Unexpectedly, however, when the C-terminal domain of the gris ARO/CYC was expressed in a context where aromatase activity was absent, it could modulate the chain length specificity of the tetracenomycin (tcm) minimal PKS, leading to the formation of a novel 18-carbon product in addition to the expected 20-carbon one. It was also found that monodomain ARO/CYCs such as tcmN cannot substitute for the the N-terminal domain of didomain ARO/CYCs, even though they exhibit high sequence similarity with the N-terminal domain. Together, these results illustrate the utility of protein engineering approaches for dissecting the structure-function relationships of PKS subunits and for the generation of mutant alleles with novel biosynthetic properties.

Generation of polyketide libraries via combinatorial biosynthesis.

Polyketides are a family of structurally complex natural products that include a number of important pharmaceuticals. Motivated by the value of these natural products, there has been much research focused on developing guidelines for engineering polyketide synthases (PKSs) to generate novel polyketides. Recent studies have provided interesting insights into the enzymatic specificity of the polyketide synthesis pathway, and have demonstrated that various PKSs can be genetically manipulated to synthesize 'unnatural' polyketide natural products. In this article, we discuss the synthesis of polyketides and polyketide libraries by combinatorial biosynthesis.