Heuristics for the rational design of aromatic polyketides were recently proposed and tested via the engineered biosynthesis of two novel products. Here we have applied these rules to a previously untested subclass of aromatic polyketides, the unreduced molecules. A recombinant strain of Streptomyces coelicolor expressing the genes for the frenolicin (fren) minimal polyketide synthase (PKS) and the TcmN subunit (a putative aromatase/ cyclase) from the tetracenomycin PKS was constructed, and its principal product, the nonaketide PK8, was characterized by spectroscopic and isotope labeling methods. The structure of PK8 was exactly as predicted by the design rules. Surprisingly however, no major octaketide product was isolated from this strain. In contrast, a strain expressing the fren minimal PKS genes alone produced octaketides to the exclusion of nonaketides. These results differ from earlier reports of both octaketide and nonaketide products from strains containing the fren minimal PKS and a regiospecific ketoreductase. We therefore propose a model for bacterial aromatic polyketide biosynthesis in which auxiliary PKS subunits such as ketoreductases, aromatases, and cyclases can modulate the intrinsic specificity of the minimal PKS with respect to both the folding pattern and the chain length of the final product.
Urea is the major nitrogenous end product of protein metabolism in mammals. Here, we describe a quantitative, sensitive method for urea determination using a modified Jung reagent. This assay is specific for urea and is unaffected by ammonia, a common interferent in tissue and cell cultures. We demonstrate that this convenient colorimetric microplate-based, room temperature assay can be applied to determine urea synthesis in cell culture.
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.
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.
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