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.
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