Luteoreticulin

The unusual nitro-substituted polyketides aureothin, neoaureothin (spectinabilin), and luteoreticulin, which are produced by diverse Streptomyces species, point to a joint evolution. Through rational genetic recombination and domain exchanges we have successfully reprogrammed the modular (type I) aur polyketide synthase (PKS) into a synthase that generates luteoreticulin. This is the first rational transformation of a modular PKS to produce a complex polyketide that was initially isolated from a different bacterium. A unique aspect of this synthetic biology approach is that we exclusively used genes from a single biosynthesis gene cluster to design the artificial pathway, an avenue that likely emulates natural evolutionary processes. Furthermore, an unexpected, context-dependent switch in the regiospecificity of a pyrone methyl transferase was observed. We also describe an unprecedented scenario where an AT domain iteratively loads an extender unit onto the cognate ACP and the downstream ACP. This aberrant function is a novel case of non-colinear behavior of PKS domains.

The use of supercritical fluids for the extraction of biologically active compounds from the biomass of microbial fermentations has been compared with extraction using the organic solvents methanol and dichloromethane. Compounds representing a range of structural types were selected for investigation. All the extracts obtained were examined using reversed-phase high-performance liquid chromatography. The extractability of metabolites using unmodified and methanol-modified supercritical-fluid carbon dioxide was examined in particular detail for six microbial metabolites: chaetoglobosin A, mycolutein, luteoreticulin, 7,8-dihydro-7,8-epoxy-1-hydroxy-3-hydroxymethyl-xanthone-8-carboxyl ic acid methyl ester, sydowinin B and elaiophylin. The extraction strength of supercritical-fluid carbon dioxide alone appeared to be lower than that of dichloromethane. All the components of interest that were extractable with dichloromethane and methanol were also extractable with methanol-modified carbon dioxide.

The structural wealth of complex polyketide metabolites produced by bacteria results from intricate, highly evolved biosynthetic programs of modular assembly lines, in which the number of modules defines the size of the backbone, and the domain composition controls the degree of functionalization. We report a remarkable case where polyketide chain length and scaffold depend on the function of a single β-keto processing domain: A ketoreductase domain represents a switch between diverging biosynthetic pathways leading either to the antifungal aureothin or to the nematicidal luteoreticulin. By a combination of heterologous expression, mutagenesis, metabolite analyses, and in vitro biotransformation we elucidate the factors governing non-colinear polyketide assembly involving module skipping and demonstrate that a simple point mutation in type I polyketide synthase (PKS) can have a dramatic effect on the metabolic profile. This finding sheds new light on possible evolutionary scenarios and may inspire future synthetic biology approaches.