No abstract

SummaryConglobatin is an unusual C2-symmetrical macrodiolide from the bacterium Streptomyces conglobatus with promising antitumor activity. Insights into the genes and enzymes that govern both the assembly-line production of the conglobatin polyketide and its dimerization are essential to allow rational alterations to be made to the conglobatin structure. We have used a rapid, direct in vitro cloning method to obtain the entire cluster on a 41-kbp fragment, encoding a modular polyketide synthase assembly line. The cloned cluster directs conglobatin biosynthesis in a heterologous host strain. Using a model substrate to mimic the conglobatin monomer, we also show that the conglobatin cyclase/thioesterase acts iteratively, ligating two monomers head-to-tail then re-binding the dimer product and cyclizing it. Incubation of two different monomers with the cyclase produces hybrid dimers and trimers, providing the first evidence that conglobatin analogs may in future become accessible through engineering of the polyketide synthase.

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.

The α,β-epoxyketone proteasome inhibitor TMC-86A was discovered as a previously unreported metabolite of Streptomyces chromofuscus ATCC49982, and the gene cluster responsible for its biosynthesis was identified via genome sequencing. Incorporation experiments with [(13)C-methyl]l-methionine implicated an α-dimethyl-β-keto acid intermediate in the biosynthesis of TMC-86A. Incubation of the chemically synthesized α-dimethyl-β-keto acid with a purified recombinant flavin-dependent enzyme that is conserved in all known pathways for epoxyketone biosynthesis resulted in formation of the corresponding α-methyl-α,β-epoxyketone. This transformation appears to proceed via an unprecedented decarboxylation-dehydrogenation-monooxygenation cascade. The biosynthesis of the TMC-86A warhead is completed by cytochrome P450-mediated hydroxylation of the α-methyl-α,β-epoxyketone.

Say “when”: Analysis of the biosynthetic gene cluster for the polyether antibiotic and anticancer agent salinomycin (1) shows that its core structure is synthesised by a nine‐multienzyme modular polyketide synthase. Deletion of the salC gene, which is required for oxidative cyclisation, has led to the detection of a novel metabolite whose structure reveals the timing of a key dehydration step.

A novel mechanism is proposed for ring formation in the biosynthetic pathway to thiotetronate antibiotics thiolactomycin and Tü 3010.

Highlights d The gene cluster for the antifungal C-nucleoside malayamycin A has been identified d 5 0 -Pseudouridine monophosphate (5 0 -J-MP) is an essential precursor d 3 0 -Enoylpyruvyltransferase MalO specifically acts on 5 0 -J-MP, not UMP d An engineered strain produces the non-natural N-linked analog of malayamycin A

Für die Agglomerin‐Biosynthese wurden die Identität und Reaktivität der Zwischenstufen bestimmt und die Rollen der Acetyltransferase Agg4 und des Eliminierungsenzyms Agg5 aufgeklärt (siehe Schema). Es wird vorgeschlagen, dass zu Agg4 und Agg5 homologe Enzyme die Dehydratisierungsschritte in allen Spirotetronat‐Biosynthesen ausführen. Dies eröffnet Möglichkeiten für die gezielte Manipulation dieser Biosynthesewege.