No abstract

Chemoenzymatic synthesis of SAM analogs This study highlights a broadly applicable platform for the facile syntheses of SAM analogs that is directly compatible with downstream SAM utilizing enzymes. The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with rapid SAM analog decomposition and thereby opens the door to the further interrogation of a wide range of SAM utilizing enzymes. As a proof of concept for the feasibility of natural product ‘alkylrandomization’, the coupled strategy was used to generate a small set of indolocarbazole analogs in conjunction with the rebeccamycin O-methyltransferase RebM.

GilOII has been unambiguously identified as the key enzyme performing the crucial C-C bond cleavage reaction responsible for the unique rearrangement of a benz[a]anthracene skeleton to the benzo[d]naphthopyranone backbone typical for the gilvocarcin type natural anticancer antibiotics. Further investigations of this enzyme led to the isolation of a hydroxy-oxepinone intermediate which allowed important conclusions regarding the cleavage mechanism.

We recently described a new method to activate antibiotic production in bacteria by introducing a mutation conferring resistance to a drug such as streptomycin, rifampin, paromomycin, or gentamicin. This method, however, enhanced antibiotic production by only up to an order of magnitude. Working with Streptomyces coelicolor A3(2), we established a method for the dramatic activation of antibiotic production by the sequential introduction of multiple drug resistance mutations. Septuple and octuple mutants, C7 and C8, thus obtained by screening for resistance to seven or eight drugs, produced huge amounts (1.63 g/liter) of the polyketide antibiotic actinorhodin, 180-fold higher than the level produced by the wild type. This dramatic overproduction was due to the acquisition of mutant ribosomes, with aberrant protein and ppGpp synthesis activity, as demonstrated by in vitro protein synthesis assays and by the abolition of antibiotic overproduction with relA disruption. This new approach, called "ribosome engineering," requires less time, cost, and labor than other methods and may be widely utilized for bacterial strain improvement.

Despite their importance in the chemical industry, the significance of rare earths in biology has been largely overlooked. Here, it is reported that the rare earth, scandium (Sc), causes antibiotic overproduction by 2-25-fold when added at a low concentration (10-100 microM) to cultures of Streptomyces coelicolor A3(2) (actinorhodin producer), Streptomyces antibioticus (actinomycin producer), and Streptomyces griseus (streptomycin producer). Not just for enhancement of antibiotic production, scandium was also effective in activating the dormant ability to produce actinorhodin in Streptomyces lividans. The effects of scandium were exerted at the level of transcription of pathway-specific positive regulatory genes, as demonstrated by marked up-regulation of actII-ORF4 in S. coelicolor cells exposed to this element. The bacterial alarmone, guanosine 5'-diphosphate 3'-diphosphate, was essential for actinorhodin overproduction provoked by scandium.