Covering: 1997 to 2010. The angucycline group is the largest group of type II PKS-engineered natural products, rich in biological activities and chemical scaffolds. This stimulated synthetic creativity and biosynthetic inquisitiveness. The synthetic studies used five different strategies, involving Diels-Alder reactions, nucleophilic additions, electrophilic additions, transition-metal mediated cross-couplings and intramolecular cyclizations to generate the angucycline frames. Biosynthetic studies were particularly intriguing when unusual framework rearrangements by post-PKS tailoring oxidoreductases occurred, or when unusual glycosylation reactions were involved in decorating the benz[a]anthracene-derived cores. This review follows our previous reviews, which were published in 1992 and 1997, and covers new angucycline group antibiotics published between 1997 and 2010. However, in contrast to the previous reviews, the main focus of this article is on new synthetic approaches and biosynthetic investigations, most of which were published between 1997 and 2010, but go beyond, e.g. for some biosyntheses all the way back to the 1980s, to provide the necessary context of information.
Rebeccamycin and staurosporine are natural products with antitumor properties, which belong to the family of indolocarbazole alkaloids. An intense effort currently exists for the generation of indolocarbazole derivatives for the treatment of several diseases, including cancer and neurodegenerative disorders. Here, we report a biological process based on combinatorial biosynthesis for the production of indolocarbazole compounds (or their precursors) in engineered microorganisms as a complementary approach to chemical synthesis. We have dissected and reconstituted the entire biosynthetic pathway for rebeccamycin in a convenient actinomycete host, Streptomyces albus. This task was achieved by coexpressing different combinations of genes isolated from the rebeccamycin-producing microorganism. Also, a gene (staC) was identified in staurosporine-producing microbes and was shown to have a key role to differentiate the biosynthetic pathways for the two indolocarbazoles. Last, incorporation of the pyrH and thal genes, encoding halogenases from different microorganisms, resulted in production of derivatives with chlorine atoms at novel positions. We produced >30 different compounds by using the recombinant strains generated in this work. (Fig. 1). Various biological activities have been reported for indolocarbazoles, but the greatest interest is focused on compounds that possess antitumor and neuroprotective properties (2-4). These activities may be due to different mechanisms of action, including DNA intercalation, inhibition of DNA topoisomerases, and inhibition of protein kinases. Great efforts are made to generate indolocarbazole derivatives with improved properties for the treatment of cancer, neurodegenerative disorders, and diabetes-associated pathologies, and several analogs have entered clinical trials (2-7).Studies on the biosynthesis of rebeccamycin and staurosporine in the producing microorganisms have shown that the indolocarbazole core is formed by decarboxylative fusion of two tryptophan-derived units, whereas the sugar moiety is derived from glucose (8, 9). Recently, we cloned and characterized the rebeccamycin biosynthetic gene cluster from the actinomycete Lechevalieria aerocolonigenes (formerly Saccharotrix aerocolonigenes) (10). Expression of the entire gene cluster and of different subsets of genes in a heterologous host yielded rebeccamycin and three biosynthetic intermediates (10). The same cluster was later isolated by other researchers (11, 12) and expressed at a low level in Escherichia coli (12), and different insertional inactivation mutants were generated in the producer organism (11). The entire staurosporine gene cluster has been isolated from Streptomyces sp. TP-A0274 (13), although a previous patent application reported the identification of some genes involved in biosynthesis of the staurosporine sugar moiety in Streptomyces longisporoflavus (14).Combinatorial biosynthesis is a recent addition to the metabolic engineering toolbox by which genes responsible for individual metabolic reacti...
Ribosome stalling at polyproline stretches is common and fundamental. In bacteria, translation elongation factor P (EF-P) rescues such stalled ribosomes, but only when it is post-translationally activated. In Escherichia coli, activation of EF-P is achieved by (R)-β-lysinylation and hydroxylation of a conserved lysine. Here we have unveiled a markedly different modification strategy in which a conserved arginine of EF-P is rhamnosylated by a glycosyltransferase (EarP) using dTDP-l-rhamnose as a substrate. This is to our knowledge the first report of N-linked protein glycosylation on arginine in bacteria and the first example in which a glycosylated side chain of a translation elongation factor is essential for function. Arginine-rhamnosylation of EF-P also occurs in clinically relevant bacteria such as Pseudomonas aeruginosa. We demonstrate that the modification is needed to develop pathogenicity, making EarP and dTDP-l-rhamnose-biosynthesizing enzymes ideal targets for antibiotic development.
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