SUMMARY From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and noninfective diseases. Procedures for in vivo and in vitro genetic manipulations in S. rimosus have been developed since the 1950s and applied to study the genetic instability of S. rimosus strains and for the molecular cloning and characterization of genes involved in oxytetracycline biosynthesis. Recent advances in the methodology of genome sequencing bring the realistic prospect of obtaining the genome sequence of S. rimosus in the near term.
Oxytetracycline (OTC) is a 19-carbon polyketide antibiotic made by Streptomyces rimosus. The otcC gene encodes an anhydrotetracycline oxygenase that catalyzes a hydroxylation of the anthracycline structure at position C-6 after biosynthesis of the polyketide backbone is completed. A recombinant strain of S. rimosus that was disrupted in the genomic copy of otcC synthesized a novel C-17 polyketide. This result indicates that the absence of the otcC gene product significantly influences the ability of the OTC "minimal" polyketide synthase to make a polyketide product of the correct chain length. A mutant copy of otcC was made by site-directed mutagenesis of three essential glycine codons located within the putative NADPH-binding domain. The mutant gene was expressed in Escherichia coli, and biochemical analysis confirmed that the gene product was catalytically inactive. When the mutant gene replaced the ablated gene in the chromosome of S. rimosus, the ability to make a 19-carbon backbone was restored, indicating that OtcC is an essential partner in the quaternary structure of the synthase complex. Oxytetracycline (OTC)4 and related tetracycline polyketide compounds are potent inhibitors of bacterial protein synthesis displaying broad-spectrum activity against both Gram-positive and Gram-negative pathogens. Although the clinical use of the tetracyclines has declined in recent years due to the emergence of resistance in these bacteria, OTC is still the first drug of choice for the treatment of intracellular infections caused by Rickettsia, Chlamydia, and mycoplasma in penicillin-sensitive patients incapable of tolerating macrolides (1). Glycylcyclines are a new class of semi-synthetic tetracycline derivatives that exemplify how modification of a known antibiotic can be a successful strategy to obtain new compounds effective against pan-drug-resistant strains (2). To this end we have been elucidating the biosynthetic pathway leading to OTC in the producing actinomycete Streptomyces rimosus, as a potential route to clinically useful OTC products by combinatorial biosynthesis (3).Polyketide synthases (PKSs) catalyzing the formation of aromatic products such as OTC are dissociable enzyme complexes of largely monofunctional proteins that follow a mechanistic pathway similar to fatty acid biosynthesis (4). The synthesis of aromatic Type II polyketides usually begins with the condensation of an acetate unit derived from the decarboxylation of a malonyl-S-acyl carrier protein (ACP) to a malonyl-S-ACP extender unit. This reaction is catalyzed by a heterodimeric ketosynthase chain length factor (KS␣-KS) (5, 6). Preference for nonacetate primers has also been recognized in a number of aromatic PKSs including OTC, although presumably decarboxylative chain initiation catalyzed by the heterodimer can still occur in the absence of these non-acetate starter units (7). Manipulating the mechanism of non-acetate priming has lead to biosynthesis of novel polyketides (8, 9). The active site of the KS␣ also catalyzes iterative elongati...
Natural products from symbiotic or commensal associations between marine invertebrate and microbial organisms show exceptional promise as pharmaceuticals in many therapeutic areas. An economic and sustainable global market supply due to difficulty of synthesis is cited as the main obstacle for exploitation of these otherwise exciting marine bioactive compounds. Different strategies have been evoked to overcome this impediment as long-term harvesting of wild stocks from the environment is considered unsound, and other modes of production based on biosynthesis, such as aquaculture, have not yet been proven as reliable. One option is to clone the genes encoding the biosynthetic expression of a lead metabolite into a surrogate host suitable for industrial-scale fermentation. To facilitate this goal we are developing a universal system to clone and express genes responsible for biosynthesis of natural products from both eukaryotic and prokaryotic partners of marine symbioses. The ability to harness the complete meta-transcriptome of entire biosynthetic pathways is particularly valuable where the biogenesis of a target natural product occurring within a complex symbiotic association is unclear.
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