Production of distinct labdane-type diterpenoids using a novel cryptic labdane-like cluster from Streptomyces thermocarboxydus K155.

Bioinformatic mining of the Streptomyces thermocarboxydus K155 genome predicted the presence of four synthases for the production of geosmin, hopene, albaflavenone, and a type B-type A diterpenoid system like that described for labdane-related diterpenoids (LRD). The lrd cluster was comprised by an operon of four genes (lrdABDC). This cluster seemed to be silent in the wild-type strain, as neither labdane nor terpene-like compounds were detected by UPLC-TOF-MS and GC-MS analyses in both culture supernatants and mycelial extracts. Heterologous expression of the lrdABDC cluster in a defective cyslabdan producer (Streptomyces cyslabdanicus K04-0144Δcld) generated 8,17-epoxy-7-hydroxy labda-12,14-diene and cyslabdan. The same cluster expressed in the strains Streptomyces coelicolor M1152, Streptomyces peucetius var. caesius, and Streptomyces avermitilis SUKA22 produced the general intermediary labda-8(17), 12(E),14-triene [(E)-biformene]. Besides (E)-biformene, S. coelicolor M1152 and S. avermitilis SUKA22 produced two and three different labdane-type diterpenoids, underlying the relevance of the genetic background of the Streptomyces host in product formation.

The structure of (E)-biformene synthase provides insights into the biosynthesis of bacterial bicyclic labdane-related diterpenoids.

The labdane-related diterpenoids (LRDs) are a large group of natural products with a broad range of biological activities. They are synthesized through two consecutive reactions catalyzed by class II and I diterpene synthases (DTSs). The structural complexity of LRDs mainly depends on the catalytic activity of class I DTSs, which catalyze the formation of bicyclic to pentacyclic LRDs, using as a substrate the catalytic product of class II DTSs. To date, the structural and mechanistic details for the biosynthesis of bicyclic LRDs skeletons catalyzed by class I DTSs remain unclear. This work presents the first X-ray crystal structure of an (E)-biformene synthase, LrdC, from the soil bacterium Streptomyces sp. strain K155. LrdC was identified as a part of an LRD cluster of five genes and was found to be a class I DTS that catalyzes the Mg2+-dependent synthesis of bicyclic LRD (E)-biformene by the dephosphorylation and rearrangement of normal copalyl pyrophosphate (CPP). Structural analysis of LrdC coupled with docking studies suggests that Phe189 prevents cyclization beyond the bicyclic LRD product through a strong stabilization of the allylic carbocation intermediate, while Tyr317 functions as a general base catalyst to deprotonate the CPP substrate. Structural comparisons of LrdC with homology models of bacterial bicyclic LRD-forming enzymes (CldD, RmnD and SclSS), as well as with the crystallographic structure of bacterial tetracyclic LRD ent-kaurene synthase (BjKS), provide further structural insights into the biosynthesis of bacterial LRD natural products.

Carbon catabolite regulation in Streptomyces: new insights and lessons learned.

One of the most significant control mechanisms of the physiological processes in the genus Streptomyces is carbon catabolite repression (CCR). This mechanism controls the expression of genes involved in the uptake and utilization of alternative carbon sources in Streptomyces and is mostly independent of the phosphoenolpyruvate phosphotransferase system (PTS). CCR also affects morphological differentiation and the synthesis of secondary metabolites, although not all secondary metabolite genes are equally sensitive to the control by the carbon source. Even when the outcome effect of CCR in bacteria is the same, their essential mechanisms can be rather different. Although usually, glucose elicits this phenomenon, other rapidly metabolized carbon sources can also cause CCR. Multiple efforts have been put through to the understanding of the mechanism of CCR in this genus. However, a reasonable mechanism to explain the nature of this process in Streptomyces does not yet exist. Several examples of primary and secondary metabolites subject to CCR will be examined in this review. Additionally, recent advances in the metabolites and protein factors involved in the Streptomyces CCR, as well as their mechanisms will be described and discussed in this review.

Synthetic biology era: Improving antibiotic's world.

The emergence of antibiotic-resistant pathogen microorganisms is problematic in the context of the current spectrum of available medication. The poor specificity and the high toxicity of some available molecules have made imperative the search for new strategies to improve the specificity and to pursue the discovery of novel compounds with increased bioactivity. Using living cells as platforms, synthetic biology has counteracted this problem by offering novel pathways to create synthetic systems with improved and desired functions. Among many other biotechnological approaches, the advances in synthetic biology have made it possible to design and construct novel biological systems in order to look for new drugs with increased bioactivity. Advancements have also been made in the redesigning of RNA and DNA molecules in order to engineer antibiotic clusters for antibiotic overexpression. As for the production of these antibacterial compounds, yeasts and filamentous fungi as well as gene therapy are utilized to enhance protein solubility. Specific delivery is achieved by creating chimeras using plant genes into bacterial hosts. Some of these synthetic systems are currently in clinical trials, proving the proficiency of synthetic biology in terms of both pharmacological activities as well as an increase in the biosafety of treatments. It is possible that we may just be seeing the tip of the iceberg, and synthetic biology applications will overpass expectations beyond our present knowledge.

Carbon source regulation of antibiotic production.

Antibiotics are low-molecular-mass products of secondary metabolism, nonessential for the growth of producing organisms, but very important for human health. They have unusual structures and are most often formed during the late growth phase of the producing microorganisms. Their production arises from intracellular intermediates, which are condensed into more complex structures through defined biochemical pathways. Their synthesis can be influenced by manipulating the type and concentration of nutrients formulating the culture media. Among them, the effect of the carbon source has been the subject of continuous studies for both industry and research groups. Glucose and other carbohydrates have been reported to interfere with antibiotic synthesis and this effect depends on the rapid utilization of the preferred carbon source. Different mechanisms have been described in bacteria and fungi to explain the negative effects of carbon catabolites on antibiotic production. They show important differences depending on the microbe being considered. Their understanding and manipulation have been useful for both perfecting fermentation conditions to produce anti-infectives and for strain improvement. To improve the production of antibiotics, carbon source repression can be decreased or abolished by mutations resulting in antimetabolite resistance. Enzymes reported as regulated by the carbon source have been used as targets for strain improvement. During the last few years, important advances have been reported elucidating the essential aspects of carbon source regulation on antibiotic production at biochemical and molecular levels. The aim of this review is to describe these advances, giving special emphasis to those reported for the genus Streptomyces.