Secondary Metabolite Production In Streptomyces Research Articles

Salinomycin biosynthesis reversely regulates the β-oxidation pathway in Streptomyces albus by carrying a 3-hydroxyacyl-CoA dehydrogenase gene in its biosynthetic gene cluster.

Streptomyces is well known for synthesis of many biologically active secondary metabolites, such as polyketides and non-ribosomal peptides. Understanding the coupling mechanisms of primary and secondary metabolism can help develop strategies to improve secondary metabolite production in Streptomyces. In this work, Streptomyces albus ZD11, an oil-preferring industrial Streptomyces strain, was proved to have a remarkable capability to generate abundant acyl-CoA precursors for salinomycin biosynthesis with the aid of its enhanced β-oxidation pathway. It was found that the salinomycin biosynthetic gene cluster contains a predicted 3-hydroxyacyl-CoA dehydrogenase (FadB3), which is the third enzyme of β-oxidation cycle. Deletion of fadB3 significantly reduced the production of salinomycin. A variety of experimental evidences showed that FadB3 was mainly involved in the β-oxidation pathway rather than ethylmalonyl-CoA biosynthesis and played a very important role in regulating the rate of β-oxidation in S. albus ZD11. Our findings elucidate an interesting coupling mechanism by which a PKS biosynthetic gene cluster could regulate the β-oxidation pathway by carrying β-oxidation genes, enabling Streptomyces to efficiently synthesize target polyketides and economically utilize environmental nutrients.

System-Level Analysis of Transcriptional and Translational Regulatory Elements in Streptomyces griseus.

Bacteria belonging to Streptomyces have the ability to produce a wide range of secondary metabolites through a shift from primary to secondary metabolism regulated by complex networks activated after vegetative growth terminates. Despite considerable effort to understand the regulatory elements governing gene expression related to primary and secondary metabolism in Streptomyces, system-level information remains limited. In this study, we integrated four multi-omics datasets from Streptomyces griseus NBRC 13350: RNA-seq, ribosome profiling, dRNA-seq, and Term-Seq, to analyze the regulatory elements of transcription and translation of differentially expressed genes during cell growth. With the functional enrichment of gene expression in different growth phases, one sigma factor regulon and four transcription factor regulons governing differential gene transcription patterns were found. In addition, the regulatory elements of transcription termination and post-transcriptional processing at transcript 3′-end positions were elucidated, including their conserved motifs, stem-loop RNA structures, and non-terminal locations within the polycistronic operons, and the potential regulatory elements of translation initiation and elongation such as 5′-UTR length, RNA structures at ribosome-bound sites, and codon usage were investigated. This comprehensive genetic information provides a foundational genetic resource for strain engineering to enhance secondary metabolite production in Streptomyces.

Synthetic Inducible Regulatory Systems Optimized for the Modulation of Secondary Metabolite Production in Streptomyces.

Biosynthesis of secondary metabolites is a highly complex process that often requires tight control of their production, as overproduction of metabolites could be toxic and also may cause metabolic burden to their hosts. Tight control of metabolite production could be achieved by expressing key biosynthetic genes under control of an inducible regulatory system. In this study, we employed the modular design approach to build a high performance synthetic inducible regulatory system that displays a large dynamic range and thus is well-suited for the modulation of secondary metabolite production in Streptomyces. To this end, an inducible regulatory system was divided into three separate functional modules: (1) the induction module, (2) the target expression module, and (3) the repressor expression module. Then, these three separate modules were individually optimized in a stepwise manner and assembled to a new system. First, the cumate (CMT) induction module was chosen as the best performing induction module based on the large dynamic range and moderate inducer sensitivity. Then the CMT induction module maintained its performance when combined with diverse constitutive target expression modules, in which overall dynamic ranges varied depending on maximum promoter strengths. Lastly, the repressor expression module was optimized to achieve complete elimination of leaky expression, further increasing the dynamic range of the system. We also demonstrate that any strong constitutive regulatory system could be converted into an inducible regulatory system by simple CRISPR/Cas9-aided markerless insertion of an operator sequence whenever tight control of gene expression is required. We believe that the synthetic inducible regulatory system we report here would become a useful tool in modulating secondary metabolite production in Streptomyces.

Interplay between carbon, nitrogen and phosphate utilization in the control of secondary metabolite production in Streptomyces.

Streptomyces species are a wide and diverse source of many therapeutic agents (antimicrobials, antineoplastic and antioxidants, to name a few) and represent an important source of compounds with potential applications in medicine. The effect of nitrogen, phosphate and carbon on the production of secondary metabolites has long been observed, but it was not until recently that the molecular mechanisms on which these effects rely were ascertained. In addition to the specific macronutrient regulatory mechanisms, there is a complex network of interactions between these mechanisms influencing secondary metabolism. In this article, we review the recent advances in our understanding of the molecular mechanisms of regulation exerted by nitrogen, phosphate and carbon sources, as well as the effects of their interconnections, on the synthesis of secondary metabolites by members of the genus Streptomyces.

Characterization of LnmO as a pathway-specific Crp/Fnr-type positive regulator for leinamycin biosynthesis in Streptomyces atroolivaceus and its application for titer improvement.

The cyclic adenosine monophosphate (cAMP) receptor protein/fumarate and nitrate reductase regulatory protein (Crp/Fnr) family of transcriptional regulators are pleiotropic transcriptional regulators that control a broad range of cellular functions. Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. We previously cloned and characterized the lnm biosynthetic gene cluster from S. atroolivaceus S-140. We here report inactivation of lnmO in S. atroolivaceus S-140 and overexpression of lnmO in the S. atroolivaceus S-140 wild-type and ∆lnmE mutant SB3033 to investigate its role in LNM biosynthesis. Bioinformatics analysis revealed LnmO as the only regulator within the lnm gene cluster, exhibiting high sequence similarity to known Crp/Fnr family regulators. The inactivation of lnmO in S. atroolivaceus S-140 completely abolished LNM production but caused no apparent morphological changes, supporting that LnmO is indispensable and specific to LNM biosynthesis. Overexpression of lnmO in S. atroolivaceus S-140 and SB3033 resulted in three- and fourfold increase in LNM and LNM E1 production, respectively, supporting that LnmO acts as a positive regulator. While all of the Crp/Fnr family regulators studied to date appeared to be pleiotropic, our results support LnmO as the first Crp/Fnr family regulator that is pathway-specific. LnmO joins the growing list of regulators that could be exploited to improve secondary metabolite production in Streptomyces. Engineered strains overproducing LNM and LNM E1 will facilitate further mechanistic studies and clinical evaluation of LNM and LNM E1 as novel anticancer drugs.

Effective Production of Aromatic Polyketides in Streptomyces using a Combined-Culture Method

Combinedculture is a fermentation method which efficiently induces secondary metabolite production in Streptomyces by co-culturing them with mycolic acid-containing bacteria. As a result of combined-culture screening -of our terrestrial Streptomyces collection using UV-HPLC, one of the tested strains, Streptoinyces. sp. TAKO-2, produced two known aromatic polyketides, julichrome Q6 (1) and julichrome Q₈.₈ (2), when co-cultured with the mycolic acid- containing bacterium Tsukamurella pulmonis TP-B0596. The structures of 1 and 2 were confirmed by spectroscopic analysis and literature data.

Improvement of secondary metabolite production in Streptomyces by manipulating pathway regulation

Titer improvement is a constant requirement in the fermentation industry. The traditional method of "random mutation and screening" has been very effective despite the considerable amount of time and resources it demands. Rational metabolic engineering, with the use of recombinant DNA technology, provides a novel, alternative strategy for titer improvement that complements the empirical method used in industry. Manipulation of the specific regulatory systems that govern secondary metabolite production is an important aspect of metabolic engineering that can efficiently improve fermentation titers. In this review, we use examples from Streptomyces secondary metabolism, the most prolific source of clinically used drugs, to demonstrate the power and utility of exploiting natural regulatory networks, in particular pathway-specific regulators, for titer improvement. Efforts to improve the titers of fredericamycin, C-1027, platensimycin, and platencin in our lab are highlighted.

New genetic methods to improve secondary metabolite production in Streptomyces

The most potent chemical mutagens commonly used for yield enhancement in streptomycetes induce a limited spectrum of base-pair substitutions, heavily dominated by GC to AT transitions. The AT to CG transversion pathway complements the GC to AT pathway, but no strong mutagens with this specificity are available. However, mutT mutations in Escherichia coli enhance spontaneous AT to CG transversions about 1000-fold, so such mutations in streptomycetes could be very beneficial for random mutagenesis. Rate-limiting steps in secondary metabolite biosynthesis, on the other hand, can be best addressed by cloning and insertion of extra copies of the appropriate gene(s) into a neutral genomic site. This approach has been used successfully to improve the production of tylosin and pristinomycin. Transposon mutagenesis can be used to identify and clone neutral genomic sites, as demonstrated in Streptomyces fradiae to improve tylosin yields. Transposon mutagenesis can also be used to activate or enhance the transcription of genes important for secondary metabolite production, and to identify and clone both positive and negative regulatory elements. In some cases it may be advantageous to amplify the complete biosynthetic pathway for secondary metabolite production. In Streptomyces rimosus, a highly productive oxytetracycline producer was shown to contain the complete biosynthetic pathway near the end of the linear chromosome, and on a hybrid linear plasmid that had undergone recombination with the chromosome. This concept of linear replicon fusion might be applicable to other secondary metabolite pathway genes that are located near one end of the linear chromosome.