Huarong Tan

Genome sequencing has revealed that microorganisms have the potential to produce many more natural products than previously thought; the challenge is to establish efficient ways to “wake up” those “sleeping” biosynthetic pathways or genes, which are undoubtedly expressed in nature under specific conditions that are not normally reproduced in the laboratory. To activate these cryptic natural products, co-cultivation of cross-kingdom microorganisms between Candida albicans and Streptomyces longshengensis was performed in this study. A novel peak generated through co-culture was isolated and analyzed by a high-performance liquid chromatograph (HPLC), and its chemical structure was further determined by using mass spectrum (MS) and nuclear magnetic resonance (NMR) analyses. Bioassays of antimicrobial and antitumor activities were performed, and heterologous expression in Escherichia coli was attempted. The chemical structure was identified as tryptophol, and the bioassays revealed that tryptophol showed antitumor activity with IC₅₀ values of 154.5, 144.3, 122.6, and 110.7 μg/mL against A549, MC38, HepG2, and MCF-7 cells, respectively. As a valuable compound, tryptophol was also heterologously expressed in E. coli C41 to address the drawbacks of chemical synthesis. These findings combine co-cultivation with genetic engineering for tryptophol biosynthesis, expanding its antitumor application and laying a foundation for its industrial and sustainable production.

To cope with the growing number of severe diseases and intractable pathogens, drug innovation in both chemical structures and pharmacological efficiency has become an imperative global mission. Oxazolomycins are a unique family of polyketide-polypeptide antibiotics from Streptomyces with diverse functional groups in their structures, conferring them multifarious activities. But further development into clinical applications has been hindered for decades for many reasons. Among them, the yield improvement is a critical basis for activity evaluation and drug-like property optimization. This study aims to enhance the production of oxazolomycins in Streptomyces longshengensis through metabolic engineering and evaluate their bioactivity against clinically relevant pathogens. Co-transcriptional analyses suggested that two operons (the transcriptional unit from gene oxaG to oxaB, and that from gene oxaH to oxaQ) could be included in the oxazolomycin biosynthetic gene cluster (oxa BGC) of S. longshengensis. So a strategy was designed to replace the native promoter regions between oxaG and oxaH with constitutive promoters Pneo and PkasO* following functional module evaluation. In the resultant strain (SLOE), the production of oxazolomycin component Toxa5 was increased to 4-fold of that in the wild-type strain. Accordingly, the transcription of all related genes in oxa was clearly promoted. SLOE was then subjected to sublethal dose of gentamicin to induce mutagenesis for optimizing the genetic background, generating a resistant mutant SLROE. With the introduction of transporter genes (ozmS and oxaA) into SLROE, 175 mg/L of Toxa5 was achieved, representing the highest yield in shake-flask fermentation to the best of our knowledge. Finally, the purified Toxa5 showed significant inhibition on the growth of clinically important Gram-negative pathogenic bacterium, Pseudomonas aeruginosa, and the biofilm formation of Bacillus subtilis. Intriguingly, an unprecedented antioxidant activity was also demonstrated. An oxazolomycin high-producing system of S. longshengensis was established by employing genetic engineering strategies to facilitate the bioactivity exploitation. Oxazolomycin Toxa5 showed interesting inhibitory effects against multiple Gram-negative and -positive pathogens as well as antioxidant capacity, indicating its great potential in clinical applications. The findings provide an efficient strategy for the overproduction and activity evaluation of oxazolomycins.

In general, the initiation or closure of antibiotic biosynthesis is determined by regulatory proteins, but most of their mechanisms of action remain unknown. The 2-deoxystreptamine-containing aminoglycosides (2-DOS AGs) form a unique category among antibiotics. Genomic analysis revealed that a group of hypothetical regulatory genes represented by neoI are widely distributed in the biosynthetic gene clusters (BGCs) of natural products from Streptomyces species, including several 2-DOS AGs. Only limited knowledge is available for the roles of NeoI-type regulators although neomycin and some of the related AGs have been developed as therapeutic drugs for decades. This study focuses on the functional determination of neoI and its homologues situated in the BGCs of six AGs. We found that the yield of neomycin in neoI disruption mutant (ΔneoI) increased by 50% compared to the wild-type (WT) strain ((420.6±44.1) mg L⁻¹), while it was partially restored by the complementation of neoI, demonstrating that NeoI acted as a repressor in neomycin biosynthesis. Further electrophoretic mobility shift assays (EMSAs) and DNase I footprinting assays indicated that NeoI could specifically bind to the promoter region between neoE and neoI with conserved nucleotides (5′-CVHYMRCHDKAGYGGACR-3′), as determined by site-directed mutagenesis. Interestingly, cross-bindings of the NeoI homologues from the six different BGCs to their corresponding DNA targets were manifested, and the five exogenous NeoI homologues could complement NeoI function of repressing neomycin biosynthesis. Our results suggested that NeoI-type regulators represent widespread and conservative regulatory characteristics in the biosynthesis of 2-DOS AGs, which would be significant for optimizing the biosynthetic pathways of valuable commercialized aminoglycoside antibiotics.

Signaling systems of microorganisms are responsible for regulating the physiological and metabolic processes and also play vital roles in the communications of cells. Identifying signaling molecules mediating the cross-talks is challenging yet highly desirable for comprehending the microbial interactions. Here, we demonstrate that a pathogenic Gram-negative Chromobacterium violaceum exerts significant influence on the morphological differentiation and secondary metabolism of Gram-positive Streptomyces. The physiological metabolisms are directly modulated by three novel cinnamoyl lipids (CVCL1, 2, and 3) from C. violaceum CV12472, whose biosynthesis is under the control of N-acylhomoserine lactone signaling system. Furthermore, a receptor of CVCLs in Streptomyces ansochromogenes 7100 is determined to be SabR1, the cognate receptor of γ-butenolide signaling molecules. This study reveals an unprecedented mode of microbial interactions, and the quorum sensing signaling systems in these two groups of bacteria can be bridged via CVCLs, suggesting that CVCLs can modulate the physiological metabolism of cross-phylum microorganisms.

Background: Uridyl peptide compounds are renowned as a subclass of nucleoside antibiotics for their highly specific antibacterial activity against Gram-negative bacteria and the unique target of action. We previously activated the biosynthetic gene cluster of a uridyl peptide antibiotic, mureidomycin, in Streptomyces roseosporus NRRL 15998 by introducing an exogenous positive regulator gene ssaA, and the generated strain was designated as Sr-hA. This study aims to further explore mureidomycin analogs from Sr-hA as well as the collaborative roles of two wide-spread genes, SSGG-02980 and SSGG-03002 encoding putative nuclease/phosphatase and oxidoreductase respectively, in mureidomycin diversification. Results: In order to understand how SSGG-02980 and SSGG-03002 contribute to mureidomycin biosynthesis, the gene disruption mutants and complementary strains were constructed. Mass spectrometry analyses revealed that two series of pairwise mureidomycin analogs were synthesized in Sr-hA with a two-dalton difference in molecular weight for each pair. By disruption of SSGG-03002, only mureidomycins with lower molecular weight (MRDs, 1–6) could be specifically accumulated in the mutant (∆03002-hA), whereas the other series of products with molecular weight plus 2 Da (rMRDs, 1ʹ–6ʹ) became dominant in SSGG-02980 disruption mutant (∆02980-hA). Further comprehensive NMR analyses were performed to elucidate the structures, and three MRDs (3, 4, 5) with unsaturated double bond at C5-C6 of uracil group were characterized from ∆03002-hA. In contrast, the paired rMRDs analogs (3ʹ, 4ʹ, 5ʹ) from ∆SSGG-02980 corresponding to 3, 4 and 5 were shown to contain a single bond at this position. The results verified that SSGG-03002 participates in the reduction of uracil ring, whereas SSGG-02980 antagonizes the effect of SSGG-03002, which has been rarely recognized for a phosphatase. Conclusions: Overall, this study revealed the key roles of two wide-spread families of enzymes in Streptomyces. Of them, oxidoreductase, SSGG-03002, is involved in dihydro-mureidomycin biosynthesis of S. roseosporus, whereas nuclease/phosphatase, SSGG-02980, has an adverse effect on SSGG-03002. This kind of unusual regulation model between nuclease/phosphatase and oxidoreductase is unprecedented, providing new insights into the biosynthesis of mureidomycins in Streptomyces. The findings would be of significance for structural diversification of more uridyl peptide antibiotics against Gram-negative bacteria.

Genome sequencing has revealed that actinomycetes possess the potential to produce many more secondary metabolites than previously thought. The existing challenge is to devise efficient methods to activate these silent biosynthetic gene clusters (BGCs). In Streptomyces ansochromogenes, disruption of wblA, a pleiotropic regulatory gene, activated the expression of cryptic tylosin analogues and abolished nikkomycin production simultaneously. Overexpressing pathway-specific regulatory genes tylR1 and tylR2 can also trigger the biosynthesis of silent tylosin analogues, in which TylR1 exerted its function via enhancing tylR2 expression. Bacterial one-hybrid system experiments unveiled that WblA directly inhibits the transcription of tylR1 and tylR2 to result in the silence of tylosin analogues BGC. Furthermore, WblA can activate the nikkomycin production through up-regulating the transcription of pleiotropic regulatory gene adpA. More interestingly, AdpA can activate sanG (an activator gene in nikkomycin BGC) but repress wblA. Our studies provide a valuable insight into the complex functions of pleiotropic regulators.

Streptomycetes possess numerous gene clusters and the potential to produce a large amount of natural products. Histone deacetylase (HDAC) inhibitors play an important role in the regulation of histone modifications in fungi, but their roles in prokaryotes remain poorly understood. Here, we investigated the global effects of the HDAC inhibitor, sodium butyrate (SB), on marine-derived Streptomycesolivaceus FXJ 8.021, particularly focusing on the activation of secondary metabolite biosynthesis. The antiSMASH analysis revealed 33 secondary metabolite biosynthetic gene clusters (BGCs) in strain FXJ 8.021, among which the silent lobophorin BGC was activated by SB. Transcriptomic data showed that the expression of genes involved in lobophorin biosynthesis (ge00097–ge00139) and CoA-ester formation (e.g., ge02824), as well as the glycolysis/gluconeogenesis pathway (e.g., ge01661), was significantly up-regulated in the presence of SB. Intracellular CoA-ester analysis confirmed that SB triggered the biosynthesis of CoA-ester, thereby increasing the precursor supply for lobophorin biosynthesis. Further acetylomic analysis revealed that the acetylation levels on 218 sites of 190 proteins were up-regulated and those on 411 sites of 310 proteins were down-regulated. These acetylated proteins were particularly enriched in transcriptional and translational machinery components (e.g., elongation factor GE04399), and their correlations with the proteins involved in lobophorin biosynthesis were established by protein–protein interaction network analysis, suggesting that SB might function via a complex hierarchical regulation to activate the expression of lobophorin BGC. These findings provide solid evidence that acetylated proteins triggered by SB could affect the expression of genes involved in the biosynthesis of primary and secondary metabolites in prokaryotes.

The crisis of antibiotic resistance has become an impending global problem. Genome sequencing reveals that streptomycetes have the potential to produce many more bioactive compounds that may combat the emerging pathogens. The existing challenge is to devise sensitive reporter systems for mining valuable antibiotics. Here, we report a visualization reporter system based on Gram-negative bacterial acyl-homoserine lactone quorum-sensing (VRS-bAHL). AHL synthase gene (cviI) of Chromobacterium violaceum as reporter gene is expressed in Gram-positive Streptomyces to synthesize AHL, which is detected with CV026, an AHL deficient mutant of C. violaceum, via its violacein production upon AHL induction. Validation assays prove that VRS-bAHL can be widely used for characterizing gene expression in Streptomyces. With the guidance of VRS-bAHL, a novel oxazolomycin derivative is discovered to the best of our knowledge. The results demonstrate that VRS-bAHL is a powerful tool for advancing genetic regulation studies and discovering valuable active metabolites in microorganisms.