Amycolatopsis mediterranei: A Sixty-Year Journey from Strain Isolation to Unlocking Its Potential of Rifamycin Analogue Production by Combinatorial Biosynthesis.

Ever since the isolation of Amycolatopsis mediterranei in 1957, this strain has been the focus of research worldwide. In the last 60 years or more, our understanding of the taxonomy, development of cloning vectors and conjugation system, physiology, genetics, genomics, and biosynthetic pathway of rifamycin B production in A. mediterranei has substantially increased. In particular, the development of cloning vectors, transformation system, characterization of the rifamycin biosynthetic gene cluster, and the regulation of rifamycin B production by the pioneering work of Heinz Floss have made the rifamycin polyketide biosynthetic gene cluster (PKS) an attractive target for extensive genetic manipulations to produce rifamycin B analogues which could be effective against multi-drug-resistant tuberculosis. Additionally, a better understanding of the regulation of rifamycin B production and the application of newer genomics tools, including CRISPR-assisted genome editing systems, might prove useful to overcome the limitations associated with low production of rifamycin analogues.

Transposon-based identification of genes involved in the rimocidin biosynthesis in Streptomyces rimosus M527.

The transposon mutagenesis strategy has been employed to generate random insertion mutants and analyze the correlation between genes and secondary metabolites in the genus Streptomyces. In this study, our primary objective was to identify an unknown gene involved in rimocidin biosynthesis and elucidate its role in rimocidin production in Streptomyces rimosus M527. To achieve this, we established a random mutant library of S. rimosus M527 using a Tn5 transposon-mediated random mutagenesis strategy. Among the 137 isolated mutants, M527-G10 and M527-W5 exhibited the most significant variations in antagonistic activity against the plant pathogenic fungus Fusarium oxysporum f. sp. cucumerinum. Specifically, M527-G10 displayed a 72.93% reduction, while M527-W5 showed a 49.8% increase in rimocidin production compared to the wild-type (WT) strain S. rimosus M527. Subsequently, we employed a plasmid rescue strategy to identify the insertion loci of the transposon in the genomes of mutants M527-G10 and M527-W5, revealing a response regulator transcription factor (rrt) and a hypothetical protein (hyp), respectively. The roles of rrt and hyp in rimocidin biosynthesis were determined through gene deletion, overexpression in the WT strain, and complemented expression in the transposon mutants. Notably, the gene-deletion mutants M527-ΔRRT and M527-ΔHYP exhibited similar behavior in rimocidin production compared to the corresponding transposon mutants M527-G10 and M527-W5, suggesting that transposon insertions in genes rrt and hyp led to alterations in rimocidin production. Furthermore, both gene deletion and overexpression of rrt and hyp had no discernible effects on cell growth. These results reveal that genes rrt and hyp have positive and negative impacts on rimocidin production in S. rimosus M527, respectively.

P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds via dehydrogenation of a specific acylated dipeptide substrate.

WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine (NMet-Dht) residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase (NRPS). The cytochrome P450 encoded by sas16 (P450Sas) has been shown to be essential for the formation of the alkene moiety in NMet-Dht, but the timing and mechanism of the P450Sas-mediated α,ß-dehydrogenation of Dht remained unclear. Here, we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein (PCP)-bound dipeptide intermediate (Z)-2-pent-1'-enyl-cinnamoyl-Thr-N-Me-Tyr. We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS, and further that P450Sas appears to be specific for substrates containing the (Z)-2-pent-1'-enyl-cinnamoyl group. A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates, including the substitution of the canonical active site alcohol residue with a phenylalanine (F250), which in turn is critical to P450Sas activity and WS9326A biosynthesis. Together, our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate, thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.

Double-reporter-guided targeted activation of the oxytetracycline silent gene cluster in Streptomyces rimosus M527.

In Streptomyces rimosus M527, the oxytetracycline (OTC) biosynthetic gene cluster is not expressed under laboratory conditions. In this study a reported-guided mutant selection (RGMS) procedure was used to activate the cluster. The double-reporter plasmid pAGT was constructed in which gusA encoding a ß-glucuronidase and tsr encoding a thiostrepton resistance methyltransferase were placed under the control of the native promoter of oxyA gene (PoxyA ). Plasmid pAGT was introduced and integrated into the chromosome of S. rimosus M527 by conjugation, yielding initial strain M527-pAGT. Subsequently, mutants of M527-pAGT were generated by using ribosome engineering technology. The mutants harboring activated OTC gene cluster were selected based on visual observation of GUS activity and thiostrepton resistance. Finally, mutant M527-pAGT-R7 was selected producing OTC in a concentration of 235.2 mg/L. In this mutant transcriptional levels of oxysr genes especial oxyAsr gene were increased compared to wild-type strain S. rimosus M527. The mutant M527-pAGT-R7 showed antagonistic activities against Gram-negative and Gram-positive strains. All data indicate that the OTC gene cluster was successfully activated using the RGMS method.

Identification of RimR2 as a positive pathway-specific regulator of rimocidin biosynthesis in Streptomyces rimosus M527.

BACKGROUND: Streoptomyces rimosus M527 is a producer of the polyene macrolide rimocidin which shows activity against various plant pathogenic fungi. Notably, the regulatory mechanisms underlying rimocidin biosynthesis are yet to be elucidated. RESULTS: In this study, using domain structure and amino acid alignment and phylogenetic tree construction, rimR2, which located in the rimocidin biosynthetic gene cluster, was first found and identified as a larger ATP-binding regulators of the LuxR family (LAL) subfamily regulator. The rimR2 deletion and complementation assays were conducted to explore its role. Mutant M527-ΔrimR2 lost its ability to produce rimocidin. Complementation of M527-ΔrimR2 restored rimocidin production. The five recombinant strains, M527-ER, M527-KR, M527-21R, M527-57R, and M527-NR, were constructed by overexpressing rimR2 gene using the promoters permE*, kasOp*, SPL21, SPL57, and its native promoter, respectively, to improve rimocidin production. M527-KR, M527-NR, and M527-ER exhibited 81.8%, 68.1%, and 54.5% more rimocidin production, respectively, than the wild-type (WT) strain, while recombinant strains M527-21R and M527-57R exhibited no obvious differences in rimocidin production compared with the WT strain. RT-PCR assays revealed that the transcriptional levels of the rim genes were consistent with the changes in rimocidin production in the recombinant strains. Using electrophoretic mobility shift assays, we confirmed that RimR2 can bind to the promoter regions of rimA and rimC. CONCLUSION: A LAL regulator RimR2 was identified as a positive specific-pathway regulator of rimocidin biosynthesis in M527. RimR2 regulates the rimocidin biosynthesis by influencing the transcriptional levels of rim genes and binding to the promoter regions of rimA and rimC.

Improvement of rimocidin production in Streptomyces rimosus M527 by reporter-guided mutation selection.

In this study, we employed a reporter-guided mutation selection (RGMS) strategy to improve the rimocidin production of Streptomyces rimosus M527, which is based on a single-reporter plasmid pAN and atmospheric and room temperature plasma (ARTP). In plasmid pAN, PrimA, a native promoter of the loading module of rimocidin biosynthesis (RimA) was chosen as a target, and the kanamycin resistance gene (neo) under the control of PrimA was chosen as the reporter gene. The integrative plasmid pAN was introduced into the chromosome of S. rimosus M527 by conjugation to yield the initial strain S. rimosus M527-pAN. Subsequently, mutants of M527-pAN were generated by ARTP. 79 mutants were obtained in total, of which 67 mutants showed a higher level of kanamycin resistance (Kanr) than that of the initial strain M527-pAN. The majority of mutants exhibited a slight increase in rimocidin production compared with M527-pAN. Notably, 3 mutants, M527-pAN-S34, S38, and S52, which exhibited highest kanamycin resistance among all Kanr mutants, showed 34%, 52%, and 45% increase in rimocidin production compared with M527-pAN, respectively. Quantitative RT-PCR analysis revealed that the transcriptional levels of neo and rim genes were increased in mutants M527-pAN-S34, S38, and S52 compared with M527-pAN. These results confirmed that the RGMS approach was successful in improving the rimocidin production in S. rimosus M527.

Intratumoral delivered novel circular mRNA encoding cytokines for immune modulation and cancer therapy.

The success of the two mRNA vaccines developed by Moderna and BioNTech during the COVID-19 pandemic increased research interest into the application of mRNA technologies. Compared with the canonical linear mRNA used in these vaccines, circular mRNA has been found to mediate more potent and durable protein expression and demands a simpler manufacturing procedure. However, the application of circular mRNA is still at the initiation stage, and proof of concept for its use as a future medicine or vaccine is required. In the current study, we established a novel type of circular mRNA, termed cmRNA, based on the echovirus 29-derived internal ribosome entry site element and newly designed homology arms and RNA spacers. Our results demonstrated that this type of circular mRNA could mediate strong and durable expression of various types of proteins, compared with typical linear mRNA. Moreover, for the first time, our study demonstrated that direct intratumoral administration of cmRNA encoding a mixture of cytokines achieved successful modulation of intratumoral and systematic anti-tumor immune responses and enhanced anti-programmed cell death protein 1 (PD-1) antibody-induced tumor repression in a syngeneic mouse model. This novel circular mRNA platform is thereby suitable for direct intratumoral administration for cancer therapy.

Revealing Genome-Based Biosynthetic Potential of Streptomyces sp. BR123 Isolated from Sunflower Rhizosphere with Broad Spectrum Antimicrobial Activity.

Actinomycetes, most notably the genus Streptomyces, have great importance due to their role in the discovery of new natural products, especially for finding antimicrobial secondary metabolites that are useful in the medicinal science and biotechnology industries. In the current study, a genome-based evaluation of Streptomyces sp. isolate BR123 was analyzed to determine its biosynthetic potential, based on its in vitro antimicrobial activity against a broad range of microbial pathogens, including gram-positive and gram-negative bacteria and fungi. A draft genome sequence of 8.15 Mb of Streptomyces sp. isolate BR123 was attained, containing a GC content of 72.63% and 8103 protein coding genes. Many antimicrobial, antiparasitic, and anticancerous compounds were detected by the presence of multiple biosynthetic gene clusters, which was predicted by in silico analysis. A novel metabolite with a molecular mass of 1271.7773 in positive ion mode was detected through a high-performance liquid chromatography linked with mass spectrometry (HPLC-MS) analysis. In addition, another compound, meridamycin, was also identified through a HPLC-MS analysis. The current study reveals the biosynthetic potential of Streptomyces sp. isolate BR123, with respect to the synthesis of bioactive secondary metabolites through genomic and spectrometric analysis. Moreover, the comparative genome study compared the isolate BR123 with other Streptomyces strains, which may expand the knowledge concerning the mechanism involved in novel antimicrobial metabolite synthesis.

In vitro and in silico Studies Reveal Bacillus cereus AA-18 as a Potential Candidate for Bioremediation of Mercury-Contaminated Wastewater.

Mercury (Hg) pollution is a worldwide problem and increasing day by day due to natural and anthropogenic sources. In this study, mercury-resistant (HgR) bacterial isolates were isolated from industrial wastewater of Ittehad Chemicals Ltd., Kala Shah Kaku, Lahore, Pakistan. Out of 65 bacterial isolates, five isolates were screened out based on showing resistance at 30-40 µg/ml against HgCl2. Selected Hg-resistant bacterial isolates were characterized as Bacillus subtilis AA-16 (OK562835), Bacillus cereus AA-18 (OK562834), Bacillus sp. AA-20 (OK562833), Bacillus paramycoides AA-30 (OK562836), and Bacillus thuringiensis AA-35 (OK562837). B. cereus AA-18 showed promising results in the resistance of HgCl2 (40 µg/ml) due to the presence of merA gene. Scanning electron microscopy (SEM) analysis of immobilized B. cereus AA-18 showed the accumulation Hg on the cell surface. The inoculation of immobilized B. cereus AA-18 remediated 86% Hg of industrial wastewater up to 72 h at large scale (p < 0.05). In silico analysis showed structural determination of MerA protein encoded by merA gene of B. cereus AA-18 (OK562598) using ProtParam, Pfam, ConSurf Server, InterPro, STRING, Jpred4, PSIPRED, I-TASSER, COACH server, TrRosetta, ERRAT, VERIFY3D, Ramachandran plot, and AutoDock Vina (PyRx 8.0). These bioinformatics tools predicted the structural-based functional homology of MerA protein (mercuric reductase) associated with mer operon harboring bacteria involved in Hg-bioremediation system.

Identification of Genes Essential for Sulfamate and Fluorine Incorporation During Nucleocidin Biosynthesis.

Nucleocidin is an adenosine derivative containing 4'-fluoro and 5'-O-sulfamoyl substituents. In this study, nucleocidin biosynthesis is examined in two newly discovered producers, Streptomyces virens B-24331 and Streptomyces aureorectus B-24301, which produce nucleocidin and related derivatives at titers 30-fold greater than S. calvus. This enabled the identification of two new O-acetylated nucleocidin derivatives, and a potential glycosyl-O-acetyltransferase. Disruption of nucJ, nucG, and nucI, within S. virens B-24331, specifying a radical SAM/Fe-S dependent enzyme, sulfatase, and arylsulfatase, respectively, led to loss of 5'-O-sulfamoyl biosynthesis, but not fluoronucleoside production. Disruption of nucN, nucK, and nucO specifying an amidinotransferase, and two sulfotransferases respectively, led to loss of fluoronucleoside production. Identification of S. virens B-24331 as a genetically tractable and high producing strain sets the stage for understanding nucleocidin biosynthesis and highlights the utility of using 16S-RNA sequences to identify alternative producers of valuable compounds in the absence of genome sequence data.

Protein X0P338, a GntR-type pleiotropic regulator for morphological differentiation and secondary metabolites production in Streptomyces diastatochromogenes 1628.

The nucleoside antibiotic, toyocamycin (TM) exhibits excellent potent activity against several phytopathogenic fungi. Despite its importance, little is known about key factors regulating TM biosynthesis and morphological differentiation in Streptomyces diastatochromogenes 1628. Based on proteomics data obtained from the analysis between wild-type (WT) S. diastatochromogenes 1628 strain and mutant strain 1628-T62 having a low yield of TM, we observed that the differentially expressed protein, X0P338, which was proposed to be a regulator of the GntR-family, exhibited a higher expression level in S. diastatochromogenes 1628. Therefore, in this study, to explore whether protein X0P338 was involved in morphological differentiation and biosynthesis of secondary metabolites, especially TM, the gene called the gntRsd -encoding protein X0P338 was cloned and overexpressed in WT strain 1628 and mutant strain 1628-T62, respectively. The results indicated that the overexpression of gntRsd enhanced TM production in both strain 1628 (120.6 mg/L vs. 306.6 mg/L) and strain 1628-T62 (15.6 mg/L vs. 258.9 mg/L). Besides, the overexpression of gntRsd had positive and negative effects on morphological differentiation in strain 1628 and strain 1628-T62, respectively. The results also showed opposite effects on tetraene macrolide production during the overexpression of gntRsd in strain 1628 and strain 1628-T62. Moreover, transcription levels of genes involved in morphological differentiation and secondary metabolites production were affected by the overexpression of gntRsd gene, both in strain 1628 and strain 1628-T62. These results confirm that X0P338 as a GntR-type pleiotropic regulator that regulates the morphological differentiation and biosynthesis of secondary metabolites, and especially has a positive effect on TM biosynthesis.

Improvement of Rimocidin Biosynthesis by Increasing Supply of Precursor Malonyl-CoA via Over-expression of Acetyl-CoA Carboxylase in Streptomyces rimosus M527.

Precursor engineering is an effective strategy for the overproduction of secondary metabolites. The polyene macrolide rimocidin, which is produced by Streptomyces rimosus M527, exhibits a potent activity against a broad range of phytopathogenic fungi. It has been predicted that malonyl-CoA is used as extender units for rimocidin biosynthesis. Based on a systematic analysis of three sets of time-series transcriptome microarray data of S. rimosus M527 fermented in different conditions, the differentially expressed accsr gene that encodes acetyl-CoA carboxylase (ACC) was found. To understand how the formation of rimocidin is being influenced by the expression of the accsr gene and by the concentration of malonyl-CoA, the accsr gene was cloned and over-expressed in the wild-type strain S. rimosus M527 in this study. The recombinant strain S. rimosus M527-ACC harboring the over-expressed accsr gene exhibited better performances based on the enzymatic activity of ACC, intracellular malonyl-CoA concentrations, and rimocidin production compared to S. rimosus M527 throughout the fermentation process. The enzymatic activity of ACC and intracellular concentration of malonyl-CoA of S. rimosus M527-ACC were 1.0- and 1.5-fold higher than those of S. rimosus M527, respectively. Finally, the yield of rimocidin produced by S. rimosus M527-ACC reached 320.7 mg/L, which was 34.0% higher than that of S. rimosus M527. These results confirmed that malonyl-CoA is an important precursor for rimocidin biosynthesis and suggested that an adequate supply of malonyl-CoA caused by accsr gene over-expression led to the improvement in rimocidin production.

Enhancement of toyocamycin production through increasing supply of precursor GTP in Streptomyces diastatochromogenes 1628.

The nucleoside antibiotic toyocamycin (TM), which is produced by Streptomyces diastatochromogenes 1628, exhibits potent activity against a broad range of phytopathogenic fungi. TM was synthesized through a multi-step reaction, using guanosine triphosphate (GTP) as precursor. Based on a comparison of proteomics data from S. diastatochromogenes 1628 and rifamycin-resistant mutant 1628-T15 with high yield of TM, we determined that the differentially expressed protein X0NBV6 called ribose-phosphate pyrophosphokinase (RHP), which is a rate-limiting enzyme involved in the de novo biosynthesis of GTP, exhibits a higher expression level in mutant 1628-T15. In this study, to elucidate the relationships between RHP, GTP, and TM production, the gene rhp sd encoding RHP was cloned and overexpressed in S. diastatochromogenes strain 1628. The recombinant strain S. diastatochromogenes 1628-RHP exhibited better performance at the transcriptional level of the rhp sd gene, as well as RHP enzymatic activity, intracellular GTP concentration, and TM production, compared to S. diastatochromogenes 1628. Finally, the yield of TM produced by S. diastatochromogenes 1628-RHP (340.2 mg/L) was 133.3% higher than that produced by S. diastatochromogenes1628. Moreover, the transcriptional level of toy genes involved in TM biosynthesis was enhanced due to the overexpression of the rhp sd gene.

Effects of S-adenosylmethionine on production of secondary metabolites in Streptomycesdiastatochromogenes 1628.

Streptomyces are famous for their ability to synthesize a large number of bioactive compounds as secondary metabolites containing antibiotics, enzyme inhibitors, and other small molecules with potential physiological activity (Niu et al., 2016; Song et al., 2019; Yin et al., 2019). Secondary metabolites are produced by a multi-step reaction of a primary metabolite as a precursor (Liu et al., 2013; Li et al., 2021). Therefore, it is of great research significance to increase the overall synthesis level of antibiotics by increasing the amount of synthesis of precursors.

Production of a broad spectrum streptothricin like antibiotic from halotolerant Streptomyces fimbriatus isolate G1 associated with marine sediments.

Streptomyces have been reported as a remarkable source for bioactive secondary metabolites with complex structural and functional diversity. In this study, 35 isolates of genus Streptomyces were purified from rhizospheric and marine soils collected from previously unexplored habitats and screened for antimicrobial activities. One of these isolates, G1, when tested in vitro, was found highly active against wide range of microbes including Gram-positive, Gram-negative bacteria, and different fungal pathogens. It was identified as mesophilic, alkaliphilic, and moderately halotolerant as it showed optimum growth at temperature 30 °C, pH 8.0 in casein-starch-peptone-yeast extract-malt extract medium supplemented with 5% NaCl. Sequence analysis of the 16S rRNA gene indicated 100% identity of this isolate to Streptomyces fimbriatus. Moreover, maximum antimicrobial activity was achieved in starch nitrate medium supplemented with 1% glycerol as carbon and 0.03% soy meal as nitrogen source. The antimicrobial compounds produced by this isolate were extracted in methanol. Bioassay-guided fractionation through thin layer chromatography of methanolic extract resulted in the separation of a most active fraction with an Rf value of 0.46. This active fraction was characterized by FTIR and LCMS analysis and found similar to streptothricin D like antibiotic with m/z 758.42.

Draft Genome Sequence of Marine Actinobacterium Streptomyces spinoverrucosus SNB-032.

Actinobacteria represent a large source of diverse bioactive compounds of medical and economic importance. Here, we report the 8.8-Mb draft genome of the marine bacterium Streptomyces spinoverrucosus SNB-032. Bioinformatic sequence analysis proved similarities to known Streptomyces strains and revealed the capacity for the production of various secondary metabolites.

Biosynthesis of the Tricyclic Aromatic Type II Polyketide Rishirilide: New Potential Third Ring Oxygenation after Three Cyclization Steps.

Rishirilides are a group of PKS II secondary metabolites produced by Streptomyces bottropensis Gö C4/4. Biosynthetic studies in the past have elucidated early and late steps of rishirilide biosynthesis. This work is aiming to solve the remaining steps in the rishirilide biosynthesis. Inactivation of the cyclase gene rslC3 in Streptomyces bottropensis resulted in an interruption of rishirilide production. Instead, accumulation of the tricyclic aromatic galvaquinones was observed. Similar results were observed after deletion of rslO4. Closer inspection into RslO4 crystal structure in addition to site-directed mutagenesis and molecular dynamic simulations revealed that RslO4 might be responsible for quinone formation on the third ring. The RslO1 three-dimensional structure shows a high similarity to FMN-dependent luciferase-like monooxygenases such as the epoxy-forming MsnO8 which acts with the flavin reductase MsnO3 in mensacarcin biosynthesis in the same strain. The high sequence similarity between RslO2 and MsnO3 suggests that RslO2 provides RslO1 with reduced FMN to form an epoxide that serves as substrate for RslO5.

Regulatory Control of Rishirilide(s) Biosynthesis in Streptomyces bottropensis.

Streptomycetes are well-known producers of numerous bioactive secondary metabolites widely used in medicine, agriculture, and veterinary. Usually, their genomes encode 20-30 clusters for the biosynthesis of natural products. Generally, the onset and production of these compounds are tightly coordinated at multiple regulatory levels, including cluster-situated transcriptional factors. Rishirilides are biologically active type II polyketides produced by Streptomyces bottropensis. The complex regulation of rishirilides biosynthesis includes the interplay of four regulatory proteins encoded by the rsl-gene cluster: three SARP family regulators (RslR1-R3) and one MarR-type transcriptional factor (RslR4). In this work, employing gene deletion and overexpression experiments we revealed RslR1-R3 to be positive regulators of the biosynthetic pathway. Additionally, transcriptional analysis indicated that rslR2 is regulated by RslR1 and RslR3. Furthermore, RslR3 directly activates the transcription of rslR2, which stems from binding of RslR3 to the rslR2 promoter. Genetic and biochemical analyses demonstrated that RslR4 represses the transcription of the MFS transporter rslT4 and of its own gene. Moreover, DNA-binding affinity of RslR4 is strictly controlled by specific interaction with rishirilides and some of their biosynthetic precursors. Altogether, our findings revealed the intricate regulatory network of teamworking cluster-situated regulators governing the biosynthesis of rishirilides and strain self-immunity.

Differential mass spectrometry-based proteome analyses unveil major regulatory hubs in rifamycin B production in Amycolatopsis mediterranei.

Rifamycin B is produced by Amycolatopsis mediterranei S699 as a secondary metabolite. Its semi-synthetic derivatives have been used for curing tuberculosis caused by Mycobacterium tuberculosis. But the emergence of rifampicin-resistant strains required analogs of rifamycin B to be developed by rifamycin biosynthetic gene cluster manipulation. In 2014 genetic engineering of the rifamycin polyketide synthase gene cluster in S699 led to a mutant, A. mediterranei DCO#34, that produced 24-desmethylrifamycin B. Unfortunately, the productivity was strongly reduced to 20 mgL-1 as compared to 50 mgL-1 of rifamycin B. To understand the mechanisms leading to reduced productivity and rifamycin biosynthesis by A. mediterranei S699 during the early and late growth phase we performed a proteome study for wild type strain S699, mutant DCO#34, and the non-producer strain SCO2-2. Proteins identification and relative label-free quantification were performed by nLC-MS/MS. Data are available via ProteomeXchange with identifier PXD016416. Also, in-silico protein-protein interaction approach was used to determine the relationship between different structural and regulatory proteins involved in rifamycin biosynthesis. Our studies revealed RifA, RifK, RifL, Rif-Orf19 as the major regulatory hubs. Relative abundance expression values revealed that genes encoding RifC-RifI and the transporter RifP, down-regulated in DCO#34 and genes encoding RifR, RifZ, other regulatory proteins up-regulated. SIGNIFICANCE: The study is designed mainly to understand the underlying mechanisms of rifamycin biosynthesis in Amycolatopsis mediterranei. This resulted in the identification of regulatory hubs which play a crucial role in regulating secondary metabolism. It elucidates the complex mechanism of secondary metabolite biosynthesis and their conversion and extracellular transportation in temporal correlation with the different growth phases. The study also elucidated the mechanisms leading to reduced production of analog, 24-desmethylrifamycin B by the genetically modified strain DCO#34, derivatives of which have been found effective against rifampicin-resistant strains of Mycobacterium tuberculosis. These results can be useful while carrying out genetic manipulations to improve the strains of Amycolatopsis to produce better analogs/drugs and promote the eradication of TB. Thus, this study is contributing significantly to the growing knowledge in the field of the crucial drug, rifamycin B biosynthesis by an economically important bacterium Amycolatopsis mediterranei.

Identification and Characterization of Four c-di-GMP-Metabolizing Enzymes from Streptomyces ghanaensis ATCC14672 Involved in the Regulation of Morphogenesis and Moenomycin A Biosynthesis.

Diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) are essential enzymes deputed to maintain the intracellular homeostasis of the second messenger cyclic dimeric (3'→5') GMP (c-di-GMP). Recently, c-di-GMP has emerged as a crucial molecule for the streptomycetes life cycle, governing both morphogenesis and secondary metabolite production. Indeed, in Streptomyces ghanaensis ATCC14672 c-di-GMP was shown to be involved in the regulatory cascade of the peptidoglycan glycosytransferases inhibitor moenomycin A (MmA) biosynthesis. Here, we report the role of four c-di-GMP-metabolizing enzymes on MmA biosynthesis as well as morphological progression in S. ghanaensis. Functional characterization revealed that RmdAgh and CdgAgh are two active PDEs, while CdgEgh is a DGC. In vivo, overexpression of rmdAgh and cdgAgh led to precocious sporulation, whereas overexpression of cdgEgh and cdgDgh (encoding a predicted DGC) caused an arrest of morphological development. Furthermore, we demonstrated that individual deletion of rmdAgh, cdgAgh, and cdgDgh enhances MmA accumulation, whereas deletion of cdgEgh has no impact on antibiotic production. Conversely, an individual deletion of each studied gene does not affect morphogenesis. Altogether, our results show that manipulation of c-di-GMP-metabolizing enzymes represent a useful approach to improving MmA production titers in S. ghanaensis.