Effects of Streptomyces melanosporofaciens X216 on microbial diversity in oilseed rape soil.

Introduction: Clubroot disease is a devastating soil borne disease caused by infection with Plasmodiophora brassicae, which primarily affects cruciferous plants. The microbial diversity of the soil is an essential indicator of its quality. Methods: This study measured the physicochemical properties of the soil to study the effect of its microbial diversity on the infection of oilseed rape with P. brassicae. High-throughput sequences of the soil bacteria and fungi in the inter-root soils of P. brassicae were analyzed under different treatment conditions. Results: In the study, it was found that the efficiency of strain X216 in preventing and controlling the root disease of rapeseed was positively correlated with the amount of solution used to irrigate the root system. The results of the greenhouse and field trials showed that the efficiency of strain X216 against the root disease of rapeseed was 43.16% in the field and 62.14% in the greenhouse. Proteobacteria, Chloroflexi, Rozellomycota, and Basidiomycota are critical phylum in the development of clubroot disease. The application of biocontrol increased the relative abundance of Actinobacteria, Bacillus, Mesorhizobium, Mycobacterium, Streptomyces and Filobasidium, which affected the structure and abundance of microbial communities. A principal coordinate analysis showed that the microbial structure in the soil varied substantially in the bacterial community, and there was no significant difference in soil structure in the fungal community. Discussion: The occurrence of clubroot disease affected the structure of inter-root microbial community composition in the soil, which resulted in a decrease in its community diversity. The application of the biocontrol bacterium X216 increased the soil microbial diversity. It effectively reduced the occurrence of P. brassicae, and this study provides a basis to study the microbial diversity in cruciferous crops.

Identification of a Novel Ibuprofen Biotransformation Pathway in Streptomyces sp. D218 and Detoxification as Indicated by the Green Algae Scenedesmus obliquus.

Ibuprofen, a widely used nonsteroidal anti-inflammatory drug, contaminates agricultural products and potentially threatens human health due to its frequent detection and poor biodegradability. Microbial metabolism dominates the elimination of residual ibuprofen in the environment. In mineral salt medium at pH 6 with 5 mM glucose, Streptomyces sp. D218 transformed ibuprofen concentrations ranging from 0.05 to 0.40 mM in 24 h. The optimal temperature, pH, and initial OD600 nm for ibuprofen transformation by strain D218 were 25-37 °C, 5.0-6.0, and 1.0-1.5, respectively. Strain D218 could simultaneously transform ibuprofen into the intermediates 2-hydroxyibuprofen and ibuprofen amide (IBUA). The two intermediates were further metabolized to 2-hydroxyibuprofen amide (2HIBUA), thus relieving the growth inhibition of ibuprofen in Scenedesmus obliquus. This is the first complete pathway reported for the detoxification of ibuprofen transformation by a Gram-positive strain. These findings further our understanding of the microbial catabolism of the IBU.

Isolation and characterization of the new Streptomyces phages Kamino, Geonosis, Abafar, and Scarif infecting a broad range of host species.

Streptomyces, a multifaceted genus of soil-dwelling bacteria belonging to the phylum Actinomycetota, features intricate phage-host interactions shaped by its complex life cycle and the synthesis of a diverse array of specialized metabolites. Here, we describe the isolation and characterization of four novel Streptomyces phages infecting a variety of different host species. While phage Kamino, isolated on Streptomyces kasugaensis, is predicted to be temperate and encodes a serine integrase in its genome, phages Geonosis (isolated on Streptomyces griseus) and Abafar and Scarif, isolated on Streptomyces albidoflavus, are virulent phages. Phages Kamino and Geonosis were shown to amplify well in liquid culture leading to a pronounced culture collapse already at low titers. Determination of the host range by testing >40 different Streptomyces species identified phages Kamino, Abafar, and Scarif as broad host-range phages. Overall, the phages described in this study expand the publicly available portfolio of phages infecting Streptomyces and will be instrumental in advancing the mechanistic understanding of the intricate antiviral strategies employed by these multicellular bacteria.IMPORTANCEThe actinobacterial genus Streptomyces is characterized by multicellular, filamentous growth and the synthesis of a diverse range of bioactive molecules. These characteristics also play a role in shaping their interactions with the most abundant predator in the environment, bacteriophages-viruses infecting bacteria. In this study, we characterize four new phages infecting Streptomyces. Out of those, three phages feature a broad host range infecting up to 15 different species. The isolated phages were characterized with respect to plaque and virion morphology, host range, and amplification in liquid culture. In summary, the phages reported in this study contribute to the broader collection of publicly available phages infecting Streptomyces, playing a crucial role in advancing our mechanistic understanding of phage-host interactions of these multicellular bacteria.

Genome Mining for Diazo-Synthesis-Related Genes in Streptomyces sp. CS057 Unveiled the Cryptic Biosynthetic Gene Cluster crx for the Novel 3,4-AHBA-Derived Compound Crexazone 2.

Natural products play a crucial role in drug development, addressing the escalating microbial resistance to antibiotics and the treatment of emerging diseases. Progress in genome sequencing techniques, coupled with the development of bioinformatics tools and the exploration of uncharted habitats, has highlighted the biosynthetic potential of actinomycetes. By in silico screening for diazo-related gene genomes from twelve Streptomyces strains isolated from Attini leaf-cutting ants, the new crx biosynthetic gene cluster (BGC) was identified in Streptomyces sp. CS057. This cluster, highly conserved in several Streptomyces strains, contains genes related to diazo group formation and genes for the biosynthesis of 3,4-AHBA. By overexpressing the LuxR-like regulatory gene crxR1, we were able to activate the crx cluster, which encodes the biosynthesis of three 3,4-AHBA-derived compounds that we named crexazones (CRXs). The chemical structure of crexazones (CRXs) was determined by LC-DAD-HRMS-based dereplication and NMR spectroscopic analyses and was found to correspond to two known compounds, 3-acetamido-4-hydroxybenzoic acid (CRX1) and the phenoxazinone texazone (CRX3), and a novel 3,4-AHBA-containing compound herein designated as CRX2. Experimental proof linking the crx BGC to their encoded compounds was achieved by generating mutants in selected crx genes.

ActO, a positive cluster-situated regulator for actinomycins biosynthesis in Streptomyces antibioticus ZS.

Actinomycins are a class of cyclic lipopeptide antibiotics produced by Streptomyces, which have rich biological activities and demonstrate great potential value. Among them, actinomycin D is currently the effective drug for some malignant tumor diseases. Although the chemical properties, biological activities and biosynthesis of actinomycins have been extensively studied, the regulation of their biosynthesis remains poorly understood. Streptomyces antibioticus ZS isolated from deep-sea corals is a producer of actinomycin D and actinomycin V. Here, we reported the characterization of a cluster-situated regulator ActO in actinomycins biosynthetic gene cluster (act cluster) of S. antibioticus ZS, which belongs to LmbU family. Deletion of actO completely blocked the synthesis of actinomycins. Overexpression of actO increased the yields of actinomycin D and actinomycin V by 4.4 fold and 2.6 fold, respectively. The result of RT-qPCR showed that ActO activates the transcription of all genes in act cluster. However, no specific binding of His6-ActO to the promoters of target genes was observed after electrophoretic mobility shift assay (EMSA). These results proved that ActO serves as a positive regulator involved in the biosynthesis of actinomycins, affecting the transcription of all genes related to the synthesis of intermediates, skeleton modification and extracellular transportation of final products. Moreover, we demonstrated that overexpression of actO is a novel strategy to increase the yields of actinomycins.

Improving the production of natamycin in Streptomyces natalensis HW-2 by L-valine feeding.

L-valine (L-Val) was previously confirmed to promote natamycin biosynthesis in S. natalensis HW-2. In this study, natamycin yield was 1.9-fold increase with 0.5 g/L L-Val feeding. The level of free amino acids in the broth was significantly affected. Transcriptome analysis showed that 646 and 189 genes were significantly differential expression at 48 h and 60 h, respectively. 7 differential expression genes in branched-chain amino acids (BCAAs) degradation were up-regulated. To further investigate the role of BCAAs degradation on natamycin biosynthesis, the gene ilvE, which encoded branched-chain amino acid aminotransferase (BCAT), was homologously overexpressed. The optimal mutant, S. natalensis LY08, was obtained, and its natamycin production was increased by 179%. With the optimized L-Val supplementation concentration, natamycin yield was increased to 2.02 g/L by strain LY08. This finding indicated the roles of BCAAs degradation on natamycin biosynthesis, and provided an efficient strategy to improve natamycin production in S. natalensis. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-024-01570-8.

Characterization of Lomofungin Gene Cluster Enables the Biosynthesis of Related Phenazine Derivatives.

Phenazine-based small molecules are nitrogen-containing heterocyclic compounds with diverse bioactivities and electron transfer properties that exhibit promising applications in pharmaceutical and electrochemical industries. However, the biosynthetic mechanism of highly substituted natural phenazines remains poorly understood. In this study, we report the direct cloning and heterologous expression of the lomofungin biosynthetic gene cluster (BGC) from Streptomyces lomondensis S015. Reconstruction and overexpression of the BGCs in Streptomyces coelicolor M1152 resulted in eight phenazine derivatives including two novel hybrid phenazine metabolites, and the biosynthetic pathway of lomofungin was proposed. Furthermore, gene deletion suggested that NAD(P)H-dependent oxidoreductase gene lomo14 is a nonessential gene in the biosynthesis of lomofungin. Cytotoxicity evaluation of the isolated phenazines and lomofungin was performed. Specifically, lomofungin shows substantial inhibition against two human cancer cells, HCT116 and 5637. These results provide insights into the biosynthetic mechanism of lomofungin, which will be useful for the directed biosynthesis of natural phenazine derivatives.

Antimicrobial potential of Streptomyces sp. NP73 isolated from the forest soil of Northeast India against multi-drug resistant Escherichia coli.

This study reports the isolation and characterization of a Streptomyces sp. from soil, capable of producing bioactive secondary metabolites active against a variety of bacterial human pathogens. We targeted the antimicrobial activity against Escherichia coli ATCC-BAA 2469, a clinically relevant strain of bacteria harbouring resistance genes for carbapenems, extended spectrum beta-lactams, tetracyclines, fluoroquinones, etc. Preliminary screening using the spot inoculation technique identified Streptomyces sp. NP73 as the potent strain among the 74 isolated Actinomycetia strain. 16S rRNA gene and whole genome sequencing (WGS) confirmed its taxonomical identity and helped in the construction of the phylogenetic tree. WGS revealed the predicted pathways and biosynthetic gene clusters responsible for producing various types of antibiotics including the isolated compound. Bioactivity guided fractionation and chemical characterization of the active fraction, carried out using liquid chromatography, gas chromatography-mass spectrometry, infra-red spectroscopy, and nuclear magnetic resonance spectroscopy, led to the tentative identification of the active compound as Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-, a diketopiperazine molecule. This compound exhibited excellent antimicrobial and anti-biofilm properties against E. coli ATCC-BAA 2469 with an MIC value of 15.64 µg ml-1, and the low cytotoxicity of the compound identified in this study provides hope for future drug development.

Inhibitory mechanism of 4-ethyl-1,2-dimethoxybenzene produced by Streptomyces albidoflavus strain ML27 against Colletotrichum gloeosporioides.

Actinomycetes have emerged as significant biocontrol resources due to their rich array of bioactive natural products. While much research has historically focused on secondary metabolites isolated from their fermentation broth, there remains a dearth of reports on their volatile organic compounds (VOCs). Here, strain ML27, isolated from soil, was identified as Streptomyces albidoflavus based on morphological features, physiological, biochemical, and molecular characteristics (16S rRNA, atpD, recA, and rpoB gene sequences). VOCs from S. albidoflavus strain ML27 were effectively captured using solid-phase microextraction (SPME) and tentatively identified through gas chromatography-mass spectrometry (GC/MS). Among these compounds, 4-ethyl-1,2-dimethoxybenzene exhibited broad-spectrum antifungal activity and demonstrated efficacy in controlling citrus anthracnose, with a control efficacy of 86.67%. Furthermore, the inhibitory mechanism of 4-ethyl-1,2-dimethoxybenzene against Colletotrichum gloeosporioides was revealed. Results indicated that 4-ethyl-1,2-dimethoxybenzene induced swelling, deformity, and breakage in C. gloeosporioides mycelia, and significantly inhibited spore germination. Transcriptome analysis revealed that 4-ethyl-1,2-dimethoxybenzene inhibited the growth and development of C. gloeosporioides primarily by disrupting energy metabolism and the integrity of the cell wall and membrane. Based on these results, it is promising to develop 4-ethyl-1,2-dimethoxybenzene as a novel biopesticide for controlling citrus anthracnose.

Activity of Streptomyces globosus OPF-9 against the important pathogen Alternaria longipes and biocontrol mechanisms revealed by multi-omic analyses.

Plant diseases caused by fungal pathogens represent main threats to the yield and quality of agricultural products, and Alternaria longipes is one of the most important pathogens in agricultural systems. Biological control is becoming increasingly prevalent in the management of plant diseases due to its environmental compatibility and sustainability. In the present study, a bacterial strain, designated as OPF-9, was shown to effectively inhibit the pathogen A. longipes, which was identified as Streptomyces globosus. The culture conditions for OPF-9 were optimized through a stepwise approach and the fermentation broth acquired displayed an excellent inhibitory activity against A. longipes in vitro and in vivo. Further investigations suggested that the fermentation broth exhibited strong stability under a range of adverse environmental conditions. To reveal the molecular bases of OPF-9 in inhibiting pathogens, the whole-genome sequencing and assembly were conducted on this strain. It showed that the genome size of OPF-9 was 7.668 Mb, containing a chromosome and two plasmids. Multiple clusters of secondary metabolite synthesis genes were identified by genome annotation analysis. In addition, the fermentation broth of strain OPF-9 was analyzed by LC-MS/MS non-target metabolomic assay and the activity of potential antifungal substances was determined. Among the five compounds evaluated, pyrogallol displayed the most pronounced inhibitory activity against A. longipes, which was found to effectively inhibit the mycelial growth of this pathogen. Overall, this study reported, for the first time, a strain of S. globosus that effectively inhibits A. longipes and revealed the underlying biocontrol mechanisms by genomic and metabolomic analyses.

Genomic Exploration of a Chitinolytic Streptomyces albogriseolus PMB5 Strain from European mantis (Mantis religiosa).

The genus Streptomyces is renowned not only for its natural antibiotic production but also for its abundant chitinolytic enzymes, which break down stubborn chitin into chitooligosaccharides. Despite this, there have been limited studies utilizing whole-genome sequencing to explore the repertoire of chitin degradation and utilization genes in Streptomyces. A particularly compelling source of novel antimicrobials and enzymes lies in the microbiota of insects, where bacterial symbionts produce antimicrobials to protect against opportunistic pathogens and enzymes to adapt to the environment. In this study, we present the chitinolytic strain Streptomyces albogriseolus PMB5, isolated from the insectivorous Mantis religiosa (European mantis). Whole-genome sequencing revealed that PMB5 harbors a linear chromosome of 7,211,961 bp and a linear plasmid of 327,989 bp. The genome comprises 6683 genes, including 6592 protein-coding sequences and 91 RNA genes. Furthermore, genome analysis revealed 19 biosynthetic gene clusters covering polyketides, terpenes, and RiPPs, with 10 clusters showing significant gene similarity (>80%) to known clusters like antimycin, hopene, and geosmin. In the genome of S. albogriseolus PMB5, we were able to identify several antibiotic resistance genes; these included cml (resistance to phenicol), gimA (resistance to macrolides), parY (resistance to aminocoumarin), oleC/oleD (resistance to macrolides), novA (resistance to aminocoumarin) and bla/blc (resistance to beta-lactams). Additionally, three clusters displayed no similarity to known sequences, suggesting novel bioactive compound discovery potential. Remarkably, strain PMB5 is the first reported S. albogriseolus capable of thriving on a medium utilizing chitin as a carbon source, with over 50 chitin-utilizing genes identified, including five AA10 family LPMOs, five GH18 chitinases, and one GH19 chitinase. This study significantly enhances the genomic understanding of S. albogriseolus, a species previously underrepresented in research, paving the way to further exploration of the biotechnological potential of the species.

Antibacterial and cytotoxic metabolites produced by Streptomyces tanashiensis BYF-112 isolated from Odontotermes formosanus.

Chemical investigations of the termite-associated Streptomyces tanashiensis BYF-112 resulted in the discovery of four novel alkaloid derivatives: vegfrecines A and B (1 and 2), exfoliazone A (3), and venezueline H (7), in addition to nine known metabolites (4-6, 8-13). The structures of these compounds were elucidated through comprehensive spectroscopic analysis and comparison with existing literature data. Antibacterial assays revealed that viridomycin A (11) exhibited potent antibacterial activity against Staphylococcus aureus, with a zone of inhibition (ZOI) of 12.67 mm, in comparison to a ZOI of 17.67 mm for the positive control gentamicin sulfate. Viridomycin A (11) showed moderate activity against Micrococcus tetragenus and Pseudomonas syringae pv. actinidae, with ZOI values of 15.50 and 14.33 mm, respectively, which were inferior to those of gentamicin sulfate (34.67 and 24.00 mm). Viridomycin F (12) also exhibited moderate antibacterial effects against S. aureus, M. tetragenus, and P. syringae pv. actinidae, with ZOI values of 8.33, 16.50, and 10.83 mm, respectively. Cytotoxicity assays demonstrated that viridobruunine A (5), exfoliazone (6), viridomycin A (11), and X-14881E (13) exhibited significant cytotoxicity against human malignant melanoma (A375), ovarian cancer (SKOV-3), and gastric cancer (MGC-803) cell lines, with IC50 values ranging from 4.61 to 19.28 µmol·L-1. Furthermore, bioinformatic analysis of the complete genome of S. tanashiensis suggested a putative biosynthetic gene cluster (BGC) responsible for the production of compounds 1-12. These findings indicate that the secondary metabolites of insect-associated S. tanashiensis BYF-112 hold promise as potential sources of novel antibacterial and anticancer agents.

Effects of α-sitosterol on growth, hematobiochemical profiles, immune-antioxidant resilience, histopathological features and expression of immune apoptotic genes of Nile tilapia, Oreochromis niloticus, challenged with Candida albicans.

In this study, the effect of the Streptomyces misakiensis metabolite (α- sitosterol, 0, 20, 40, 60, and 80 mg/kg) dietary supplementation on growth performance, antioxidant-immune stability and Candida albicans resistance of Nile tilapia was evaluated. The results revealed that the incorporation of α-sitosterol at doses of 60 and 80 mg/kg into the diet significantly improved the growth rate of Nile tilapia. The fish receiving 80 mg/kg showed an increased level of high-density lipoprotein, total protein, globulin, and albumin, and significantly reduced levels of indicators of hepato-renal damage, glucose, triglycerides, low-density lipoprotein, and total cholesterol. Dietary α-sitosterol induced a considerable increase in hepatopancreas glutathione peroxidase, superoxide dismutase and catalase activities and a significant drop in malondialdehyde levels. Supplementing the diet with 80 mg/kg of α-sitosterol increased nitric oxide, complement-3, nitro blue tetrazolium levels, lysozyme, and phagocytic activities. In particular, supplementing with α-sitosterol at 60-80 mg/kg of diet significantly enhanced the expression of pro/anti-inflammatory markers (il1b, il10, tgfb, ifng, tnfa and il8) after the C. albicans challenge. Also, there was a decrease in cumulative mortality percent, pro-apoptotic markers (casp3, bax and hsp70) and an increase in anti-apoptotic indicators (bcl2). Interestingly, following the C. albicans challenge, fish that received 0 and 20 mg α-sitosterol/kg exhibited significant inflammation in the hepatopancreas, spleen, and intestine. On the other hand, inflammation could be alleviated by feeding 60-80 mg α-sitosterol/kg. Due to these findings, α-sitosterol could be an innovative option to enhance growth, general physiological status, immune service, and antifungal resistance of Nile tilapia against C. albicans.

Natural products with γ-pyrone scaffold from Streptomyces.

The Streptomyces sp. is considered the vast reservoir of bioactive natural products belonging to different classes like polyketides, terpenoids, lanthipeptides, and non-ribosomal peptides to name a few. The ubiquitous distribution of the genus makes them capable of producing distinct compounds. Many of those compounds contain a unique γ-pyrone with various chemical structures and exhibit different bioactivities. One such class, nitrophenyl-γ-pyrone, constitutes different bioactive compounds isolated from Streptomyces sp. from different sources ranging from soil to marine environments. In addition, such compounds have antinematodal, cytotoxicity activities, and inhibition of adipogenesis. These compounds include aureothin (3), spectinabilin (7), and their derivatives. Moreover, there are other compounds like actinopyrones (11-16), benwamycins (22-23), and peucemycin and its derivatives (24-26) that also have antibacterial and anticancer activities. The other group classified as anthra-γ-pyrone has various bioactive natural products. For instance, tetrahydroanthra-γ-pyrone, shellmycin A-D (27-30) possess antibacterial as well as anticancer activities. In addition, the pluramycin family compounds belonging to anthra-γ-pyrone group also possess cytotoxic activity, for instance, kidamycin (31), rubiflavin, and their derivatives (33-37). Xanthones are another important group of natural products that also contain γ-pyrone ring producing different bioactivities. Albofungin (42) and its derivatives (43-46) belong to subgroup polycyclic tetrahydro xanthones that possess antibacterial, anticancer, and antibiofilm, antimacrofouling activities. Similarly, other compounds, belonging to this subgroup, exhibit different bioactivities like antifungal, antimalarial, and antibacterial activities and block transient receptor potential vanilloid 1 (TRPV1). These compounds include cervinomycins (48-55), citreamycins (56-57), sattahipmycin (59), and chrexanthomycins (60-63). This review gives succinct information on the γ-pyrone containing natural products isolated from Streptomyces sp. focusing on their structure and bioactivities. KEY POINTS: • The Streptomyces sp. is the producer of various bioactive natural products including the one with γ-pyrone ring. • These γ-pyrone compounds are structurally different and possess different bioactivities. • The Streptomyces has the potential to produce such compounds and the reservoir of these compounds is expected to increase in the future.

Combined effect of polyphosphate kinase and lon protease in Streptomyces coelicolor A3(2) antibiotic production.

The bacterial stringent response is a global regulatory process in which polyphosphate kinase (Ppk) and lon protease are important players. Previous studies have shown that overexpression of the lon gene and deletion of the ppk gene significantly increased actinorhodin production in Streptomyces coelicolor (SCO). In this study, a recombinant SCOΔppk-lon cell, expressing the extra lon gene in Δppk cells, was simulated using a modified in silico (computational) model, ecSco-GEM, and the negative effect of Ppk on actinorhodin production was confirmed. In addition, we identified key enzymes that play a positive role in actinorhodin production. Of these, NADH dehydrogenase/complex-I, beta-ketoacyl-[acyl-carrier-protein] synthase III, glycine cleavage system, and superoxide dismutase were identified as the most significant. By confirming these results with experiments, we have shown that GEMs can be a reliable starting point for in vitro (lab-based) studies of Streptomyces..

Modular Cloning Tools for Streptomyces spp. and Application to the De Novo Biosynthesis of Flavokermesic Acid.

The filamentous Streptomyces are among the most prolific producers of bioactive natural products and are thus attractive chassis for the heterologous expression of native and designed biosynthetic pathways. Although suitable Streptomyces hosts exist, including genetically engineered cluster-free mutants, the approach is currently limited by the relative paucity of synthetic biology tools facilitating the de novo assembly of multicomponent gene clusters. Here, we report a modular system (MoClo) for Streptomyces including a set of adapted vectors and genetic elements, which allow for the construction of complete genetic circuits. Critical functional validation of each of the elements was obtained using the previously reported ß-glucuronidase (GusA) reporter system. Furthermore, we provide proof-of-principle for the toolbox inS. albus, demonstrating the efficient assembly of a biosynthetic pathway to flavokermesic acid (FK), an advanced precursor of the commercially valuable carminic acid.

Discovery of a novel methionine biosynthetic route via O-phospho-l-homoserine.

Methionine (Met), a sulfur-containing amino acid, is essential for the underlying biological processes in living organisms. In addition to its importance as a starting building block for peptide chain elongation in protein biosynthesis, Met is a direct precursor of S-adenosyl-l-methionine, an indispensable methyl donor molecule in primary and secondary metabolism. Streptomyces bacteria are well known to produce diverse secondary metabolites, but many strains lack canonical Met pathway genes for l-homocysteine, a direct precursor of Met in bacteria, plants, and archaea. Here, we report the identification of a novel gene (metM) responsible for the Met biosynthesis in Streptomyces strains and demonstrate the catalytic function of the gene product, MetM. We further identified the metO gene, a downstream gene of metM, and showed that it encodes a sulfur-carrier protein (SCP). In in vitro analysis, MetO was found to play an important role in a sulfur donor by forming a thiocarboxylated SCP. Together with MetO (thiocarboxylate), MetM directly converted O-phospho-l-homoserine to l-homocysteine. O-Phospho-l-homoserine is also known as an intermediate for threonine biosynthesis in bacteria and plants, and MetM shares sequence homology with threonine synthase. Our findings thus revealed that MetM seizes O-phospho-l-homoserine from the threonine biosynthetic pathway and uses it as an intermediate of the Met biosynthesis to generate the sulfur-containing amino acid. Importantly, this MetM/MetO pathway is highly conserved in Streptomyces bacteria and distributed in other bacteria and archaea.IMPORTANCEMethionine (Met) is a sulfur-containing proteinogenic amino acid. Moreover, Met is a direct precursor of S-adenosyl-l-methionine, an indispensable molecule for expanding the structural diversity of natural products. Because Met and its derivatives benefit humans, the knowledge of Met biosynthesis is important as a basis for improving their fermentation. Streptomyces bacteria are well known to produce diverse and valuable natural products, but many strains lack canonical Met pathway genes. Here, we identified a novel l-homocysteine synthase (MetM) in Streptomyces and demonstrated that it converts O-phospho-L-homoserine to l-homocysteine using a thiocarboxylated sulfur-carrier protein as a sulfur donor. Since the metM is distributed in other bacteria and archaea, our pioneering study contributes to understanding Met biosynthesis in these organisms.

Increasing the flexibility of the substrate binding pocket of Streptomyces phospholipase D can enhance its catalytic efficiency in soybean phosphatidylcholine.

The catalytic efficiency of Streptomyces klenkii phospholipase D (SkPLD) in soybean phosphatidylcholine (soy-PC) processing is constrained by its acyl chain specificity. To address this limitation, we engineered the substrate-binding pocket of SkPLD to increase its flexibility. The mutant P343A/Y383L exhibited a 7.14-fold increase in catalytic efficiency toward soy-PC compared to the wild type. This enhancement was attributed to improved substrate-binding pocket flexibility, as evidenced by the significantly higher specific activity of the mutant toward PCs with various acyl chains (58.20-327.76 U/mg vs. 13.56-76.67 U/mg). Monomolecular film experiments demonstrated that the P343A/Y383L mutant reduced the energy barrier for PC binding, facilitating favorable interactions with the soy-PC monolayer. Molecular dynamics simulations revealed that the mutant's increased flexibility allowed for easier diffusion and penetration into the soy-PC monolayer, while the non-polar amino acids in the substrate-binding pocket promoted rapid interactions with the acyl chains of PC, ultimately leading to enhanced catalytic activity.

Streptomyces small laccase expressed in Aspergillus Niger as a new addition for the lignocellulose bioconversion toolbox.

Laccases are multi-copper oxidases that are usually composed of three Cu-oxidase domains. Domains one and three house the copper binding sites, and the second domain is involved in forming a substrate-binding cleft. However, Streptomyces species are found to have small laccases (SLAC) that lack one of the three Cu-oxidase domains. This type of SLAC with interesting lignocellulose bioconversion activities has not been reported in Aspergillus niger. In our research, we explored the expression and engineering of the SLAC from Streptomyces leeuwenhoekii C34 in A. niger. Genes encoding two versions of the SLAC were expressed. One encoding the SLAC in its native form and a second encoding the SLAC fused to two N-terminal CBM1 domains. The latter is a configuration also known for specific yeast laccases. Both SLAC variants were functionally expressed in A. niger as shown by in vitro activity assays and proteome analysis. Laccase activity was also analyzed toward bioconversion of lignocellulosic rice straw. From this analysis it was clear that the SLAC activity improved the efficiency of saccharification of lignocellulosic biomass by cellulase enzyme cocktails.

Deciphering domain structures of Aspergillus and Streptomyces GH3-ß-Glucosidases: a screening system for enzyme engineering and biotechnological applications.

The glycoside hydrolase family 3 (GH3) ß-glucosidases from filamentous fungi are crucial industrial enzymes facilitating the complete degradation of lignocellulose, by converting cello-oligosaccharides and cellobiose into glucose. Understanding the diverse domain organization is essential for elucidating their biological roles and potential biotechnological applications. This research delves into the variability of domain organization within GH3 ß-glucosidases. Two distinct configurations were identified in fungal GH3 ß-glucosidases, one comprising solely the GH3 catalytic domain, and another incorporating the GH3 domain with a C-terminal fibronectin type III (Fn3) domain. Notably, Streptomyces filamentous bacteria showcased a separate clade of GH3 proteins linking the GH3 domain to a carbohydrate binding module from family 2 (CBM2). As a first step to be able to explore the role of accessory domains in ß-glucosidase activity, a screening system utilizing the well-characterised Aspergillus niger ß-glucosidase gene (bglA) in bglA deletion mutant host was developed. Based on this screening system, reintroducing the native GH3-Fn3 gene successfully expressed the gene allowing detection of the protein using different enzymatic assays. Further investigation into the role of the accessory domains in GH3 family proteins, including those from Streptomyces, will be required to design improved chimeric ß-glucosidases enzymes for industrial application.