Biosynthesis Gene Cluster Research Articles

Uncovering the genetic basis of antiviral polyketide limocrocin biosynthesis through heterologous expression

BackgroundStreptomyces roseochromogenes NRRL 3504 produces clorobiocin, an aminocoumarin antibiotic that inhibits DNA replication. No other natural products have been isolated from this bacterium so far, despite the presence of a rich repertoire of specialized metabolite biosynthesis gene clusters (smBGCs) within its genome. Heterologous expression of smBGCs in suitable chassis speeds up the discovery of the natural products hidden behind these sets of genes.ResultsIn this work we focus on one intriguing smBGC of NRRL 3504 bearing some similarity to gene clusters involved in production of manumycin family polyketides. Through heterologous expression in Streptomyces chassis strains S. albus Del14 and S. lividans ΔYA9, this smBGC (hereafter referred to as lim BGC) was shown to direct the production of unusual polyketide limocrocin (LIM) known for its ability to interfere with viral reverse transcriptases. The organization of lim BGC, data on the structures of revealed metabolites as well as manipulations of lim genes allowed us to put forward an initial hypothesis about a biosynthetic pathway leading to LIM. We provide initial data on two LIM derivatives as well as updated NMR spectra for the main product.ConclusionThis study reveals the genetic control of biosynthesis of LIM that remained hidden for the last 70 years. This, in turn, opens the door to biological routes towards overproduction of LIM as well as generation of its derivatives.

Mitochondrial Porin Is Required for Versatile Biocontrol Trait-Involved Biological Processes in a Filamentous Insect Pathogenic Fungus.

The mitochondrial voltage-dependent anion channel (VDAC) is the major channel in the mitochondrial outer membrane for metabolites and ions. VDACs also regulate a variety of biological processes, which vary in the number of VDAC isoforms across different eukaryotes. However, little is known about VDAC-mediated biocontrol traits in biocontrol fungi. Here, the only VDAC isoform (BbOmm1) in the filamentous insect pathogenic fungus Beauveria bassiana was characterized, which was crucial for maintenance of mitochondrial homeostasis and function and important biocontrol traits. Besides serious impairment of fungal growth, conidiation, and germination, inactivation of BbOmm1 led to increased sensitivity/tolerance to oxidative and osmotic stresses and production of oosporein and other secondary metabolites, which corresponded to the mitochondrial damage, downregulation of membrane lipid and cell wall homeostasis-involved genes, and upregulation of detoxification genes and biosynthesis gene clusters of those metabolites. These results enrich our understanding of VDAC-mediated biocontrol traits in insect fungal pathogens.

Enhancement of FK520 production in Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 by overexpressing the regulatory gene fkbR2.

Ascomycin (FK520) is a 23-membered macrolide antibiotic primarily produced by the Streptomyces hygroscopicus var. ascomyceticus. Structurally similar to tacrolimus and rapamycin, it serves as an effective immunosuppressant widely used in the treatment of rejection reactions after organ transplantation and certain autoimmune diseases. Currently, FK520 is mainly produced through microbial fermentation, but its yield remains low. Since the gene fkbR2 is a regulatory gene within the FK520 biosynthesis gene cluster that has not been studied, this paper focuses on the overexpression of the gene fkbR2 in Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 (WT). By constructing a strain with overexpressed fkbR2 gene, we initially obtained a high-yield strain R2-17 through shake flask fermentation, with a 28% increase in yield compared to WT. In the process of further improving the stability of the high-yield strain, this paper defines two indices: high-yield index and stability index. After two consecutive rounds of natural breeding, strain R2-17 achieved a high-yield index of 100% and a stability index of 80%. Finally, the high-yield strain R2-17-3-10 was successfully screened, and the yield was increased by 34% compared with the strain WT, reaching 686.47mg/L. A comparative analysis between the high-yield strain R2-17-3-10 and the original strain WT revealed differences in fermentation process parameters such as FK520 synthesis rate, pH, bacterial growth, glycerol consumption rate, ammonia nitrogen level, and ammonium ion concentration. In addition, the transcription levels of genes involved in the synthesis of precursors 4,5-dihydroxycyclohex-1-enecarboxylic acid (fkbO), ethylmalonyl-CoA (fkbE, fkbU, fkbS), and pipecolic acid (fkbL), as well as pathway-specific regulatory genes (fkbN, fkbR1), were significantly increased at different time points in the high-yield strain R2-17-3-10. EMSAs analysis showed that the FkbR2 protein could not bind to the promoter region of above genes. This suggests that the gene fkbR2 may enhance the supply of FK520 synthetic precursors by indirectly regulating the transcription levels of these genes, thereby promoting an increase in FK520 production. These results demonstrate that modifying genes within the biosynthetic gene clusters of natural products can be successfully applied to increase the yields of industrially and clinically important compounds. However, it is found that fkbR2 gene is a regulatory gene that has not been fully studied, and it is worth further studying its regulatory mechanism.

Description of Hyphococcus formosus sp. nov. and Hyphococcus lacteus sp. nov., isolated from coastal sediment, and reclassification of Marinicaulis flavus as Hyphococcus luteus nom. nov. and Marinicaulis aureus as Hyphococcus aureus comb. nov.

During a study on sediment bacterial diversity in coastal China, three bacterial strains, DH-69T, EH-24, and ECK-19T, were isolated from coastal sediments off Xiaoshi Island, Weihai. These strains were Gram-staining-negative, aerobic, and coccoid to rod-shaped with prosthecae and flagella. Comparison of the 16S rRNA gene showed that they shared the highest identity values with Hyphococcus flavus MCCC 1K03223T (96.2-97.6%), followed by Marinicaulis flavus SY-3-19T (95.2-96.8%) and Marinicaulis aureus HHTR114T (95.2-96.2%). Genome comparisons using average nucleotide identity (ANI) and average amino acid identity (AAI) suggested that the three novel strains and the three related strains belonged to the same genus, with strains DH-69T, EH-24, and ECK-19T identified as two distinct novel species. Pan-genome analysis revealed that 995 core genes were shared among 23 Hyphococcus genomes/MAGs. Secondary metabolites analysis identified a biosynthesis gene cluster for microsclerodermin, a potent antifungal peptide, in the novel strains. Moreover, these newly isolated strains were detected in various ecosystems, with a particular prevalence in marine environments, based on analysis of 500,048 amplicon datasets, underscoring their ecological preference. Based on polyphasic characterizations, strains DH-69T and EH-24 represent a novel species of the genus Hyphococcus, for which the name Hyphococcus formosus sp. nov. is proposed with the type strain DH-69T (= MCCC 1H00436T=KCTC 8010T). Strain ECK-19T represents another novel Hyphococcus species, for which the name Hyphococcus lacteus sp. nov. is proposed with the type strain ECK-19T (= MCCC 1H00435T=KCTC 8009T). Furthermore, Marinicaulis flavus and Marinicaulis aureus are proposed to be reclassified as Hyphococcus luteus nom. nov. and Hyphococcus aureus comb. nov., respectively, accompanied by an emended description of the genus Hyphococcus.

Yatakemycin biosynthesis requires two deoxyribonucleases for toxin self-resistance.

The highly active natural product yatakemycin (YTM) from Streptomyces sp. TP-A0356 is a potent DNA damaging agent with antimicrobial and antitumor properties. The YTM biosynthesis gene cluster (ytk) contains several toxin self-resistance genes. Of these, ytkR2 encodes a DNA glycosylase that is important for YTM production and host survival by excising lethal YTM-adenine lesions from the genome, presumably initiating a base excision repair (BER) pathway. However, the genes involved in repair of the resulting apurinic/apyrimidinic (AP) site as the second BER step have not been identified. Here, we show that ytkR4 and ytkR5 are essential for YTM production and encode deoxyribonucleases related to other known DNA repair nucleases. Purified YtkR4 and YtkR5 exhibit AP endonuclease activity specific for YtkR2-generated AP sites, providing a basis for BER of the toxic AP intermediate produced from YTM-adenine excision and consistent with co-evolution of ytkR2, ytkR4, and ytkR5. YtkR4 and YtkR5 also exhibit 3'-5' exonuclease activity with differing substrate specificities. The YtkR5 exonuclease is capable of digesting through a YTM-DNA lesion and may represent an alternative repair mechanism to BER. We also show that ytkR4 and ytkR5 homologs are often clustered together in putative gene clusters related to natural product production, consistent with non-redundant roles in repair of other DNA adducts derived from genotoxic natural products.

Genomic insights into the probiotic potential and genes linked to gallic acid metabolism in Pediococcus pentosaceus MBBL6 isolated from healthy cow milk.

Pediococcus pentosaceus is well known for its probiotic properties, including roles in improving health, antimicrobial production, and enhancing fermented food quality. This study aimed to comprehensively analyze the whole genome of P. pentosaceus MBBL6, isolated from healthy cow milk, to assess its probiotic and antimicrobial potentials. P. pentosaceus MBBL6, isolated from a healthy cow milk at BSMRAU dairy farm, Gazipur, Bangladesh, underwent comprehensive genomic analysis, including whole genome sequencing, assembly, annotation, phylogenetic comparison, and assessment of metabolic pathways and secondary metabolites. Antimicrobial efficacy was evaluated through in-vitro and in-vivo studies, alongside in-silico exploration for potential mastitis therapy. We predicted 1,906 genes and 204 SEED sub-systems involved in carbohydrate metabolism and vitamin B complex biosynthesis, with a focus on lactose metabolism in MMBL6. Notably, 43 putative carbohydrate-active enzyme genes, including lysozymes, suggest the ability of MBBL6 for carbohydrate biotransformation and antimicrobial activity. The genome also revealed primary metabolic pathways for arginine and gallic acid metabolism and secondary metabolite gene clusters, including T3PKS and RiPP-like regions. Importantly, two bacteriocin biosynthesis gene clusters namely bovicin_255_variant and penocin_A, were identified in MBBL6. The safety assessment of MBBL6 genome revealed no virulence genes and a low pathogenicity score (0.196 out of 1.0). Several genes related to survival in gastrointestinal tract and colonization were also identified. Furthermore, MBBL6 exhibited susceptibility to a wide range of antibiotics in-vitro, and effectively suppressed mastitis pathogens in an in-vivo mouse mastitis model trial. The observed bacteriocin, particularly bovicin, demonstrated the ability to disrupt the function of an essential protein, Rho factor of mastitis pathogens by blocking transcription termination process. Taken together, our in-depth genomic analysis underscores the metabolic versatility, safety profile, and antimicrobial potential of P. pentosaceus MBBL6, suggesting its promise for applications in therapeutics, bioremediation, and biopreservation.

Characterization of Siderophores Produced by Bacillus velezensis YL2021 and Its Application in Controlling Rice Sheath Blight and Rice Blast.

Bacillus velezensis YL2021 has extensive antimicrobial activities against phytopathogens, and its genome harbors a catechol-type siderophore biosynthesis gene cluster. Here, we describe the characterization of siderophores produced by strain YL2021 and its antimicrobial activity in vitro and in vivo. A few types of siderophores were detected by chrome azurol S plates coupled with Arnow's test, purified, and identified by reversed-phase high-performance liquid chromatography. We found that strain YL2021 can produce different antimicrobial compounds under low-iron M9 medium or iron-sufficient Luria-Bertani medium, although antimicrobial activities can be easily observed on the two media as described above in vitro. Strain YL2021 can produce at least three catechol-type siderophores in low-iron M9 medium, whereas no siderophores were produced in Luria-Bertani medium. Among them, the main antimicrobial siderophore produced by strain YL2021 was bacillibactin, with m/z 882, based on the liquid chromatography-tandem mass spectrometry analysis, which has broad-spectrum antimicrobial activities against gram-positive and gram-negative bacteria, the oomycete Phytophthora capsici, and phytopathogenic fungi. Moreover, the antifungal activity of siderophores, including bacillibactin, observed in vitro was correlated with control efficacies against rice sheath blight disease caused by Rhizoctonia solani and rice blast disease caused by Magnaporthe oryzae in vivo. Collectively, the results demonstrate that siderophores, including bacillibactin, produced by B. velezensis YL2021 are promising biocontrol agents for application in rice disease control.

The ABC transporter SmABCG1 mediates tanshinones export from the peridermic cells of Salvia miltiorrhiza root.

Plants have mechanisms to transport secondary metabolites from where they are biosynthesized to the sites where they function, or to sites such as the vacuole for detoxification. However, current research has mainly focused on metabolite biosynthesis and regulation, and little is known about their transport. Tanshinone, a class diterpenoid with medicinal properties, is biosynthesized in the periderm of Salvia miltiorrhiza roots. Here, we discovered that tanshinone can be transported out of peridermal cells and secreted into the soil environment and that the ABC transporter SmABCG1 is involved in the efflux of tanshinone ⅡA and tanshinone Ⅰ. The SmABCG1 gene is adjacent to the diterpene biosynthesis gene cluster in the S. miltiorrhiza genome. The temporal-spatial expression pattern of SmABCG1 is consistent with tanshinone accumulation profiles. SmABCG1 is located on the plasma membrane and preferentially accumulates in the peridermal cells of S. miltiorrhiza roots. Heterologous expression in Xenopus laevis oocytes demonstrated that SmABCG1 can export tanshinone ⅡA and tanshinone Ⅰ. CRISPR/Cas9-mediated mutagenesis of SmABCG1 in S. miltiorrhiza hairy roots resulted in a significant decrease in tanshinone contents in both hairy roots and the culture medium, whereas overexpression of this gene resulted in increased tanshinone contents. CYP76AH3 transcript levels increased in hairy roots overexpressing SmABCG1 and decreased in knockout lines, suggesting that SmABCG1 may affect the expression of CYP76AH3, indirectly regulating tanshinone biosynthesis. Finally, tanshinone ⅡA showed cytotoxicity to Arabidopsis roots. These findings offer new perspectives on plant diterpenoid transport and provide a new genetic tool for metabolic engineering and synthetic biology research.

Description of the first marine-isolated member of the under-represented phylum Gemmatimonadota, and the environmental distribution and ecogenomics of Gaopeijiales ord. nov.

The phylum Gemmatimonadota is widespread but rarely cultured and, in fact, there are only six described species isolated from soil, freshwater, and wastewater treatment. However, no isolates of Gemmatimonadota from marine environment have been described; thus, little is known about the physiology and metabolism of members of the marine lineages. In this study, four novel facultatively anaerobic bacterial strains belonging to Gemmatimonadota were isolated from marine sediments collected from Xiaoshi Island in Weihai, China, using an aerobic enrichment method. The integrated results of phylogenetic and phenotypic characteristics supported that these four strains represent one novel species in a novel genus, for which the name Gaopeijia maritima gen. nov., sp. nov. is proposed, as the first representative of novel taxa, Gaopeijiales ord. nov., Gaopeijiaceae fam. nov. in the class Longimicrobiia. Gaopeijiales was detected in 22,884 out of 95,549 amplicon data sets, mainly from soil. However, the highest mean relative abundances were in sponge (0.7%) and marine sediment (0.35%), showing salt-related character. Most of the Gaopeijiales subgroups potentially belong to the rare bacterial biosphere. The aerobic enrichment in this study could significantly increase the relative abundance of Gaopeijiales (from 0.37% to 2.6%). Furthermore, the metabolic capabilities inferred from high-quality representative Gaopeijiales genomes/MAGs suggest that this group primarily performs chemoorganoheterotrophic metabolism with facultatively anaerobic characteristics and possesses various secondary metabolite biosynthesis gene clusters (BGCs), mirroring those observed in the four novel strains.IMPORTANCEDespite rapid advances in molecular and sequencing technologies, obtaining pure cultures remains a crucial research goal in microbiology, as it is essential for a deeper understanding of microbial metabolism. Gemmatimonadota is a widespread but rarely cultured bacterial phylum. Currently, there are only six cultured strains of this interesting group, all isolated from non-marine environments. Little is known about the physiology and metabolism of members of the marine lineages. Here we isolated and characterized four novel marine strains, and proposed a new order Gaopeijiales within Gemmatimonadota. Furthermore, the global distribution, environmental preference, and metabolic potential of Gaopeijiales are analyzed using public data. Our work enriches the resources available for the under-represented phylum Gemmatimonadota and provides insights into the physiological and metabolic characteristics of the marine lineage (Gaopeijiales) through culturology and omics.

Capsular polysaccharide structure of Acinetobacter baumannii K58 from clinical isolate MRSN31468

Capsular polysaccharides (CPS) of Acinetobacter baumannii is a virulence factor with diverse structures. CPS are produced by the CPS biosynthesis gene cluster in their K locus (KL). However, CPS variations may occur due to insertion of additional genes from external sources, e.g., prophages. Recently, the CPS structure from a clinical isolate, BAL062 which includes KL58 locus, was found to have a pseudaminic acid isomer (8ePse5NAc7NAc) as a result of prophage inserted epaA/epaB genes. Here, we report a CPS structure produced by A. baumannii strain MRSN31468 which also belongs to a KL58 type. The K58 CPS structure was determined by 1D and 2D NMR analysis of the oligosaccharides derived from the CPS by a phage depolymerase, and supported by the sugar composition analysis. The K58 CPS structure has the following tetra saccharide repeating unit. [Display omitted] The K58 CPS differs from the CPS from BAL062 only by replacing 8-epimerized β-8ePse5NAc7NAc with β-Pse5NAc7NAc.

Insights into the Genome of a New Strain Serratia rubidaea XU1 Isolated from Radioactive Soil and its Prodigiosin Production and Antimicrobial Properties.

The genus Serratia is a typical red bacterium involved in prodigiosin synthesis. Here, we report the genome sequence of Serratia rubidaea XU1, which was isolated from radiation-contaminated soil in Xinjiang, China. The genome of XU1 is composed of 4,972,898 base pairs with a GC content of 59.25%. The genome sequence contains 4707 genes and encodes 4573 proteins, 79 tRNAs, and 17 rRNAs. The prodigiosin biosynthesis gene cluster was identified and analyzed, showing a sequence similarity of 85.55-96.02% with Serratia rubidaea. After optimizing the biosynthesis process, XU1 was able to achieve a maximum titer of 574 units/cell of prodigiosin at a pH of 7.5 and a temperature of 25°C for 36h. Glycerol at 20g/L and beef extract at 5g/L were used as the carbon and nitrogen sources, respectively. Prodigiosin extracted from XU1 demonstrated inhibition of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. The availability of the sequenced genome of XU1 will be greatly beneficial and contribute to complementary studies on the biosynthetic mechanisms of prodigiosin.

Whole-genome sequence of Bacillus subtilis TR6 strain isolated from tomato (Solanum lycopersicum L.) roots.

Here, we announce the sequence of endophyte Bacillus subtilis TR6 strain isolated from tomato roots growing in a greenhouse in St. Petersburg, Russia. With a length of 4,071,622 bp, this strain contained 4,073 potential coding sequences and 10 predicted secondary metabolite biosynthesis gene clusters.

Acquisition and evolution of the neurotoxin domoic acid biosynthesis gene cluster in Pseudo-nitzschia species

Of the hitherto over 60 taxonomically identified species in the genus of Pseudo-nitzschia, 26 have been confirmed to be toxigenic. Nevertheless, the acquisition and evolution of the toxin biosynthesis (dab) genes by this extensive group of Pseudo-nitzschia species remains unclear. Through constructing chromosome-level genomes of three Pseudo-nitzschia species and draft genomes of ten additional Pseudo-nitzschia species, putative genomic integration sites for the dab genes in Pseudo-nitzschia species were explored. A putative breakpoint was observed in syntenic regions in the dab gene cluster-lacking Pseudo-nitzschia species, suggesting potential independent losses of dab genes. The breakpoints between this pair of conserved genes were also identified in some dab genes-possessing Pseudo-nitzschia species, suggesting that the dab gene clusters transposed to other loci after the initial integration. A “single acquisition, multiple independent losses (SAMIL)” model is proposed to explain the acquisition and evolution of the dab gene cluster in Pseudo-nitzschia species.

Genomic analysis of secondary metabolite biosynthesis gene clusters and structural characterization of terpene synthase and cytochrome P450 enzymes in Zingiber officinale Roscoe

This study uses bioinformatics approaches to elucidate the genetic basis of secondary metabolite biosynthesis in Zingiber officinale (Z. officinale). To this end, it identifies 44 secondary metabolite biosynthetic gene clusters and maps onto individual chromosomes, with chromosomes 1A and 8A exhibiting higher concentrations. Here, protein homology modeling provided insights into the structural characteristics of terpene synthases and Cytochrome P450 enzymes, shedding light on their potential roles in stress response and secondary metabolite production. Moreover, the identification of enzymes, such as (-)-kolavenyl diphosphate synthase TPS28 and cytochrome P450 93A3-like, opens up new possibilities for investigating the intricate pathways involved in terpene diversity and stress response mechanisms within Z. officinale. This study highlights the importance of understanding the molecular mechanisms underlying plant-derived bioactive compounds for pharmaceutical applications.

Role of AplaeA in the regulation of spore production and poly(malic acid) synthesis in Aureobasidium pullulans

Poly(malic acid) (PMLA) as one of the important metabolites in Aureobasidium pullulans has great application in a variety of fields. LaeA, a global regulator capable of controlling fungal secondary metabolites, has been identified in A. pullulans. The involvement of LaeA in the biosynthesis of PMLA through direct or indirect means and its role in A. pullulans is currently unknown. Here we extracted and characterized the AplaeA gene from A. pullulans HIT-LCY3T and resolved it by bioinformatics. The function of AplaeA was subsequently characterized by Rnai-LaeA and OEX-LaeA mutants. Based on phenotypic observations of the mutant strains, it was found that the gene had a large effect on the morphology and melanin production aspects of the strain, and that overexpression of the AplaeA gene resulted in a substantial increase in spore numbers. RT-qPCR combined with protein interactions results analyzed that ApLaeA positively regulates PMLA biosynthesis by controlling the expression of core genes of PMLA biosynthesis gene cluster and other related regulatory factors. Finally, OEX-LaeA was used as the starting strain to obtain the optimal fermentation conditions. These findings illustrate the regulatory mechanism of a hypothesized global regulator, LaeA, in A. pullulans HIT-LCY3T, suggesting its potential application in industrial-scale PMLA biosynthesis.

Unlocking the Siderophore Biosynthesis Pathway and Its Biological Functions in the Fungal Insect Pathogen Beauveria bassiana.

Siderophores are small molecule iron chelators. The entomopathogenic fungus Beauveria bassiana produces a plethora of siderophores under iron-limiting conditions. In this study, a siderophore biosynthesis pathway, akin to the general pathway observed in filamentous fungi, was revealed in B. bassiana. Among the siderophore biosynthesis genes (SID), BbSidA was required for the production of most siderophores, and the SidC and SidD biosynthesis gene clusters were indispensable for the production of ferricrocin and fusarinine C, respectively. Biosynthesis genes play various roles in siderophore production, vegetative growth, stress resistance, development, and virulence, in which BbSidA plays the most important role. Accordingly, B. bassiana employs a cocktail of siderophores for iron metabolism, which is essential for fungal physiology and host interactions. This study provides the initial network for the genetic modification of siderophore biosynthesis, which not only aims to improve the efficacy of biocontrol agents but also ensures the efficient production of siderophores.

Nucleic acid demethylase MpAlkB1 regulates the growth, development, and secondary metabolite biosynthesis in Monascus purpureus.

Nucleic acid demethylases of α-ketoglutarate-dependent dioxygenase (AlkB) family can reversibly erase methyl adducts from nucleobases, thus dynamically regulating the methylation status of DNA/RNA and playing critical roles in multiple cellular processes. But little is known about AlkB demethylases in filamentous fungi so far. The present study reports that Monascus purpureus genomes contain a total of five MpAlkB genes. The MpAlkB1 gene was disrupted and complemented through homologous recombination strategy to analyze its biological functions in M. purpureus. MpAlkB1 knockout significantly accelerated the growth of strain, increased biomass, promoted sporulation and cleistothecia development, reduced the content of Monascus pigments (Mps), and strongly inhibited citrinin biosynthesis. The downregulated expression of the global regulator gene LaeA, and genes of Mps biosynthesis gene cluster (BGC) or citrinin BGC in MpAlkB1 disruption strain supported the pleiotropic trait changes caused by MpAlkB1 deletion. These results indicate that MpAlkB1-mediated demethylation of nucleic acid plays important roles in regulating the growth and development, and secondary metabolism in Monascus spp.

Deletions of conserved extracytoplasmic function sigma factors-encoding genes in Streptomyces have a major impact on secondary metabolism

BackgroundEthanol shock significantly affects expression of over 1200 genes in Streptomyces venezuelae NRRL B-65,442, including those involved in secondary metabolite biosynthesis and a cryptic gene pepX, which encodes a 19-amino acid peptide with an unknown function.ResultsTo establish a possible correlation between the PepX peptide and secondary metabolism in S. venezuelae, its gene was deleted, followed by analyses of the transcriptome and secondary metabolome of the mutant. Although the secondary metabolome of the pepX mutant was not strongly affected, pepX deletion, similar to ethanol shock, mostly resulted in downregulated expression of secondary metabolite biosynthesis gene clusters (BGCs). At the same time, there was a reverse correlation between the expression of certain extracytoplasmic function sigma factors (ECFs) and several BGCs. Individual deletions of three selected ECF-coding genes conserved in Streptomyces that were upregulated upon both pepX deletion and ethanol shock, had a profound positive effect on the expression of BGCs, which also correlated with the overproduction of specific secondary metabolites. Deletion of one such ECF-coding gene in a marine sponge-derived Streptomyces sp. also significantly altered the secondary metabolite profile, suggesting an important role of this ECF in the regulation of secondary metabolism.ConclusionsThese findings pave the way for the activation or upregulation of BGCs in Streptomyces bacteria harboring genes for ECFs homologous to those identified in this study, hereby assisting in the discovery of novel bioactive secondary metabolites.

Получение гетерологичных продуцентов рамнолипидов для промышленного производства высокоэффективного биосурфактанта

Rhamnolipids are promising microbial glycolipid biosurfactants that can be used as preparations for soil bioremediation in oil pollution. The expression of the rhamnolipid biosynthesis gene cluster in the main producer, Pseudomonas aeruginosa, is regulated by quorum sensing signals (QS). Complex biosynthesis regulation does not allow achieving high product yields. The production of heterologous rhamnolipid producers is a promising way to overcome the complex QS-dependent regulation of rhamnolipid biosynthesis, and also avoids the use of opportunistic Pseudomonas aeruginosa for the industrial production of rhamnolipids. Heterologous Escherichia coli BLWT-based producers with a transformant E14 productivity of 3.40±0.96 g/L were obtained on the basis of the plasmid vector pAl2–T with a construct from the native promoter and rhlAB genes, and Pseudomonas viridiflava AlC1223-based ones with the productivity of transformants P5, P6, P8 332.00±0.10, 200.00±0.10 and 180.00±0.10 g/L, respectively. The presence of rhlAB genes in the expression vector allows the obtaining monorhamnolipids for further use as an agent for the solubilization of soil hydrophobic pollutant (e. g. petroleum products), which will intensify bioremediation processes.

Metabolome and Genome Analysis of a Novel Endophytic Fungus Aureobasidium pullulans KB3: Discovery of Polyketones and Polyketone Biosynthesis Pathway.

Endophytic fungi associated with plants may contain undiscovered bioactive compounds. Under standard laboratory conditions, most undiscovered compounds are inactive, whereas their production could be stimulated under different cultivation conditions. In this study, six endophytic fungi were isolated from the bark of Koelreuteria paniculata in Quancheng Park, Jinan City, Shandong Province, one of which was identified as a new subspecies of Aureobasidium pullulans, named A. pullulans KB3. Additionally, metabolomic tools were used to screen suitable media for A. pullulans KB3 fermentation, and the results showed that peptone dextrose medium (PDM) was more beneficial to culture A. pullulans KB3 for isolation of novel compounds. Sphaerolone, a polyketone compound, was initially isolated from A. pullulans KB3 via scaled up fermentation utilizing PDM. Additionally, the whole-genome DNA of A. pullulans KB3 was sequenced to facilitate compound isolation and identify the biosynthesis gene clusters (BGCs). This study reports the multi-omics (metabolome and genome) analysis of A. pullulans KB3, laying the foundation for discovering novel compounds of silent BGCs and identifying their biosynthesis pathway.