Streptomyces Albus Research Articles

Heterologous Expression of Type II PKS Gene Cluster Leads to Diversified Angucyclines in Streptomyces albus J1074

Heterologous expression has emerged as an effective strategy in activating Streptomyces cryptic gene clusters or improving yield. Eight compounds were successfully obtained by heterologous expression of the type II PKS gene cluster spi derived from marine Streptomyces sp. HDN155000 in the chassis host Streptomyces albus J1074. The structures with absolute configurations were elucidated using extensive MS and NMR spectroscopic methods, as well as theoretical NMR calculations and electronic circular dichroism (ECD) calculations. Interestingly, compound WS009 Z (2) contains a rare thiomethyl group, angumycinone T (4) has a novel oxo-bridge formed between C12a and C4, and angumycinone X (3) showed cytotoxicity toward K562 and NCI-H446/EP cell lines.

Innovative synthesis and molecular modeling of actinomycetes-derived silver nanoparticles for biomedical applications

The rising demand for innovative antimicrobial solutions has shifted focus towards silver nanoparticles (AgNPs), especially those produced through eco-friendly methods. This study introduces a novel approach utilizing actinomycetes strains—Streptomyces albus, Micromonospora maris, and Arthrobacter crystallopoietes—to biosynthesize AgNPs with remarkable antibacterial properties. Through molecular characterization, we identified unique features of these nanoparticles, and computational modeling suggested significant ion-ligand interactions with proteins 6REV and 3K07. Our research highlights the promise of these biogenically synthesized nanoparticles in advancing biomedical applications. Actinomycetes were sourced and screened for their ability to produce metallic nanoparticles, revealing that among 35 samples, only six showed this capability. Notably, Streptomyces albus strain smmdk14 (OR685674), Micromonospora maris strain smmdk13 (OR685672), and Arthrobacter crystallopoietes strain smmdk12 (OR685674) were identified as effective silver nanoparticle producers. The synthesized nanoparticles demonstrated potent antibacterial activity against common pathogens including E. coli, Pseudomonas aeruginosa, Klebsiella spp., Enterococcus faecalis, Staphylococcus aureus, and Acinetobacter spp. The data obtained from color change observation, UV–visible spectrophotometry, Zeta potential, FTIR spectroscopy, and transmission electron microscopy (TEM) characterized AgNPs potentiality. The nanoparticles were spherical, with sizes ranging from 6.46 nm to 24.7 nm. Optimization of production conditions, comparison of antimicrobial effects with antibiotics, evaluation of potential toxicity, and assessment of wound-healing capabilities were also conducted. The biosynthesized AgNPs exhibited superior antibacterial properties compared to traditional antibiotics and significantly accelerated wound healing by approximately 66.4 % in fibroblast cell cultures. Additionally, computational analysis predicted interactions between various metal ions and specific amino acid residues in proteins 6REV and 3K07. Overall, this study demonstrates the successful creation of AgNPs with notable antibacterial and wound-healing properties, underscoring their potential for medical applications.

Effective Synthesis of C20-epi-Isothiocyanato-Salinomycin and Its Thiourea Derivatives as Potential Anticancer Agents.

Salinomycin, a naturally occurring polyether ionophore antibiotic isolated from Streptomyces albus, has been demonstrated potent cytotoxic activity against a variety of cancer cell lines. In particular, it exhibits selective targeting of cancer stem cells. However, systemic toxicity, drug resistance and low bioavailability of the drug significantly limit its potential applications. In this study, the C20-epi-isothiocyanate of salinomycin was designed and synthesized, and then reacted with amines as a versatile synthon to assemble a series of salinomycin thiourea derivatives, which improved the druggability of salinomycin. The antiproliferative activities of the compounds were evaluated in vitro against A549, HepG2, Hela, 4T1, and MCF-7 cancer cell lines using the CCK-8 assay. The pharmacological results showed that some salinomycin thiourea derivatives exhibited excellent inhibitory activity against at least one of the tested tumor cells and high selectivity. Further mechanistic studies showed that compound 9f, containing a 3,5-difluorobenzyl moiety, could directly induce apoptosis, probably by increasing caspase-9 protein expression and cell cycle arrest in G1 phase in a concentration dependent manner.

Albusamides A-G: Hydroxylated Fatty Amine Derivatives from the Deep-Sea-Derived Actinomycete Streptomyces albus 228DD-066 and Their Cytotoxic Activity.

A series of new hydroxylated fatty amine derivatives, albusamides A-G (1-7), along with four known compounds (8-11), which are reported for the first time from a natural source, were isolated from the culture broth of Streptomyces albus 228DD-066 derived from a deep-sea sediment sample gathered off the coast of Dokdo Island, Republic of Korea. Their structures were elucidated through the comprehensive analysis of 1D and 2D NMR spectra and HRESIMS, and absolute configurations were determined using the modified Mosher's method. Biological evaluations against solid and blood cancer cell lines revealed that these new metabolites have moderate to strong cytotoxic activity. Compound 3 exhibited high cytotoxic activity with GI50 values ranging from 0.4 to 0.6 μM against solid cancer cell lines and exhibited the strongest cytotoxicity (GI50 value = 0.2 μM) against the WSU-DLCL2 blood cancer cell line.

Engineering the TetR-family transcriptional regulator XNR_0706 to enhance heterologous spinosad production in Streptomyces albus B4 chassis

The TetR family of regulators are an important group of transcription regulators that regulate diverse cellular processes in prokaryotes. In this study, we found that XNR_0706, a TetR family regulator, controlled the expression of XNR_0345, XNR_0454, XNR_0513 and XNR_1438 putatively involved in fatty acid β-oxidation by interacting with the promoter regions in Streptomyces albus B4. The transcription level of these four genes was downregulated in XNR_0706 deletion strain (ΔXNR_0706) and restored by XNR_0706 complementation in Δ0706/pIB-0706, demonstrating that XNR_0706 was a positive transcriptional regulator of the genes. With toxic long-chain fatty acids addition in TSB media, deletion of XNR_0706 caused significantly poor growth, whereas XNR_0706 complementation increased the utilization of additional fatty acids, resulting in restored growth. Fatty acid β-oxidation is one source of acetyl- and malonyl-CoA precursors for polyketides biosynthesis in actinobacteria. Overexpression of XNR_0706 in B4/spnNEW, a spinosad heterologous expression strain derived from S. albus B4, increased spinosad yield by 20.6%. Additionally, supplement of 0.3 g/L fatty acids resulted in a further 42.4% increase in spinosad yield. Our study reveals a regulatory mechanism in long-chain fatty acids metabolism in S. albus and these insights into the molecular regulation of β-oxidation by XNR_0706 are instrumental for increasing secondary metabolites in actinobacteria.

Generation of lysolipin derivatives by genetic engineering

Lysolipin I is a halogenated, polycyclic xanthone natural product belonging to the polyketide class of antibiotics, naturally produced by Streptomyces violaceoniger TÜ96 and Streptomyces tendae TÜ4042. The biosynthesis is encoded on a 43 kb-spanning biosynthetic gene cluster. Heterologous expression of the gene cluster has been established in previous work by using the cosmid 4H04, which was transferred to Streptomyces albus. In the current study, we demonstrate the optimization of production yields of therapeutically interesting lysolipin derivatives with extended activity against Gram-negatives and less cytotoxic bioactivities, respectively, by using mutated heterologous S. albus producer strains. Production yields were significantly increased by adapting cultivation conditions as well as by inactivating the transcriptional repressor gene llpRI, which lead to increased and consistent lysolipin (derivatives) production. Furthermore, cultivation of a S. albus 4H04∆llpOI mutant strain in bromide-containing fermentation medium resulted in the production of a new brominated lysolipin derivative (C28H20BrNO9).

Engineering of a TetR family transcriptional regulator BkdR enhances heterologous spinosad production in Streptomyces albus B4 chassis.

In this study, we describe a pccAB operon involved in short-chain acyl-CoA carboxylation in S. albus B4 chassis. The TetR family regulator, BkdR, represses this operon. Our results show that BkdR regulates the precursor supply needed for heterologous spinosad biosynthesis by controlling acetyl- and propionyl-CoA assimilation. The deletion of the BkdR-encoding gene exerts an increase in heterologous spinosad yield. Our research reveals a regulatory mechanism in short-chain acyl-CoA metabolism and suggests new possibilities for S. albus chassis engineering to enhance heterologous polyketide yield.

Engineering Streptomyces albus B4 for Enhanced Production of (R)-Mellein: A High-Titer Heterologous Biosynthesis Approach.

The natural compound (R)-(-)-mellein exhibits antiseptic and fungicidal activities. We investigated its biosynthesis using the polyketide synthase encoded by SACE_5532 (pks8) from Saccharopolyspora erythraea heterologously expressed in Streptomyces albus B4, a chassis chosen for its fast growth, genetic manipulability, and ample large short-chain acyl-CoA precursor supply. High-level heterologous (R)-(-)-mellein yield was achieved by pks8 overexpression and duplication. The precursor supply pathways were strengthened by overexpression of SACE_0028 (encoding acetyl-CoA carboxylase) and four genes involved in β-oxidation (fadD, fadE, fadB, and fadA). Cell growth inhibition by (R)-(-)-mellein production at high concentration was relieved by in situ adsorption using Amberlite XAD16 resin. The final strain, B4mel12, produced (R)-(-)-mellein at 6395.2 mg/L in shake-flask fermentation. Overall, this is the first report of heterologous (R)-(-)-mellein synthesis in microorganism with a high titer. (R)-(-)-mellein prototype in this study opens a possibility for the overproduction of valuable melleins in S. albus B4.

Discovery, characterization, and engineering of an advantageous Streptomyces host for heterologous expression of natural product biosynthetic gene clusters

BackgroundStreptomyces is renowned for its robust biosynthetic capacity in producing medically relevant natural products. However, the majority of natural products biosynthetic gene clusters (BGCs) either yield low amounts of natural products or remain cryptic under standard laboratory conditions. Various heterologous production hosts have been engineered to address these challenges, and yet the successful activation of BGCs has still been limited. In our search for a valuable addition to the heterologous host panel, we identified the strain Streptomyces sp. A4420, which exhibited rapid initial growth and a high metabolic capacity, prompting further exploration of its potential.ResultsWe engineered a polyketide-focused chassis strain based on Streptomyces sp. A4420 (CH strain) by deleting 9 native polyketide BGCs. The resulting metabolically simplified organism exhibited consistent sporulation and growth, surpassing the performance of most existing Streptomyces based chassis strains in standard liquid growth media. Four distinct polyketide BGCs were chosen and expressed in various heterologous hosts, including the Streptomyces sp. A4420 wild-type and CH strains, alongside Streptomyces coelicolor M1152, Streptomyces lividans TK24, Streptomyces albus J1074, and Streptomyces venezuelae NRRL B-65442. Remarkably, only the Streptomyces sp. A4420 CH strain demonstrated the capability to produce all metabolites under every condition outperforming its parental strain and other tested organisms. To enhance visualization and comparison of the tested strains, we developed a matrix-like analysis involving 15 parameters. This comprehensive analysis unequivocally illustrated the significant potential of the new strain to become a popular heterologous host.ConclusionOur engineered Streptomyces sp. A4420 CH strain exhibits promising attributes for the heterologous expression of natural products with a focus on polyketides, offering an alternative choice in the arsenal of heterologous production strains. As genomics and cloning strategies progress, establishment of a diverse panel of heterologous production hosts will be crucial for expediting the discovery and production of medically relevant natural products derived from Streptomyces.

Heterologous activation and metabolites identification of the pks7 gene cluster from Saccharopolyspora erythraea

The microbial genome remains a huge treasure trove for the discovery of diverse natural products. Saccharopolyspora erythraea NRRL23338, the industry producer of erythromycin, has a dozen of biosynthetic gene clusters whose encoding products are unidentified. Heterologous expression of one of the polyketide clusters pks7 in Streptomyces albus B4 chassis resulted in the characterization of its function responsible for synthesizing both 6-methylsalicyclic acid and 6-ethylsalicyclic acid. Meanwhile, two new 6-ethylsalicyclic acid ester derivatives were isolated as shunt metabolites. Their structures were identified by comprehensive analysis of MS and NMR experiments. Putative functions of genes within the pks7 BGC were also discussed.

Engineered Biosynthesis and Anticancer Studies of Ring-Expanded Antimycin-Type Depsipeptides.

Respirantins are 18-membered antimycin-type depsipeptides produced by Streptomyces sp. and Kitasatospora sp. These compounds have shown extraordinary anticancer activities against a panel of cancer cell lines with nanomolar levels of IC50 values. However, further investigation has been impeded by the low titers of the natural producers and the challenging chemical synthesis due to their structural complexity. The biosynthetic gene cluster (BGC) of respirantin was previously proposed based on a bioinformatic comparison of the four members of antimycin-type depsipeptides. In this study, we report the first successful reconstitution of respirantin in Streptomyces albus using a synthetic BGC. This heterologous system serves as an accessible platform for the production and diversification of respirantins. Through polyketide synthase pathway engineering, biocatalysis, and chemical derivatization, we generated nine respirantin compounds, including six new derivatives. Cytotoxicity screening against human MCF-7 and Hela cancer cell lines revealed a unique biphasic dose-response profile of respirantin. Furthermore, a structure-activity relationship study has elucidated the essential functional groups that contribute to its remarkable cytotoxicity. This work paves the way for respirantin-based anticancer drug discovery and development.

Antibacterial activity of Nocardia spp. and Streptomyces sp. on multidrug-resistant pathogens causing neonatal sepsis.

Neonatal sepsis leads to severe morbidity and occasionally death among neonates within the first week following birth, particularly in low- and middle-income countries. Empirical therapy includes antibiotics recommended by WHO. However, these have been ineffective against antimicrobial multidrug-resistant bacterial strains such as Klebsiella spp, Escherichia coli, and Staphylococcus aureus species. To counter this problem, new molecules and alternative sources of compounds with antibacterial activity are sought as options. Actinobacteria, particularly pathogenic strains, have revealed a biotechnological potential still underexplored. This study aimed to determine the presence of biosynthetic gene clusters and the antimicrobial activity of actinobacterial strains isolated from clinical cases against multidrug-resistant bacteria implicated in neonatal sepsis. In total, 15 strains isolated from clinical cases of actinomycetoma were used. PCR screening for the PKS-I, PKS-II, NRPS-I, and NRPS-II biosynthetic systems determined their secondary metabolite-producing potential. The strains were subsequently assayed for antimicrobial activity by the perpendicular cross streak method against Escherichia fergusonii Sec 23, Klebsiella pneumoniae subsp. pneumoniae H1064, Klebsiella variicola H776, Klebsiella oxytoca H793, and Klebsiella pneumoniae subsp. ozaenae H7595, previously classified as multidrug-resistant. Finally, the strains were identified by 16S rRNA gene sequence analysis. It was found that 100% of the actinobacteria had biosynthetic systems. The most frequent biosynthetic system was NRPS-I (100%), and the most frequent combination was NRPS-I and PKS-II (27%). All 15 strains showed antimicrobial activity. The strain with the highest antimicrobial activity was Streptomyces albus 94.1572, as it inhibited the growth of the five multidrug-resistant bacteria evaluated.

Development of a bacterial gene transcription activating strategy based on transcriptional activator positive feedback

IntroductionTranscription of biological nitrogen fixation (nif) genes is activated by the NifA protein which recognizes specific activating sequences upstream of σ54-dependent nif promoters. The large quantities of nitrogenase which can make up 20% of the total proteins in the cell indicates high transcription activating efficiency of NifA and high transcription level of nifHDK nitrogenase genes. ObjectivesDevelopment of an efficient gene transcription activating strategy in bacteria based on positive transcription regulatory proteins and their regulating DNA sequences. MethodsWe designed a highly efficient gene transcription activating strategy in which the nifA gene was placed directly downstream of its regulating sequences. The NifA protein binds its regulating sequences and stimulates transcription of itself and downstream genes. Overexpressed NifA causes transcription activation by positive reinforcement. ResultsWhen this gene transcription activating strategy was used to overexpress NifA in Pseudomonas stutzeri DSM4166 containing the nif gene cluster, the nitrogenase activity was increased by 368 folds which was 16 times higher than that obtained by nifA driven by the strongest endogenous constitutive promoter. When this strategy was used to activate transcription of exogenous biosynthetic genes for the plant auxin indole-3-acetic acid and the antitumor alkaloid pigment prodigiosin in DSM4166, both of them resulted in better performance than the strongest endogenous constitutive promoter and the highest reported productions in heterologous hosts to date. Finally, we demonstrated the universality of this strategy using the positive transcriptional regulator of the psp operon, PspF, in E. coli and the pathway-specific positive transcription regulator of the polyene antibiotic salinomycin biosynthesis, SlnR, in Streptomyces albus. ConclusionMany positive transcription regulatory proteins and their regulating DNA sequences have been identified in bacteria. The gene transcription activating strategy developed in this study will have broad applications in molecular biology and biotechnology.

Discovery and overproduction of novel highly bioactive pamamycins through transcriptional engineering of the biosynthetic gene cluster

BackgroundPamamycins are a family of highly bioactive macrodiolide polyketides produced by Streptomyces alboniger as a complex mixture of derivatives with molecular weights ranging from 579 to 705 Daltons. The large derivatives are produced as a minor fraction, which has prevented their isolation and thus studies of chemical and biological properties.ResultsHerein, we describe the transcriptional engineering of the pamamycin biosynthetic gene cluster (pam BGC), which resulted in the shift in production profile toward high molecular weight derivatives. The pam BGC library was constructed by inserting randomized promoter sequences in front of key biosynthetic operons. The library was expressed in Streptomyces albus strain with improved resistance to pamamycins to overcome sensitivity-related host limitations. Clones with modified pamamycin profiles were selected and the properties of engineered pam BGC were studied in detail. The production level and composition of the mixture of pamamycins was found to depend on balance in expression of the corresponding biosynthetic genes. This approach enabled the isolation of known pamamycins and the discovery of three novel derivatives with molecular weights of 663 Da and higher. One of them, homopamamycin 677A, is the largest described representative of this family of natural products with an elucidated structure. The new pamamycin 663A shows extraordinary activity (IC50 2 nM) against hepatocyte cancer cells as well as strong activity (in the one-digit micromolar range) against a range of Gram-positive pathogenic bacteria.ConclusionBy employing transcriptional gene cluster refactoring, we not only enhanced the production of known pamamycins but also discovered novel derivatives exhibiting promising biological activities. This approach has the potential for broader application in various biosynthetic gene clusters, creating a sustainable supply and discovery platform for bioactive natural products.

Larvicidal potential, toxicological assessment, and molecular docking studies of four Egyptian bacterial strains against Culex pipiens L. (Diptera: Culicidae)

Mosquito control in Egypt depends on applying chemical synthetic pesticides that impact negatively on human health and the environment as well as the development of antibiotic and chemical resistance. This study aims to control the 3rd and 4th instars of Culex pipiens larvae using four bacterial strains. According to Phenotypic and molecular identification, the four isolates were identified as Bacillus subtilis MICUL D2023, Serratia marcescens MICUL A2023, Streptomyces albus LARVICID, and Pseudomonas fluorescens MICUL B2023. All strains were deposited in GenBank under accession numbers OQ764791, OQ729954, OQ726575, and OQ891356, respectively. Larvicidal activity of all microbial strain metabolites against a field strain of C. pipiens explored low LC50 results and reached its lowest values on the 3rd day with values of 6.40%, 38.4%, and 46.33% for P. fluorescens, S. albus, and S. marcescens, respectively. In addition, metabolites of P. fluorescence were more toxic than those of S. albus, followed by S. marcescens. B. subtilis shows no larvicidal effect on both field and lab mosquito strains. Microscopic alterations of 3rd and 4th instars showed toxic effects on different body parts (thorax, midgut, and anal gills), including losing external hairs, abdominal breakage, and larvae shrinkage, as well as different histological malformations in the digestive tract, midgut, and cortex. GC–MS analysis detected 51, 30, and 32 different active compounds from S. albus, S. marcescens, and P. fluorescens, respectively. GC detected 1, 2-BENZEA2:A52NEDICARBOXYLIC ACID, 2-Cyclohexene-1-carboxylic-acid-5-2-butenyl-methyl ester, and 3 octadecahydro2R3S4Z9Z-11R-12S from S. albus, S. marcesens, and P. fluorescens, respectively. Total protein, Total carbohydrate, and Acetylcholine esterase activity indicated significantly low levels on the 3rd day. All strain metabolites were safe against HSF cell lines. The docking results confirmed the role of the produced metabolites as larvicidal agents and Acetylcholine esterase inhibition. Such a problem need more studies on applying more and more natural pesticides.

Metabolic and genomic analysis deciphering biocontrol potential of endophytic Streptomyces albus RC2 against crop pathogenic fungi.

Plant diseases caused by phytopathogenic fungi are one of the leading factors affecting crop loss. In the present study, sixty-one Streptomyces strains were screened for their antifungal activity against relevant wide range fungal pathogens prominent in Vietnam, namely Lasiodiplodia theobromae, Fusarium fujikuroi, and Scopulariopsis gossypii. Endophytic strain RC2 was the most effective strain in the mycelial inhibition of the tested fungi. Based on phenotypic characteristics, 16S rDNA gene analysis, and genomic analysis, strain RC2 belonged to Streptomyces albus. An ethyl acetate extract of S. albus RC2 led to the strong growth inhibition of S. gossypii Co1 and F. fujikuroi L3, but not L. theobromae N13. The crude extract also suppressed the spore germination of S. gossypii Co1 and F. fujikuroi L3 to 92.4 ± 3.2% and 87.4% ± 1.9%, respectively. In addition, the RC2 extract displayed potent and broad-spectrum antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, and the phytopathogenic bacteria Ralstonia solanacearum and Xanthomonas oryzae. The genome of strain RC2 was sequenced and revealed the presence of 15 biosynthetic gene clusters (BGCs) with similarities ≥ 45% to reference BGCs available in the antiSMASH database. The UPLC-HRMS analysis led to the identification of 8 other secondary metabolites, which have not been reported in S. albus. The present study indicated that RC2 could be a potent biocontrol agent against phytopathogenic fungi. Further attention should be paid to antifungal metabolites without functional annotation, development of product prototypes, and greenhouse experiments to demonstrate effective control of the plant diseases.

A review on Industrially important metabolite from Actinomycetes

Microbial biotic compounds such as enzymes, pigments, antibiotics, and antimicrobial chemicals play an important function as metabolites, resulting in a wide range of applications and uses in a variety of sectors. Nowadays, the entire world is migrating away from synthetic and chemical products in favor of natural ones. Consistent study for novel microbial metabolites that could be employed in industrial processes, thereby driving profit growth in a variety of businesses. Actinomycetes are a major microbial population found in soil, plant tissues, fresh water, and marine settings. Many useful extracellular enzymes, pigments, antibiotics, and antibacterial compounds are produced by actinomycetes. Enzymes like cellulases from Streptomyces albus; pigments like Violacein from Streptomyces violaceus; antibiotics like Tetracycline from Streptomyces aureofaciens, produced by Actinomycetes and applied in different industries. Actinomycetes found in severe conditions are known to create novel bioactive compounds with high industrial potential. This article attempts to summarize Actinomycetes' ability to create bioactive secondary metabolites from actinomycetes and its applications in various industries.

4-Methoxy-2,2′-bipyrrole-5-carbaldehyde, a biosynthetic intermediate of bipyrrole-containing natural products from the Streptomyces culture, arrests the strobilation of moon jellyfish Aurelia coerulea

Streptomyces spp. are well-known producers of secondary metabolites with diverse biological activities. We screened the substances that regulate polyp-to-jellyfish transition, called strobilation, of the moon jellyfish (Aurelia coerulea) from the Streptomyces culture library. Among the culture extracts of the strains tested, Streptomyces albus HUT6047 inhibited the strobilation of A. coerulea. The active component in strain HUT6047 was purified. Based on structure elucidation, this component was identified as 4-methoxy-2,2′-bipyrrole-5-carbaldehyde (MBC), a possible common biosynthetic intermediate of pyrrole-containing natural products including prodigiosins and tambjamines. Synthetic MBC arrested strobilation without inducing cytotoxicity and generated abnormal tentacle-like structures in a dose-dependent manner. Synthetic MBC also exhibited a minimum activity of 6.3 µM. To our knowledge, this study provides the first example of a biological activity of MBC.

Antimicrobial and Cytotoxic Characteristics of Antibiotic Streptofungin

Background. Streptomycetes were and remain a source of new antimicrobial compounds of various nature. A new wave of interest in such research is associated with the possibilities of applied genomics to reveal the hidden biosynthetic potential of streptomycetes, and therefore the discovery of new practically valuable antimicrobial products. The problem of antibiotic resistance of pathogens can be solved by creating compositions of active substances of different nature to overcome the protective mechanisms of pathogens.
 Objective. The purpose of the work was to establish and evaluate the antimicrobial and cytotoxic characteristics of the new antibiotic streptofungin, synthesized by Streptomyces albus 2435 (CMIM-S-668) and its mutant strains 2435/М, UN44, 4S, US101, AE6, 105, 80/5.
 Methods. To establish the characteristics of streptofungin, the antagonistic activity of selected S. albus producer strains was determined (by the radial streak method), the minimum inhibitory concentration of the antibiotic (by the serial dilution method), and cytotoxicity was determined by the MTT test with resazurin.
 Results. The activity of the antibiotic streptofungin against Candida fungi (C. albicans, C. utilis) is shown. Minimum inhibitory concentrations of streptofungin were determined for C. albicans ATCC 10231 (10 μg/ml), B. subtilis ATCC 6633 (200 μg/ml) and P. aeruginosa ATCC 9027 (500 μg/ml). According to the resazurin test, streptofungin does not show a cytotoxic effect in a wide range of concentrations from 2.5 to 500 μg/ml, and therefore can be considered potentially permissible for humans and animals in the studied concentrations.
 Conclusions. The antagonistic activity of mutant strains of S. albus culture is due to the action of a complex of antimicrobial products that have a different antimicrobial spectrum and mechanism of action. The obtained results give reasons to consider streptofungin as a promising pharmaceutical substance with antifungal action, as well as to consider the possibility of its combination with the bacteriolytic enzyme complex of the same culture for the development of an antimicrobial agent with a wide spectrum of action.

High-titer production of staurosporine by heterologous expression and process optimization.

Staurosporine is the most well-known member of the indolocarbazole alkaloid family; it can induce apoptosis of many types of cells as a strong protein kinase inhibitor, and is used as an important lead compound for the synthesis of the antitumor drugs. However, the low fermentation level of the native producer remains the bottleneck of staurosporine production. Herein, integration of multi-copy biosynthetic gene cluster (BGC) in well characterized heterologous host and optimization of the fermentation process were performed to enable high-level production of staurosporine. First, the 22.5kb staurosporine BGC was captured by CRISPR/Cas9-mediated TAR (transformation-associated recombination) from the native producer (145mg/L), and then introduced into three heterologous hosts Streptomyces avermitilis (ATCC 31267), Streptomyces lividans TK24 and Streptomyces albus J1074 to evaluate the staurosporine production capacity. The highest yield was achieved in S. albus J1074 (750mg/L), which was used for further production improvement. Next, we integrated two additional staurosporine BGCs into the chromosome of strain S-STA via two different attB sites (vwb and TG1), leading to a double increase in the production of staurosporine. And finally, optimization of fermentation process by controlling the pH and glucose feeding could improve the yield of staurosporine to 4568mg/L, which was approximately 30-fold higher than that of the native producer. This is the highest yield ever reported, paving the way for the industrial production of staurosporine. KEYPOINTS: • Streptomyces albus J1074 was the most suitable heterologous host to express the biosynthetic gene cluster of staurosporine. • Amplification of the biosynthetic gene cluster had obvious effect on improving the production of staurosporine. • The highest yield of staurosporine was achieved to 4568mg/L by stepwise increase strategy.