Systems metabolic engineering of the primary and secondary metabolism of Streptomyces albidoflavus enhances production of the reverse antibiotic nybomycin against multi-resistant Staphylococcus aureus.

Nybomycin is an antibiotic compound with proven activity against multi-resistant Staphylococcus aureus, making it an interesting candidate for combating these globally threatening pathogens. For exploring its potential, sufficient amounts of nybomycin and its derivatives must be synthetized to fully study its effectiveness, safety profile, and clinical applications. As native isolates only accumulate low amounts of the compound, superior producers are needed. The heterologous cell factory S. albidoflavus 4N24, previously derived from the cluster-free chassis S. albidoflavus Del14, produced 860 µg L-1 of nybomycin, mainly in the stationary phase. A first round of strain development modulated expression of genes involved in supply of nybomycin precursors under control of the common Perm* promoter in 4N24, but without any effect. Subsequent studies with mCherry reporter strains revealed that Perm* failed to drive expression during the product synthesis phase but that use of two synthetic promoters (PkasOP* and P41) enabled strong constitutive expression during the entire process. Using PkasOP*, several rounds of metabolic engineering successively streamlined expression of genes involved in the pentose phosphate pathway, the shikimic acid pathway, supply of CoA esters, and nybomycin biosynthesis and export, which more than doubled the nybomycin titer to 1.7 mg L-1 in the sixth-generation strain NYB-6B. In addition, we identified the minimal set of nyb genes needed to synthetize the molecule using single-gene-deletion strains. Subsequently, deletion of the regulator nybW enabled nybomycin production to begin during the growth phase, further boosting the titer and productivity. Based on RNA sequencing along the created strain genealogy, we discovered that the nyb gene cluster was unfavorably downregulated in all advanced producers. This inspired removal of a part and the entire set of the four regulatory genes at the 3'-end nyb of the cluster. The corresponding mutants NYB-8 and NYB-9 exhibited marked further improvement in production, and the deregulated cluster was combined with all beneficial targets from primary metabolism. The best strain, S. albidoflavus NYB-11, accumulated up to 12 mg L-1 nybomycin, fifteenfold more than the basic strain. The absence of native gene clusters in the host and use of a lean minimal medium contributed to a selective production process, providing an important next step toward further development of nybomycin.

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

BACKGROUND: Pamamycins 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. RESULTS: Herein, 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. CONCLUSION: By 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.

Systems biology of industrial oxytetracycline production in Streptomyces rimosus: the secrets of a mutagenized hyperproducer.

BACKGROUND: Oxytetracycline which is derived from Streptomyces rimosus, inhibits a wide range of bacteria and is industrially important. The underlying biosynthetic processes are complex and hinder rational engineering, so industrial manufacturing currently relies on classical mutants for production. While the biochemistry underlying oxytetracycline synthesis is known to involve polyketide synthase, hyperproducing strains of S. rimosus have not been extensively studied, limiting our knowledge on fundamental mechanisms that drive production. RESULTS: In this study, a multiomics analysis of S. rimosus is performed and wild-type and hyperproducing strains are compared. Insights into the metabolic and regulatory networks driving oxytetracycline formation were obtained. The overproducer exhibited increased acetyl-CoA and malonyl CoA supply, upregulated oxytetracycline biosynthesis, reduced competing byproduct formation, and streamlined morphology. These features were used to synthesize bhimamycin, an antibiotic, and a novel microbial chassis strain was created. A cluster deletion derivative showed enhanced bhimamycin production. CONCLUSIONS: This study suggests that the precursor supply should be globally increased to further increase the expression of the oxytetracycline cluster while maintaining the natural cluster sequence. The mutagenized hyperproducer S. rimosus HP126 exhibited numerous mutations, including large genomic rearrangements, due to natural genetic instability, and single nucleotide changes. More complex mutations were found than those typically observed in mutagenized bacteria, impacting gene expression, and complicating rational engineering. Overall, the approach revealed key traits influencing oxytetracycline production in S. rimosus, suggesting that similar studies for other antibiotics could uncover general mechanisms to improve production.

Heterologous Production and Biosynthesis of Threonine-16:0dioic acids with a Hydroxamate Moiety.

Dereplication and genome mining in Streptomyces aureus LU18118 combined with heterologous expression of selected biosynthetic gene clusters (BGCs) led to the discovery of various threonine-16:0dioic acids named lipothrenins. Lipothrenins consist of the core elements l-Thr, d-allo-Thr, or Dhb, which are linked to hexadecanedioic acid by an amide bond. The main compound lipothrenin A (1) carries the N-hydroxylated d-allo form of threonine and expresses a siderophore activity. The lipothrenin BGC was analyzed by a series of deletion experiments. As a result, a variety of interesting genes involved in the recruitment and selective activation of linear 16:0dioic acids, amide bond formation, and the epimerization of l-Thr were revealed. Furthermore, a diiron N-oxygenase was identified that may be directly involved in the monooxygenation of the amide bond. This is divergent from the usual hydroxamate formation mechanism in siderophores, which involves hydroxylation of the free amine prior to amide bond formation. Siderophore activity was observed for all N-hydroxylated lipothrenins by application of the CAS assay method.

Properties of Multidrug-Resistant Mutants Derived from Heterologous Expression Chassis Strain Streptomyces albidoflavus J1074.

Streptomyces albidoflavus J1074 is a popular platform to discover novel natural products via the expression of heterologous biosynthetic gene clusters (BGCs). There is keen interest in improving the ability of this platform to overexpress BGCs and, consequently, enable the purification of specialized metabolites. Mutations within gene rpoB for the ß-subunit of RNA polymerase are known to increase rifampicin resistance and augment the metabolic capabilities of streptomycetes. Yet, the effects of rpoB mutations on J1074 remained unstudied, and we decided to address this issue. A target collection of strains that we studied carried spontaneous rpoB mutations introduced in the background of the other drug resistance mutations. The antibiotic resistance spectra, growth, and specialized metabolism of the resulting mutants were interrogated using a set of microbiological and analytical approaches. We isolated 14 different rpoB mutants showing various degrees of rifampicin resistance; one of them (S433W) was isolated for the first time in actinomycetes. The rpoB mutations had a major effect on antibiotic production by J1074, as evident from bioassays and LC-MS data. Our data support the idea that rpoB mutations are useful tools to enhance the ability of J1074 to produce specialized metabolites.

Development of a Streptomyces-based system for facile thioholgamide library generation and analysis.

Derivatizing natural products (NPs) is essential in structure-activity relationship (SAR) studies, compound optimization, and drug development. Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent one of the major classes of natural products. Thioholgamide represents thioamitide - a recently emerged family of RiPPs with unique structures and great potential in anticancer drug development. Although the method for generating the RiPP library by codon substitutions in the precursor peptide gene is straightforward, the techniques to perform RiPP derivatization in Actinobacteria remain limited and time-consuming. Here, we report a facile system for producing a library of randomized thioholgamide derivatives utilizing an optimized Streptomyces host. This technique enabled us to access all possible amino acid substitutions of the thioholgamide molecule, one position at a time. Out of 152 potential derivatives, 85 were successfully detected, revealing the impact of amino acid substitutions on thioholgamide post-translational modifications (PTMs). Moreover, new PTMs were observed among thioholgamide derivatives: thiazoline heterocycles, which have not yet been reported for thioamitides, and S-methylmethionine, which is very rare in nature. The obtained library was subsequently used for thioholgamide SAR studies and stability assays.

Diversity and bioactive potential of Actinomycetia from the rhizosphere soil of Juniperus excelsa.

Microbial natural products are among the main sources of compounds used in the medical biotechnology field for the purpose of drug development. However, as antibiotic resistance in pathogenic microorganisms is known to be increasing dramatically, there exists a need to develop new antibiotics. Actinomycetia have proven to be a good source of biologically active compounds, although the rediscovery of previously known compounds significantly slows down the introduction of new antibiotics. As a consequence, increasing attention is being paid to the isolation of actinomycete strains from previously unexplored sources, which can significantly increase the likelihood of discovering new biologically active compounds. This study investigated the diversity and bioactive potential of 372 actinomycete strains isolated from the rhizosphere soil of Juniperus excelsa M. Bieb. The examined actinomycete strains belonged to 11 genera, namely, Actinoplanes, Actinorectispora, Amycolatopsis, Kribbella, Micrococcus, Micromonospora, Nocardia, Promicromonospora, Rhodococcus, Saccharopolyspora and Streptomyces. The bioactive potential of each isolated actinomycete strain was determined on the basis of its ability to produce antimicrobial metabolites against Gram-positive and Gram-negative bacteria and yeast. Some 159 strains (42.74%) exhibited antimicrobial activity against at least one of the tested microbial strains. The dereplication analysis of the extract of the Streptomyces sp. Je 1-651 strain, which exhibited strong antimicrobial activity, led to the annotation of spiramycins and stambomycins. Moreover, the phylogenetic analysis based on the 16S rRNA gene sequence of the Je 1-651 strain revealed it to be close to the S. ambofaciens.

Mutations within gene XNR_2147 for TetR-like protein enhance lincomycin resistance and endogenous specialized metabolism of Streptomyces albus J1074.

Streptomyces albus J1074 is one of the most popular heterologous expression platforms among streptomycetes. Identification of new genes and mutations that influence specialized metabolism in this species is therefore of great applied interest. Here, we describe S. albus KO-1304 that was isolated as a spontaneous lincomycin-resistant variant of double rpsLR94G rsmGR15SG40E mutant KO-1295. Besides altered antibiotic resistance profile, KO-1304 exhibited increased antibiotic activity as compared to its parental strains. KO-1304 genome sequencing revealed mutations within gene XNR_2147 encoding putative TetR-like protein. Gene XNR_2146 for efflux protein is the most likely target of repressing action of Xnr_2147. Our data agree with the scenario where lincomycin resistance phenotype of KO-1304 arose from inability of mutated Xnr_2147 protein to repress XNR_2146. Introduction of additional copy of XNR_2146 into wild type strain increased antibiotic activity of the latter, attesting to the practical value of transporter genes for strain improvement.

Flavacol and Its Novel Derivative 3-ß-Hydroxy Flavacol from Streptomyces sp. Pv 4-95 after the Expression of Heterologous AdpA.

Actinomycetes are one of the main producers of biologically active compounds. However, their capabilities have not been fully evaluated due to the presence of many unexpressed silent clusters; moreover, actinomycetes can probably produce new or previously discovered natural products under certain conditions. Overexpressing the adpA gene into streptomycetes strains can unlock silent biosynthetic gene clusters. Herein, we showed that by applying this approach to Streptomyces sp. Pv 4-95 isolated from Phyllostachys viridiglaucescens rhizosphere soil, two new mass peaks were identified. NMR structure analysis identified these compounds as flavacol and a new 3-ß-hydroxy flavacol derivative. We suggest that the presence of heterologous AdpA has no direct effect on the synthesis of flavacol and its derivatives in the Pv 4-95 strain. However, AdpA affects the synthesis of precursors by increasing their quantity, which then condenses into the resulting compounds.

Furaquinocins K and L: Novel Naphthoquinone-Based Meroterpenoids from Streptomyces sp. Je 1-369.

Actinomycetes are the most prominent group of microorganisms that produce biologically active compounds. Among them, special attention is focused on bacteria in the genus Streptomyces. Streptomycetes are an important source of biologically active natural compounds that could be considered therapeutic agents. In this study, we described the identification, purification, and structure elucidation of two new naphthoquinone-based meroterpenoids, furaquinocins K and L, from Streptomyces sp. Je 1-369 strain, which was isolated from the rhizosphere soil of Juniperus excelsa (Bieb.). The main difference between furaquinocins K and L and the described furaquinocins was a modification in the polyketide naphthoquinone skeleton. In addition, the structure of furaquinocin L contained an acetylhydrazone fragment, which is quite rare for natural compounds. We also identified a furaquinocin biosynthetic gene cluster in the Je 1-369 strain, which showed similarity (60%) with the furaquinocin B biosynthetic gene cluster from Streptomyces sp. KO-3988. Furaquinocin L showed activity against Gram-positive bacteria without cytotoxic effects.

Screening of Thiopeptide-Producing Streptomycetes Isolated From the Rhizosphere Soil of Juniperus excelsa.

The identification of an increasing number of drug-resistant pathogens has stimulated the development of new therapeutic agents to combat them. Microbial natural products are among the most important elements when it comes to drug discovery. Today, thiopeptide antibiotics are receiving increasing research attention due to their potent activity against Gram-positive bacteria. In this study, we demonstrated the successful use of a whole-cell microbial biosensor (Streptomyces lividans TK24 pMO16) for the specific detection of thiopeptide antibiotics among the native actinomycete strains isolated from the rhizosphere soil of Juniperus excelsa (Bieb.). Among the native strains, two strains of Streptomyces, namely sp. Je 1-79 and Je 1-613, were identified that were capable of producing thiopeptide antibiotics. A multilocus sequence analysis of five housekeeping genes (gyrB, atpD, recA, rpoB, and trpB) classified them as representatives of two different species of the genus Streptomyces. The thiopeptide antibiotics berninamycin A and B were identified in the extracts of the two strains by means of a dereplication analysis. The berninamycin biosynthetic gene cluster was also detected in the genome of the Streptomyces sp. Je 1-79 strain and showed a high level of similarity (93%) with the ber cluster from S. bernensis. Thus, the use of this whole-cell biosensor during the first stage of the screening process could serve to accelerate the specific detection of thiopeptide antibiotics.

Miramides A-D: Identification of Detoxin-like Depsipeptides after Heterologous Expression of a Hybrid NRPS-PKS Gene Cluster from Streptomyces mirabilis Lu17588.

Natural products derived from plants, fungi or bacteria have been used for years in the medicine, agriculture and food industries as they exhibit a variety of beneficial properties, such as antibiotic, antifungal, anticancer, herbicidal and immunosuppressive activities. Compared to synthetic compounds, natural products possess a greater chemical diversity, which is a reason why they are profitable templates for developing pharmaceutical drug candidates and ongoing research on them is inevitable. Performing heterologous expression with unknown gene clusters is the preferred method to activate gene clusters that are not expressed in the wild-type strain under laboratory conditions; thus, this method offers a way to discover new interesting metabolites. Here, we report the gene cluster assembly of a hybrid NRPS-PKS gene cluster from Streptomyces mirabilis Lu17588, which was heterologously expressed in Streptomyces albus Del14. Four new compounds were produced by the obtained strain, which were named miramides A-D. Isolation and structure elucidation revealed similarity of the isolated compounds to the known depsipeptides rimosamides/detoxins.

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

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

Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway.

Thioholgamides are ribosomally synthesized and posttranslationally modified peptides (RiPPs), with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing. Thr dehydration follows thioamidation, and our studies reveal that both these modifications require the formation of protein complexes─ThoH/I and ThoC/D. Harnessing the power of AlphaFold, we deduce that ThoD acts as a lyase and also proposes putative catalytic residues. ThoF catalyzes the oxidative decarboxylation of the terminal Cys, and the subsequent macrocyclization is facilitated by ThoE. This is followed by Ser dehydration, which is also carried out by ThoC/D. ThoG is responsible for histidine bis-N-methylation, which is a prerequisite for His ß-hydroxylation─a modification carried out by ThoJ. The last step of the pathway is the removal of the leader peptide by ThoK to afford mature thioholgamide.

Novel Biosynthetic Route to the Isoquinoline Scaffold.

Isoquinoline alkaloids are a large class of natural products with a broad range of biological activities, including antimicrobial, antitumor, antileukemic and anti-inflammatory properties. Although mostly found in plants, isoquinolines can also be found in the extracts of bacterial and fungal cultures. Regardless of the origin, most of the reported biosynthetic routes for isoquinolines use tyrosine as a main biosynthetic precursor. Here, we report the identification of a new biosynthetic pathway for production of isoquinolinequinone alkaloid mansouramycin D in Streptomyces albus Del14. Using feeding, mass spectrometry, and nuclear magnetic resonance spectroscopy, we demonstrate that tryptophan serves instead of tyrosine as a main mansouramycin biosynthetic precursor. The biosynthetic genes were identified in the chromosome of the strain by using gene inactivation and heterologous expression. Insights into the biosynthesis of mansouramycins are also presented.

Scandium-microorganism interactions in new biotechnologies.

Scandium (Sc) plays a special role in high-tech industries because of its wide application in green, space, and defense technologies. However, Sc mining and purification are problematic due to political, technological, and environmental difficulties. The deficit of this element limits global technological development. One sustainable solution to this problem is to use microorganisms to extract Sc from ore and waste, as well as to concentrate and separate it from other elements. Sc also demonstrates attractive metabolic effects on microbes that is of great interest in white biotechnology. Sc increases the production of proteins and secondary metabolites and activates poorly expressed genes. This review offers a comprehensive analysis of current knowledge on the application of Sc-microorganism interactions in promising biotechnologies, its perspectives, and future challenges.

New Kendomycin Derivative Isolated from Streptomyces sp. Cl 58-27.

In the course of screening new streptomycete strains, the strain Streptomyces sp. Cl 58-27 caught our attention due to its interesting secondary metabolite production profile. Here, we report the isolation and characterization of an ansamycin natural product that belongs structurally to the already known kendomycins. The structure of the new kendomycin E was elucidated using NMR spectroscopy, and the corresponding biosynthetic gene cluster was identified by sequencing the genome of Streptomyces sp. Cl 58-27 and conducting a detailed analysis of secondary metabolism gene clusters using bioinformatic tools.

A Promiscuous Halogenase for the Derivatization of Flavonoids.

Halogenation often improves the bioactive properties of natural products and is used in pharmaceutical research for the generation of new potential drug leads. High regio- and stereospecificity, simple reaction conditions and straightforward downstream processing are the main advantages of halogenation using enzymatic biocatalysts compared to chemical synthetic approaches. The identification of new promiscuous halogenases for the modification of various natural products is of great interest in modern drug discovery. In this paper, we report the identification of a new promiscuous FAD-dependent halogenase, DklH, from Frankia alni ACN14a. The identified halogenase readily modifies various flavonoid compounds, including those with well-studied biological activities. This halogenase has been demonstrated to modify not only flavones and isoflavones, but also flavonols, flavanones and flavanonols. The structural requirements for DklH substrate recognition were determined using a feeding approach. The homology model of DklH and the mechanism of substrate recognition are also proposed in this paper.

New Scabimycins A-C Isolated from Streptomyces acidiscabies (Lu19992).

Peptide natural products displaying a wide range of biological activities have become important drug candidates over the years. Microorganisms have been a powerful source of such bioactive peptides, and Streptomyces have yielded many novel natural products thus far. In an effort to uncover such new, meaningful compounds, the metabolome of Streptomyces acidiscabies was analyzed thoroughly. Three new compounds, scabimycins A-C (1-3), were discovered, and their chemical structures were elucidated by NMR spectroscopy. The relative and absolute configurations were determined using ROESY NMR experiments and advanced Marfey's method.

Composition of nutrient media and temperature of cultivation imposes effect on the content of secondary metabolites of Nocardiopsis sp. isolated from a Siberian Cave.

Growth of human population leads to many global and medical problems. The problems include the crisis of health, antibiotic resistance, drug discovery, etc. Increasing antimicrobial resistance of microorganisms results in the need to screen natural products (incl. antibiotics and antimicrobial peptides) and their producers in different ecological niches. The purpose of this study was to estimate antibiotic activity and biotechnological potential of rare actinobacteria Nocardiopsis sp. The strain was isolated from Okhotnichya cave located in Siberia. Here, we cultivated the strain at 3 temperature modes (13 °C, 28 °C, 37 °C) in 11 liquid nutrient (rich and poor) media. Using modern assays of liquid chromatography and high-resolution mass spectrometry, we estimated the content and number of produced natural products, distribution of their masses, and potential rate of novel secondary metabolites. We demonstrated that minimal nutrient media with l-asparagine and SM25 media with malt extract were less productive at current experimental parameters. As it was shown, this strain was characterized by antibiotic properties against Bacillus subtilis when cultivated at 28 °C. Also, weak antibiotic activity of crude extracts was found in strain cultivation at 13 °C. Also, we detected a high number of novel amphiphilic and hydrophobic NPs produced by this strain. We demonstrated both the influence of the nutrient media composition and cultivation temperature on biosynthetic capabilities of rare strain Nocardiopsis sp. Finally, high level of natural products that were predicted as novel confirms high biotechnological value of rare genera of Actinobacteria that could be explained by the evolution of microorganisms in the isolated environment of cave ecosystem.