A novel synthetic inhibitor of polyamine utilization in Streptomyces coelicolor.

In this work, we present the first inhibitor of GlnA2Sc, a gamma-glutamylpolyamine synthetase, which allows Streptomyces coelicolor to detoxify high concentrations of polyamines and to utilize them as a carbon or nitrogen source. GlnA2 belongs to the class of glutamine synthetase-like (GS-like) enzymes that catalyze the glutamylation of different nitrogen-containing compounds. Whereas a number of inhibitors for GS are known, none of them are known to inhibit GlnA2. In this work, PPU268, an inhibitor for GlnA2 is presented that is structurally derived from the prototypic GS inhibitor-methionine sulfoximine (MSO). It combines two features: the binding mechanism of MSO and the amine substrate specificity of GlnA2Sc. This inhibitor is a novel compound to block the polyamine utilization in bacteria resulting in the inability to detoxify polyamines. This may offer a possibility to develop novel therapeutic strategies to combat actinobacterial human pathogens that encounter polyamines in the course of the infection processes.

Metabolic engineering of the shikimate pathway in Amycolatopsis strains for optimized glycopeptide antibiotic production.

Glycopeptide antibiotics (GPA) consist of a glycosylated heptapeptide backbone enriched in aromatic residues originating from the shikimate pathway. Since the enzymatic reactions within the shikimate pathway are highly feedback-regulated, this raises the question as to how GPA producers control the delivery of precursors for GPA assembly. We chose Amycolatopsis balhimycina, the producer of balhimycin, as a model strain for analyzing the key enzymes of the shikimate pathway. A. balhimycina contains two copies each of the key enzymes of the shikimate pathway, deoxy-d-arabino-heptulosonate-7-phosphate synthase (Dahp) and prephenate dehydrogenase (Pdh), with one pair (Dahpsec and Pdhsec) encoded within the balhimycin biosynthetic gene cluster and one pair (Dahpprim and Pdhprim) in the core genome. While overexpression of the dahpsec gene resulted in a significant (>4-fold) increase in balhimycin yield, no positive effects were observed after overexpression of the pdhprim or pdhsec genes. Investigation of allosteric enzyme inhibition revealed that cross-regulation between the tyrosine and phenylalanine pathways plays an important role. Tyrosine, a key precursor of GPAs, was found to be a putative activator of prephenate dehydratase (Pdt), which catalyzes the first step reaction from prephenate to phenylalanine in the shikimate pathway. Surprisingly, overexpression of pdt in A. balhimycina led to an increase in antibiotic production in this modified strain. In order to demonstrate that this metabolic engineering approach is generally applicable to GPA producers, we subsequently applied this strategy to Amycolatopsis japonicum and improved the production of ristomycin A, which is used in diagnosis of genetic disorders. Comparison of "cluster-specific" enzymes with the isoenzymes from the primary metabolism's pathway provided insights into the adaptive mechanisms used by producers to ensure adequate precursor supply and GPA yields. These insights further demonstrate the importance of a holistic approach in bioengineering efforts that takes into account not only peptide assembly but also adequate precursor supply.

Optimization of FK-506 production in Streptomyces tsukubaensis by modulation of Crp-mediated regulation.

FK-506 is a potent immunosuppressive macrocyclic polyketide with growing pharmaceutical interest, produced by Streptomyces tsukubaensis. However, due to low levels synthesized by the wild-type strain, biotechnological production of FK-506 is rather limited. Optimization strategies to enhance the productivity of S. tsukubaensis by means of genetic engineering have been established. In this work primarily global regulatory aspects with respect to the FK-506 biosynthesis have been investigated with the focus on the global Crp (cAMP receptor protein) regulator. In expression analyses and protein-DNA interaction studies, the role of Crp during FK-506 biosynthesis was elucidated. Overexpression of Crp resulted in two-fold enhancement of FK-506 production in S. tsukubaensis under laboratory conditions. Further optimizations using fermentors proved that the strategy described in this study can be transferred to industrial scale, presenting a new approach for biotechnological FK-506 production. KEY POINTS: • The role of the global Crp (cAMP receptor protein) regulator for FK-506 biosynthesis in S. tsukubaensis was demonstrated • Crp overexpression in S. tsukubaensis was applied as an optimization strategy to enhance FK-506 and FK-520 production resulting in two-fold yield increase.

A Whole-Cell Assay for Detection of Antibacterial Activity in Actinomycete Culture Supernatants.

Whole-cell antibacterial assays are particularly suitable for fast detection and semi-quantification of bioactivities in extracts or other solutions such as microbial culture supernatants. As Actinomycetales, including the members of the genus Streptomyces, are one of the most potent "suppliers" of antibiotics and other bioactive compounds, there is a strong interest in the development of useful assays enabling early identification of such valuable producers. Furthermore, such assays facilitate the screening of a large collection of clones for the detection of engineered "super-producers" that are essential for industrial manufacturing of the respective product.In this protocol, we describe a whole-cell assay for a fast detection of antimicrobial agents in culture supernatants. As model, Streptomyces fradiae DSM 41546, the producer of the antibiotic tylosin, and the test strain Bacillus subtilis ATCC 6051 are used. Culture supernatants of S. fradiae DSM 41546 and controls are diluted, and their antibiotic activity is tested against B. subtilis ATCC 6051. For the dilutions of tylosin-containing culture supernatant, a clear concentration-dependent growth inhibition effect on B. subtilis ATCC 6051 is demonstrated. In contrast, dilutions of the culture supernatant lacking tylosin do not inhibit the growth of B. subtilis. Moreover, including defined concentrations of tylosin facilitates the semi-quantification of tylosin in the culture supernatants.Our results confirm the applicability of the assay in fast screenings for antimicrobial products in culture supernatants. The protocol can be used as positive control within screening campaigns in the drug discovery field.

Optimization of the precursor supply for an enhanced FK506 production in Streptomyces tsukubaensis.

Tacrolimus (FK506) is a macrolide widely used as immunosuppressant to prevent transplant rejection. Synthetic production of FK506 is not efficient and costly, whereas the biosynthesis of FK506 is complex and the level produced by the wild type strain, Streptomyces tsukubaensis, is very low. We therefore engineered FK506 biosynthesis and the supply of the precursor L-lysine to generate strains with improved FK506 yield. To increase FK506 production, first the intracellular supply of the essential precursor lysine was improved in the native host S. tsukubaensis NRRL 18488 by engineering the lysine biosynthetic pathway. Therefore, a feedback deregulated aspartate kinase AskSt* of S. tsukubaensis was generated by site directed mutagenesis. Whereas overexpression of AskSt* resulted only in a 17% increase in FK506 yield, heterologous overexpression of a feedback deregulated AskCg* from Corynebacterium glutamicum was proven to be more efficient. Combined overexpression of AskCg* and DapASt, showed a strong enhancement of the intracellular lysine pool following increase in the yield by approximately 73% compared to the wild type. Lysine is coverted into the FK506 building block pipecolate by the lysine cyclodeaminase FkbL. Construction of a ∆fkbL mutant led to a complete abolishment of the FK506 production, confirming the indispensability of this enzyme for FK506 production. Chemical complementation of the ∆fkbL mutant by feeding pipecolic acid and genetic complementation with fkbL as well as with other lysine cyclodeaminase genes (pipAf, pipASt, originating from Actinoplanes friuliensis and Streptomyces pristinaespiralis, respectively) completely restored FK506 production. Subsequently, FK506 production was enchanced by heterologous overexpression of PipAf and PipASp in S. tsukubaensis. This resulted in a yield increase by 65% compared to the WT in the presence of PipAf from A. friuliensis. For further rational yield improvement, the crystal structure of PipAf from A. friuliensis was determined at 1.3 Å resolution with the cofactor NADH bound and at 1.4 Å with its substrate lysine. Based on the structure the Ile91 residue was replaced by Val91 in PipAf, which resulted in an overall increase of FK506 production by approx. 100% compared to the WT.

Mechanism-Based Design of the First GlnA4-Specific Inhibitors.

γ-Glutamylamine synthetases are an important class of enzymes that play a key role in glutamate-based metabolism. Methionine sulfoximine (MSO) is a well-established inhibitor for the archetypal glutamine synthetase (GS) but inhibitors for most GS-like enzymes are unknown. Assuming a conserved catalytic mechanism for GS and GS-like enzymes, we explored if subtype-selective inhibitors can be obtained by merging MSO with the cognate substrates of the respective GS-like enzymes. Using GlnA4Sc from Streptomyces coelicolor, an enzyme recently shown to produce γ-glutamylethanolamine, we demonstrate that MSO can be reengineered in a straightforward fashion into potent and selective GlnA4Sc inhibitors. Linkage chemistry as well as linker length between the MSO moiety and the terminal hydroxyl group derived from ethanolamine were in agreement with the postulated phosphorylated catalytic intermediate. The best GlnA4 inhibitor 7 b potently blocked S. coelicolor growth in the presence of ethanolamine as the sole nitrogen source. Our results provide the first GlnA4Sc -specific inhibitors and suggest a general strategy to develop mechanism-based inhibitors for GS-like enzymes.

Polyamine and Ethanolamine Metabolism in Bacteria as an Important Component of Nitrogen Assimilation for Survival and Pathogenicity.

Nitrogen is an essential element required for bacterial growth. It serves as a building block for the biosynthesis of macromolecules and provides precursors for secondary metabolites. Bacteria have developed the ability to use various nitrogen sources and possess two enzyme systems for nitrogen assimilation involving glutamine synthetase/glutamate synthase and glutamate dehydrogenase. Microorganisms living in habitats with changeable availability of nutrients have developed strategies to survive under nitrogen limitation. One adaptation is the ability to acquire nitrogen from alternative sources including the polyamines putrescine, cadaverine, spermidine and spermine, as well as the monoamine ethanolamine. Bacterial polyamine and monoamine metabolism is not only important under low nitrogen availability, but it is also required to survive under high concentrations of these compounds. Such conditions can occur in diverse habitats such as soil, plant tissues and human cells. Strategies of pathogenic and non-pathogenic bacteria to survive in the presence of poly- and monoamines offer the possibility to combat pathogens by using their capability to metabolize polyamines as an antibiotic drug target. This work aims to summarize the knowledge on poly- and monoamine metabolism in bacteria and its role in nitrogen metabolism.

Genome Assembly and Genetic Traits of the Pleuromutilin-Producer Clitopilus passeckerianus DSM1602.

The gilled mushroom Clitopilus passeckerianus (Entolomataceae, Agaricales, Basidiomycota) is well known to produce the terpenoid pleuromutilin, which is the biotechnological basis for medically important antibiotics such as lefamulin and retapamulin. Their unique mode of action and good tolerance entails an increasing demand of pleuromutilin-derived antibiotics in veterinary and human health care. Surprisingly, despite their pharmaceutical importance, no genome sequence is available of any pleuromutilin-producing fungus. Here, we present the high-quality draft genome sequence of the pleuromutilin-producer C. passeckerianus DSM1602 including functional genome annotation. More precisely, we employed a hybrid assembly strategy combining Illumina sequencing and Nanopore sequencing to assemble the mitochondrial genome as well as the nuclear genome. In accordance with the dikaryotic state of the fungus, the nuclear genome has a diploid character. Interestingly, the mitochondrial genome appears duplicated. Bioinformatic analysis revealed a versatile secondary metabolism with an emphasis on terpenoid biosynthetic enzymes in C. passeckerianus and also in related strains. Two alleles of biosynthetic gene clusters for pleuromutilin were found in the genome of C. passeckerianus. The pleuromutilin genes were reassembled with yeast-specific elements for heterologous expression in Saccharomyces cerevisiae. Our work lays the foundation for metabolic strain engineering towards higher yields of the valuable compound pleuromutilin.

A Second Gamma-Glutamylpolyamine Synthetase, GlnA2, Is Involved in Polyamine Catabolism in Streptomyces coelicolor.

Streptomyces coelicolor is a soil bacterium living in a habitat with very changeable nutrient availability. This organism possesses a complex nitrogen metabolism and is able to utilize the polyamines putrescine, cadaverine, spermidine, and spermine and the monoamine ethanolamine. We demonstrated that GlnA2 (SCO2241) facilitates S. coelicolor to survive under high toxic polyamine concentrations. GlnA2 is a gamma-glutamylpolyamine synthetase, an enzyme catalyzing the first step in polyamine catabolism. The role of GlnA2 was confirmed in phenotypical studies with a glnA2 deletion mutant as well as in transcriptional and biochemical analyses. Among all GS-like enzymes in S. coelicolor, GlnA2 possesses the highest specificity towards short-chain polyamines (putrescine and cadaverine), while its functional homolog GlnA3 (SCO6962) prefers long-chain polyamines (spermidine and spermine) and GlnA4 (SCO1613) accepts only monoamines. The genome-wide RNAseq analysis in the presence of the polyamines putrescine, cadaverine, spermidine, or spermine revealed indication of the occurrence of different routes for polyamine catabolism in S. coelicolor involving GlnA2 and GlnA3. Furthermore, GlnA2 and GlnA3 are differently regulated. From our results, we can propose a complemented model of polyamine catabolism in S. coelicolor, which involves the gamma-glutamylation pathway as well as other alternative utilization pathways.

The Endophytic Fungus Cyanodermella asteris Influences Growth of the Nonnatural Host Plant Arabidopsis thaliana.

Cyanodermella asteris is a fungal endophyte from Aster tataricus, a perennial plant from the northern part of Asia. Here, we demonstrated an interaction of C. asteris with Arabidopsis thaliana, Chinese cabbage, rapeseed, tomato, maize, or sunflower resulting in different phenotypes such as shorter main roots, massive lateral root growth, higher leaf and root biomass, and increased anthocyanin levels. In a variety of cocultivation assays, it was shown that these altered phenotypes are caused by fungal CO2, volatile organic compounds, and soluble compounds, notably astins. Astins A, C, and G induced plant growth when they were individually included in the medium. In return, A. thaliana stimulates the fungal astin C production during cocultivation. Taken together, our results indicate a bilateral interaction between the fungus and the plant. A stress response in plants is induced by fungal metabolites while plant stress hormones induced astin C production of the fungus. Interestingly, our results not only show unidirectional influence of the fungus on the plant but also vice versa. The plant is able to influence growth and secondary metabolite production in the endophyte, even when both organisms do not live in close contact, suggesting the involvement of volatile compounds.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

A Regulator Based "Semi-Targeted" Approach to Activate Silent Biosynthetic Gene Clusters.

By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel "semi-targeted" approach focusing on activating "silent" BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.

Engineering of Streptoalloteichus tenebrarius 2444 for Sustainable Production of Tobramycin.

Tobramycin is a broad-spectrum aminoglycoside antibiotic agent. The compound is obtained from the base-catalyzed hydrolysis of carbamoyltobramycin (CTB), which is naturally produced by the actinomycete Streptoalloteichus tenebrarius. However, the strain uses the same precursors to synthesize several structurally related aminoglycosides. Consequently, the production yields of tobramycin are low, and the compound's purification is very challenging, costly, and time-consuming. In this study, the production of the main undesired product, apramycin, in the industrial isolate Streptoalloteichus tenebrarius 2444 was decreased by applying the fermentation media M10 and M11, which contained high concentrations of starch and dextrin. Furthermore, the strain was genetically engineered by the inactivation of the aprK gene (∆aprK), resulting in the abolishment of apramycin biosynthesis. In the next step of strain development, an additional copy of the tobramycin biosynthetic gene cluster (BGC) was introduced into the ∆aprK mutant. Fermentation by the engineered strain (∆aprK_1-17L) in M11 medium resulted in a 3- to 4-fold higher production than fermentation by the precursor strain (∆aprK). The phenotypic stability of the mutant without selection pressure was validated. The use of the engineered S. tenebrarius 2444 facilitates a step-saving, efficient, and, thus, more sustainable production of the valuable compound tobramycin on an industrial scale.

Mining Indonesian Microbial Biodiversity for Novel Natural Compounds by a Combined Genome Mining and Molecular Networking Approach.

Indonesia is one of the most biodiverse countries in the world and a promising resource for novel natural compound producers. Actinomycetes produce about two thirds of all clinically used antibiotics. Thus, exploiting Indonesia's microbial diversity for actinomycetes may lead to the discovery of novel antibiotics. A total of 422 actinomycete strains were isolated from three different unique areas in Indonesia and tested for their antimicrobial activity. Nine potent bioactive strains were prioritized for further drug screening approaches. The nine strains were cultivated in different solid and liquid media, and a combination of genome mining analysis and mass spectrometry (MS)-based molecular networking was employed to identify potential novel compounds. By correlating secondary metabolite gene cluster data with MS-based molecular networking results, we identified several gene cluster-encoded biosynthetic products from the nine strains, including naphthyridinomycin, amicetin, echinomycin, tirandamycin, antimycin, and desferrioxamine B. Moreover, 16 putative ion clusters and numerous gene clusters were detected that could not be associated with any known compound, indicating that the strains can produce novel secondary metabolites. Our results demonstrate that sampling of actinomycetes from unique and biodiversity-rich habitats, such as Indonesia, along with a combination of gene cluster networking and molecular networking approaches, accelerates natural product identification.

Poly- and Monoamine Metabolism in Streptomyces coelicolor: The New Role of Glutamine Synthetase-Like Enzymes in the Survival under Environmental Stress.

Soil bacteria from the genus Streptomyces, phylum Actinobacteria, feature a complex metabolism and diverse adaptations to environmental stress. These characteristics are consequences of variable nutrition availability in the soil and allow survival under changing nitrogen conditions. Streptomyces coelicolor is a model organism for Actinobacteria and is able to use nitrogen from a variety of sources including unusual compounds originating from the decomposition of dead plant and animal material, such as polyamines or monoamines (like ethanolamine). Assimilation of nitrogen from these sources in S. coelicolor remains largely unstudied. Using microbiological, biochemical and in silico approaches, it was recently possible to postulate polyamine and monoamine (ethanolamine) utilization pathways in S. coelicolor. Glutamine synthetase-like enzymes (GS-like) play a central role in these pathways. Extensive studies have revealed that these enzymes are able to detoxify polyamines or monoamines and allow the survival of S. coelicolor in soil containing an excess of these compounds. On the other hand, at low concentrations, polyamines and monoamines can be utilized as nitrogen and carbon sources. It has been demonstrated that the first step in poly-/monoamine assimilation is catalyzed by GlnA3 (a γ-glutamylpolyamine synthetase) and GlnA4 (a γ-glutamylethanolamide synthetase), respectively. First insights into the regulation of polyamine and ethanolamine metabolism have revealed that the expression of the glnA3 and the glnA4 gene are controlled on the transcriptional level.

Genetic Engineering in Combination with Semi-Synthesis Leads to a New Route for Gram-Scale Production of the Immunosuppressive Natural Product Brasilicardin†A.

Brasilicardin A (1) consists of an unusual anti/syn/anti-perhydrophenanthrene skeleton with a carbohydrate side chain and an amino acid moiety. It exhibits potent immunosuppressive activity, yet its mode of action differs from standard drugs that are currently in use. Further pre-clinical evaluation of this promising, biologically active natural product is hampered by restricted access to the ready material, as its synthesis requires both a low-yielding fermentation process using a pathogenic organism and an elaborate, multi-step total synthesis. Our semi-synthetic approach included a) the heterologous expression of the brasilicardin A gene cluster in different non-pathogenic bacterial strains producing brasilicardin A aglycone (5) in excellent yield and b) the chemical transformation of the aglycone 5 into the trifluoroacetic acid salt of brasilicardin A (1 a) via a short and straightforward five-steps synthetic route. Additionally, we report the first preclinical data for brasilicardin A.

A novel LysR-type regulator negatively affects biosynthesis of the immunosuppressant brasilicardin.

Brasilicardin A (BraA) is a promising immunosuppressive compound produced naturally by the pathogenic bacterium Nocardia terpenica IFM 0406. Heterologous host expression of brasilicardin gene cluster showed to be efficient to bypass the safety issues, low production levels and lack of genetic tools related with the use of native producer. Further improvement of production yields requires better understanding of gene expression regulation within the BraA biosynthetic gene cluster (Bra-BGC); however, the only so far known regulator of this gene cluster is Bra12. In this study, we discovered the protein LysRNt, a novel member of the LysR-type transcriptional regulator family, as a regulator of the Bra-BGC. Using in vitro approaches, we identified the gene promoters which are controlled by LysRNt within the Bra-BGC. Corresponding genes encode enzymes involved in BraA biosynthesis as well as the key Bra-BGC regulator Bra12. Importantly, we provide in vivo evidence that LysRNt negatively affects production of brasilicardin congeners in the heterologous host Amycolatopsis japonicum. Finally, we demonstrate that some of the pathway related metabolites, and their chemical analogs, can interact with LysRNt which in turn affects its DNA-binding activity.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Maßgeschneiderte Polyketidsynthasen zur Herstellung von Polyketid-Derivaten.

Polyketides (PKs) are secondary metabolites with valuable properties, including antibiotic, antifungal, antiviral, anticancer, and immunosuppressive activity. The biosynthesis of PKs is accomplished by multifunctional megaenzymes, the polyketide synthases (PKSs). The molecular architecture of those remarkable assembly lines provides opportunities for their engineering and generation of PK derivatives with potentially improved pharmacokinetics and/or expanded spectrum of bioactivity.