P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds via dehydrogenation of a specific acylated dipeptide substrate.

WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine (NMet-Dht) residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase (NRPS). The cytochrome P450 encoded by sas16 (P450Sas) has been shown to be essential for the formation of the alkene moiety in NMet-Dht, but the timing and mechanism of the P450Sas-mediated α,ß-dehydrogenation of Dht remained unclear. Here, we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein (PCP)-bound dipeptide intermediate (Z)-2-pent-1'-enyl-cinnamoyl-Thr-N-Me-Tyr. We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS, and further that P450Sas appears to be specific for substrates containing the (Z)-2-pent-1'-enyl-cinnamoyl group. A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates, including the substitution of the canonical active site alcohol residue with a phenylalanine (F250), which in turn is critical to P450Sas activity and WS9326A biosynthesis. Together, our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate, thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.

Structural diversity, biosynthesis, and biological functions of lipopeptides from Streptomyces.

Covering: up to 2022Streptomyces are ubiquitous in terrestrial and marine environments, where they display a fascinating metabolic diversity. As a result, these bacteria are a prolific source of active natural products. One important class of these natural products is the nonribosomal lipopeptides, which have diverse biological activities and play important roles in the lifestyle of Streptomyces. The importance of this class is highlighted by the use of related antibiotics in the clinic, such as daptomycin (tradename Cubicin). By virtue of recent advances spanning chemistry and biology, significant progress has been made in biosynthetic studies on the lipopeptide antibiotics produced by Streptomyces. This review will serve as a comprehensive guide for researchers working in this multidisciplinary field, providing a summary of recent progress regarding the investigation of lipopeptides from Streptomyces. In particular, we highlight the structures, properties, biosynthetic mechanisms, chemical and chemoenzymatic synthesis, and biological functions of lipopeptides. In addition, the application of genome mining techniques to Streptomyces that have led to the discovery of many novel lipopeptides is discussed, further demonstrating the potential of lipopeptides from Streptomyces for future development in modern medicine.

Enhanced Rishirilide Biosynthesis by a Rare In-Cluster Phosphopantetheinyl Transferase in Streptomyces xanthophaeus.

Phosphopantetheinyl transferases (PPTases) play important roles in activating apo-acyl carrier proteins (apo-ACPs) and apo-peptidyl carrier proteins (apo-PCPs) in both primary and secondary metabolism. PPTases catalyze the posttranslational modifications of those carrier proteins by covalent attachment of the 4'-phosphopantetheine group to a conserved serine residue. The protein-protein interactions between a PPTase and a cognate acyl or peptidyl carrier protein have important regulatory functions in microbial biosynthesis, but the molecular mechanism underlying their specific recognition remains elusive. In this study, we identified a new rishirilide biosynthetic gene cluster with a rare in-cluster PPTase from Streptomyces xanthophaeus no2. The function of this Sfp-type PPTase, SxrX, in rishirilide production was confirmed using genetic mutagenesis and biochemical characterization. We applied molecular modeling and site-directed mutagenesis to identify key residues mediating the protein-protein interaction between SxrX and its cognate ACP. In addition, six natural products were isolated from wild-type S. xanthophaeus no2 and the ΔsxrX mutant, including rishirilide A and lupinacidin A, that exhibited antimicrobial and anticancer activities, respectively. SxrX is the first Sfp-type PPTase identified from an aromatic polyketide biosynthetic gene cluster and shown to be responsible for high-level production of rishirilide derivatives. IMPORTANCE Genome mining has been a vital means for natural product drug discovery in the postgenomic era. The rishirilide-type polyketides have attracted attention due to their potent bioactivity, but the poor robustness of production hosts has limited further research and development. This study not only identifies a hyperproducer of rishirilides but also reveals a rare, in-cluster PPTase SxrX that plays an important role in boosting rishirilide biosynthesis. Experimental and computational investigations revealed new insights on the protein-protein interaction between SxrX and its cognate ACP with wide implications for understanding polyketide biosynthesis.

Directed evolution of a cyclodipeptide synthase with new activities via label-free mass spectrometric screening.

Directed evolution is a powerful approach to engineer enzymes via iterative creation and screening of variant libraries. However, assay development for high-throughput mutant screening remains challenging, particularly for new catalytic activities. Mass spectrometry (MS) analysis is label-free and well suited for untargeted discovery of new enzyme products but is traditionally limited by slow speed. Here we report an automated workflow for directed evolution of new enzymatic activities via high-throughput library creation and label-free MS screening. For a proof of concept, we chose to engineer a cyclodipeptide synthase (CDPS) that synthesizes diketopiperazine (DKP) compounds with therapeutic potential. In recombinant Escherichia coli, site-saturation mutagenesis (SSM) and error-prone PCR (epPCR) libraries expressing CDPS mutants were automatically created and cultivated on an integrated work cell. Culture supernatants were then robotically processed for matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS analysis at a rate of 5 s per sample. The resulting mass spectral data were processed via custom computational algorithms, which performed a multivariant analysis of 108 theoretical mass-to-charge (m/z) values of 190 possible DKP molecules within a mass window of 115-373 Da. An F186L CDPS mutant was isolated to produce cyclo(l-Phe-l-Val), which is undetectable in the product profile of the wild-type enzyme. This robotic, label-free MS screening approach may be generally applicable to engineering other enzymes with new activities in high throughput.

Novel Antioxidants and α-Glycosidase and Protein Tyrosine Phosphatase 1B Inhibitors from an Endophytic Fungus Penicillium brefeldianum F4a.

Oxidative stress plays a very important role in the progression of diabetes and its complications. A therapeutic agent that is both antidiabetic and antioxidant would be the preferred choice for the treatment of diabetes. The crude extract of the endophytic fungus Penicillium brefeldianum F4a has significant antioxidant and α-glycosidase and protein tyrosine phosphatase 1B (PTP1B) inhibition activities. Chemical investigation of P. brefeldianum F4a using an activity-guided isolation led to the discovery of three new compounds called peniorcinols A-C (1-3) along with six known compounds: penialidins A (4), penialidin F (5), myxotrichin C (6), riboflavin (7), indole-3-acetic acid (8), and 2-(4-hydroxy-2-methoxy-6-methylphenyl) acetic acid (9). Their chemical structures were established by their NMR and HRESIMS. The absolute configurations of 1 and 3 were determined by experimental and calculated electronic circular dichroism (ECD). Their antioxidant activities were evaluated by DPPH• and ABTS•+ scavenging assays. Compounds 1-6 and 8-9 showed moderate to strong free radical scavenging activities. Significantly, 4-6 exhibited more potent ABTS•+ scavenging activity than that of the positive control. Their α-glycosidase and PTP1B inhibition activities were tested. Among them, compound 3 showed α-glucosidase inhibition activity, and compounds 7 and 8 showed PTP1B inhibitory activity for the first time. It is worth noting that 3 and 8 displayed both antioxidant and α-glycosidase or PTP1B inhibition activities. These finding suggest that compounds 3 and 8 could be used as lead compounds to generate new potent drugs for the treatment of oxidative stress-related diabetes.

Secreted Expression of mRNA-Encoded Truncated ACE2 Variants for SARS-CoV-2 via Lipid-Like Nanoassemblies.

The transfer of foreign synthetic messenger RNA (mRNA) into cells is essential for mRNA-based protein-replacement therapies. Prophylactic mRNA COVID-19 vaccines commonly utilize nanotechnology to deliver mRNA encoding SARS-CoV-2 vaccine antigens, thereby triggering the body's immune response and preventing infections. In this study, a new combinatorial library of symmetric lipid-like compounds is constructed, and among which a lead compound is selected to prepare lipid-like nanoassemblies (LLNs) for intracellular delivery of mRNA. After multiround optimization, the mRNA formulated into core-shell-structured LLNs exhibits more than three orders of magnitude higher resistance to serum than the unprotected mRNA, and leads to sustained and high-level protein expression in mammalian cells. A single intravenous injection of LLNs into mice achieves over 95% mRNA translation in the spleen, without causing significant hematological and histological changes. Delivery of in-vitro-transcribed mRNA that encodes high-affinity truncated ACE2 variants (tACE2v mRNA) through LLNs induces elevated expression and secretion of tACE2v decoys, which is able to effectively block the binding of the receptor-binding domain of the SARS-CoV-2 to the human ACE2 receptor. The robust neutralization activity in vitro suggests that intracellular delivery of mRNA encoding ACE2 receptor mimics via LLNs may represent a potential intervention strategy for COVID-19.

Genomic and Metabolomic Investigation of a Rhizosphere Isolate Streptomyces netropsis WLXQSS-4 Associated with a Traditional Chinese Medicine.

Rhizosphere microorganisms play important ecological roles in promoting herb growth and producing abundant secondary metabolites. Studies on the rhizosphere microbes of traditional Chinese medicines (TCMs) are limited, especially on the genomic and metabolic levels. In this study, we reported the isolation and characterization of a Steptomyces netropsis WLXQSS-4 strain from the rhizospheric soil of Clematis manshurica Rupr. Genomic sequencing revealed an impressive total of 40 predicted biosynthetic gene clusters (BGCs), whereas metabolomic profiling revealed 13 secondary metabolites under current laboratory conditions. Particularly, medium screening activated the production of alloaureothin, whereas brominated and chlorinated pimprinine derivatives were identified through precursor-directed feeding. Moreover, antiproliferative activities against Hela and A549 cancer cell lines were observed for five compounds, of which two also elicited potent growth inhibition in Enterococcus faecalis and Staphylococcus aureus, respectively. Our results demonstrated the robust secondary metabolism of S. netropsis WLXQSS-4, which may serve as a biocontrol agent upon further investigation.

Isolation and characterization of a new cyclic lipopeptide surfactin from a marine-derived Bacillus velezensis SH-B74.

A marine-sediment-derived bacterium Bacillus velezensis SH-B74 can produce cyclic lipopeptides (CLPs). This study presented the isolation, characterization, and activity evaluation of a new CLP from the bacterial cultures of the strain SH-B74. Multiple chromatographic methods (solid-phase extraction and reversed-phase high-performance liquid chromatography) were applied to the purifying procedure of CLP, and the structural characterization of the new CLP was conducted by various spectroscopy (1D and 2D nuclear magnetic resonance together with Fourier transform infrared spectroscopy) and spectrometry (liquid chromatography-mass spectrometry, high-resolution mass spectrometry and tandem mass spectrometry) techniques as well as Marfey's method. The results displayed that the new CLP (anteiso-C15 Ile2,7 surfactin, 1) consists of a peptidic backbone of L-Glu1, L-Ile2, D-Leu3, L-Val4, L-Asp5, D-Leu6, L-Ile7, and an anteiso-C15 type saturated fatty acid chain. Further activity assay showed that the new CLP displays activity on the inhibition of the appressoria formation of rice blast causal pathogen Magnaporthe oryzae. To sum up, the results presented the perspective of potential application of the new CLP as a green agrichemical to control M. oryzae.

Isolation and characterization of a new cyclic lipopeptide orfamide H from Pseudomonas protegens CHA0.

A new cyclic lipopeptide (CLP) orfamide H (1) was purified and identified from the cultural broth of the bacterial strain Pseudomonas protegens CHA0. The crude extract of the strain CHA0 was obtained by an acid-aided precipitation process, then the compound 1 was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). Subsequently, the chemical structure of orfamide H was determined by 1D and 2D nuclear magnetic resonance (NMR) and mass spectrometry (MS). Further biological assays indicate that the new CLP orfamide H shows the activity on inhibiting the appressoria formation of the fungus Magnaporthe oryzae, the causal agent of the blast disease in rice. Taken all together, these results indicated that the new CLP orfamide H has the capacity to be developed as an agrichemical to control blast disease in rice.

Rational Design of Hybrid Natural Products by Utilizing the Promiscuity of an Amide Synthetase.

WS9326A and annimycin are produced by Streptomyces asterosporus DSM 41452. WS9326A is a nonribosomal peptide synthetase-(NRPS-) derived depsipeptide containing a cinnamoyl moiety, while annimycin is a linear polyketide bearing a 2-amino-3-hydroxycyclopent-2-enone (C5N) group. Both gene clusters have been sequenced and annotated. In this study, we show that the amide synthetase Ann1, responsible for attaching the C5N unit during annimycin biosynthesis, has the ability to catalyze fortuitous side reactions to polyenoic acids in addition to its main reaction. Novel compounds were generated by feeding experiments and in vitro studies. We also rationally designed a hybrid natural product consisting of the cinnamoyl moiety of WS9326A and the C5N moiety of annimycin by creating a mutant of S. asterosporus that retains genes encoding biosynthesis of the C5N unit of annimycin and the cinnamoyl group of WS9326A. The promiscuity of Ann1 also proved useful for trapping compounds that arise from acyl-ACP intermediates, which occur in the biosynthesis of the cinnamoyl moiety of WS9326A, by hydrolysis. In this pathway, we postulate that sas27 and sas28 genes are involved in the biosynthesis of the cinnamoyl moiety in WS9326A.

A Potential Biocontrol Agent Streptomyces violaceusniger AC12AB for Managing Potato Common Scab.

Potato common scab (PCS) is an economically important disease worldwide. In this study we demonstrated the possible role of Streptomyces violaceusniger AC12AB in controlling PCS. Isolates of Streptomyces scabies were obtained from CS infected tubers collected from Maine United States, which were confirmed by morphological and molecular analysis including 16S rRNA sequencing and RFLP analysis of amplified 16S-23S ITS. Pathogenicity assays related genes including txtAB, nec1, and tomA were also identified in all S. scabies strains through PCR reaction. An antagonistic bacterial strain was isolated from soil in Punjab and identified as S. violaceusniger AC12AB based on 16S rRNA sequencing analysis. Methanolic extract of S. violaceusniger AC12AB contained azalomycin RS-22A which was confirmed by 1H and 13C-NMR, 1H/1H-COSY, HMBC and HMQC techniques. S. violaceusniger AC12AB exhibited plant growth promotion attributes including Indole-3-acetic acid production with 17 µgmL-1 titers, siderophores production, nitrogen fixation and phosphates solubilization potential. When tubers were inoculated with S. violaceusniger AC12AB, significant (P < 0.05) PCS disease reduction up to 90% was observed in greenhouse and field trials, respectively. Likewise, S. violaceusniger AC12AB significantly (P < 0.05) increased potato crop up to 26.8% in field trial. Therefore, plant growth promoting S. violaceusniger AC12AB could provide a dual benefit by decreasing PCS disease severity and increasing potato yield as an effective and inexpensive alternative strategy to manage this disease.

Genome mining reveals the origin of a bald phenotype and a cryptic nucleocidin gene cluster in Streptomyces asterosporus DSM 41452.

Streptomyces asterosporus DSM 41452 is a producer of the polyketide annimycin and the non-ribosomal depsipeptide WS9326A. This strain is also notable for exhibiting a bald phenotype that is devoid of spores and aerial mycelium when grown on solid media. Based on the similarity of the 16S rRNA sequence to Streptomyces calvus, the only known producer of the fluorometabolite nucleocidin, the genome of S. asterosporus DSM 41452 was sequenced and analyzed. Twenty-nine natural product gene clusters were detected in the genome, including a gene cluster predicted to encode the fluorometabolite nucleocidin. Through genome analysis and gene complementation experiments, we demonstrate that the bald phenotype arises from a transposon gene inserted within the promoter sequence for the pleiotropic regulator adpA. Complementation of S. asterosporus DSM 41452 with a functional adpA sequence restored morphological differentiation and promoted the production of nucleocidin.

Biological Control of Potato Common Scab With Rare Isatropolone C Compound Produced by Plant Growth Promoting Streptomyces A1RT.

Potato is prone to many drastic diseases like potato common scab (CS). As no highly effective methods exist for managing CS, this study explored the possibility of using biological control. Ten bacterial strains were isolated from CS-infected potato tubers from four different locations of Punjab, Pakistan, and identified based on biochemical and molecular analysis. Analysis of 16s rDNA sequences amplified by PCR revealed the isolated bacterial strains to be Streptomyces scabies, S. turgidiscabies and S. stelliscabiei. Pathogenic islands were also confirmed among the isolates after identification of txtAB, nec1, and tomA genes with PCR amplification. One strain isolated from soil was antagonistic to the pathogenic Streptomyces spp., and determined to be Streptomyces A1RT on the basis of 16s rRNA sequencing. A methanolic extract of Streptomyces A1RT contained Isatropolone C, which was purified and structurally determined by 1H- and 13C-NMR, 1H/1H-COSY, HMQC, and HMBC techniques. Streptomyces A1RT also produced the plant growth hormone indole-3-acetic acid (IAA) with a titer of 26 µg ml-1 as confirmed by spectrophotometry and HPLC. In a greenhouse assay, disease severity index was established from 0 to 500. Average disease severity indexes were recorded as 63, 130.5, and 78 for Streptomyces scabies, S. turgidiscabies and S. stelliscabiei, respectively. When Streptomyces A1RT was applied in soil that contained one of these pathogenic isolates, the average disease severity indexes were significantly (P < 0.05) reduced to 11.1, 5.6 and 8.4, respectively. A significant increase in tuber weight and shoot development was also observed with the tubers treated with Streptomyces A1RT. The use of the plant growth-promoting Streptomyces A1RT against potato CS thus provides an alternative strategy to control the disease without affecting environmental, plants, animals and human health.

New WS9326A Derivatives and One New Annimycin Derivative with Antimalarial Activity are Produced by Streptomyces asterosporus DSM 41452 and Its Mutant.

In this study, we report that Streptomyces asterosporus DSM 41452 is a producer of new molecules related to the nonribosomal cyclodepsipeptide WS9326A and the polyketide annimycin. S. asterosporus DSM 41452 is shown to produce six cyclodepsipeptides and peptides, WS9326A to G. Notably, the compounds WS9326F and WS9326G have not been described before. The genome of S. asterosporus DSM 41452 was sequenced, and a putative WS9326A gene cluster was identified. Gene-deletion experiments confirmed that this cluster was responsible for the biosynthesis of WS9326A to G. Additionally, a gene-deletion experiment demonstrated that sas16 encoding a cytochrome P450 monooxygenase was involved in the synthesis of the novel (E)-2,3-dehydrotyrosine residue found in WS9326A and its derivatives. An insertion mutation within the putative annimycin gene cluster led to the production of a new annimycin derivative, annimycin B, which exhibited modest inhibitory activity against Plasmodium falciparum.

Wide Distribution of Foxicin Biosynthetic Gene Clusters in Streptomyces Strains - An Unusual Secondary Metabolite with Various Properties.

Streptomyces diastatochromogenes Tü6028 is known to produce the polyketide antibiotic polyketomycin. The deletion of the pokOIV oxygenase gene led to a non-polyketomycin-producing mutant. Instead, novel compounds were produced by the mutant, which have not been detected before in the wild type strain. Four different compounds were identified and named foxicins A-D. Foxicin A was isolated and its structure was elucidated as an unusual nitrogen-containing quinone derivative using various spectroscopic methods. Through genome mining, the foxicin biosynthetic gene cluster was identified in the draft genome sequence of S. diastatochromogenes. The cluster spans 57 kb and encodes three PKS type I modules, one NRPS module and 41 additional enzymes. A foxBII gene-inactivated mutant of S. diastatochromogenes Tü6028 ΔpokOIV is unable to produce foxicins. Homologous fox biosynthetic gene clusters were found in more than 20 additional Streptomyces strains, overall in about 2.6% of all sequenced Streptomyces genomes. However, the production of foxicin-like compounds in these strains has never been described indicating that the clusters are expressed at a very low level or are silent under fermentation conditions. Foxicin A acts as a siderophore through interacting with ferric ions. Furthermore, it is a weak inhibitor of the Escherichia coli aerobic respiratory chain and shows moderate antibiotic activity. The wide distribution of the cluster and the various properties of the compound indicate a major role of foxicins in Streptomyces strains.

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028.

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC. Furthermore, the extracts are tested for their bioactivity by different assays. For structure elucidation the compound of interest is purified by a combination of different chromatography methods. Genome sequencing coupled with genome mining allows the identification of a natural product biosynthetic gene cluster using different computer programs. To confirm that the correct gene cluster has been identified, gene inactivation experiments have to be performed. The resulting mutants are analyzed for the production of the particular natural product. Once the correct gene cluster has been inactivated, the strain should fail to produce the compound. The workflow is shown for the antibacterial compound polyketomycin produced by Streptomyces diastatochromogenes Tü6028. Around ten years ago, when genome sequencing was still very expensive, the cloning and identification of a gene cluster was a very time-consuming process. Fast genome sequencing combined with genome mining accelerates the trial of cluster identification and opens up new ways to explore biosynthesis and to generate novel natural products by genetic methods. The protocol described in this paper can be assigned to any other compound derived from a Streptomyces strain or another microorganism.

A new polyketide, penicillolide from the marine-derived fungus Penicillium sacculum.

A new polyketide, penicillolide (1) was isolated from the fermentation broth of the marine-derived fungus Penicillium sacculum GT-308. Compound 1 is a polyketide with a unique carbon skeleton. The structure of this compound was established via extensive spectroscopic analyses including 1D-, 2D-NMR, and HRESI-MS.

Changing Biosynthetic Profiles by Expressing bldA in Streptomyces Strains.

Recently we described an unusual way of activating a cryptic gene cluster when we explored the origin of the bald phenotype of Streptomyces calvus. Complementation of S. calvus with a correct copy of bldA restored sporulation and additionally promoted production of a new natural products. In this study we report on the expression of bldA in several Streptomyces strains that have been described as "poorly sporulating" strains. In seven out of 15 cases, HPLC profiling revealed the production of new compounds, and in two cases the overproduction of known compounds. Two compounds were isolated and their structures were determined.

Two new amides from a halotolerant fungus, Myrothecium sp. GS-17.

Two new amides, named N-acetyl-2,4,10,17-tetrahydroxyheptadecylamine (1) and N-acetyl-3,5,11,18-tetrahydroxyoctadecyl-2-amine (2), were isolated from a halotolerant fungus, Myrothecium sp. GS-17. Their structures were identified on the basis of spectroscopic characteristics. The cancer cell cytotoxicities of two compounds were evaluated, and compound 2 exhibited weak cytotoxicity in HL-60 cell line.

Polyketides from the halotolerant fungus Myrothecium sp. GS-17.

Two new polyketides, myrothecol (1) and 5-hydroxy-3-methyl-4-(1-hydroxylethyl)-furan-2(5H)-one (2), were isolated from the fermentation broth of the halotolerant fungus Myrothecium sp. GS-17 along with three known compounds, 5-hydroxyl-3-[(1S)-1-hydroxyethyl]-4-methylfuran-2(5H)-one (3), 3,5-dimethyl-4- hydroxylmethyl-5-methoxyfuran-2(5H)-one (4), and 3,5-dimethyl-4-hydroxymethyl-5- hydroxyfuran-2(5H)-one (5). Compound 1 is the first natural occurring polyketide with a unique furylisobenzofuran skeleton. The structures of these compounds were established via extensive spectroscopic analyses including 1D-, 2D-NMR, HRESI-MS, and crystal X-ray diffraction analysis.