Heterologous biosynthesis of myxobacterial lanthipeptides melittapeptins.

The myxobacteria are an attractive bioresource for bioactive compounds since the large size genome contains many biosynthetic gene clusters of secondary metabolites. The genome of the myxobacterium Melittangium boletus contains three biosynthetic gene clusters for lanthipeptide production. One of the gene clusters includes genes coding lanthipeptide precursor (melA), class II lanthipeptide synthetase (melM), and transporter (melT). The amino acid sequence of melA indicated similarity with that of known lanthipeptides mersacidin and lichenicidin A1 by the alignment. To perform heterologous production of new lanthipeptides, the expression vector containing the essential genes (melA and melM) was constructed by utilizing codon-optimized synthetic genes. The co-expression of two genes in the host bacterial cells of Escherichia coli BL21 (DE3) afforded new lanthipeptides named melittapeptins A-C. The structures of melittapeptins A-C including lanthionine/methyllanthionine bridge pattern were proposed based on protease digestion and MS/MS experiments. The native strain of M. boletus did not produce melittapeptins A-C, so heterologous production using the biosynthetic gene cluster was effective in obtaining the lanthipeptides. Melittapeptins A-C showed specific and potent antibacterial activity to the Gram-positive bacterium Micrococcus luteus. To the best of our knowledge, this is the first report of antibacterial lanthipeptides derived from myxobacterial origin. KEY POINTS: • New lanthipeptides melittapeptins were heterologously produced in Escherichia coli. • Melittapeptins showed specific antibacterial activity against Micrococcus luteus. • Melittapeptins were the first antibacterial lanthipeptides of myxobacterial origin.

Unique Physiological and Genetic Features of Ofloxacin-Resistant Streptomyces Mutants.

New antimicrobial agents are urgently needed to combat the emergence and spread of multidrug-resistant bacteria. Activating the cryptic biosynthetic gene clusters for actinomycete secondary metabolites can provide essential clues for research into new antimicrobial agents. An effective method for this purpose is based on drug resistance selection. This report describes interesting results for drug resistance selection using antibiotics that target DNA replication and can effectively potentiate secondary metabolite production by actinomycetes. Ofloxacin-resistant mutants were isolated from five different streptomycetes. Ofloxacin is an antibiotic that binds to DNA complexes and type II topoisomerase, causing double-stranded breaks in bacterial chromosomes. Physiological and genetic characterization of the mutants revealed that the development of ofloxacin resistance in streptomycetes leads to the emergence of various types of secondary metabolite-overproducing strains. In Streptomyces coelicolor A3(2), ofloxacin-resistant mutants that overproduced actinorhodin, undecylprodigiosin, or carotenoid were identified. An ofloxacin-resistant mutant that overproduces methylenomycin A, whose biosynthetic gene cluster is located on the endogenous plasmid, SCP1, also was isolated. These observations indicate that ofloxacin resistance activates biosynthetic genes on both chromosomes and endogenous plasmids. We also identified the mutations that are probably involved in the phenotype of ofloxacin resistance and secondary metabolite overproduction in S. coelicolor A3(2). Furthermore, we observed an interesting phenomenon in which several ofloxacin-resistant mutants overproduced antibiotics in the presence of ofloxacin. Based on these results, we present the unique physiological and genetic characteristics of ofloxacin-resistant Streptomyces mutants and discuss the importance and potential development of the new findings. IMPORTANCE The abuse or overuse of antibacterial agents for therapy and animal husbandry has caused an increased population of antimicrobial-resistant bacteria in the environment. Consequently, fewer effective antimicrobials are now available. Due to the depleted antibiotic pipeline, pandemic outbreaks caused by antimicrobial-resistant bacteria are deeply concerning, and the development of new antibiotics is now an urgent issue. Promising sources of antimicrobial agents include cryptic biosynthetic gene clusters for secondary metabolites in streptomycetes and rare actinomycetes. This study's significance is the development of an unprecedented activation method to accelerate drug discovery research on a global scale. The technique developed in this study could allow for simultaneous drug discovery in different countries, maximizing the world's microbial resources.

Heterologous expression of a cryptic gene cluster from a marine proteobacterium Thalassomonas actiniarum affords new lanthipeptides thalassomonasins A and B.

AIMS: The aim of this study was to utilize a cryptic biosynthetic gene cluster (BGC) of a marine proteobacterium Thalassomonas actiniarum for production of new lanthipeptides by heterologous expression system. METHODS AND RESULTS: Based on genome mining, a new BGC of class I lanthipeptide was found in the genome sequence of a marine proteobacterium T. actiniarum. Molecular cloning was performed to construct an expression vector derived from commercially available plasmid pET-41a(+). Heterologous production of new lanthipeptides named thalassomonasins A and B was performed using the host Escherichia coli BL21(DE3) harbouring the expression vector. The structure of thalassomonasin A was determined by the interpretation of NMR and MS data. As a result, thalassomonasin A was determined to be a lanthipeptide with three units of lanthionine. The bridging pattern of the lanthionine rings in thalassomonasin A was determined by interpretation of NOESY data. The structure of thalassomonasin B was proposed by MS/MS experiment. CONCLUSIONS: We succeeded in heterologous production of new class I lanthipeptides using a BGC of a marine proteobacterium T. actiniarum. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of heterologous production of lanthipeptides derived from proteobacterial origin. There are many cryptic biosynthetic gene clusters (BCGs) of this class of lanthipeptides in proteobacterial genomes. This study may lead to the production of new lanthipeptides by utilizing the BCGs.

Heterologous production of new lanthipeptides hazakensins A and B using a cryptic gene cluster of the thermophilic bacterium Thermosporothrix hazakensis.

The thermophilic bacterium Thermosporothrix hazakensis belongs to a class of Ktedonobacteria in the phylum Chloroflexota. Lanthipeptides are a naturally occurring peptide group that contains antibacterial compounds such as nisin. To find a new lanthipeptide that is a possible candidate for an antibacterial reagent, we performed genome-mining of T. hazakensis and heterologous expression experiments. Based on genome-mining, the presence of a total of ten putative biosynthetic gene clusters for class I and class II lanthipeptides was indicated from the genome sequence of T. hazakensis. New lanthipeptides named hazakensins A and B were produced by heterologous expression of a class I lanthipeptide biosynthetic gene cluster in the expression host Escherichia coli. Co-expression of the biosynthetic gene cluster with tRNA-Glu and glutamyl-tRNA synthetase coding genes derived from T. hazakensis increased the production yield of both lanthipeptides by about 4-6 times. The chemical structures of hazakensins A and B including the bridging pattern of lanthionine/methyllanthionine rings were determined by NMR and MS experiments. Since production of hazakensins A and B was not observed in the native strain T. hazakensis, heterologous production was an effective method to obtain the lanthipeptides derived from the biosynthetic gene cluster. This is the first report of heterologous production of class I lanthipeptides originating from the filamentous green non-sulfur bacteria, to the best of our knowledge. The success of heterologous production of hazakensins may lead to the discovery and development of new lanthipeptides derived from the origins of bacteria in the phylum Chloroflexota.

Heterologous expression of a cryptic gene cluster from Marinomonas fungiae affords a novel tricyclic peptide marinomonasin.

The ω-ester-containing peptides (OEPs) are a group of ribosomally synthesized and post-translationally modified peptides (RiPPs). The biosynthetic gene clusters of ω-ester-containing peptides commonly include ATP-grasp ligase coding genes and are distributed over the genomes of a wide variety of bacteria. A new biosynthetic gene cluster of ω-ester-containing peptides was found in the genome sequence of the marine proteobacterium Marinomonas fungiae. Heterologous production of a new tricyclic peptide named marinomonasin was accomplished using the biosynthetic gene cluster in Escherichia coli expression host strain BL21(DE3). By ESI-MS and NMR experiments, the structure of marinomonasin was determined to be a tricyclic peptide 18 amino acids in length with one ester and two isopeptide bonds in the molecule. The bridging patterns of the three intramolecular bonds were determined by the interpretation of HMBC and NOESY data. The bridging pattern of marinomonasin was unprecedented in the ω-ester-containing peptide group. The results indicated that the ATP-grasp ligase for the production of marinomonasin was a novel enzyme possessing bifunctional activity to form one ester and two isopeptide bonds. KEY POINTS: • New tricyclic peptide marinomonasin was heterologously produced in Escherichia coli. • Marinomonasin contained one ester and two isopeptide bonds in the molecule. • The bridging pattern of intramolecular bonds was novel.

Isolation and structure determination of new linear azole-containing peptides spongiicolazolicins A and B from Streptomyces sp. CWH03.

Linear azole-containing peptides are a class of ribosomally synthesized and post-translationally modified peptides. We performed a chemical investigation on marine actinomycetes, and new linear azole-containing peptides named spongiicolazolicins A and B were found in the MeOH extracts of a newly isolated strain Streptomyces sp. CWH03 (NBRC 114659) and two strains of S. spongiicola (strain HNM0071T: DSM 103383T and strain 531S: NBRC 113560). The strain Streptomyces sp. CWH03 was indicated to be a new species closely related to S. spongiicola by phylogenetic analysis using the genome sequence. The new peptides named spongiicolazolicins A and B were isolated from the cell of Streptomyces sp. CWH03. The partial structure of spongiicolazolicin A was determined by 2D NMR experiments. Based on data of MS/MS experiments, the chemical structures of spongiicolazolicins A and B were proposed using the amino acid sequence deduced from the precursor-encoding gene, which was found from whole-genome sequence data of Streptomyces sp. CWH03. The biosynthetic gene cluster of spongiicolazolicins was proposed based on comparative analysis with that of a known linear azole peptide goadsporin. KEY POINTS: • Streptomyces sp. CWH03 was a new species isolated from marine sediment. • New linear azole-containing peptides named spongiicolazolicins A and B were isolated. • Biosynthetic pathway of spongiicolazolicins was proposed.

Heterologous production of new protease inhibitory peptide marinostatin E.

Bicyclic peptides, marinostatins, are protease inhibitors derived from the marine bacterium Algicola sagamiensis. The biosynthetic gene cluster of marinostatin was previously identified, although no heterologous production was reported. In this report, the biosynthetic gene cluster of marinostatin (mstA and mstB) was cloned into the expression vector pET-41a(+). As a result of the coexpression experiment, a new analogous peptide named marinostatin E was successfully produced using Escherichia coli BL21(DE3). The structure of marinostatin E was determined by a combination of chemical treatments and tandem mass spectrometry experiments. Marinostatin E exhibited inhibitory activities against chymotrypsin and subtilisin with an IC50 of 4.0 and 39.6 µm, respectively.

Heterologous expression of a cryptic gene cluster from Grimontia marina affords a novel tricyclic peptide grimoviridin.

Microviridins are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that have been isolated from a wide variety of cyanobacterial strains. There are similar gene clusters of RiPPs distributed in the genomes of bacteria belonging to the phyla Proteobacteria and Bacteroidetes. A cryptic gene cluster for the production of microviridin-type peptide was found in the genome of the marine γ-Proteobacterium Grimontia marina. Heterologous production of new microviridin-type peptide named grimoviridin was accomplished in Escherichia coli using the biosynthetic gene cluster of G. marina. The structure of grimoviridin was determined by analysis of MS and NMR data. Grimoviridin contained one isopeptide and two ester bonds, which had exactly the same bridging pattern as other microviridin-type peptides. The absolute stereochemistries of constituent amino acids were determined to be all L-forms by modified Marfey's method. Grimoviridin showed potent inhibitory activity against trypsin with an IC50 value of 238 nM. This is the first report of heterologous production of microviridin-type peptide using a biosynthetic gene cluster from a Proteobacterium. Key points • Heterologous production afforded new microviridin-type peptide named grimoviridin. • This is the first report of microviridin-type peptide from proteobacterial origin. • Grimoviridin showed potent inhibitory activity against trypsin.

How to harness biosynthetic gene clusters of lasso peptides.

Lasso peptides produced by bacteria have a very unique cyclic structure ("lasso" structure) and are resistant to protease. To date, a number of lasso peptides have been isolated from proteobacteria and actinobacteria. Many lasso peptides exhibit various biological activities, such as antibacterial activity, and are expected to have various applications. Based on study of genome mining, large numbers of biosynthetic gene cluster of lasso peptides are revealed to distribute over genomes of proteobacteria and actinobacteria. However, the biosynthetic gene clusters are cryptic in most cases. Therefore, the combination of genome mining and heterologous production is efficient method for the production of lasso peptides. To utilize lasso peptide as fine chemical, there have been several attempts to add new function to lasso peptide by genetic engineering. Currently, a more efficient lasso peptide production system is being developed to harness cryptic biosynthetic gene clusters of lasso peptide. In this review, the overview of lasso peptide study is discussed.

Isolation and structure determination of a new lasso peptide specialicin based on genome mining.

Based on genome mining, a new lasso peptide specialicin was isolated from the extract of Streptomyces specialis. The structure of specialicin was established by ESI-MS and NMR analyses to be a lasso peptide with the length of 21 amino acids, containing an isopeptide bond and two disulfide bonds in the molecule. The stereochemistries of the constituent amino acids except for Trp were determined to be L and the stereochemistry of Trp at C-terminus was determined to be D. Three dimensional structure of specialicin was determined based on NOE experimental data, which indicated that specialicin possessed the similar conformational structure with siamycin I. Specialicin showed the antibacterial activity against Micrococcus luteus and the moderate anti-HIV activity against HIV-1 NL4-3. The biosynthetic gene cluster of specialicin was proposed from the genome sequence data of S. specialis.

Heterologous production of a new lasso peptide brevunsin in Sphingomonas subterranea.

A shuttle vector pHSG396Sp was constructed to perform gene expression using Sphingomonas subterranea as a host. A new lasso peptide biosynthetic gene cluster, derived from Brevundimonas diminuta, was amplified by PCR and integrated to afford a expression vector pHSG396Sp-12697L. The new lasso peptide brevunsin was successfully produced by S. subterranea, harboring the expression vector, with a high production yield (10.2 mg from 1 L culture). The chemical structure of brevunsin was established by NMR and MS/MS experiments. Based on the information obtained from the NOE experiment, the three-dimensional structure of brevunsin was determined, which indicated that brevunsin possessed a typical lasso structure. This expression vector system provides a new heterologous production method for unexplored lasso peptides that are encoded by bacterial genomes.

Isolation and structure determination of a new lantibiotic cinnamycin B from Actinomadura atramentaria based on genome mining.

New lantibiotic cinnamycin B was isolated from the extract of Actinomadura atramentaria NBRC 14695(T), based on genome mining and chemical investigation. The partial structure of cinnamycin B was established by 2D NMR experiments, which indicated that cinnamycin B had same methyl lanthionine bridging pattern with cinnamycin. The reduction with NaBH4-NiCl2 afforded the reduced cinnamycin B, and MS/MS experiment indicated the presence of hydroxy asparatic acid in the molecule. Cinnamycin B showed an antibacterial activity against Streptomyces antibioticus with dosage of 5 µg (0.5µL, 10 mg/mL solution) at spot-on-lawn testing method. The gene cluster of cinnamycin B on the genome of A. atramentaria was identified and discussed in comparison with that of cinnamycin.

Structure determination of a siderophore peucechelin from Streptomyces peucetius.

Previously, Park et al. isolated a new siderophore from Streptomyces peucetius ATCC 27952 based on information of the genome sequence and the structure of the siderophore was deduced to be a cyclic peptide based on MS/MS analysis. To clarify the structure of the siderophore, we cultured S. peucetius with iron deficient medium. Through several chromatographic procedures, the siderophore named peucechelin was isolated with the yield enough to perform NMR experiments. The planar structure of peucechelin was elucidated by the combination of ESI-MS experiment and NMR spectroscopic analyses of the gallium (III) complex. Unlike the previously deduced cyclic structure, the structure was determined to be a linear peptide which was similar to a known siderophore foroxymithine. The stereochemistries of amino acids constituting peucechelin were determined by applying modified Marfey method to the hydrolysate. Since the biosynthetic gene of peucechelin was formerly determined by Park et al. the similar genes were searched using genome data of other streptomycetes. As a result, the similar genes were found in the genome data of S. venezuelae and S. purpureus. Isolation and identification of siderophore was performed from the iron deficient culture of S. venezuelae. The siderophore of S. venezuelae was identified to be known compound foroxymithine by analysis ESI-MS and NMR spectra in the similar manner with peucechelin. Production of foroxymithine was also observed in the iron deficient culture of S. purpureus. Based on the genome data, comparison of the biosynthetic genes of structurally related siderophores peucechelin and foroxymithine was accomplished in discussion.

Isolation and structure determination of new siderophore albachelin from Amycolatopsis alba.

A new siderophore named albachelin was isolated from iron deficient culture of Amycolatopsis alba. The planar structure of albachelin was elucidated by the combination of ESI-MS/MS experiment and NMR spectroscopic analyses of the gallium (III) complex. The structure of albachelin was determined to be a linear peptide consisting of 6 mol of amino acids including 3 mol of serine, one mol each of N-α-acethyl-N-δ-hydroxy-N-δ-formylornithine, N-α-methyl-N-δ-hydroxyornithine, and cyclic N-hydroxyornithine. The stereochemistries of amino acids constituting albachelin were analyzed by applying modified Marfey method to the hydrolysate of albachelin. Based on bioinformatics, we deduced and discussed the possible biosynthetic gene cluster involved in albachelin biosynthesis from the genome sequence of A. alba. By prediction of substrates for adenylation domains, a non-ribosomal peptide biosynthetase gene (AMYAL_RS0130210) was proposed to be the main biosynthetic gene for albachelin biosynthesis. The related genes including transporter for siderophore were found near the NRPS gene as a gene cluster.

Isolation and structure determination of a new depsipeptide crocapeptin C from the myxobacterium Melittangium boletus.

A new cyclic depsipeptide, crocapeptin C (1), containing 3-amino-6-hydroxy-2-piperidone (Ahp) was isolated from the methanol extract of the myxobacterium Melittangium boletus. The chemical structure of crocapeptin C (1) was determined through NMR and ESI-MS analysis. The stereochemistries of the constituent amino acids in crocapeptin C (1) were determined using the advanced Marfey's method and ROESY spectrum data. Crocapeptin C (1) exhibited potent inhibitory activity against chymotrypsin with an IC50 value of 0.5 µM.

Heterologous production of a new lanthipeptide boletupeptin using a cryptic biosynthetic gene cluster of the myxobacterium Melittangium boletus.

Myxobacteria have comparatively large genomes that contain many biosynthetic genes with the potential to produce secondary metabolites. Based on genome mining, we discovered a new biosynthetic gene cluster of class III lanthipeptide in the genome of the myxobacterium Melittangium boletus. The biosynthetic gene cluster contained a precursor peptide-coding gene bolA, and a class III lanthipeptide synthetase-coding gene bolKC. The expression vector containing bolA and bolKC was constructed using synthetic DNA with codon-optimized sequences based on the commercially available vector pET29b. Co-expression of the two genes in the host Escherichia coli BL21(DE3) yielded a new class III lanthipeptide named boletupeptin. The structure of boletupeptin was proposed to have one unit of labionin, as determined by mass spectrometry experiments after reductive cleavage. This is the first report of a class III lanthipeptide from a myxobacterial origin.

Structure and biosynthesis of scabichelin, a novel tris-hydroxamate siderophore produced by the plant pathogen Streptomyces scabies 87.22.

Scabichelin and turgichelin, novel tris-hydroxamate siderophores, were isolated from Streptomyces antibioticus NBRC 13838/Streptomyces scabies JCM 7914 and Streptomyces turgidiscabies JCM 10429, respectively. The planar structures of scabichelin and turgichelin were elucidated by mass spectrometry, and 1- and 2-D NMR spectroscopic analyses of their gallium(III) complexes. The relative and absolute stereochemistry of the metabolites was determined by the modified Marfey's method in conjunction with computational modelling and NOESY NMR analysis of Ga-scabichelin and Ga-turgichelin. Genome sequence analysis of the plant pathogen Streptomyces scabies 87.22 identified a gene cluster containing a gene encoding a nonribosomal peptide synthetase (NRPS) that was predicted to direct the production of a pentapeptide with structural similarities to scabichelin and turgichelin. Comparative LC-MS/MS analyses of iron-deficient culture supernatants from wild type S. scabies 87.22 and a mutant in which the NRPS gene had been disrupted, and scabichelin purified from S. antibioticus, showed that scabichelin is the metabolic product of the cryptic gene cluster, strongly suggesting that it functions as a siderophore.

Isolation and structural determination of makinolide B from Streptomyces sp. MK-19.

A new 16-membered macrolide named makinolide B (1) was isolated from Streptomyces sp. MK-19. The structure of makinolide B (1) was determined on the basis of 2D NMR experiments involving DQF-COSY, TOCSY, HSQC, and HMBC methods. Application of the paper disk diffusion method to makinolide B (1) showed weak antibacterial activity against Staphylococcus aureus at the dose of 100 µg/disk.

Heterologous Production and Structure Determination of a New Lanthipeptide Sinosporapeptin Using a Cryptic Gene Cluster in an Actinobacterium Sinosporangium siamense.

Lipolanthine is a subclass of lanthipeptide that has the modification of lipid moiety at the N-terminus. A cryptic biosynthetic gene cluster comprising four genes (sinA, sinKC, sinD, and sinE) involved in the biosynthesis of lipolanthine was identified in the genome of an actinobacterium Sinosporangium siamense. Heterologous coexpression of a precursor peptide coding gene sinA and lanthipeptide synthetase coding gene sinKC in the host Escherichia coli strain BL21(DE3) resulted in the synthesis of a new lanthipeptide, sinosporapeptin. It contained unusual amino acids, including one labionin and two dehydrobutyrine residues, as determined using NMR and MS analyses. Another coexpression experiment with two additional genes of decarboxylase (sinD) and N-acetyl transferase (sinE) resulted in the production of a lipolanthine-like modified sinosporapeptin.

Streptomyces pacificus sp. nov., a novel spongiicolazolicin-producing actinomycete isolated from a coastal sediment.

A polyphasic approach was used to determine the taxonomic position of a marine actinomycete, designated isolate CWH03T, which we previously reported to produce new linear azole-containing peptides spongiicolazolicins A and B. Strain CWH03T is mesophilic, neutrophilic, and halotolerant streptomycete that forms spiral spore chains on aerial mycelium. Comparative 16S rRNA gene sequencing showed that CWH03T was most closely related to Streptomyces tirandamycinicus HNM0039T (99.7%), Streptomyces spongiicola HNM0071T (99.4%), 'Streptomyces marianii' ICN19T (99.1%) and Streptomyces wuyuanensis CGMCC4.7042T (99.0%). The phylogenetic tree prepared using the 16S rRNA gene, as well as the phylogenomic tree using the genome BLAST distance phylogeny method and 81 core housekeeping genes, respectively, showed that the closest relative of strain CWH03T was S. spongiicola HNM0071T. The average nucleotide identity and digital DNA-DNA hybridization values between strains CWH03T and S. spongiicola HNM0071T were 91.46% and 44.2%, respectively, which were below the thresholds of 96% and 70% for prokaryotic conspecific assignation. The G+C content of the genomic DNA of strain CWH03T was 72.3%. Whole-cell hydrolysates of strain CWH03T contained LL-diaminopimelic acid. The predominant menaquinone was MK-9(H8) (88.3%), and the major fatty acids were iso-C16:0 (28.4%), anteiso-C15:0 (15.0%) and iso-C15:0 (12.9%). The major phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid. Based on data obtained from phenotypic, phylogenetic, genomic, and chemotaxonomic analyses, strain CWH03T represents a novel species of the genus Streptomyces, for which the proposed name is Streptomyces pacificus sp. nov. The type strain is CWH03T ( = NBRC 114659T = TBRC 15780T).