Based on genome mining, a new antibacterial peptide named actinokineosin was isolated from a rare actinomycete Actinokineospora spheciospongiae. The amino acid sequence of the C‐terminus of ...actinokineosin was established by TOF‐MS/MS experiments. The amino acid sequence in the macrolactam ring was determined by TOF‐MS/MS analyses after cleavage with BNPS‐skatole and successive trypsin treatment. As a result of an antibacterial assay using a paper disk, actinokineosin showed antibacterial activity against Micrococcus luteus at a dosage of 50 μg per disk. From the genome sequence data of A. spheciospongiae, the biosynthetic gene cluster of actinokineosin was found and was indicated to consist of 10 genes. Among the genes, the gene aknA encoded the precursor of actinokineosin and the genes including aknC, aknB1 and aknB2 were proposed as modification enzymes to give mature actinokineosin.
Significance and Impact of the Study
Genome mining is a powerful tool to find new bioactive compounds from the genome database. In this report, we succeeded in isolation and structure determination of a new antibacterial peptide named actinokineosin based on genome mining.
Significance and Impact of the Study: Genome mining is a powerful tool to find new bioactive compounds from the genome database. In this report, we succeeded in isolation and structure determination of a new antibacterial peptide named actinokineosin based on genome mining.
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
.
A new antibacterial peptide named pentaminomycin C was isolated from an extract of Streptomyces cacaoi subsp. cacaoi NBRC 12748
, along with a known peptide BE-18257A. Pentaminomycin C was determined ...to be a cyclic pentapeptide containing an unusual amino acid, Nδ-hydroxyarginine (5-OHArg), by a combination of ESI-MS and NMR analyses. The structure of pentaminomycin C was determined to be cyclo(-L-Leu-D-Val-L-Trp-L-5-OHArg-D-Phe-). Pentaminomycin C exhibited antibacterial activities against Gram-positive bacteria including Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus. The biosynthetic gene cluster for pentaminomycin C and BE-18257A was identified from the genome sequence data of S. cacaoi subsp. cacaoi.
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 IC
50
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.
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.
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.
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