Isolation and identification of a new lasso peptide cattlecin from Streptomyces cattleya based on genome mining

Lasso peptides are ribosomally synthesized and posttranslationally modified peptides with diverse biological functions. Recent genome mining has revealed that many species of actinomycetes possibly contain biosynthetic gene clusters of lasso peptides. With genome mining for lasso peptide biosynthesis, we screened several actinomycetes for lasso peptide production using high-performance liquid chromatography and electrospray ionization–mass spectrometry. Consequently, Streptomyces cattleya was identified as a producer of a new lasso peptide named cattlecin. Analysis of amino acid content on cattlecin indicated the presence of four moles each of Asp and His, three moles each of Gly and Tyr, and one mole of Ser. Tandem mass spectrometry (MS/MS) analysis of cattlecin revealed C-terminal sequence of WHHGWYGWWDD. The peptide sequence (SYHWGDYHDWHHGWYGWWDD) was the expected amino acid sequence of cattlecin based on genome mining. As a result of MS/MS analysis, the amine residue of the first Ser was proposed to form a macrolactam ring with the β-carboxyl residue of the ninth Asp. The biosynthetic gene cluster of cattlecin comprised four genes: catA, catC, catB1, and catB2, which is typical of a lasso peptide biosynthetic gene cluster in actinomycetes.


Introduction
Lasso peptides are ribosomally synthesized and posttranslationally modified peptides with biological activities, including antibacterial properties, and have the unique knot structure of a ''lasso'' in common [1]. The amino group of the N-terminal amino acid of lasso peptides forms a peptide bond with bor c-carboxyl group of Asp or Glu in the eighth or the ninth position from the N terminus, resulting in the formation of a macrolactam ring. The macrolactam ring appears as a loop of a ''lasso,'' with a tail of the C-terminal linear peptide that normally locates through the macrolactam ring. Due to this unique structure, lasso peptides are stable against proteolytic degradation and high temperature. Recently, genome mining approaches have been used to identify new lasso peptides. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)-MS analysis identified the lasso peptide SRO15-2005 from the extract of Streptomyces roseosporus NRRL 15998 based on genome mining [2]. This prompted us to find new lasso peptides in actinomycetes using the genome mining approach. Thus, we identified a new lasso peptide cattlecin in Streptomyces cattleya. The structure of cattlecin was analyzed by a combination of amino acid content analysis

Isolation of cattlecin
Streptomyces cattleya was cultured using 10 L of ISP2 agar medium for 7 days at 30°C. The aerial hyphae and spore cells on the agar surface were harvested with steel spatula. Double volume of MeOH was added to the harvested cells, followed by filtration with filter paper (Whatman No. 1, GE Healthcare Life Sciences, Little Chalfont, UK). The MeOH extract was concentrated to an aqueous residue using rotary evaporator. The aqueous residue was subjected to opencolumn chromatography using hydrophobic resin CHP-20P (Mitsubishi Chemical, Tokyo, Japan), eluted with 10% MeOH, 60% MeOH, and 100% MeOH. The 60% MeOH fraction was subjected to high-performance liquid chromatography (HPLC) purification using ODS column (4.6 9 250 mm, Wakopak Handy-ODS, Wako Pure Chemical Industries, Ltd., Osaka, Japan) with gradient elution from 10 to 60% MeCN containing 0.05% trifluoroacetic acid for 20 min with UV detector set at 220 nm to yield 2.0 mg of cattlecin (retention time 14.9 min).

Mass spectrometry
The electrospray ionization (ESI) TOF mass spectrum was recorded using a JEOL JMS-T100LP mass spectrometer (JEOL, Tokyo, Japan). MALDI-TOF/TOF mass spectra were recorded on a 4800 plus MALDI-TOF/TOF analyzer (AB SCIEX, Redwood City, CA, USA) using a-cyano-4hydroxycinnamic acid (Bruker Daltonics, Billerica, MA, USA) as the matrix in the positive-ion mode with an acceleration voltage of 20 kV. The mass spectrometer was tuned and calibrated using a commercially available standard peptide mixture (Peptide Calibration Standard II, Bruker Daltonics) prior to the measurement. The ESI Fourier-transform ion cyclotron resonance mass spectrum was recorded on the ApexII 70e mass spectrometer (Bruker Daltonics). The sample solution was infused into the ESI source directly at 2-5 lL min -1 using a syringe pump after dilution by 50% methanol. The accurate mass measurement was recorded in the presence of the internal standard YOKUDELNA (JEOL, Tokyo, Japan).

Amino acid analysis
Following the previous report [3], amino acid content of cattlecin was determined.

Antimicrobial test
Following the previous report [3], antimicrobial activity of cattlecin was evaluated.

Results
A lasso peptide sviceucin was recently identified by genome mining, and it was isolated via heterologous expression [4]. Using BLAST similarity search of the amino acid sequence of sviceucin (accession number: EDY58505.1), we found five sviceucin analog peptides, as shown in Fig. 1. The leader peptide sequences (underlined in Fig. 1) that were deduced to be cleaved off during posttranslational modification had the conserved amino acid motif, indicated in boldface in Fig. 1. As a comparison of amino acid sequences of the peptides in Fig. 1, the structural peptide parts started with Cys, Ser, or Gly, and Asp was present at the seventh, eighth, or ninth position. Among the strains of possible lasso peptide producers shown in Fig. 1 To isolate sufficient quantity of cattlecin for structural determination, S. cattleya was cultivated with 1 L of ISP2 agar media. After 7 days of cultivation, spore cells and aerial hyphae were harvested using a steel spatula. The harvested cells were extracted with double the volume of MeOH, followed by centrifugation. After condensation using a rotary evaporator, the extract was purified via opencolumn chromatography using hydrophobic resin CHP-20P (Mitsubishi Chemical), eluted with 10, 60, and 100% of MeOH. The 60% MeOH fraction was repeatedly purified using HPLC to obtain pure cattlecin.
The molecular formula of cattlecin was determined to be C 131 H 139 N 33 O 32 by analysis using ESI-FTCR mass spectrometry ([M ? 3H] 3? at m/z 896.3489; the calculated value was 896.3494). The amino acid content of cattlecin was analyzed as described previously [3]. As a result, amino acid content analysis of cattlecin indicated the presence of 4 mol each of Asp and His, 3 mol each of Gly and Tyr, and 1 mol of Ser at mole rate. The attempt to measure NMR spectra using DMSO-d 6 as a solvent was unsuccessful due to ambiguous broad peaks. To determine the peptide sequence, MALDI-TOF MS/MS analysis was used as described previously [5]. The product ions formed by cattlecin as determined by MALDI-TOF MS/MS were of b-series peptides b9-b18 and of y-series peptides y9 and y10 (Fig. 2). The macrolactam ring structure was reported not to produce fragmented ions [2], so we deduced the structure of cattlecin as shown in Fig. 2, considering the amino acid sequence of the prepeptide gene (CCB72812.1). Considering that the cattlecin-analogous peptide sviceucin [4] contains an isopeptide bond between the amine residue of Cys1 and the b-carboxyl residue of Asp8, we assumed that cattlecin may also possess a similar topology in terms of biosynthesis.
The antibiotic paper disk method was performed as an antimicrobial test for cattlecin against many microorganisms as described previously [3]. The testing microorganisms included bacterial strains, such as E. coli, P. aeruginosa, B. subtilis, S. aureus, and M. luteus; yeast strains, such as S. cerevisiae and K. apiculata; and fungal strains, such as A. niger, A. oryzae, and M. hiemalis. However, cattlecin, at a dose of 50 lg disk -1 , did not show antimicrobial activity against any of the testing microorganisms.

Discussion
Cattlecin was a highly hydrophobic peptide that contained total of eight hydrophobic amino acids (5 mol of Trp and 3 mol of Tyr) in 20 amino acid residues. In addition to that, cattlecin contained 4 units of Asp in the molecule, which gave cattlecin the characteristic of acidic peptide at biological pH. To find similar peptide to cattlecin, BLASTP search using core peptide amino acid sequence of cattlecin (SYHWGDYHDWHHGWYGWWDD) was performed. However, no similar peptide was found in the database. Although leader peptide sequence was conserved among lasso peptides of actinomycetes ( Fig. 1), the core peptide sequences were very diverse. A lasso peptide microcin J25 was isolated from E. coli [6], and its biosynthesis has been well studied as a model for lasso peptide biosynthesis. The biosynthetic gene cluster of microcin J25 consists of four genes, including a precursor peptide ( [12]. Normally, lasso peptide biosynthetic genes of proteobacteria have the same set of the above-mentioned four genes, although some gene clusters lack the transporter gene D [8]. The protein McjC is involved in the formation of the macrolactam ring, and the function of protein McjB was found to be cleaving of the leader peptide from the prepeptide, as deduced via in vitro experiments [9]. In actinomycetes, lasso peptide biosynthetic genes consist of a similar gene set, except that a maturation enzyme B is encoded by split-B genes: gene B1 and gene B2 [8]. So far, biosynthetic gene clusters of lasso peptides, such as lariatin, [10] SRO15-2005 [2], lassomycin [11], sviceucin [4], and streptomonomicin [12], have been identified. By referring to such lasso peptide biosynthetic genes [10], we found the possible biosynthetic gene cluster of cattlecin, which consists of four genes: catA (accession number: CCB72812.1, 44 aa), catC (CCB72813.1, 605 aa), catB1 (CCB72814.1, 67 aa), and catB2 (CCB72815.1, 143 aa) from the genome sequence of S. cattleya. Among the lasso peptides of actinomycetes, the function of modification genes in lariatin has been well studied [10,13]. Based on the similarity of each cattlecin gene to lariatin biosynthetic genes, larA, larB, larC, and larD, we proposed the functions of the genes as shown in Fig. 3. The gene catA encodes a precursor of cattlecin, and catB1, catB2, and catC encode modification enzymes that produce the mature lasso peptide. The gene catC encodes a putative asparagine synthetase possibly responsible for the formation of the Gly1-Glu9 isopeptide bond, which showed high similarity to larB, as determined via a BLAST homology search (28% identity; 45% positive matches). The gene catB1 showed a high similarity to larC as seen via BLAST homology search (37% identity; 53% positive matches). Although larC has been indicated to be essential for lariatin biosynthesis, as deduced by a gene-disruption experiment [10], its function remains unclear. BLAST homology search also showed high similarity between catB2 and larD (43% identity; 55% positive matches). We deduce that catB2 may be involved in the processing of the leader peptide because the sequence possesses the Cys-His-Asp catalytic triad, which is essential for serine protease similar to larD [10]. The gene for mature peptide exporter corresponding to larE was not found in/near the biosynthetic gene cluster of cattlecin.