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PreprintCited by 3Microbial Natural Products and BiosynthesisAntimicrobial Peptides and ActivitiesChemical Synthesis and Analysis

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Genome Mining Reveals the Structure of the Clostrisin and Cellulosin Biosynthetic Gene Clusters, Two Novel Lantibiotics

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Alejo Hernandez, Moisés Alejandro, Villavicencio Sánchez, Katia Pamela, Sánchez Morales, Rosendo, Hernández-Magro Gil, Karla Georgina, Moreno-Gutiérrez, David Silverio, Sanchez-Rueda, Eddie Guillermo, Teresa-Cruz, Yanet, Hernández Garcia, Armando, Romero-Rodríguez, Alba, Juárez, Oscar, Martínez-Caballero, Siseth, Figueroa, Mario, Ceapa, Corina Diana·2023·Beilstein Journal of Organic Chemistry
DOI 10.3762/bxiv.2023.56.v1PMC PMC11301040

Preprint — published as 10.3762/bjoc.20.159 · Paper page

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Abstract

Antimicrobial resistance is an issue of enormous proportions that poses a significant threat to global public health. This study focuses on lanthipeptides, ribosomally encoded peptides which display significant structural diversity and hold promising potential as antibiotics. Genome mining was employed to locate biosynthetic gene clusters (BGC) for class II lanthipeptides. A phylogenetic study analyzing homologous sequences of functional LanM sequences revealed a unique evolutionary clade of 39 LanMs associated with 28 Clostridial bacterial genomes. In silico exploration identified nine complete BGCs, including one from Clostridium cellulovorans 743B, that harbors two new lanthipeptides: Clostrisin and Cellulosin. The lanthipeptide BGCs were heterologously expressed in Escherichia coli. Molecular weights associated with the expected post-translational modifications of the purified lanthipeptides were confirmed by MALDI-TOF. Both peptides demonstrated antimicrobial activity against multidrug-resistant bacteria, such as a clinical strain of Staphylococcus epidermidis MIQ43 and a reference strain of resistant Pseudomonas aeruginosa PA14. This study showcases the immense potential of genome mining in identifying new class II lanthipeptides.

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Figures

Figure 1 Phylogenetic trees of the LanM synthetase amino acid sequences. Unrooted phylogenetic tree of all the homologous LanM sequences identified by BLAST in our study and those in MIBiG (previously characterized). This analysis involved 343 amino acid sequences; there were a total of 1964 positions in the final dataset. The phylogenetic tree was built from the alignments using the Neighbor-Joining [43], and the evolutionary distances were calculated using the Poisson correction method. A LanL class IV lanthionine synthetase from the venezuelin cluster [44] was used as an outgroup. Square symbols refer to previously characterized LanM sequences from the MIBiG database.
Figure 1 Phylogenetic trees of the LanM synthetase amino acid sequences. Unrooted phylogenetic tree of all the homologous LanM sequences identified by BLAST in our study and those in MIBiG (previously characterized). This analysis involved 343 amino acid sequences; there were a total of 1964 positions in the final dataset. The phylogenetic tree was built from the alignments using the Neighbor-Joining [43], and the evolutionary distances were calculated using the Poisson correction method. A LanL class IV lanthionine synthetase from the venezuelin cluster [44] was used as an outgroup. Square symbols refer to previously characterized LanM sequences from the MIBiG database.CC BY · PubMed Central
Figure 2 The phylogenetic tree was built by concatenating 1000 shared clostridial genes (left) between the studied genomes, and the lanthipeptide class II synthetase clusters from the selected BGCs of this taxonomic clade are presented (right). The highlighted cluster was chosen for heterologous expression.
Figure 2 The phylogenetic tree was built by concatenating 1000 shared clostridial genes (left) between the studied genomes, and the lanthipeptide class II synthetase clusters from the selected BGCs of this taxonomic clade are presented (right). The highlighted cluster was chosen for heterologous expression.CC BY · PubMed Central
Figure 3 A. Similarity network created with the ESI web tool with the precursor peptide amino acid sequences. In the similarity network of precursor peptides, each blue node represents a characterized lanthipeptide, and each pink node represents the precursor peptides identified in this work. Each line represents a minimum of 27% of sequence identity (EFI – enzyme similarity tool [51]). B. Alignment of clostrisin with FlvA.2g [43], enterocin Wβ [44], plantaricin Wβ [45], and thusin α [46]. C. Alignment of cellulosin with lichenicidin A1 [47], FlvA.1 [43], FlvA.2 [43], BhtA α [48], SmB β [49], Smb α [49], and thusin β [46]. The lanthionine bridges placed on clostrisin and cellulosin are based on the experimental results of the MS2 analyses.
Figure 3 A. Similarity network created with the ESI web tool with the precursor peptide amino acid sequences. In the similarity network of precursor peptides, each blue node represents a characterized lanthipeptide, and each pink node represents the precursor peptides identified in this work. Each line represents a minimum of 27% of sequence identity (EFI – enzyme similarity tool [51]). B. Alignment of clostrisin with FlvA.2g [43], enterocin Wβ [44], plantaricin Wβ [45], and thusin α [46]. C. Alignment of cellulosin with lichenicidin A1 [47], FlvA.1 [43], FlvA.2 [43], BhtA α [48], SmB β [49], Smb α [49], and thusin β [46]. The lanthionine bridges placed on clostrisin and cellulosin are based on the experimental results of the MS2 analyses.CC BY · PubMed Central
Figure 4 Mass-spectrometric analysis of purified clostrisin and cellulosin (ESIMS spectra). A1 and A2: CloA2, the precursor peptide of cellulosin with a 6xHis tag, was analyzed. A1 shows the ion pattern of CloA2. A2 displays the specific ions with 8 charges of CloA2. GSSHHHHHHSQDPNSSSARLQKNYEELFNEVNENASLQAELNGGSIATTIVCTIAQSLLGCVGSYVLGNKGYGCTVTNECMSNCRC, average mass: 9225.12 Da − 6H2O 9147.12 Da. B1 and B2: The tryptic fragment from CloA2 was analyzed. B1 shows the ion pattern of the tryptic fragment. B2 displays the specific ions with 8 charges of the tryptic fragment. C shows the ion pattern of the tryptic fragment after iodoacetamide derivatization NYEELFNEVNENASLQAELNGGSIATTIVCTIAQSLLGCVGSYVLGNKGYGCTVTNECMSNCRC. Average mass: 6771.58 Da − 6H2O 663.58 Da + IAA 6720.58 Da. D. CloA1, precursor peptide of clostrisin with 6xHis, was analyzed: GSSHHHHHHSQDPNSSSARLQQNYESKAGFISEMELDELVSNKTVGGATTVPCAIAIIGITLSAGICPTSACSKDCPWNNC. Isotopic mass: 5843.88 Da.
Figure 4 Mass-spectrometric analysis of purified clostrisin and cellulosin (ESIMS spectra). A1 and A2: CloA2, the precursor peptide of cellulosin with a 6xHis tag, was analyzed. A1 shows the ion pattern of CloA2. A2 displays the specific ions with 8 charges of CloA2. GSSHHHHHHSQDPNSSSARLQKNYEELFNEVNENASLQAELNGGSIATTIVCTIAQSLLGCVGSYVLGNKGYGCTVTNECMSNCRC, average mass: 9225.12 Da − 6H2O 9147.12 Da. B1 and B2: The tryptic fragment from CloA2 was analyzed. B1 shows the ion pattern of the tryptic fragment. B2 displays the specific ions with 8 charges of the tryptic fragment. C shows the ion pattern of the tryptic fragment after iodoacetamide derivatization NYEELFNEVNENASLQAELNGGSIATTIVCTIAQSLLGCVGSYVLGNKGYGCTVTNECMSNCRC. Average mass: 6771.58 Da − 6H2O 663.58 Da + IAA 6720.58 Da. D. CloA1, precursor peptide of clostrisin with 6xHis, was analyzed: GSSHHHHHHSQDPNSSSARLQQNYESKAGFISEMELDELVSNKTVGGATTVPCAIAIIGITLSAGICPTSACSKDCPWNNC. Isotopic mass: 5843.88 Da.CC BY · PubMed Central
Figure 5 Growth curves of the strains with the bacterial activity of the samples. A. Precursor peptide for CloA1 and clostrisin and B. precursor peptide for CloA2 and cellulosin with the bacteria: 1. S. epidermidis MIQ43 and the 2. P. aeruginosa ATCC PA14 and 3. P. aeruginosa MIQPA25. Tests were performed in sterile polystyrene 96-well flat-bottom-shaped microtiter plates. Inoculum densities were adjusted at an OD600nm of 0.1. The optical density (OD) was measured at 600 nm after incubating the plates at 36 °C for 16 hours at 220 rpm. Three independent experiments were performed, each with three repetitions of every peptide concentration. The statistical significance was calculated with a t-test in GraphPad 8.0.2.
Figure 5 Growth curves of the strains with the bacterial activity of the samples. A. Precursor peptide for CloA1 and clostrisin and B. precursor peptide for CloA2 and cellulosin with the bacteria: 1. S. epidermidis MIQ43 and the 2. P. aeruginosa ATCC PA14 and 3. P. aeruginosa MIQPA25. Tests were performed in sterile polystyrene 96-well flat-bottom-shaped microtiter plates. Inoculum densities were adjusted at an OD600nm of 0.1. The optical density (OD) was measured at 600 nm after incubating the plates at 36 °C for 16 hours at 220 rpm. Three independent experiments were performed, each with three repetitions of every peptide concentration. The statistical significance was calculated with a t-test in GraphPad 8.0.2.CC BY · PubMed Central
Figure 6 Atomic force microscopy images (peak force mode) of S. epidermidis MIQ43 incubated with the different samples for 1 hour and 5 hours. From left to right: no treatment, CloA2 precursor peptide, cellulosin, CloA1 precursor peptide, clostrisin. All samples were evaluated at an estimated concentration of 22.4 ng/µL. Scale bar: 1 μm.
Figure 6 Atomic force microscopy images (peak force mode) of S. epidermidis MIQ43 incubated with the different samples for 1 hour and 5 hours. From left to right: no treatment, CloA2 precursor peptide, cellulosin, CloA1 precursor peptide, clostrisin. All samples were evaluated at an estimated concentration of 22.4 ng/µL. Scale bar: 1 μm.CC BY · PubMed Central

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Authors
Alejo Hernandez, M. A., Villavicencio Sánchez, K. P., Sánchez Morales, R., Hernández-Magro Gil, K. G., Moreno-Gutiérrez, D. S., Sanchez-Rueda, E. G., Teresa-Cruz, Y., Hernández Garcia, A., Romero-Rodríguez, A., Juárez, O., Martínez-Caballero, S., Figueroa, M., & Ceapa, C. D.
Affiliations
  • Universidad Nacional Autónoma de México (MX)
  • Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (MX)
  • Illinois Institute of Technology (US)
ORCID
Venue
Beilstein Journal of Organic Chemistry
Publisher
Beilstein Institut
Published
2023-12-06
Volume
20
Pages
1800-1816
ISSN
1860-5397 (ISSN-L)
DOI
10.3762/bxiv.2023.56.v1
PMC
PMC11301040
OpenAlex
W4401126041
Cited by
3
References
1
Citations by year
2026: 2 · 2025: 1
FWCI
0.85
Type
journal article
Language
en
Open access
Licence
Locations
  • Beilstein Journal of Organic Chemistry (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Consejo Nacional de Ciencia y Tecnología (CF-2022/319816, CF-2022/319596)
  • Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (project number IA201721, project number IA205122)
Topics
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
  • Life Sciences › Immunology and Microbiology › Microbiology › Antimicrobial Peptides and Activities
  • Physical Sciences › Chemistry › Organic Chemistry › Chemical Synthesis and Analysis
Keywords
lanthipeptides, Clostridium, biosynthetic gene clusters, genome mining, multidrug resistance, antimicrobial activity, Staphylococcus epidermidis, Pseudomonas aeruginosa, Clostridium cellulovorans, heterologous expression, post-translational modification, peptide structure

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CoChat. (2026). Citation check: Genome Mining Reveals the Structure of the Clostrisin and Cellulosin Biosynthetic Gene Clusters, Two Novel Lantibiotics. https://app.cochat.ai/papers/10.3762/bxiv.2023.56.v1
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Alejo Hernandez, M. A., Villavicencio Sánchez, K. P., Sánchez Morales, R., Hernández-Magro Gil, K. G., Moreno-Gutiérrez, D. S., Sanchez-Rueda, E. G., Teresa-Cruz, Y., Hernández Garcia, A., Romero-Rodríguez, A., Juárez, O., Martínez-Caballero, S., Figueroa, M., & Ceapa, C. D. (2023). Genome Mining Reveals the Structure of the Clostrisin and Cellulosin Biosynthetic Gene Clusters, Two Novel Lantibiotics. Beilstein Journal of Organic Chemistry. https://doi.org/10.3762/bxiv.2023.56.v1

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