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1.
Front Microbiol ; 15: 1337647, 2024.
Article in English | MEDLINE | ID: mdl-38435696

ABSTRACT

Circular bacteriocins form a distinct group of antimicrobial peptides (AMPs) characterized by their unique head-to-tail ligated circular structure and functional properties. They belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family. The ribosomal origin of these peptides facilitates rapid diversification through mutations in the precursor genes combined with specific modification enzymes. In this study, we primarily explored the bacteriocin engineering potential of circularin A, a circular bacteriocin produced by Clostridium beijerinckii ATCC 25752. Specifically, we employed strategies involving α-helix replacements and disulfide bond introductions to investigate their effects on both biosynthesis and bioactivity of the bacteriocin. The results show the feasibility of peptide engineering to introduce certain structural properties into circularin A through carefully designed approaches. The introduction of cysteines for potential disulfide bonds resulted in a substantial reduction in bacteriocin biosynthesis and/or bioactivity, indicating the importance of maintaining dynamic flexibility of α-helices in circularin A, while reduction of the potential disulfide in one case increased the activity. The 5 α-helices of circularin A were respectively replaced by corresponding helices from another circular peptide, enterocin AS-48, and modestly active peptides were obtained in a few cases. Overall, this study provides valuable insights into the engineering potential of circular bacteriocins as antimicrobial agents, including their structural and functional restrictions and their suitability as peptide engineering scaffolds. This helps to pave the way for the development of novel antimicrobial peptides with tailored properties based on circular bacteriocins.

2.
ACS Synth Biol ; 12(3): 852-862, 2023 03 17.
Article in English | MEDLINE | ID: mdl-36857413

ABSTRACT

Circularin A is a circular bacteriocin belonging to a subgroup of the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. The post-translational biosynthesis of circular bacteriocins primarily consists of leader cleavage, core peptide circularization, and bacteriocin secretion. However, none of these processes have been fully elucidated due to the complex biosynthesis of such bacteriocins and the lack of homology to the functions of other known biosynthetic enzymes. In this study, we investigated the leader- and terminal residue requirements for the biosynthesis of circularin A by systematic mutational analyses, including the mutational effects of variable leader lengths, as well as site-directed substitutions of residues at positions near the leader cleavage site and the circularization site. Results show that the leader with only one Met residue, the shortest leader possible, is sufficient to produce mature circularin A; helix-forming short-sidechain hydrophobic residues are required at positions Val1 and Ala2 of the N-terminus to form active peptide derivatives, indicating the possible steric hindrance effect at these two positions; and an aromatic residue is required at the C-terminal Tyr69 position to produce a mature circular derivative. However, the requirements for residues at position Ala68 are much more relaxed relative to the positions of Val1 and Ala2, since even substitution with the largest possible residue, i.e., tryptophan, still allows the generation of an active Ala68Trp derivative. Our findings provide new perspectives for the biosynthesis of this short-leader circular bacteriocin, which enables the application of circular bacteriocin biosynthesis in rational modified peptide engineering.


Subject(s)
Bacteriocins , Amino Acid Sequence , Bacteriocins/pharmacology , Peptides/genetics , Protein Processing, Post-Translational , Protein Sorting Signals/genetics
3.
Front Microbiol ; 13: 1026290, 2022.
Article in English | MEDLINE | ID: mdl-36504829

ABSTRACT

Circular bacteriocins, also known as bacterial head-to-tail cyclized peptides, are a subgroup of ribosomally synthesized and post-translationally modified peptides (RiPPs). Compared with their conventional linear counterparts, circular bacteriocins are highly stable over a broad temperature and pH range, and circularization decreases proteolytic degradation by exopeptidases. These features render them great potential as scaffold candidates to withstand strident conditions in food- and pharmaceutical applications. However, the biosynthesis and bioactivity of circular bacteriocins still remain largely unknown. To investigate and gain more insights into the biosynthesis of circular bacteriocins and to achieve efficient production and characterization of bacteriocin variants, we developed an efficient cloning and heterologous expression system for clostridial circularin A and successfully produced this circular peptide in Lactococcus lactis NZ9000. We report three system formats with single plasmid or plasmid combinations to achieve successful cloning and functional production of circularin A in L. lactis. These systematic varieties enabled us to choose the appropriate method to efficiently obtain various constructs with desired properties. With the established heterologous systems in L. lactis, we performed several mutagenesis studies in the precursor peptide to study its structure/function relationships. The overlay activity assay revealed that these mutant variants had variable effects on different indicator strains: lysine substitution for certain glutamine residue(s) greatly decreased its bioactivity against Clostridium perfringens and L. lactis NZ9000, and alanine replacement for the cationic residues significantly reduced the activity against Lactobacillus sake ATCC 15521, whereas alanine substitution for the aromatic residues decreased its bioactivity against all three testing strains dramatically. Moreover, the conditions for bacteriocin production were optimized. Results show that supplementing the minimal medium with extra glucose (or sucrose) and immediate nisin-induction improved the peptide yield significantly. Briefly, we developed an excellent system for the production of circularin A and a wide range of variant peptides in a convenient host, as well as a method for fast detection of peptide production and activity. This system facilitated our mutagenesis studies which provided valuable insights into the effects of mutating specific residues on its biosynthesis and bioactivity, and will eventually enable more complex research into the biosynthesis of circularin A.

4.
Microb Biotechnol ; 15(5): 1633-1651, 2022 05.
Article in English | MEDLINE | ID: mdl-34856064

ABSTRACT

A form of lateral membrane compartmentalization in bacteria is represented by functional membrane microdomains (FMMs). FMMs are important for various cellular processes and offer application possibilities in microbial biotechnology. We designed a lipidomics method to directly measure relative abundances of lipids in detergent-resistant and detergent-sensitive membrane fractions of the model bacterium Bacillus subtilis 168 and the biotechnologically attractive miniBacillus PG10 strain. Our study supports previous work suggesting that cardiolipin and prenol lipids are enriched in FMMs of B. subtilis. Additionally, structural analysis of acyl chains of major phospholipids indicated that FMMs display increased order and thickness compared with the surrounding bilayer. Despite the 36% genome reduction, membrane and FMM integrity are largely preserved in miniBacillus PG10, as supported by analysis of membrane fluidity, flotillin distribution and gene expression data. The novel insights in FMM architecture reported here will contribute to further explore the biological significance of FMMs and the means by which FMMs can be exploited as heterologous production platforms. Moreover, our lipidomics method enables comparative FMM lipid profiling between different bacteria.


Subject(s)
Bacillus subtilis , Detergents , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Detergents/analysis , Detergents/metabolism , Membrane Fluidity , Membrane Microdomains/chemistry , Membrane Microdomains/genetics , Membrane Microdomains/metabolism
5.
ACS Synth Biol ; 10(10): 2767-2771, 2021 10 15.
Article in English | MEDLINE | ID: mdl-34587446

ABSTRACT

To better understand cellular life, it is essential to decipher the contribution of individual components and their interactions. Minimal genomes are an important tool to investigate these interactions. Here, we provide a database of 105 fully annotated genomes of a series of strains with sequential deletion steps of the industrially relevant model bacterium Bacillus subtilis starting with the laboratory wild type strain B. subtilis 168 and ending with B. subtilis PG38, which lacks approximately 40% of the original genome. The annotation is supported by sequencing of key intermediate strains as well as integration of literature knowledge for the annotation of the deletion scars and their potential effects. The strain compendium presented here represents a comprehensive genome library of the entire MiniBacillus project. This resource will facilitate the more effective application of the different strains in basic science as well as in biotechnology.


Subject(s)
Bacillus subtilis/genetics , Genome, Bacterial
6.
Appl Environ Microbiol ; 87(18): e0112321, 2021 08 26.
Article in English | MEDLINE | ID: mdl-34232062

ABSTRACT

Cell chaining in Bacillus subtilis is naturally observed in a subset of cells during exponential growth and during biofilm formation. However, the recently constructed large-scale genome-minimized B. subtilis strain PG10 displays a severe and permanent defect in cell separation, as it exclusively grows in the form of long filaments of nonseparated cells. In this study, we investigated the underlying mechanisms responsible for the incomplete cell division of PG10 by genomic and transcriptomic analyses. Repression of the SigD regulon, including the major autolysin gene lytF, was identified as the cause for the cell separation problem of PG10. It appeared that SigD-regulated genes are downregulated in PG10 due to the absence of the flagellar export apparatus, which normally is responsible for secretion of FlgM, the anti-sigma factor of SigD. Although mild negative effects on growth and cell morphology were observed, deletion of flgM could revert the aberrant cell-chaining phenotype and increased transformation efficiency. Interestingly, our work also demonstrates the occurrence of increased antisense transcription of slrR, a transcriptional repressor of autolysin genes, in PG10 and provides further understanding for this observation. In addition to revealing the molecular basis of the cell separation defect in PG10, our work provides novel targets for subsequent genome reduction efforts and future directions for further optimization of miniBacillus PG10. IMPORTANCE Reduction of the size of bacterial genomes is relevant for understanding the minimal requirements for cellular life as well as from a biotechnological point of view. Although the genome-minimized Bacillus subtilis strain PG10 displays several beneficial traits as a microbial cell factory compared to its parental strain, a defect at the final stage of cell division was introduced during the genome reduction process. By genetic and transcriptomic analyses, we identified the underlying reasons for the cell separation problem of PG10. In addition to enabling PG10 to grow in a way similar to that of B. subtilis wild-type strains, our work points toward subsequent targets for fine-tuning and further reduction of the genome of PG10. Moreover, solving the cell separation defect facilitates laboratory handling of PG10 by increasing the transformation efficiency, among other means. Overall, our work contributes to understanding and improving biotechnologically attractive minimal bacterial cell factories.


Subject(s)
Bacillus subtilis/cytology , Bacillus subtilis/genetics , Cell Division , N-Acetylmuramoyl-L-alanine Amidase/genetics , Bacterial Proteins/genetics , Gene Expression Profiling , Industrial Microbiology
7.
mBio ; 12(4): e0121921, 2021 08 31.
Article in English | MEDLINE | ID: mdl-34281399

ABSTRACT

A membrane-associated lanthipeptide synthetase complex, consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT, has been described for nisin biosynthesis in the coccoid bacterium Lactococcus lactis. Here, we used advanced fluorescence microscopy to visualize the functional nisin biosynthesis machinery in rod-shaped cells and analyzed its spatial distribution and dynamics employing a platform we developed for heterologous production of nisin in Bacillus subtilis. We observed that NisT, as well as NisB and NisC, were all distributed in a punctate pattern along the cell periphery, opposed to the situation in coccoid cells. NisBTC proteins were found to be highly colocalized, being visualized at the same spots by dual fluorescence microscopy. In conjunction with the successful isolation of the biosynthetic complex NisBTC from the cell membrane, this corroborated that the visual bright foci were the sites for nisin maturation and transportation. A strategy of differential timing of expression was employed to demonstrate the in vivo dynamic assembly of NisBTC, revealing the recruitment by NisT of NisBC to the membrane. Additionally, by use of mutated proteins, the nucleotide binding domain (NBD) of NisT was found to function as a membrane anchor for NisB and/or NisC. We also show that the nisin biosynthesis sites are static and likely associated with proteins residing in lipid rafts. Based on these data, we propose a model for a three-phase production of modified precursor nisin in rod-shaped bacteria, presenting the assembly dynamics of NisBTC and emphasizing the crucial role of NisBC, next to NisT, in the process of precursor nisin translocation. IMPORTANCE Nisin is a model antimicrobial peptide for LanBC-modified lantibiotics that are modified and transported by a membrane synthetase complex. Although the subcellular localization and the assembly process of such a complex in L. lactis have been described in our recent work (J. Chen, A. J. van Heel, and O. P. Kuipers, mBio 11:e02825-20, 2020, https://doi.org/10.1128/mBio.02825-20), it proved difficult to gain a more detailed insight into the exact LanBTC assembly in the L. lactis system. Rod-shaped cells, especially B. subtilis, are better suited to study the assembly dynamics of these protein complexes. In this work, we present evidence for the existence of the lanthipeptide biosynthetic complex by visualizing and isolating the machinery in vivo. The dynamic behavior of the modification machinery and the transporter within the cells was characterized in depth, revealing the dependence of first LanB and LanC on each other and subsequent recruitment of them by LanT during the machinery assembly. Importantly, the elucidation of the dynamic assembly of the complex will facilitate future studies of lanthipeptide transport mechanisms and the structural characterization of the complete complex.


Subject(s)
Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Biosynthetic Pathways , Nisin/biosynthesis , Nisin/genetics , Antimicrobial Peptides/biosynthesis , Antimicrobial Peptides/genetics , Antimicrobial Peptides/metabolism , Bacterial Proteins/genetics , Bacteriocins/biosynthesis , Bacteriocins/genetics , Bacteriocins/metabolism , Biosynthetic Pathways/genetics , Biosynthetic Pathways/physiology , Microscopy, Fluorescence/methods , Nisin/analysis
8.
Nat Prod Rep ; 38(1): 130-239, 2021 01 01.
Article in English | MEDLINE | ID: mdl-32935693

ABSTRACT

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.


Subject(s)
Computational Biology/methods , Enzymes/metabolism , Peptides/chemistry , Peptides/metabolism , Protein Engineering/methods , Biological Products/chemistry , Biological Products/classification , Biological Products/metabolism , Enzymes/chemistry , Hydroxylation , Methylation , Peptides/classification , Peptides/genetics , Phosphorylation , Protein Processing, Post-Translational , Protein Sorting Signals/physiology , Ribosomes/metabolism
9.
mBio ; 11(6)2020 11 10.
Article in English | MEDLINE | ID: mdl-33173006

ABSTRACT

Nisin, a class I lantibiotic, is synthesized as a precursor peptide by a putative membrane-associated lanthionine synthetase complex consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT. Here, we characterize the subcellular localization and the assembly process of the nisin biosynthesis machinery in Lactococcus lactis by mutational analyses and fluorescence microscopy. Precursor nisin, NisB, and NisC were found to be mainly localized at the cell poles, with a preference for the old poles. They were found to be colocalized at the same spots in these old pole regions, functioning as a nisin modification complex. In contrast, the transporter NisT was found to be distributed uniformly and circumferentially in the membrane. When nisin secretion was blocked by mutagenesis of NisT, the nisin biosynthesis machinery was also visualized directly at a polar position using fluorescence microscopy. The interactions between NisB and other components of the machinery were further studied in vivo, and therefore, the "order of assembly" of the complex was revealed, indicating that NisB directly or indirectly plays the role of a polar "recruiter" in the initial assembly process. Additionally, a potential domain that is located at the surface of the elimination domain of NisB was identified to be crucial for the polar localization of NisB. Based on these data, we propose a model wherein precursor nisin is first completely modified by the nisin biosynthesis machinery, preventing the premature secretion of partially modified peptides, and subsequently secreted by recruited NisT, preferentially at the old pole regions.IMPORTANCE Nisin is the model peptide for LanBC-modified lantibiotics that are commonly modified and exported by a putative synthetase complex. Although the mechanism of maturation, transport, immunity, and regulation is relatively well understood, and structural information is available for some of the proteins involved (B. Li, J. P. J. Yu, J. S. Brunzelle, G. N. Moll, et al., Science 311:1464-1467, 2006, https://doi.org/10.1126/science.1121422; M. A. Ortega, Y. Hao, Q. Zhang, M. C. Walker, et al., Nature 517:509-512, 2015, https://doi.org/10.1038/nature13888; C. Hacker, N. A. Christ, E. Duchardt-Ferner, S. Korn, et al., J Biol Chem 290:28869-28886, 2015, https://doi.org/10.1074/jbc.M115.679969; Y. Y. Xu, X. Li, R. Q. Li, S. S. Li, et al., Acta Crystallogr D Biol Crystallogr 70:1499-1505, 2014, https://doi.org/10.1107/S1399004714004234), the subcellular localization and assembly process of the biosynthesis complex remain to be elucidated. In this study, we determined the spatial distribution of nisin synthesis-related enzymes and the transporter, revealing that the modification and secretion of the precursor nisin mainly occur at the old cell poles of L. lactis and that the transporter NisT is probably recruited later to this spot after the completion of the modification reactions by NisB and NisC. Fluorescently labeled nisin biosynthesis machinery was visualized directly by fluorescence microscopy. To our knowledge, this is the first study to provide direct evidence of the existence of such a complex in vivo Importantly, the elucidation of the "order of assembly" of the complex will facilitate future endeavors in the investigation of the nisin secretion mechanism and even the isolation and structural characterization of the complete complex.


Subject(s)
Lactococcus lactis/metabolism , Nisin/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Transport , Lactococcus lactis/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism
10.
ACS Synth Biol ; 9(7): 1833-1842, 2020 07 17.
Article in English | MEDLINE | ID: mdl-32551553

ABSTRACT

Efficient bacterial cell factories are important for the screening and characterization of potent antimicrobial peptides such as lantibiotics. Although lantibiotic production systems have been established in Lactococcus lactis and Escherichia coli, the industrial workhorse Bacillus subtilis has been left relatively unexplored as a lantibiotic production host. Therefore, we tested different B. subtilis strains for their ability to produce lantibiotic peptides by using the subtilin modification and transport enzymes derived from the natural subtilin producer B. subtilis ATCC 6633. Our study shows that although B. subtilis ATCC 6633 and 168 are able to produce various processed lantibiotic peptides, an evident advantage of using either the 8-fold protease-deficient strain WB800 or the genome-minimized B. subtilis 168 strain PG10 is the lack of extracellular serine protease activity. Consequently, leader processing of lantibiotic precursor peptides is circumvented and thus potential toxicity toward the production host is prevented. Furthermore, PG10 provides a clean secondary metabolic background and therefore appears to be the most promising B. subtilis lantibiotic production host. We demonstrate the production of various lantibiotic precursor peptides by PG10 and show different options for their in vitro activation. Our study thus provides a convenient B. subtilis-based lantibiotic production system, which facilitates the search for novel antimicrobial peptides.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Bacteriocins/biosynthesis , Metabolic Engineering/methods , Nisin/biosynthesis , Serine Proteases/deficiency , Anti-Bacterial Agents/pharmacology , Bacillus subtilis/drug effects , Bacteriocins/genetics , Bacteriocins/pharmacology , Bioreactors , Gene Expression , Genes, Bacterial , Genome, Bacterial , Microbial Sensitivity Tests , Nisin/genetics , Nisin/pharmacology , Peptide Biosynthesis/genetics , Plasmids/genetics , Serine Proteases/genetics
11.
Nucleic Acids Res ; 46(W1): W278-W281, 2018 07 02.
Article in English | MEDLINE | ID: mdl-29788290

ABSTRACT

Interest in secondary metabolites such as RiPPs (ribosomally synthesized and posttranslationally modified peptides) is increasing worldwide. To facilitate the research in this field we have updated our mining web server. BAGEL4 is faster than its predecessor and is now fully independent from ORF-calling. Gene clusters of interest are discovered using the core-peptide database and/or through HMM motifs that are present in associated context genes. The databases used for mining have been updated and extended with literature references and links to UniProt and NCBI. Additionally, we have included automated promoter and terminator prediction and the option to upload RNA expression data, which can be displayed along with the identified clusters. Further improvements include the annotation of the context genes, which is now based on a fast blast against the prokaryote part of the UniRef90 database, and the improved web-BLAST feature that dynamically loads structural data such as internal cross-linking from UniProt. Overall BAGEL4 provides the user with more information through a user-friendly web-interface which simplifies data evaluation. BAGEL4 is freely accessible at http://bagel4.molgenrug.nl.


Subject(s)
Bacteriocins/genetics , Protein Biosynthesis/genetics , Software , Bacteriocins/biosynthesis , Databases, Protein , Humans , Internet , Peptides/genetics
12.
Front Microbiol ; 9: 160, 2018.
Article in English | MEDLINE | ID: mdl-29479343

ABSTRACT

Lantibiotics are ribosomally produced and posttranslationally modified peptides containing several lanthionine residues. They exhibit substantial antimicrobial activity against Gram-positive bacteria, including relevant pathogens. The production of the model lantibiotic nisin minimally requires the expression of the modification and export machinery. The last step during nisin maturation is the cleavage of the leader peptide. This liberates the active compound and is catalyzed by the cell wall-anchored protease NisP. Here, we report the production and purification of a soluble variant of NisP. This has enabled us to study its specificity and test its suitability for biotechnological applications. The ability of soluble NisP to cleave leaders from various substrates was tested with two sets of nisin variants. The first set was designed to investigate the influence of amino acid variations in the leader peptide or variations around the cleavage site. The second set was designed to study the influence of the lanthionine ring topology on the proteolytic efficiency. We show that the substrate promiscuity is higher than has previously been suggested. Our results demonstrate the importance of the arginine residue at the end of the leader peptide and the importance of lanthionine rings in the substrate for specific cleavage. Collectively, these data indicate that NisP is a suitable protease for the activation of diverse heterologously expressed lantibiotics, which is required to release active antimicrobial compounds.

13.
Microb Genom ; 3(10): e000134, 2017 10.
Article in English | MEDLINE | ID: mdl-29177092

ABSTRACT

The need for novel antibiotics in an era where antimicrobial resistance is on the rise, and the number of new approved antimicrobial drugs reaching the market is declining, is evident. The underused potential of post-translationally modified peptides for clinical use makes this class of peptides interesting candidates. In this study, we made use of the vast amounts of available genomic data and screened all publicly available prokaryotic genomes (~3000) to identify 394 novel head-to-tail cyclized antimicrobial peptides. To verify these in silico results, we isolated and characterized a novel antimicrobial peptide from Bacillus pumilus that we named pumilarin. Pumilarin was demonstrated to have a circular structure and showed antimicrobial activity against several indicator strains, including pathogens.


Subject(s)
Bacillus pumilus/genetics , Bacillus pumilus/metabolism , Bacteriocins , Genomic Library , Animals , Anti-Bacterial Agents , Bacterial Infections/therapy , Bacteriocins/genetics , Bacteriocins/isolation & purification , Bacteriocins/pharmacology , Drug Resistance, Microbial , Humans
14.
Microb Cell Fact ; 16(1): 56, 2017 Apr 04.
Article in English | MEDLINE | ID: mdl-28376879

ABSTRACT

BACKGROUND: Bacillus subtilis is widely used as a cell factory for numerous heterologous proteins of commercial value and medical interest. To explore the possibility of further enhancing the secretion potential of this model bacterium, a library of engineered strains with modified cell surface components was constructed, and the corresponding influences on protein secretion were investigated by analyzing the secretion of α-amylase variants with either low-, neutral- or high- isoelectric points (pI). RESULTS: Relative to the wild-type strain, the presence of overall anionic membrane phospholipids (phosphatidylglycerol and cardiolipin) increased dramatically in the PssA-, ClsA- and double KO mutants, which resulted in an up to 47% higher secretion of α-amylase. Additionally, we demonstrated that the appropriate net charge of secreted targets (AmyTS-23, AmyBs and AmyBm) was beneficial for secretion efficiency as well. CONCLUSIONS: In B. subtilis, the characteristics of cell membrane phospholipid bilayer and the pIs of heterologous α-amylases appear to be important for their secretion efficiency. These two factors can be engineered to reduce the electrostatic interaction between each other during the secretion process, which finally leads to a better secretion yield of α-amylases.


Subject(s)
Bacillus subtilis/metabolism , Cell Membrane/metabolism , Metabolic Engineering/methods , alpha-Amylases/metabolism , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Cardiolipins/metabolism , Cell Membrane/chemistry , Cell Membrane/genetics , Isoelectric Point , Phosphatidylglycerols/metabolism , Secretory Pathway/genetics , Secretory Pathway/physiology , Static Electricity , alpha-Amylases/biosynthesis , alpha-Amylases/genetics
15.
FEMS Microbiol Rev ; 41(1): 5-18, 2017 01.
Article in English | MEDLINE | ID: mdl-27591436

ABSTRACT

As the number of new antibiotics that reach the market is decreasing and the demand for them is rising, alternative sources of novel antimicrobials are needed. Lantibiotics are potent peptide antimicrobials that are ribosomally synthesized and stabilized by post-translationally introduced lanthionine rings. Their ribosomal synthesis and enzymatic modifications provide excellent opportunities to design and engineer a large variety of novel antimicrobial compounds. The research conducted in this area demonstrates that the modularity present in both the peptidic rings as well as in the combination of promiscuous modification enzymes can be exploited to further increase the diversity of lantibiotics. Various approaches, where the modifying enzymes and corresponding leader peptides are decoupled from their natural core peptide and integrated in designed plug-and-play production systems, enable the production of modified peptides that are either derived from vast genomic data or designed using functional parts from a wide diversity of core peptides. These approaches constitute a powerful discovery platform to develop novel antimicrobials with high therapeutic potential.


Subject(s)
Anti-Infective Agents , Bacteriocins/biosynthesis , Bioengineering , Drug Design
16.
ACS Synth Biol ; 5(10): 1146-1154, 2016 10 21.
Article in English | MEDLINE | ID: mdl-27294279

ABSTRACT

To find the right conditions to isolate natively expressed antimicrobial peptides from a wide range of different microorganisms can be a challenge. Here, we exploited a heterologous expression system to produce and characterize several novel lantibiotics. We identified 54 novel putative class I and class II lantibiotics after inspecting all publicly available prokaryotic genomes using the in-house developed mining tool BAGEL3. The genes encoding these new lantibiotics fused to the nisin leader peptide gene sequence were synthesized, and the constructs were plugged into the nisin expression and modification system. Using this approach 30 peptides could be expressed, 27 of which were dehydrated by NisBC on at least 1 predicted position. Good antimicrobial activity against several pathogenic bacteria could be demonstrated for 5 novel heterologously modified lantibiotics. Lantibiotics from Corynebacterium lipophiloflavum DSM 44291 and Streptococcus agalactiae ATCC 13813, named flavucin and agalacticin, respectively, were fully modified and displayed high antimicrobial activity. The efficiency of functional expression was significantly enhanced when we made use of the native nisin leader cleavage site, instead of an artificial factor Xa site. Thus, we describe an efficient way for heterologous production of active lantibiotics, facilitating a rapid identification of promising molecules.


Subject(s)
Bacteriocins/chemistry , Gene Expression Regulation, Bacterial , Nisin/chemistry , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteriocins/pharmacology , Cloning, Molecular , Corynebacterium/chemistry , Corynebacterium/genetics , Multigene Family , Protein Sorting Signals/genetics , Streptococcus agalactiae/chemistry , Streptococcus agalactiae/genetics
17.
Genome Announc ; 4(3)2016 May 05.
Article in English | MEDLINE | ID: mdl-27151781

ABSTRACT

Here, we report the draft genomes of five strains of Geobacillus spp., one Caldibacillus debilis strain, and one draft genome of Anoxybacillus flavithermus, all thermophilic spore-forming Gram-positive bacteria.

19.
Genome Announc ; 4(2)2016 Mar 17.
Article in English | MEDLINE | ID: mdl-26988043

ABSTRACT

Here, we report the draft genome sequences of 10 isolates of Bacillus subtilis, a spore forming Gram-positive bacterium. The strains were selected from food products and produced spores with either high or low heat resistance.

20.
Amino Acids ; 48(5): 1309-18, 2016 May.
Article in English | MEDLINE | ID: mdl-26872656

ABSTRACT

Lantibiotics are posttranslationally modified peptides with efficient inhibitory activity against various Gram-positive bacteria. In addition to the original modifications, incorporation of non-canonical amino acids can render new properties and functions to lantibiotics. Nisin is the most studied lantibiotic and contains no tryptophan residues. In this study, a system was constructed to incorporate tryptophan analogues into nisin, which included the modification machinery (NisBTC) and the overexpression of tryptophanyl-tRNA synthetase (TrpRS). Tryptophan and three different tryptophan analogues (5-fluoroTrp (5FW), 5-hydroxyTrp (5HW) and 5-methylTrp (5MeW)) were successfully incorporated at four different positions of nisin (I1W, I4W, M17W and V32W). The incorporation efficiency of tryptophan analogues into mutants I1W, M17W and V32W was over 97 %, while the mutant I4W showed relatively low incorporation efficiency (69-93 %). The variants with 5FW showed relatively higher production yield, while 5MeW-containing variants showed the lowest yield. The dehydration efficiency of serines or threonines was affected by the tryptophan mutants of I4W and V32W. The affinity of the peptides for the cation-ion exchange and reverse phase chromatography columns was significantly reduced when 5HW was incorporated. The antimicrobial activity of IIW and its 5FW analogue both decreased two times compared to that of nisin, while that of its 5HW analogue decreased four times. The 5FW analogue of I4W also showed two times decreased activity than nisin. However, the mutant M17W and its 5HW analogue both showed 32 times reduced activity relative to that of nisin.


Subject(s)
Bacteriocins/chemistry , Nisin/chemistry , Nisin/pharmacology , Tryptophan/pharmacology , Bacteriocins/genetics , Bacteriocins/pharmacology , Lactococcus lactis/drug effects , Molecular Structure , Nisin/genetics , Tryptophan/analogs & derivatives , Tryptophan/genetics
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