Class II two-peptide lanthipeptide proteases: exploring LicTP for biotechnological applications.

The enzymatic machinery involved in the biosynthesis of lantibiotic is an untapped source of proteases with different specificities. Lanthipeptide biosynthesis requires proteolysis of specific target sequences by known proteases, which are encoded by contiguous genes. Herein, the activity of lichenicidin A2 (LicA2) trimming proteases (LicP and LicT) was investigated in vivo. Firstly, the impact of some residues and the size of the peptide were evaluated. Then followed trials in which LicA2 leader was evaluated as a tag to direct production and secretion of other relevant peptides. Our results show that a negatively charged residue (preferably Glu) at cleavage site is important for LicP efficacy. Some mutations of the lichenicidin hexapeptide such as Val-4Ala, Asp-5Ala, Asn-6Ser, and the alteration of GG-motif to GA resulted in higher processing rates, indicating the possibility of improved lichenicidin production in Escherichia coli. More importantly, insulin A, amylin (non-lanthipeptides), and epidermin were produced and secreted to E. coli supernatant, when fused to the LicA2 leader peptide. This work aids in clarifying the activity of lantibiotic-related transporters and proteases and to evaluate their possible application in industrial processes of relevant compounds, taking advantage of the potential of microorganisms as biofactories. KEY POINTS: • LicM2 correct activity implies a negatively charged residue at position -1. • Hexapeptide mutations can increase the amount of fully processed Bliß. • LicA2 leader peptide directs LicTP cleavage and secretion of other peptides.

Insights into the mode of action of the two-peptide lantibiotic lichenicidin.

Lantibiotics are promising candidates to address the worldwide problem of antibiotic resistance. They belong to a class of natural compounds exhibiting strong activity against clinically relevant Gram-positive bacterial strains, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). Lichenicidin is a class II two-peptide lantibiotic. The presence of the two mature peptides, Bliα and Bliß, is necessary for full activity against target bacteria. This work aims at clarifying the synergistic activity of both peptides in their interaction with the target membranes. The effect of lichenicidin was tested against S. aureus cells and large unilamellar vesicles. Lichenicidin increases the net surface charge of S. aureus, as shown by zeta-potential measurements, without reaching electroneutralization. In addition, lichenicidin causes cell surface perturbations that culminate in the leakage of its internal contents, as observed by atomic force microscopy. Bliα seems to have low affinity for S. aureus, however, it contributes to increase the affinity of Bliß, because together they present higher affinity than separately. In contrast, Bliα seems to provide an anchoring site for lichenicidin in lipid II-containing membranes. Interestingly, Bliß alone can induce high levels of membrane leakage, but this effect appears to be faster in the presence of Bliα. Based on this information, we propose a mechanism of action of lichenicidin.

Assessing the potential of the two-peptide lantibiotic lichenicidin as a new generation antimicrobial.

Lantibiotics are a promising class of natural antimicrobial peptides. Lichenicidin is a two-peptide lantibiotic in which two mature peptides act synergistically to exhibit full bioactivity. Considering the two-peptide lantibiotics described so far, only cytolysin has been deeply characterized in terms of toxicity towards eukaryotic cells and it was found to be hemolytic and cytotoxic. This work aimed to improve the production of lichenicidin in vivo and characterize its antibacterial activity and toxicity against human cells. Peptides were purified and minimal inhibitory concentration (MIC) was determined against several strains; a time-kill assay was performed with Staphylococcus aureus. The hemolytic effect of lichenicidin was evaluated on blood samples from healthy donors and its toxicity towards human fibroblasts. The quantity of purified peptides was 1 mg/l Bliα and 0.4 mg/l Bliß. MIC for methicillin-sensitive and resistant S. aureus (MSSA and MRSA) strains were 16-32 µg/ml and 64-128 µg/ml, respectively. At the MIC, lichenicidin took less than 3 h to eliminate MSSA, indicating a strong bactericidal effect. It induces cell lysis at the highest concentration, an effect that might be potentiated by Bliß. Lichenicidin was not cytotoxic to human erythrocytes and fibroblasts. In this work, we evaluated the therapeutic potential of lichenicidin as a possible antimicrobial alternative.

Eliciting the silent lucensomycin biosynthetic pathway in Streptomyces cyanogenus S136 via manipulation of the global regulatory gene adpA.

Actinobacteria are among the most prolific sources of medically and agriculturally important compounds, derived from their biosynthetic gene clusters (BGCs) for specialized (secondary) pathways of metabolism. Genomics witnesses that the majority of actinobacterial BGCs are silent, most likely due to their low or zero transcription. Much effort is put into the search for approaches towards activation of silent BGCs, as this is believed to revitalize the discovery of novel natural products. We hypothesized that the global transcriptional factor AdpA, due to its highly degenerate operator sequence, could be used to upregulate the expression of silent BGCs. Using Streptomyces cyanogenus S136 as a test case, we showed that plasmids expressing either full-length adpA or its DNA-binding domain led to significant changes in the metabolome. These were evident as changes in the accumulation of colored compounds, bioactivity, as well as the emergence of a new pattern of secondary metabolites as revealed by HPLC-ESI-mass spectrometry. We further focused on the most abundant secondary metabolite and identified it as the polyene antibiotic lucensomycin. Finally, we uncovered the entire gene cluster for lucensomycin biosynthesis (lcm), that remained elusive for five decades until now, and outlined an evidence-based scenario for its adpA-mediated activation.

Lichenicidin rational site-directed mutagenesis library: A tool to generate bioengineered lantibiotics.

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that arise as an alternative to the traditional antibiotics. Lichenicidin is active against clinically relevant bacteria and it was the first lantibiotic to be fully produced in vivo in the Gram-negative host Escherichia coli. Here, we present the results of a library of lichenicidin mutants, in which the mutations were generated based on the extensive bibliographical search available for other lantibiotics. The antibacterial activity of two-peptide lantibiotics, as is lichenicidin, requires the synergistic activity of two peptides. We established a method that allows screening for bioactivity which does not require the purification of the complementary peptide. It is an inexpensive, fast and user-friendly method that can be scaled up to screen large libraries of bioengineered two-peptide lantibiotics. The applied system is reliable and robust because, in general, the results obtained corroborate structure-activity relationship studies carried out for other lantibiotics.

A Self-Sacrificing N-Methyltransferase Is the Precursor of the Fungal Natural Product Omphalotin.

Research on ribosomally synthesized and posttranslationally modified peptides (RiPPs) has led to an increasing understanding of biosynthetic mechanisms, mostly drawn from bacterial examples. In contrast, reports on RiPPs from fungal producers, apart from the amanitins and phalloidins, are still scarce. The fungal cyclopeptide omphalotin A carries multiple N-methylations on the peptide backbone, a modification previously known only from nonribosomal peptides. Mining the genome of the omphalotin-producing fungus for a precursor peptide led to the identification of two biosynthesis genes, one encoding a methyltransferase OphMA that catalyzes the automethylation of its C-terminus, which is then released and cyclized by the protease OphP. Our findings suggest a novel biosynthesis mechanism for a RiPP in which a modifying enzyme bears its own precursor peptide.

Engineering the heterologous expression of lanthipeptides in Escherichia coli by multigene assembly.

Lantibiotics are an important class of ribosomally synthesised peptide antibiotics with a remarkable pharmacological potential. Structural variants of lantibiotics generated by peptide engineering in vivo are an important aspect for improving the peptide's efficacy, stability and bioavailability as well as production titre, which severely impacts the potential exploitation in pharmaceutical applications. Therefore, expression systems are needed which allow for a robust genetic access for ample mutagenesis experiments. Based on previous heterologous expression of the two-component lanthipeptide lichenicidin (Bliα and Bliß) in Escherichia coli BLic5, we now employ a multigene assembly strategy for recombinant lantibiotic peptide production in the Gram-negative host. Two E. coli high copy plasmids for separate and increased expression of a two-component lantibiotic were cloned and tested for expression. From these E. coli HP expression strains, an up to 100 times increased expression was found compared with Bacillus licheniformis I89 and E. coli BLic5. Total expression yields reach 4 mg L(-1) for Bliα and 6 mg L(-1) for Bliß. The expression system developed in this study constitutes an important cornerstone for future in vivo peptide engineering studies and is of significance for potential applications aiming at higher production titres of ribosomally synthesised, post translationally modified peptides.

Paenilamicin: structure and biosynthesis of a hybrid nonribosomal peptide/polyketide antibiotic from the bee pathogen Paenibacillus larvae.

The spore-forming bacterium Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a fatal disease of honey bees that occurs worldwide. Previously, we identified a complex hybrid nonribosomal peptide/polyketide synthesis (NRPS/PKS) gene cluster in the genome of P. larvae. Herein, we present the isolation and structure elucidation of the antibacterial and antifungal products of this gene cluster, termed paenilamicins. The unique structures of the paenilamicins give deep insight into the underlying complex hybrid NRPS/PKS biosynthetic machinery. Bee larval co-infection assays reveal that the paenilamicins are employed by P. larvae in fighting ecological niche competitors and are not directly involved in killing the bee larvae. Their antibacterial and antifungal activities qualify the paenilamicins as attractive candidates for drug development.

Synthesis and structural characterization of hexacoordinate silicon, germanium, and titanium complexes of the E. coli siderophore enterobactin.

The E. coli siderophore enterobactin, one of the strongest Fe(III) chelators known to date, is also capable of binding Si(IV) under physiological conditions. We report on the synthesis and structural characterization of the tris(catecholate) Si(IV) -enterobactin complex and its Ge(IV) and Ti(IV) analogues. Comparative structural analysis, supported by quantum-chemical calculations, reveals the correlation between the ionic radius and the structural changes in enterobactin upon complexation.

Lichenicidin biosynthesis in Escherichia coli: licFGEHI immunity genes are not essential for lantibiotic production or self-protection.

This study demonstrated, for the first time, that immunity genes licFGEHI are not essential for self-protection and production of the two-component lantibiotic lichenicidin in the Gram-negative heterologous host Escherichia coli BLic5. Additionally, it was experimentally demonstrated that lichenicidin lantibiotics are active against the E. coli imp4213 strain, a mutant strain possessing a permeable outer membrane.

Heterologous expression, biosynthesis, and mutagenesis of type II lantibiotics from Bacillus licheniformis in Escherichia coli.

Lichenicidin is a class II two-component lantibiotic produced by Bacillus licheniformis. It is composed of the two peptides Bliα and Bliß, which act synergistically against various Gram-positive bacteria. The lichenicidin gene cluster was successfully expressed in Escherichia coli, thus constituting the first report to our knowledge of a full reconstitution of a lantibiotic biosynthetic pathway in vivo by a Gram-negative host. This system was further exploited to characterize and assign the function of proteins encoded in the biosynthetic gene cluster in the maturation of lichenicidin peptides. Moreover, a trans complementation system was developed for expression of Bliα and Bliß variants in vivo. This contribution will spur future studies in the heterologous expression and engineering of lantibiotics.

Anti-Candida metabolites from endophytic fungi.

Submerged cultures of some 1500 Ascomycota and Basidiomycota isolated from their fruit-bodies or as soil-borne, coprophilous or endophytic fungi were screened for activity against Candida albicans and a range of other pathogenic and saprotrophic fungi. Considerably more Ascomycota (11-16%) than Basidiomycota (3.5%) produced metabolites with activity against C. albicans. From five species of endophytes, six bioactive compounds were isolated and identified, viz. cerulenin (1), arundifungin (2), sphaeropsidin A (3), 5-(1,3-butadiene-1-yl)-3-(propene-1-yl)-2-(5H)-furanone (4), ascosteroside A (formerly called ascosteroside; 5) and a derivative of 5, ascosteroside B (6). 1, 3 and 5 were isolated from fungi belonging to different orders than previously described producers. Antifungal activities of 2 and 4-6 in the agar diffusion test were comparable with those of amphotericin B. Compound 6 exhibited a similar antifungal activity as 5 but its cytotoxicity towards Hep G2 cells was considerably lower. This study points to endophytic fungi related to hemibiotrophic or latent plant pathogens as an important source of bio- and chemodiversity.