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.info:eu-repo/semantics/publishedVersio

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 Bliβ, 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 Bliβ variants in vivo. This contribution will spur future studies in the heterologous expression and engineering of lantibiotics.T.C. was supported by Fundação para a Ciência e Tecnologia and Medinfar Pharmaceuticals SA grant (SFRH/BDE/15559/2005). The work was supported by Centre for Environmental and Marine Studies (CESAM) of University of Aveiro and the Cluster of Excellence “Unifying concepts in catalysis” granted by the DFG, coordinated by TU Berlin. The authors acknowledge Dr. José C. Duarte for supplying B. licheniformis I89 strain, and Professor Dr. Klaus Hantke from University of Tübingen for supplying E. coli BW25113 and BW25113ΔtolC:kan strains.publishe

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

© 2021 Elsevier B.V. All rights reserved.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 Bliβ, 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 Bliβ, 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, Bliβ 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.This work was supported by Fundação para a Ciência e a Tecnologia – Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal), Programa Operacional Potencial Humano (POPH, Portugal) and European Union fellowships (SFRH/BD/97099/2013, PD/BD/128290/2017, PD/BD/136880/2018, and SFRH/BPD/77900/2011, to JCB, MM, ICS and TC, respectively); by national funds (OE) through FCT – Fundação para a Ciência e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19 (CEECIND/01463/2017); by the Cluster of Excellence Unifying Systems in Catalysis (Germany); by Coli4Lan Project (Portugal) (FCOMP-01-0124-FEDER-027569); FCT-MCTES (Programa de Investimento e Despesas de Desenvolvimento da Administração Central – PIDDAC, Portugal); Fundo Europeu de Desenvolvimento Regional (FEDER, Portugal), through the COMPETE – Programa Operacional Fatores de Competitividade (POFC); by CESAM (Aveiro, Portugal; UIDP/50017/2020 and UIDB/50017/2020) and FCT-MCTES through national funds, to the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020.info:eu-repo/semantics/publishedVersio

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.TU Berlin, Open-Access-Mittel – 202

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