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

Production of the Catechol Type Siderophore Bacillibactin by the Honey Bee Pathogen Paenibacillus larvae

The Gram-positive bacterium Paenibacillus larvae is the etiological agent of American Foulbrood. This bacterial infection of honey bee brood is a notifiable epizootic posing a serious threat to global honey bee health because not only individual larvae but also entire colonies succumb to the disease. In the recent past considerable progress has been made in elucidating molecular aspects of host pathogen interactions during pathogenesis of P. larvae infections. Especially the sequencing and annotation of the complete genome of P. larvae was a major step forward and revealed the existence of several giant gene clusters coding for non-ribosomal peptide synthetases which might act as putative virulence factors. We here present the detailed analysis of one of these clusters which we demonstrated to be responsible for the biosynthesis of bacillibactin, a P. larvae siderophore. We first established culture conditions allowing the growth of P. larvae under iron-limited conditions and triggering siderophore production by P. larvae. Using a gene disruption strategy we linked siderophore production to the expression of an uninterrupted bacillibactin gene cluster. In silico analysis predicted the structure of a trimeric trithreonyl lactone (DHB-Gly-Thr)3 similar to the structure of bacillibactin produced by several Bacillus species. Mass spectrometric analysis unambiguously confirmed that the siderophore produced by P. larvae is identical to bacillibactin. Exposure bioassays demonstrated that P. larvae bacillibactin is not required for full virulence of P. larvae in laboratory exposure bioassays. This observation is consistent with results obtained for bacillibactin in other pathogenic bacteria

Heterologous Expression and Engineering Studies of Labyrinthopeptins, Class III Lantibiotics from Actinomadura namibiensis

SummaryLabyrinthopeptins are class III lantibiotics produced by the actinomycete Actinomadura namibiensis. The most characteristic structural feature is the posttranslationally installed triamino triacid labionin with a quaternary α-carbon. In addition to the unique structure, labyrinthopeptin A2 possess remarkable antiviral and antiallodynic biological activities. To harness the substrate tolerance of the biosynthetic machinery, we developed an efficient system for the generation of labyrinthopeptin analogs. Streptomyces lividans was used as a heterologous host since the natural producer Actinomadura namibiensis remained genetically intractable. Generation of a library of 39 mutants allowed identification of variable and invariable regions in the labyrinthopeptin structures. Additional data on the flexibility of the biosynthetic machinery were provided by in vitro experiments. This study is detailed investigation on the potential to generate analogs of class III lantibiotics by genetic engineering

Two heterologously expressed Planobispora rosea proteins cooperatively induce Streptomyces lividans thiostrepton uptake and storage from the extracellular medium

<p>Abstract</p> <p>Background</p> <p>A bacterial artificial chromosomal library of <it>Planobispora rosea</it>, a genetically intractable actinomycete strain, was constructed using <it>Escherichia coli</it>-<it>Streptomyces </it>artificial chromosome (ESAC) and screened for the presence of genes known to be involved in the biosynthesis of antibiotics.</p> <p>Results</p> <p>One clone with a 40 kb insert showed antimicrobial activity against Gram positive bacteria. Insert sequence analysis and subcloning experiments revealed that the bioactivity was due to a 3.5 kb DNA fragment containing two open reading frames. These <it>orfs </it>encode two proteins with high similarity to a putative membrane protein of <it>Streptomyces coelicolor </it>and to the nogalamycin resistance protein SnorO of <it>Streptomyces nogalater</it>, respectively. The role of these two Orfs is unknown in <it>Planobispora. </it>Disruption and complementation experiments revealed that both proteins are necessary for the antibacterial activity and chemical analysis demonstrated that the antibiotic activity was due to thiostrepton, antibiotic used as recombinant clone selection marker.</p> <p>Conclusion</p> <p>Two <it>Planobispora rosea orfs </it>are responsible for increasing intracellular amounts and storage of thiostrepton in <it>Streptomyces lividans</it>.</p

The occurrence of ansamers in the synthesis of cyclic peptides

α-Amanitin is a bicyclic octapeptide composed of a macrolactam with a tryptathionine cross-link forming a handle. Previously, the occurrence of isomers of amanitin, termed atropisomers has been postulated. Although the total synthesis of α-amanitin has been accomplished this aspect still remains unsolved. We perform the synthesis of amanitin analogs, accompanied by in-depth spectroscopic, crystallographic and molecular dynamics studies. The data unambiguously confirms the synthesis of two amatoxin-type isomers, for which we propose the term ansamers. The natural structure of the P-ansamer can be ansa-selectively synthesized using an optimized synthetic strategy. We believe that the here described terminology does also have implications for many other peptide structures, e.g. norbornapeptides, lasso peptides, tryptorubins and others, and helps to unambiguously describe conformational isomerism of cyclic peptides

The Thioesterase Bhp is Involved in the Formation of β-Hydroxytyrosine during Balhimycin Biosynthesis in Amycolatopsis balhimycina

The putative hydrolase gene bhp from the balhimycin biosynthetic gene cluster has been cloned and overexpressed in Escherichia coli. The corresponding enzyme Bhp was purified to homogeneity by nickel-chelating chromatography and characterized. Although Bhp has sequence similarities to hydrolases with “haloperoxidase”/perhydrolase activity, it did not show any enzymatic activity with standard “haloperoxidase”/perhydrolase substrates (e.g., monochlorodimedone and phenol red), nonspecific esterase substrates (such as p-nitrophenyl acetate, p-nitrophenyl phosphate and S-thiophenyl acetate) or the model lactonase substrate dihydrocoumarin. However, Bhp could be shown to catalyse the hydrolysis of S-β-hydroxytyrosyl-N-acetyl cysteamine thioester (β-OH-Tyr-SNAC) with 15 times the efficiency of S-L-tyrosyl- N-acetyl cysteamine thioester (L-Tyr-SNAC). This is in agreement with the suggestion that Bhp is involved in balhimycin biosynthesis, during which it was supposed to catalyse the hydrolysis of β-OH-Tyr-S-PCP (PCP=peptidyl carrier protein) to free β-hydroxytyrosine (β-OH-Tyr) and strongly suggests that Bhp is a thioesterase with high substrate specificity for PCP-bound β-OH-Tyr and not a “haloperoxidase”/perhydrolase or nonspecific esterase

Streptocollin, a Type IV Lanthipeptide Produced by Streptomyces collinus Tü 365

Lanthipeptides are ribosomally synthesized and post-translationally modified microbial secondary metabolites. Here, we report the identification and isolation of streptocollin from Streptomyces collinus Tü 365, a new member of class IV lanthipeptides. Insertion of the constitutive ermE* promoter upstream of the lanthipeptide synthetase gene stcL resulted in peptide production. The streptocollin gene cluster was heterologously expressed in S. coelicolor M1146 and M1152 with 3.5- and 5.5-fold increased yields, respectively. The structure and ring topology of streptocollin were determined by high resolution MS/MS analysis. Streptocollin contains four macrocyclic rings, with one lanthionine and three methyllanthionine residues. To the best of our knowledge, this is the first report on the isolation of a class IV lanthipeptide in preparative amounts, and on the successful heterologous expression of a class IV lanthipeptide gene cluster

Molecular insights into antibiotic resistance - how a binding protein traps albicidin

The worldwide emergence of antibiotic resistance poses a serious threat to human health. A molecular understanding of resistance strategies employed by bacteria is obligatory to generate less-susceptible antibiotics. Albicidin is a highly potent antibacterial compound synthesized by the plant-pathogenic bacterium Xanthomonas albilineans. The drug-binding protein AlbA confers albicidin resistance to Klebsiella oxytoca. Here we show that AlbA binds albicidin with low nanomolar affinity resulting in full inhibition of its antibacterial activity. We report on the crystal structure of the drug-binding domain of AlbA (AlbAS) in complex with albicidin. Both α-helical repeat domains of AlbAS are required to cooperatively clamp albicidin, which is unusual for drug-binding proteins of the MerR family. Structure-guided NMR binding studies employing synthetic albicidin derivatives give valuable information about ligand promiscuity of AlbAS. Our findings thus expand the general understanding of antibiotic resistance mechanisms and support current drug-design efforts directed at more effective albicidin analogs

Hexapod Assassins’ Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications