Lipopeptide culture filtrates from Bacillus spp. provide effective protection to wheat against the foliar pathogen Zymoseptoria tritici.

AIMS: Biocontrol products based on microorganisms and natural substances are promising alternatives to chemical pesticides that could contribute to develop a more sustainable agriculture. Here, we investigated the potential of cell-free culture filtrates (CFCFs) from two strains of the Bacillus subtilis group to inhibit Zymoseptoria tritici, a major fungal pathogen of wheat. METHODS AND RESULTS: Foliar application of CFCFs from Bacillus velezensis GA1 and Bacillus sp. III1 on wheat seedlings in a greenhouse strongly reduced Z. tritici disease severity (>90%). In vitro bioassays showed that CFCFs completely inhibited the spore germination and fungal growth (100%). In planta cytological investigations revealed a significant impact of the treatments on both spore germination (∼40% inhibition) and fungal growth of Z. tritici (>80% inhibition). High Performance Liquid Chromatography (HPLC) analysis showed that the Bacillus strains displayed different lipopeptide profiles. The CFCF obtained from Bacillus GA1 contained 90 mg l-1 of iturin A + surfactins + fengycins and the CFCF obtained from Bacillus sp. III1 contained 25 mg l-1 of mojavensin A (iturin family) + surfactins + fengycins. CONCLUSIONS: Strains of the B. subtilis group producing different iturins could provide several CFCF-based solutions for the biocontrol of Z. tritici.

The Diversity of Lipopeptides in the Pseudomonas syringae Complex Parallels Phylogeny and Sheds Light on Structural Diversification during Evolutionary History.

Pseudomonas spp. colonize diverse aquatic and terrestrial habitats and produce a wide variety of secondary metabolites, including lipopeptides. However, previous studies have often examined a limited number of lipopeptide-producing strains. In this study, we performed a systematic analysis of lipopeptide production across a wide data set of strains of the Pseudomonas syringae complex (724) by using a combined bioinformatics, mass spectrometry, and phylogenetics approach. The large P. syringae complex, which is composed of 13 phylogroups, is known to produce factins (including syringafactin-like lipopeptides), mycins (including syringomycin-like lipopeptides), and peptins (such as syringopeptins). We found that 80.8% of P. syringae strains produced lipopeptides and that factins were the most frequently produced (by 96% of the producing strains). P. syringae strains were either factin monoproducers or factin, mycin, and peptin coproducers or lipopeptide nonproducers in relation to their phylogenetic group. Our analyses led to the discovery of 42 new lipopeptides, bringing the number of lipopeptides identified in the P. syringae complex to 75. We also highlighted that factins have high structural resemblance and are widely distributed among the P. syringae complex, while mycins and peptins are highly structurally diverse and patchily distributed. IMPORTANCE This study provides an insight into the P. syringae metabolome that emphasizes the high diversity of lipopeptides produced within the P. syringae complex. The production profiles of strains are closely related to their phylogenetic classification, indicating that structural diversification of lipopeptides parallels the phylogeny of this bacterial complex, thereby further illustrating the inherent importance of lipopeptides in the ecology of this group of bacteria throughout its evolutionary history. Furthermore, this overview of P. syringae lipopeptides led us to propose a refined classification that could be extended to the lipopeptides produced by other bacterial groups.

Author Correction: A GDSL lipase-like from Ipomoea batatas catalyzes efficient production of 3,5-diCQA when expressed in Pichia pastoris.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-020-01488-x.

A GDSL lipase-like from Ipomoea batatas catalyzes efficient production of 3,5-diCQA when expressed in Pichia pastoris.

The synthesis of 3,5-dicaffeoylquinic acid (3,5-DiCQA) has attracted the interest of many researchers for more than 30 years. Recently, enzymes belonging to the BAHD acyltransferase family were shown to mediate its synthesis, albeit with notably low efficiency. In this study, a new enzyme belonging to the GDSL lipase-like family was identified and proven to be able to transform chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA, CGA) in 3,5-DiCQA with a conversion rate of more than 60%. The enzyme has been produced in different expression systems but has only been shown to be active when transiently synthesized in Nicotiana benthamiana or stably expressed in Pichia pastoris. The synthesis of the molecule could be performed in vitro but also by a bioconversion approach beginning from pure 5-CQA or from green coffee bean extract, thereby paving the road for producing it on an industrial scale.

Enzymatic depolymerization of industrial lignins by laccase-mediator systems in 1,4-dioxane/water.

Lignin is the second most abundant polymer after cellulose in lignocellulosic biomass. Its aromatic composition and recalcitrant nature make its valorization a major challenge for obtaining low molecular weight aromatics compounds with high value-added from the enzymatic depolymerization of industrial lignins. The oxidation reaction of lignin polymer using laccases alone remains inefficient. Therefore, researches are focused on the use of a laccase-mediator system (LMS) to facilitate enzymatic depolymerization. Until today, the LMS system was studied using water-soluble lignin only (commercial lignins, modified lignins, or lignin model compounds). This work reports a study of three LMS systems to depolymerize the three major industrial lignins (organosolv lignin, Kraft lignin, and sodium lignosulfonate). We show that an enzymatic depolymerization of these lignins can be achieved by LMS using laccase from Trametes versicolor, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt as mediator and a cosolvent (25% of 1,4-dioxane) to enhance the solubilization of lignins.

Bio-emulsifying and biodegradation activities of syringafactin producing Pseudomonas spp. strains isolated from oil contaminated soils.

Pseudomonas strains isolated from oil contaminated soils were screened for biosurfactant production. Three out of eleven Pseudomonas isolates were selected for their high emulsifying activity (E24 value on n-hexadecane ~ 78%). These isolates (E39, E311 and E313) were identified as members of the P. putida group using phenotypical methods and a molecular approach. To identify the chemical nature of produced biosurfactants, thin layer chromatography and MALDI-ToF mass spectrometry analysis were carried out and revealed lipopeptides belonging to the syringafactin family. The activity of the produced biosurfactants was stable over a pH range of 6-12, at high salinity (10%) and after heating at 80 °C. Tests in contaminated sand micro-bioreactors showed that the three strains were able to degrade diesel. These results suggest the potential of these syringafactin producing strains for application in hydrocarbon bioremediation.

Lipopeptide biodiversity in antifungal Bacillus strains isolated from Algeria.

Several Bacillus strains have been well studied for their ability to control soil-borne plant diseases. This property is linked to the production of several families of lipopeptides. Depending of their structure, these compounds show antifungal and/or plant systemic resistance inducing activities. In this work, the biodiversity of lipopeptides produced by different antifungal Bacillus strains isolated from seeds, rhizospheric, and non-rhizospheric soils in Algeria was analyzed. Sixteen active strains were characterized by PCR for their content in genes involved in lipopeptide biosynthesis and by MALDI-ToF for their lipopeptide production, revealing a high biodiversity of products. The difficulty to detect kurstakin genes led us to design two new sets of specific primers. An interesting potential of antifungal activity and the synthesis of two forms of fengycins differing in the eighth amino acid (Gln/Glu) were found from the strain 8. Investigation of its genome led to the finding of an adenylation domain of the fengycin synthetase predicted to activate the glutamate residue instead of the glutamine one. According to the comparison of both the results of MALDI-ToF-MS and genome analysis, it was concluded that this adenylation domain could activate both residues at the same time. This study highlighted that the richness of the Algerian ecosystems in Bacillus strains is able to produce: surfactin, pumilacidin, lichenysin, kurstakin, and different types of fengycins.

Genetic engineering of the branched fatty acid metabolic pathway of Bacillus subtilis for the overproduction of surfactin C14 isoform.

Surfactin, a lipopeptide produced by Bacillus subtilis, is one of the most powerful biosurfactants known. This molecule consists of a cyclic heptapeptide linked to a ß-hydroxy fatty acid chain. The isomery and the length of the fatty acid (FA) chain are responsible for the surfactin's activities. In this study, the gene codY, which encode for the global transcriptional regulator and the gene lpdV, located in the bkd operon (lpdV, bkdAA, bkdAB and bkdB genes), which is responsible for the last step of the branched chain amino acid (BCAA) degradation in acyl-CoA were deleted. The influence of these deletions on the quantitative and qualitative surfactin production was analysed. The surfactin production was quantified by RP-HPLC and the surfactin isoforms were characterized using LC-MS-MS and GC-MS analysis. The results obtained in the mutants showed an enhancement of surfactin specific production by a factor of 5.8 for the codY mutant and 1.4 for lpdV mutant. Moreover qualitative analysis of the lpdV mutant reveals that it mainly produced surfactin C14 isoform (2 fold more than the wild type) with linear FA chain. Complete analysis of the extracellular metabolites using 1 H quantitative NMR reveals a reduced production of acetoin in this mutant. This work demonstrates for the first time an original approach to overproduce specifically surfactin with C14 FA chain.

The safe enterocin DD14 is a leaderless two-peptide bacteriocin with anti-Clostridium perfringens activity.

Enterococcus faecalis 14, a strain previously isolated from meconium, displayed activity against four Clostridium perfringens isolates when co-cultured on agar plates. The anti-Clostridium activity was ascribed to the production of enterocin DD14, which was subsequently purified. The minimum inhibitory concentration (MIC) of enterocin DD14 against one collection strain and one clinical C. perfringens strain was determined at 50 µg/mL. Furthermore, using the intestinal epithelial cell line IPEC-1, it was shown that E. faecalis 14 was not cytotoxic after 24 h of contact, and no cytotoxicity was observed when IPEC-1 cells were incubated with pure enterocin DD14 for 4 h. Enterocin DD14 was characterised using mass spectrometry and was shown to consist of two small proteins of 5200.74 Da and 5206.41 Da, respectively. The two peptides (DD14A and DD14B) have highly similar amino acid sequences and no signal peptide, which classifies enterocin DD14 as a class IIb leaderless two-peptide bacteriocin. The genes encoding DD14A and DD14B were sequenced and were shown to be 100% identical to other previously described enterocins MR10A and MR10B, in contrast to the producing strains, which are different. Consequently, the present in vitro study supports the potential of this E. faecalis 14 strain and/or its purified enterocin DD14 as putative anti-C. perfringens compounds in chickens.

Production of Bacillus amyloliquefaciens OG and its metabolites in renewable media: valorisation for biodiesel production and p-xylene decontamination.

Biosurfactants are important in many areas; however, costs impede large-scale production. This work aimed to develop a global sustainable strategy for the production of biosurfactants by a novel strain of Bacillus amyloliquefaciens. Initially, Bacillus sp. strain 0G was renamed B. amyloliquefaciens subsp. plantarum (syn. Bacillus velezensis) after analysis of the gyrA and gyrB DNA sequences. Growth in modified Landy's medium produced 3 main recoverable metabolites: surfactin, fengycin, and acetoin, which promote plant growth. Cultivation was studied in the presence of renewable carbon (as glycerol) and nitrogen (as arginine) sources. While diverse kinetics of acetoin production were observed in different media, similar yields (6-8 g·L-1) were obtained after 72 h of growth. Glycerol increased surfactin-specific production, while arginine increased the yields of surfactin and fengycin and increased biomass significantly. The specific production of fengycin increased ∼10 times, possibly due to a connecting pathway involving arginine and ornithine. Adding value to crude extracts and biomass, both were shown to be useful, respectively, for the removal of p-xylene from contaminated water and for biodiesel production, yielding ∼70 mg·g-1 cells and glycerol, which could be recycled in novel media. This is the first study considering circular bioeconomy to lower the production costs of biosurfactants by valorisation of both microbial cells and their primary and secondary metabolites.

High-throughput strategies for the discovery and engineering of enzymes for biocatalysis.

Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge, i.e., food industry, polymers and chemical industry. Key tools and technologies, such as bioinformatics tools to guide mutant library design, molecular biology tools to create mutants library, microfluidics/microplates, parallel miniscale bioreactors and mass spectrometry technologies to create high-throughput screening methods and experimental design tools for screening and optimization, allow to evolve the discovery, development and implementation of enzymes and whole cells in (bio)processes. These technological innovations are also accompanied by the development and implementation of clean and sustainable integrated processes to meet the growing needs of chemical, pharmaceutical, environmental and biorefinery industries. This review gives an overview of the benefits of high-throughput screening approach from the discovery and engineering of biocatalysts to cell culture for optimizing their production in integrated processes and their extraction/purification.

Nonribosomal peptide synthetase with a unique iterative-alternative-optional mechanism catalyzes amonabactin synthesis in Aeromonas.

Based on the exploration of data generated by genome sequencing, a bioinformatics approach has been chosen to identify the biosynthetic pathway of the siderophores produced by Aeromonas species. The amonabactins, considered as a virulence factor, represent a family of four variants of catechol peptidic siderophores containing Dhb, Lys, Gly, and an aromatic residue either Trp or Phe in a D-configuration. The synthesis operon is constituted of seven genes named amoCEBFAGH and is iron-regulated. The cluster includes genes encoding proteins involved in the synthesis and incorporation of the Dhb monomer, and genes encoding specific nonribosomal peptide synthetases, which are responsible for the building of the peptidic moiety. The amonabactin assembly line displays a still so far not described atypical mode of synthesis that is iterative, alternative, and optional. A disruption mutant in the adenylation domain of AmoG was unable to synthesize any amonabactin and to grow in iron stress conditions while a deletion of amoH resulted in the production of only two over the four forms. The amo cluster is widespread among most of the Aeromonas species, only few species produces the enterobactin siderophore.

Anti-MRSA Activities of Enterocins DD28 and DD93 and Evidences on Their Role in the Inhibition of Biofilm Formation.

Methicillin-resistant Staphylococcus aureus (MRSA) has become a worrisome superbug. This work aimed at studying the effects of two class IIb bacteriocins, enterocins DD28 and DD93 as anti-MRSA agents. Thus, these bacteriocins were purified, from the cultures supernatants of Enterococcus faecalis 28 and 93, using a simplified purification procedure consisting in a cation exchange chromatography and a reversed-phase high-performance liquid chromatography. The anti-Staphylococcal activity was shown in vitro by the assessment of the minimal inhibitory concentration (MIC), followed by a checkerboard and time-kill kinetics experiments. The data unveiled a clear synergistic effect of enterocins DD28 and DD93 in combination with erythromycin or kanamycin against the clinical MRSA-S1 strain. Besides, these combinations impeded as well the MRSA-S1 clinical strain to setup biofilms on stainless steel and glace devices.

Production of an antimicrobial peptide derived from slaughterhouse by-product and its potential application on meat as preservative.

Bovine cruor, a slaughterhouse by-product, contains mainly hemoglobin, broadly described as a rich source of antimicrobial peptides. In the current context of food safety, bioactive peptides could be of interest as preservatives in the distribution of food products. The aim of this work was to study the α137-141 fragment of hemoglobin (Thr-Ser-Lys-Tyr-Arg), a small (653Da) and hydrophilic antimicrobial peptide. Its production was fast, with more 65% finally produced at 24h already produced after 30min of hydrolysis with pepsin. Moreover, increasing substrate concentration (from 1 to 8% (w/v)) resulted in a proportional augmentation of α137-141 production (to 807.95±41.03mgL(-1)). The α137-141 application on meat as preservative (0.5%, w/w) reduced the lipid oxidation about 60% to delay meat rancidity. The α137-141 peptide also inhibited the microbial growths under refrigeration during 14days. These antimicrobial effects were close to those of the butylated hydroxytoluene (BHT).

Burkholderia genome mining for nonribosomal peptide synthetases reveals a great potential for novel siderophores and lipopeptides synthesis.

Burkholderia is an important genus encompassing a variety of species, including pathogenic strains as well as strains that promote plant growth. We have carried out a global strategy, which combined two complementary approaches. The first one is genome guided with deep analysis of genome sequences and the second one is assay guided with experiments to support the predictions obtained in silico. This efficient screening for new secondary metabolites, performed on 48 gapless genomes of Burkholderia species, revealed a total of 161 clusters containing nonribosomal peptide synthetases (NRPSs), with the potential to synthesize at least 11 novel products. Most of them are siderophores or lipopeptides, two classes of products with potential application in biocontrol. The strategy led to the identification, for the first time, of the cluster for cepaciachelin biosynthesis in the genome of Burkholderia ambifaria AMMD and a cluster corresponding to a new malleobactin-like siderophore, called phymabactin, was identified in Burkholderia phymatum STM815 genome. In both cases, the siderophore was produced when the strain was grown in iron-limited conditions. Elsewhere, the cluster for the antifungal burkholdin was detected in the genome of B. ambifaria AMMD and also Burkholderia sp. KJ006. Burkholderia pseudomallei strains harbor the genetic potential to produce a novel lipopeptide called burkhomycin, containing a peptidyl moiety of 12 monomers. A mixture of lipopeptides produced by Burkholderia rhizoxinica lowered the surface tension of the supernatant from 70 to 27 mN·m(-1) . The production of nonribosomal secondary metabolites seems related to the three phylogenetic groups obtained from 16S rRNA sequences. Moreover, the genome-mining approach gave new insights into the nonribosomal synthesis exemplified by the identification of dual C/E domains in lipopeptide NRPSs, up to now essentially found in Pseudomonas strains.

Food peptidomics of in vitro gastrointestinal digestions of partially purified bovine hemoglobin: low-resolution versus high-resolution LC-MS/MS analyses.

Consumers and governments have become aware how the daily diet may affect the human health. All proteins from both plant and animal origins are potential sources of a wide range of bioactive peptides and the large majority of those display health-promoting effects. In the meat production food chain, the slaughterhouse blood is an inevitable co-product and, today, the blood proteins remain underexploited despite their bioactive potentiality. Through a comparative food peptidomics approach we illustrate the impact of resolving power, accuracy, sensitivity, and acquisition speed of low-resolution (LR)- and high-resolution (HR)-LC-ESI-MS/MS on the obtained peptide mappings and discuss the limitations of MS-based peptidomics. From in vitro gastrointestinal digestions of partially purified bovine hemoglobin, we have established the peptide maps of each hemoglobin chain. LR technique (normal bore C18 LC-LR-ESI-MS/MS) allows us to identify without ambiguity 75 unique peptides while the HR approach (nano bore C18 LC-HR-ESI-MS/MS) unambiguously identify more than 950 unique peptides (post-translational modifications included). Herein, the food peptidomics approach using the most performant separation methods and mass spectrometers with high-resolution capabilities appears as a promising source of information to assess the health potentiality of proteins.

The Tunisian oasis ecosystem is a source of antagonistic Bacillus spp. producing diverse antifungal lipopeptides.

The use of microbial products has become a promising alternative approach to controlling plant diseases caused by phytopathogenic fungi. Bacteria isolated from the date palm tree rhizosphere of the Tunisian oasis ecosystem could provide new biocontrol microorganisms adapted to extreme conditions, such as drought, salinity and high temperature. The aim of this study was to screen bacteria isolated from the rhizosphere of the date palm tree for their ability to inhibit phytopathogenic fungi, and to identify molecules responsible for their antifungal activity. Screening for antifungal activity was performed on twenty-eight isolates. Five antagonistic isolates were selected and identified as different species of Bacillus using phenotypical methods and a molecular approach. The five antagonistic Bacillus isolated showed tolerance to abiotic stresses (high temperature, salinity, drought). Their ability to produce lipopeptides was investigated using a combination of two techniques: PCR amplification and MALDI-ToF mass spectrometry. Analyses revealed that the antagonistic isolates produced a high diversity of lipopeptides that belonged to surfactin, fengycin, iturin and kurstakin families. Their antagonistic activity, related to their capacity for producing diverse antifungal lipopeptides and their tolerance to abiotic stresses, highlighted Bacillus strains isolated from the rhizosphere of the date palm tree as potential biocontrol agents for combatting plant diseases in extreme environments.

Effect of growth temperature, surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants.

The goal of this study was to investigate the effect of the environmental conditions such as the temperature change, incubation time and surface type on the resistance of Staphylococcus aureus biofilms to disinfectants. The antibiofilm assays were performed against biofilms grown at 20 °C, 30 °C and 37 °C, on the stainless steel and polycarbonate, during 24 and 48 h. The involvement of the biofilm matrix and the bacterial membrane fluidity in the resistance of sessile cells were investigated. Our results show that the efficiency of disinfectants was dependent on the growth temperature, the surface type and the disinfectant product. The increase of growth temperature from 20 °C to 37 °C, with an incubation time of 24 h, increased the resistance of biofilms to cationic antimicrobials. This change of growth temperature did not affect the major content of the biofilm matrix, but it decreased the membrane fluidity of sessile cells through the increase of the anteiso-C19 relative amount. The increase of the biofilm resistance to disinfectants, with the rise of the incubation time, was dependent on both growth temperature and disinfectant product. The increase of the biofilm age also promoted increases in the matrix production and the membrane fluidity of sessile cells. The resistance of S. aureus biofilm seems to depend on the environment of the biofilm formation and involves both extracellular matrix and membrane fluidity of sessile cells. Our study represents the first report describing the impact of environmental conditions on the matrix production, sessile cells membrane fluidity and resistance of S. aureus biofilms to disinfectants.

Novel angiotensin I-converting enzyme inhibitory peptides from enzymatic hydrolysates of goby (Zosterisessor ophiocephalus) muscle proteins.

In recent years, food protein-derived bioactive peptides have received considerable attention because of their numerous health benefits. Amongst bioactive peptides, those with antihypertensive activity are receiving special attention due to their role in cardiovascular diseases. Goby protein hydrolysates (GPHs) prepared by treatment with five different crude bacterial proteases were found to exhibit varying degrees of angiotensin I-converting enzyme (ACE) inhibitory activity. The hydrolysate generated by the crude protease from Bacillus mojavensis A21, which displayed the highest ACE inhibitory activity, was further fractionated by size exclusion chromatography on a Sephadex G-25 and reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses and amino acid sequences of five peptides, in sub-fraction F5-2, which exhibited the highest ACE inhibitory activity, were determined using ESI-MS and ESI-MS/MS, respectively. The structures of these peptides were identified as Ala-Arg-Ser, Val-Val-Ala-Pro-Phe-Ala-His-Gly-Thr, Arg-Ser-Thr-Ala, Phe-Tyr-Pro-Pro, Arg-Cys-Ser-Ala-Gly-Val. Further, the sequences of fifteen peptides in the F5-4 sub-fraction, which exhibited high activity, were determined. Therefore, GPHs have a potential as hypotensive nutraceutical ingredients. BIOLOGICAL SIGNIFICANCE: Peptides find many outlets of application in the biotechnological field, amongst which are pharmaceutical applications. Progression amongst new small molecules deposited like substance medicamentous blows itself. In this context, large pharmaceutical companies invest in peptide research to open therapeutic new prospects. Even if they are used as therapeutic agents for nearly one century in their natural form, the use of peptides remains parsimonious although we experienced a significant development since a few tens of years, in particular thanks to the clarification of the methods of production, chemical in solid or biological phase such as in phage display. Peptides present many advantages compared to traditional drugs that have small molecules, Generation of bioactive peptides by proteolysis of food proteins, using exogenous proteases, is a new and interesting approach for the production and identification of new and potent specific hypotensive agents. From another side, compared with natural peptides isolated from different sources, there is more diversity in structure and mode of action of the derived bioactive peptides. In fact, proteolysis of protein substrates, having different amino acid composition and sequences, by proteases having different specificities may generate numerous specific peptide inhibitors, with different lengths and amino acid sequences. These bioactive peptides have received considerable attention for their effectiveness in both the prevention and the treatment of hypertension.

Self-assembly of three bacterially-derived bioactive lipopeptides.

The self-assembly in aqueous solution of three lipopeptides obtained from Bacillus subtilis has been investigated. The lipopeptides surfactin, plipastatin and mycosubtilin contain distinct cyclic peptide headgroups as well as differences in alkyl chain length, branching and chain length distribution. Cryogenic transmission electron microscopy and X-ray scattering reveal that surfactin and plipastatin aggregate into 2 nm-radius spherical micelles, whereas in complete contrast mycosubtilin self-assembles into extended nanotapes based on bilayer ordering of the lipopeptides. Circular dichroism and FTIR spectroscopy indicate the presence of turn structures in the cyclic peptide headgroup. The unexpected distinct mode of self-assembly of mycosubtilin compared to the other two lipopeptides is ascribed to differences in the surfactant packing parameter. This in turn is due to specific features of the conformation of the peptide headgroup and alkyl chain branching.