Bacillus velezensis: a versatile ally in the battle against phytopathogens-insights and prospects.

The escalating interest in Bacillus velezensis as a biocontrol agent arises from its demonstrated efficacy in inhibiting both phytopathogenic fungi and bacteria, positioning it as a promising candidate for biotechnological applications. This mini review aims to offer a comprehensive exploration of the multifaceted properties of B. velezensis, with particular focus on its beneficial interactions with plants and its potential for controlling phytopathogenic fungi. The molecular dialogues involving B. velezensis, plants, and phytopathogens are scrutinized to underscore the intricate mechanisms orchestrating these interactions. Additionally, the review elucidates the mode of action of B. velezensis, particularly through cyclic lipopeptides, highlighting their importance in biocontrol and promoting plant growth. The agricultural applications of B. velezensis are detailed, showcasing its role in enhancing crop health and productivity while reducing reliance on chemical pesticides. Furthermore, the review extends its purview in the industrial and environmental arenas, highlighting its versatility across various sectors. By addressing challenges such as formulation optimization and regulatory frameworks, the review aims to chart a course for the effective utilization of B. velezensis. KEY POINTS: • B. velezensis fights phytopathogens, boosting biotech potential • B. velezensis shapes agri-biotech future, offers sustainable solutions • Explores plant-B. velezensis dialogue, lipopeptide potential showcased.

Lipopeptides from Bacillus velezensis ZLP-101 and their mode of action against bean aphids Acyrthosiphon pisum Harris.

BACKGROUND: Natural products are important sources for the discovery of new biopesticides to control the worldwide destructive pests Acyrthosiphon pisum Harris. Here, insecticidal substances were discovered and characterized from the secondary metabolites of the bio-control microorganism Bacillus velezensis strain ZLP-101, as informed by whole-genome sequencing and analysis. RESULTS: The genome was annotated, revealing the presence of four potentially novel gene clusters and eight known secondary metabolite synthetic gene clusters. Crude extracts, prepared through ammonium sulfate precipitation, were used to evaluate the effects of strain ZLP-101 on Acyrthosiphon pisum Harris aphid pests via exposure experiments. The half lethal concentration (LC50) of the crude extract from strain ZLP-101 against aphids was 411.535 mg/L. Preliminary exploration of the insecticidal mechanism revealed that the crude extract affected aphids to a greater extent through gastric poisoning than through contact. Further, the extracts affected enzymatic activities, causing holes to form in internal organs along with deformation, such that normal physiological activities could not be maintained, eventually leading to death. Isolation and purification of extracellular secondary metabolites were conducted in combination with mass spectrometry analysis to further identify the insecticidal components of the crude extracts. A total of 15 insecticidal active compounds were identified including iturins, fengycins, surfactins, and spergualins. Further insecticidal experimentation revealed that surfactin, iturin, and fengycin all exhibited certain aphidicidal activities, and the three exerted synergistic lethal effects. CONCLUSIONS: This study improved the available genomic resources for B. velezensis and serves as a foundation for comprehensive studies of the insecticidal mechanism by Bacillus velezensis ZLP-101 in addition to the active components within biological control strains.

A cyclic lipopeptide from Fusarium graminearum targets plant membranes to promote virulence.

Microbial plant pathogens deploy amphipathic cyclic lipopeptides to reduce surface tension in their environment. While plants can detect these molecules to activate cellular stress responses, the role of these lipopeptides or associated host responses in pathogenesis are not fully clear. The gramillin cyclic lipopeptide is produced by the Fusarium graminearum fungus and is a virulence factor and toxin in maize. Here, we show that gramillin promotes virulence and necrosis in both monocots and dicots by disrupting ion balance across membranes. Gramillin is a cation-conducting ionophore and causes plasma membrane depolarization. This disruption triggers cellular signaling, including a burst of reactive oxygen species (ROS), transcriptional reprogramming, and callose production. Gramillin-induced ROS depends on expression of host ILK1 and RBOHD genes, which promote fungal induction of virulence genes during infection and host susceptibility. We conclude that gramillin's ionophore activity targets plant membranes to coordinate attack by the F. graminearum fungus.

Investigation of the influence of pH and temperature on melanization and survival under oxidative stress in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic pathogenic fungus that produces melanin during infection, an important virulence factor in Cryptococcal infections that enhances the ability of the fungus to resist immune defense. This fungus can synthesize melanin from a variety of substrates, including L-DOPA (L-3,4-dihydroxyphenylalanine). Since melanin protects the fungus from various stress factors such as oxidative, nitrosative, extreme heat and cold stress; we investigated the effects of environmental conditions on melanin production and survival. In this study, we investigated the effects of different pH values (5.6, 7.0 and 8.5) and temperatures (30 °C and 37 °C) on melanization and cell survival using a microtiter plate-based melanin production assay and an oxidative stress assay, respectively. In addition, the efficacy of compounds known to inhibit laccase involved in melanin synthesis, i.e., tunicamycin, ß-mercaptoethanol, dithiothreitol, sodium azide and caspofungin on melanization was evaluated and their sensitivity to temperature and pH changes was measured. The results showed that melanin content correlated with pH and temperature changes and that pH 8.5 and 30 °C, were best for melanin production. Besides that, melanin production protects the fungal cells from oxidative stress induced by hydrogen peroxide. Thus, changes in pH and temperature drastically alter melanin production in C. neoformans and it correlates with the fungal survival. Due to the limited antifungal repertoire and the development of resistance in cryptococcal infections, the investigation of environmental conditions in the regulation of melanization and survival of C. neoformans could be useful for future research and clinical phasing.

Microscopic and Transcriptomic Analyses to Elucidate Antifungal Mechanisms of Bacillus velezensis TCS001 Lipopeptides against Botrytis cinerea.

Botrytis cinerea is an important fungal pathogen that causes gray mold disease in plants. Previously, Bacillus velezensis TCS001 live culture presented broad-spectrum antifungal activity against various plant pathogenic fungi and oomycetes, particularly B. cinerea. Here, the bioactivity of lipopeptides produced by TCS001 against B. cinerea was investigated. The IC50 values of the crude lipopeptide extract (CLE) from TCS001 to suppress mycelial growth and conidial germination were 14.20 and 49.39 mg/L, respectively. SEM and TEM imaging revealed that CLE caused morphological deformities and ultrastructural changes in the mycelium. Transcriptomic analyses combined with ΔBcpsd mutant construction demonstrated that the CLE could confer antifungal activity via suppressing Bcpsd expression in the pathogen. In addition, the CLE activated the plant immune system by increasing the content of defense-related enzymes and the expression of marker genes in immunity signaling pathways in cucumber plants. Therefore, TCS001 CLE could be potentially developed into biopesticides for the biocontrol of gray mold disease.

Production and optimization of surfactin produced from locally isolated Bacillus halotolerans grown on agro-industrial wastes and its antimicrobial efficiency.

INTRODUCTION: Optimal exploitation of the huge amounts of agro-industrial residuals that are produced annually, which endangers the ecosystem and ultimately contributes to climate change, is one of the solutions available to produce value-added compounds. AIM AND OBJECTIVES: This study aimed at the economic production and optimization of surfactin. Therefore, the production was carried out by the microbial conversion of Potato Peel Waste (PPW) and Frying Oil Waste (FOW) utilizing locally isolated Bacillus halotolerans. Also, investigating its potential application as an antimicrobial agent towards some pathogenic strains. RESULTS: Screening the bacterial isolates for surfactin production revealed that the strain with the highest yield (49 g/100 g substrate) and efficient oil displacement activity was genetically identified as B. halotolerans. The production process was then optimized utilizing Central Composite Design (CCD) resulting in the amelioration of yield by 11.4% (from 49 to 55.3 g/100 g substrate) and surface tension (ST) by 8.3% (from 36 to 33 mN/m) with a constant level of the critical micelle concentration (CMC) at 125 mg/L. Moreover, the physiochemical characterization studies of the produced surfactin by FTIR, 1H NMR, and LC-MS/MS proved the existence of a cyclic lipopeptide (surfactin). The investigations further showed a strong emulsification affinity for soybean and motor oil (E24 = 50%), as well as the ability to maintain the emulsion stable over a wide pH (4-10) and temperature (10-100 °C) range. Interestingly, surfactin had a broad-spectrum range of inhibition activity against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, klebsiella pneumonia, and Candida albicans. CONCLUSION: Subsequently, the screening of the isolates and the utilized food-processing wastes along with the extraction technique resulted in a high yield of surfactin characterized by acceptable ST and CMC levels. However, optimization of the cultural conditions to improve the activity and productivity was achieved using Factor-At-A-Time (OFAT) and Central Composite Design (CCD). In contrast, surface activity recorded a maximum level of (33 mN/n) and productivity of 55.3 g/100 g substrate. The optimized surfactin had also the ability to maintain the stability of emulsions over a wide range of pH and temperature. Otherwise, the obtained results proved the promising efficiency of the surfactin against bacterial and fungal pathogens.

The potential of Pseudomonas fluorescens SBW25 to produce viscosin enhances wheat root colonization and shapes root-associated microbial communities in a plant genotype-dependent manner in soil systems.

Microorganisms interact with plant roots through colonization of the root surface, i.e., the rhizoplane or the surrounding soil, i.e., the rhizosphere. Beneficial rhizosphere bacteria such as Pseudomonas spp. can promote plant growth and protect against pathogens by producing a range of bioactive compounds, including specialized metabolites like cyclic lipopeptides (CLPs) known for their biosurfactant and antimicrobial activities. However, the role of CLPs in natural soil systems during bacteria-plant interactions is underexplored. Here, Pseudomonas fluorescens SBW25, producing the CLP viscosin, was used to study the impact of viscosin on bacterial root colonization and microbiome assembly in two cultivars of winter wheat (Heerup and Sheriff). We inoculated germinated wheat seeds with SBW25 wild type or a viscosin-deficient mutant and grew the plants in agricultural soil. After 2 weeks, enhanced root colonization of SBW25 wild type compared to the viscosin-deficient mutant was observed, while no differences were observed between wheat cultivars. In contrast, the impact on root-associated microbial community structure was plant-genotype-specific, and SBW25 wild type specifically reduced the relative abundance of an unclassified oomycete and Phytophthora in Sheriff and Heerup, respectively. This study provides new insights into the natural role of viscosin and specifically highlights the importance of viscosin in wheat root colonization under natural soil conditions and in shaping the root microbial communities associated with different wheat cultivars. Furthermore, it pinpoints the significance of microbial microdiversity, plant genotype, and microbe-microbe interactions when studying colonization of plant roots. IMPORTANCE: Understanding parameters governing microbiome assembly on plant roots is critical for successfully exploiting beneficial plant-microbe interactions for improved plant growth under low-input conditions. While it is well-known from in vitro studies that specialized metabolites are important for plant-microbe interactions, e.g., root colonization, studies on the ecological role under natural soil conditions are limited. This might explain the often-low translational power from laboratory testing to field performance of microbial inoculants. Here, we showed that viscosin synthesis potential results in a differential impact on the microbiome assembly dependent on wheat cultivar, unlinked to colonization potential. Overall, our study provides novel insights into factors governing microbial assembly on plant roots, and how this has a derived but differential effect on the bacterial and protist communities.

Inhibitory activity of bacterial lipopeptides against Fusarium oxysporum f.sp. Strigae.

This study investigated the influence of bacterial cyclic lipopeptides (LP; surfactins, iturins, fengycins) on microbial interactions. The objective was to investigate whether the presence of bacteria inhibits fungal growth and whether this inhibition is due to the release of bacterial metabolites, particularly LP. Selected endophytic bacterial strains with known plant-growth promoting potential were cultured in the presence of Fusarium oxysporum f.sp. strigae (Fos), which was applied as model fungal organism. The extracellular metabolome of tested bacteria, with a focus on LP, was characterized, and the inhibitory effect of bacterial LP on fungal growth was investigated. The results showed that Bacillus velezensis GB03 and FZB42, as well as B. subtilis BSn5 exhibited the strongest antagonism against Fos. Paraburkholderia phytofirmans PsJN, on the other hand, tended to have a slight, though non-significant growth promotion effect. Crude LP from strains GB03 and FZB42 had the strongest inhibitory effect on Fos, with a significant inhibition of spore germination and damage of the hyphal structure. Liquid chromatography tandem mass spectrometry revealed the production of several variants of iturin, fengycin, and surfactin LP families from strains GB03, FZB42, and BSn5, with varying intensity. Using plate cultures, bacillomycin D fractions were detected in higher abundance in strains GB03, FZB42, and BSn5 in the presence of Fos. Additionally, the presence of Fos in dual plate culture triggered an increase in bacillomycin D production from the Bacillus strains. The study demonstrated the potent antagonistic effect of certain Bacillus strains (i.e., GB03, FZB42, BSn5) on Fos development. Our findings emphasize the crucial role of microbial interactions in shaping the co-existence of microbial assemblages.

Strategies for improving fengycin production: a review.

Fengycin is an important member of the lipopeptide family with a wide range of applications in the agricultural, food, medical and cosmetic industries. However, its commercial application is severely hindered by low productivity and high cost. Therefore, numerous studies have been devoted to improving the production of fengycin. We summarize these studies in this review with the aim of providing a reference and guidance for future researchers. This review begins with an overview of the synthesis mechanism of fengycin via the non-ribosomal peptide synthetases (NRPS), and then delves into the strategies for improving the fengycin production in recent years. These strategies mainly include fermentation optimization and metabolic engineering, and the metabolic engineering encompasses enhancement of precursor supply, application of regulatory factors, promoter engineering, and application of genome-engineering (genome shuffling and genome-scale metabolic network model). Finally, we conclude this review with a prospect of fengycin production.

Enhanced surface colonisation and competition during bacterial adaptation to a fungus.

Bacterial-fungal interactions influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Here, we use a co-culture experimental evolution approach to investigate bacterial adaptation to the presence of a fungus, using a simple model of bacterial-fungal interactions encompassing the bacterium Bacillus subtilis and the fungus Aspergillus niger. We find in one evolving population that B. subtilis was selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles possibly provoking an RlmA-dependent cell wall stress. Thus, our results indicate that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates the competitive success of the bacterium.

Physiochemical interaction between osmotic stress and a bacterial exometabolite promotes plant disease.

Various microbes isolated from healthy plants are detrimental under laboratory conditions, indicating the existence of molecular mechanisms preventing disease in nature. Here, we demonstrated that application of sodium chloride (NaCl) in natural and gnotobiotic soil systems is sufficient to induce plant disease caused by an otherwise non-pathogenic root-derived Pseudomonas brassicacearum isolate (R401). Disease caused by combinatorial treatment of NaCl and R401 triggered extensive, root-specific transcriptional reprogramming that did not involve down-regulation of host innate immune genes, nor dampening of ROS-mediated immunity. Instead, we identified and structurally characterized the R401 lipopeptide brassicapeptin A as necessary and sufficient to promote disease on salt-treated plants. Brassicapeptin A production is salt-inducible, promotes root colonization and transitions R401 from being beneficial to being detrimental on salt-treated plants by disturbing host ion homeostasis, thereby bolstering susceptibility to osmolytes. We conclude that the interaction between a global change stressor and a single exometabolite from a member of the root microbiome promotes plant disease in complex soil systems.

Biosynthesis of isonitrile lipopeptides.

Isonitrile lipopeptides discovered from Actinobacteria have attracted wide attention due to their fascinating biosynthetic pathways and relevance to the virulence of many human pathogens including Mycobacterium tuberculosis. Specifically, the identification of the new class of isonitrile-forming enzymes that belong to non-heme iron (II) and α-ketoglutarate dependent dioxygenases has intrigued several research groups to investigate their catalytic mechanism. Here we summarize the recent studies on the biosynthesis of isonitrile lipopeptides from Streptomyces and Mycobacterium. The latest research on the core and tailoring enzymes involved in the pathway as well as the isonitrile metabolic enzymes are discussed in this review.

Improving salt-tolerant artificial consortium of Bacillus amyloliquefaciens for bioconverting food waste to lipopeptides.

High-salt content in food waste (FW) affects its resource utilization during biotransformation. In this study, adaptive laboratory evolution (ALE), gene editing, and artificial consortia were performed out to improve the salt-tolerance of Bacillus amyloliquefaciens for producing lipopeptide under FW and seawater. High-salt stress significantly decreased lipopeptide production in the B. amyloliquefaciens HM618 and ALE strains. The total lipopeptide production in the recombinant B. amyloliquefaciens HM-4KSMSO after overexpressing the ion transportor gene ktrA and proline transporter gene opuE and replacing the promoter of gene mrp was 1.34 times higher than that in the strain HM618 in medium containing 30 g/L NaCl. Lipopeptide production under salt-tolerant consortia containing two strains (HM-4KSMSO and Corynebacterium glutamicum) and three-strains (HM-4KSMSO, salt-tolerant C. glutamicum, and Yarrowia lipolytica) was 1.81- and 2.28-fold higher than that under pure culture in a medium containing FW or both FW and seawater, respectively. These findings provide a new strategy for using high-salt FW and seawater to produce value-added chemicals.

Non-modular fatty acid synthases yield distinct N-terminal acylation in ribosomal peptides.

Recent efforts in genome mining of ribosomally synthesized and post-translationally modified peptides (RiPPs) have expanded the diversity of post-translational modification chemistries. However, RiPPs are rarely reported as hybrid molecules incorporating biosynthetic machinery from other natural product families. Here we report lipoavitides, a class of RiPP/fatty-acid hybrid lipopeptides that display a unique, putatively membrane-targeting 4-hydroxy-2,4-dimethylpentanoyl (HMP)-modified N terminus. The HMP is formed via condensation of isobutyryl-coenzyme A (isobutyryl-CoA) and methylmalonyl-CoA catalysed by a 3-ketoacyl-(acyl carrier protein) synthase III enzyme, followed by successive tailoring reactions in the fatty acid biosynthetic pathway. The HMP and RiPP substructures are then connected by an acyltransferase exhibiting promiscuous activity towards the fatty acyl and RiPP substrates. Overall, the discovery of lipoavitides contributes a prototype of RiPP/fatty-acid hybrids and provides possible enzymatic tools for lipopeptide bioengineering.

Inhibition mechanism of crude lipopeptide from Bacillus subtilis against Aeromonas veronii growth, biofilm formation, and spoilage of channel catfish flesh.

Aeromonas veronii is associated with food spoilage and some human diseases, such as diarrhea, gastroenteritis, hemorrhagic septicemia or asymptomatic and even death. This research investigated the mechanism of the growth, biofilm formation, virulence, stress resistance, and spoilage potential of Bacillus subtilis lipopeptide against Aeromonas veronii. Lipopeptides suppressed the transmembrane transport of Aeromonas veronii by changing the cell membrane's permeability, the structure of membrane proteins, and Na+/K+-ATPase. Lipopeptide significantly reduced the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) by 86.03% and 56.12%, respectively, ultimately slowing Aeromonas veronii growth. Lipopeptides also restrained biofilm formation by inhibiting Aeromonas veronii motivation and extracellular polysaccharide secretion. Lipopeptides downregulated gene transcriptional levels related to the virulence and stress tolerance of Aeromonas veronii. Furthermore, lipopeptides treatment resulted in a considerable decrease in the extracellular protease activity of Aeromonas veronii, which restrained the decomposing of channel catfish flesh. This research provides new insights into lipopeptides for controlling Aeromonas veronii and improving food safety.

Lipopeptide C17 Fengycin B Exhibits a Novel Antifungal Mechanism by Triggering Metacaspase-Dependent Apoptosis in Fusarium oxysporum.

Fusarium wilt is a worldwide soil-borne fungal disease caused by Fusarium oxysporum that causes serious damage to agricultural products. Therefore, preventing and treating fusarium wilt is of great significance. In this study, we purified ten single lipopeptide fengycin components from Bacillus subtilis FAJT-4 and found that C17 fengycin B inhibited the growth of F. oxysporum FJAT-31362. We observed early apoptosis hallmarks, including reactive oxygen species accumulation, mitochondrial dysfunction, and phosphatidylserine externalization in C17 fengycin B-treated F. oxysporum cells. Further data showed that C17 fengycin B induces cell apoptosis in a metacaspase-dependent manner. Importantly, we found that the expression of autophagy-related genes in the TOR signaling pathway was significantly upregulated; simultaneously, the accumulation of acidic autophagy vacuoles in F. oxysporum cell indicated that the autophagy pathway was activated during apoptosis induced by C17 fengycin B. Therefore, this study provides new insights into the antifungal mechanism of fengycin.

Microbial lipopeptides: their pharmaceutical and biotechnological potential, applications, and way forward.

As lead molecules, cyclic lipopeptides with antibacterial, antifungal, and antiviral properties have garnered a lot of attention in recent years. Because of their potential, cyclic lipopeptides have earned recognition as a significant class of antimicrobial compounds with applications in pharmacology and biotechnology. These lipopeptides, often with biosurfactant properties, are amphiphilic, consisting of a hydrophilic moiety, like a carboxyl group, peptide backbone, or carbohydrates, and a hydrophobic moiety, mostly a fatty acid. Besides, several lipopeptides also have cationic groups that play an important role in biological activities. Antimicrobial lipopeptides can be considered as possible substitutes for antibiotics that are conventional to address the current drug-resistant issues as pharmaceutical industries modify the parent antibiotic molecules to render them more effective against antibiotic-resistant bacteria and fungi, leading to the development of more resistant microbial strains. Bacillus species produce lipopeptides, which are secondary metabolites that are amphiphilic and are typically synthesized by non-ribosomal peptide synthetases (NRPSs). They have been identified as potential biocontrol agents as they exhibit a broad spectrum of antimicrobial activity. A further benefit of lipopeptides is that they can be produced and purified biotechnologically or biochemically in a sustainable manner using readily available, affordable, renewable sources without harming the environment. In this review, we discuss the biochemical and functional characterization of antifungal lipopeptides, as well as their various modes of action, method of production and purification (in brief), and potential applications as novel antibiotic agents.

Lipopeptides from Bacillus velezensis induced apoptosis-like cell death in the pathogenic fungus Fusarium concentricum.

AIMS: Stem rot caused by Fusarium concentricum is a new disease of Paris polyphylla reported by our research group. The present study investigates the growth inhibitory and apoptotic effects of Bacillus velezensis FJAT-54560 lipopeptide against F. concentricum. METHODS AND RESULTS: HPLC preparation and LC-MS analysis results show that the crude lipopeptides secreted by Bacillus velezensis FJAT-54560 isolated from Jasminum sambac consist of C14-17 iturin A, C14 fengycin B, C16 fengycin A/A2, C18 fengycin A, C20 fengycin B2, C21 fengycin A2, C22-23 fengycin A, C12-16 surfactin A, and C15 surfactin A derivatives. The mass ratios (g/g) of iturin, fengycin, and surfactin in lipopeptides are 2.40, 67.51, and 30.08%, respectively. Through inhibition zone and inhibition rate experiments, we found that crude lipopeptides and purified fengycin exhibit strong antifungal activity against F. concentricum, including accumulation of reactive oxygen species, loss of mitochondrial membrane potential, DNA fragmentation, Ca2+ accumulation, chromatin condensation, and phosphatidylserine externalization. Transcriptomic analysis indicates that crude lipopeptide-induced apoptosis in F. concentricum cells may be mediated by apoptosis-inducing factors and apoptosis mediators and can serve as a metacaspase-independent model. CONCLUSION: Lipopeptides from Bacillus velezensis FJAT-54560 can control the pathogenic fungus F. concentricum by inducing apoptosis.

Crude Lipopeptides Produced by Bacillus amyloliquefaciens Could Control the Growth of Alternaria alternata and Production of Alternaria Toxins in Processing Tomato.

Alternaria spp. and its toxins are the main contaminants in processing tomato. Based on our earlier research, the current study looked into the anti-fungal capacity of crude lipopeptides from B. amyloliquefaciens XJ-BV2007 against A. alternata. We found that the crude lipopeptides significantly inhibited A. alternata growth and reduced tomato black spot disease incidence. SEM analysis found that the crude lipopeptides could change the morphology of mycelium and spores of A. alternata. Four main Alternaria toxins were detected using UPLC-MS/MS, and the findings demonstrated that the crude lipopeptides could lessen the accumulation of Alternaria toxins in vivo and in vitro. Meanwhile, under the stress of crude lipopeptides, the expression of critical biosynthetic genes responsible for TeA, AOH, and AME was substantially down-regulated. The inhibitory mechanism of the crude lipopeptides was demonstrated to be the disruption of the mycelial structure of A. alternata, as well as the integrity and permeability of the membrane of A. alternata sporocytes. Taken together, crude lipopeptides extracted from B. amyloliquefaciens XJ-BV2007 are an effective biological agent for controlling tomato black spot disease and Alternaria toxins contamination.

Biosynthetic mechanism of the yellow pigments in the marine bacterium Pseudoalteromonas sp. strain T1lg65.

The Pseudoalteromonas genus marine bacteria have attracted increasing interest because of their abilities to produce bioactive metabolites. The pigmented Pseudoalteromonas group encodes more secondary metabolite biosynthetic gene clusters (BGCs) than the non-pigmented group. Here, we report a yellow pigmented bacterium Pseudoalteromonas sp. strain T1lg65, which was isolated from a mangrove forest sediment. We showed that the yellow pigments of T1lg65 belong to the group of lipopeptide alterochromides. Further genetic analyses of the alterochromide BGC revealed that the yellow pigments are biosynthesized by aryl-polyene synthases and nonribosomal peptide synthases. Within the gene cluster, altA encodes a tyrosine ammonia acid lyase, which catalyzes synthesis of the precursor 4-hydroxycinnamic acid (4-HCA) from tyrosine in the alterochromide biosynthetic pathway. In addition, altN, encoding a putative flavin-dependent halogenase, was proven to be responsible for the bromination of alterochromides based on gene deletion, molecular docking, and site mutagenesis analyses. In summary, the biosynthetic pathway, precursor synthesis, and bromination mechanism of the lipopeptide alterochromides were studied in-depth. Our results expand the knowledge on biosynthesis of Pseudoalteromonas pigments and could promote the development of active pigments in the future.IMPORTANCEThe marine bacteria Pseudoalteromonas spp. are important biological resources because they are producers of bioactive natural products, including antibiotics, pigments, enzymes, and antimicrobial peptides. One group of the microbial pigments, alterochromides, holds a great value for their novel lipopeptide structures and antimicrobial activities. Previous studies were limited to the structural characterization of alterochromides and genome mining for the alterochromide biosynthesis. This work focused on the biosynthetic mechanism for alterochromide production, especially revealing functions of two key genes within the gene cluster for the alterochromide biosynthesis. On the one hand, our study provides a target for metabolic engineering of the alterochromide biosynthesis; on the other hand, the 4-HCA synthase AltA and brominase AltN show potential in the biocatalyst industry.