Lipopeptides Iturin Research Articles

Transcriptomic and Biochemical Analysis of the Antimicrobial Mechanism of Lipopeptide Iturin W against Staphylococcus aureus.

Staphylococcus aureus is one of the most serious pathogens threatening food safety and public health. We have previously showed that iturin W exhibited obvious antifungal activity on plant pathogens. In the present study, we found iturin W, especially C14 iturin W, showed strong antimicrobial activity against S. aureus, and the antimicrobial mechanism of C14 iturin W was further investigated by transcriptomic analysis and a related biochemical experiment. The results showed that C14 iturin W can reduce the expression levels of genes associated with the reactive oxygen species (ROS) scavenging enzyme and genes involved in arginine biosynthesis, thus leading to the increase in ROS levels of S. aureus. Furthermore, C14 iturin W can also interfere with proton dynamics, which is crucial for cells to regulate various biological possesses. Therefore, ROS accumulation and change in proton motive force are import ways for C14 iturin W to exert the antimicrobial activity. In addition, C14 iturin W can also reduce the expression levels of genes related to virulence factors and decrease the production of enterotoxins and hemolysins in S. aureus, indicating that C14 iturin W has a good potential in food and pharmaceutical fields to reduce the harm caused by S. aureus in the future.

Insights into the antifungal mechanism of Bacillus subtilis cyclic lipopeptide iturin A mediated by potassium ion channel

Fungal infections pose severe and potentially lethal threats to plant, animal, and human health. Ergosterol has served as the primary target for developing antifungal medications. However, many antifungal drugs remain highly toxic to humans due to similarity in cell membrane composition between fungal and animal cells. Iturin A, lipopeptide produced by Bacillus subtilis, efficiently inhibit various fungi, but demonstrated safety in oral administration, indicating the existence of targets different from ergosterol. To pinpoint the exact antifungal target of iturin A, we used homologous recombination to knock out and overexpress erg3, a key gene in ergosterol synthesis. Saccharomyces cerevisiae and Aspergillus carbonarius were transformed using the LiAc/SS-DNNPEG and Agrobacterium-mediated transformation (AMT), respectively. Surprisingly, increasing ergosterol content did not augment antifungal activity. Furthermore, iturin A's antifungal activity against S. cerevisiae was reduced while it pre-incubation with voltage-gated potassium (Kv) channel inhibitor, indicating that Kv activation was responsible for cell death. Iturin A was found to activate the Kv protein, stimulating K+ efflux from cell. In vitro tests confirmed interaction between iturin A and Kv protein. This study highlights Kv as one of the precise targets of iturin A in its antifungal activity, offering a novel target for the development of antifungal medications.

Exploring Mechanisms of Antifungal Lipopeptide Iturin A from Bacillus against Aspergillus niger.

The control of Aspergillus niger (A. niger) is of great significance for the agricultural economy and food safety. In this study, the antifungal effect and mechanism of iturin A from Bacillus amyloliquefaciens (CGMCC No. 8473) against A. niger (ATCC 16404) were investigated using biochemical analyses and proteomics. Changes in a mycelium treated with iturin A were observed using scanning electron microscopy and transmission electron microscopy, including mycelial twisting and collapse, organelle disintegration, and intracellular vacuolization. The cytomembrane integrity of A. niger was affected by iturin A, as detected by propidium iodide staining. In addition, the generation of excess reactive oxygen species, the hyperpolarization of the mitochondrial membrane potential and malondialdehyde accumulation also indicated that iturin A induced apoptosis in A. niger through the oxidative stress pathway. Proteomics results showed that 310 proteins were differentially expressed in the A. niger mycelium exposed to iturin A, including 159 upregulated proteins and 151 downregulated proteins, which were mainly associated with energy metabolism of A. niger. We propose that iturin A might inhibit the growth of A. niger by disrupting cytomembrane integrity, via oxidative stress, and by interfering with glycolysis/gluconeogenesis and the tricarboxylic acid cycle. Overall, iturin A is a promising antifungal agent that provides a rationale for controlling A. niger contamination in food.

Total synthesis of antifungal lipopeptide iturin A analogues and evaluation of their bioactivity against F. graminearum.

The pursuit of novel antifungal agents is imperative to tackle the threat of antifungal resistance, which poses major risks to both human health and to food security. Iturin A is a cyclic lipopeptide, produced by Bacillus sp., with pronounced antifungal properties against several pathogens. Its challenging synthesis, mainly due to the laborious synthesis of the β-amino fatty acid present in its structure, has hindered the study of its mode of action and the development of more potent analogues. In this work, a facile synthesis of bioactive iturin A analogues containing an alkylated cysteine residue is presented. Two analogues with opposite configurations of the alkylated cysteine residue were synthesized, to evaluate the role of the stereochemistry of the newly introduced amino acid on the bioactivity. Antifungal assays, conducted against F. graminearum, showed that the novel analogues are bioactive and can be used as a synthetic model for the design of new analogues and in structure-activity relationship studies. The assays also highlight the importance of the β-amino acid in the natural structure and the role of the stereochemistry of the amino fatty acid, as the analogue with the D configuration showed stronger antifungal properties than the one with the L configuration.

Bacillus subtilis engineered for topical delivery of an antifungal agent.

Fungal skin infections are a common condition affecting 20-25 percent of the world population. While these conditions are treatable with regular application of an antifungal medication, we sought to develop a more convenient, longer-lasting topical antifungal platform that could increase patient adherence to treatment regimens by using Bacillus subtilis, a naturally antifungal bacteria found on the skin, for drug production and delivery. In this study, we engineered B. subtilis for increased production of the antifungal lipopeptide iturin A by overexpression of the pleiotropic regulator DegQ. The engineered strain had an over 200% increase in iturin A production as detected by HPLC, accompanied by slower growth but the same terminal cell density as determined by absorbance measurements of liquid culture. In an in vitro antifungal assay, we found that despite its higher iturin A production, the engineered strain was less effective at reducing the growth of a plug of the pathogenic fungus Trichophyton mentagrophytes on an agar plate compared to the parent strain. The reduced efficacy of the engineered strain may be explained by its reduced growth rate, which highlights the need to address trade-offs between titers (e.g. measured drug production) and other figures of merit (e.g. growth rate) during metabolic engineering.

Production optimization of antifungal lipopeptides by Bacillus subtilis CtpxS2-1 using low-cost optimized medium

The aim of the present study was evaluated the use of low-cost optimized medium defatted soy flour and molasses (DSF40–MOL5; 40 g l−1 defatted soy flour, 5 g l−1 molasses plus mineral trace supplements) to improvement the antifungal lipopeptides production by biocontrol agent Bacillus subtilis CtpxS2-1. First, population dynamics of CtpxS2-1 in DSF40–MOL5 medium was recorded in a 5-l fermenter during 24 h, 48 h and 72 h and compared with the bacterium grown in medium optimal for lipopeptide production (MOLP) as control. Results showed that the CtpxS2-1 population dynamics, pH and oxygen consumption was similar in both media with a 4–h lag phase, followed by exponential growth and maximum production around 9.5 log CFU ml−1 after 16–18 h. Second, lipopeptides extracts quantifications, followed of high-performance thin layer chromatographic (HPTLC) and bioautography analysis against Colletotrichum acutatum showed a significant increment of fengycin and iturin lipopeptides production and their antifungal activity in DSF40–MOL5 compared with MOLP. PCR (RT-PCR)/RT q-PCR analysis during production confirmed the transcription of fengycin, iturin an surfactin lipopeptides by amplification of cDNA; and that FenB and ItuA genes were progressive expressed in higher amounts throughout in DSF40–MOL5 than MOLP. The biosynthesis of FenB gene and its antifungal activity against C. acutatum was the highest for CtpxS2-1 grown in DSF40–MOL5. Surfactin genes were also expressed during production process however their didn’t showed antifungal activity Finally, treatment of artificially infected seed of three cultivars of Lupinus mutabilis with CtpxS2-1 grown in DSF40-MOL5 reduced anthracnose up to 0% and improved seed and plant health. This evidence supports that antifungal potential of B.subtilis CtpxS2-1 to control lupin anthracnose could be enhanced using low-cost medium DSF40–MOL5 and it could provide a reliable method for scaling up the bacterial fermentation process at industrial level.

New Bacillus subtilis Strains Isolated from Prosopis glandulosa Rhizosphere for Suppressing Fusarium Spp. and Enhancing Growth of Gossypium hirsutum L.

Rhizobacteria from desert plants can alleviate biotic stress and suppress plant diseases, and consequently can enhance plant growth. Therefore, the current study was performed to isolate and identify Prosopis glandulosa-associating rhizobacteria based on their antagonistic activity against Fusarium species and plant growth-promoting properties. Three bacterial isolates were identified as Bacillus subtilis: LDA-1, LDA-2, and LDA-3. The molecular analysis suggests the biosynthesis of the bacteriocins subtilisin and subtilosin, as well as the lipopeptide iturin, by these strains. In addition, the antagonistic study by dual-culture assay showed a high efficacy of all B. subtilis strains against phytopathogenic fungi (Fusarium nygamai, F. equisseti, F. solani, F. solani ICADL1, and F. oxysporum ICADL2) with inhibition percentages ranging from 43.3 to 83.5% in comparison to the control. Moreover, atomic force microscopy (AFM) analysis showed significant differences in the cell wall topography of the F. solani ICADL1 among the treated mycelia and untreated control. As a result, these three B. subtilis strains were used as bioinoculants for cotton seedlings infected by F. solani ICADL1 in pot trials, and the results revealed that the bacterial inoculations as an individual or combined with F. solani ICADL1 significantly improved cotton root and stem length, lateral roots, indole acetic acid (IAA), and gibberellic acid (GA3) contents, as well as increased antioxidants, flavonoids, and phenols in comparison to those obtained from healthy and infected control plants. In conclusion, the three bacterial strains of B. subtilis (i.e., LDA-1, LDA-2, and LDA-3) are considered promising tools as biocontrol agents for F. solani and cotton growth promoters, and consequently can be used as bio-ertilizer in sustainable agriculture systems.

Identification of Lipopeptide Iturin A Produced by Bacillus amyloliquefaciens NCPSJ7 and Its Antifungal Activities against Fusarium oxysporum f. sp. niveum.

Bacillus amyloliquefaciens NCPSJ7 showed potential fungicidal activities for the effective control of fungal infection. From the PCR test, the key genes (srfAA, sfp, fenD, bmyB, ituD, and ituC) were detected in B. amyloliquefaciens NCPSJ7. These genes were closely related to the lipopeptides (LPs) synthesis. Next, three LPs families were identified with liquid chromatography–mass spectrometry (LC/MS), including iturin A, fengycin A, and surfactin. After purification with C18, the main active antifungal compound was proven to be C14-iturin A by ESI-HRMS, which has significant activities against fungi. These results proved that C14-iturin A played an important role in inhibiting the growth of fungi for B. amyloliquefaciens NCPSJ7. Furthermore, the isolated LP could inhibit mycelial growth and conidia germination at 30 μg/mL. SEM allowed us to observe that mycelial morphology and conidia germination were also affected. The mycelial ultrastructure TEM observations showed that the external electron-dense outer layer cell wall, which mainly consisted of glycoproteins, was affected. Furthermore, swollen mitochondria, enriched glycogen, and increased vacuoles were also found. LP also affected the intact wall and membranes, leading to their increased permeability, which was proved by propidium iodide (PI) staining and conductivity measurements. Meanwhile, the ergosterol, which has an affinity for iturin A, also increased. These results indicated that LP caused fungal dysfunction and membrane permeability increase, leading to fungal inhibition. Identifying and studying LPs is important in exploring the fungicidal activities of B. amyloliquefaciens, which promotes the use of B. amyloliquefaciens NCPSJ7 as a potential candidate for biocontrol.

Systemically engineering Bacillus amyloliquefaciens for increasing its antifungal activity and green antifungal lipopeptides production.

The biosynthesis of antifungal lipopeptides iturin and fengycin has attracted broad interest; however, there is a bottleneck in its low yield in wild strains. Because the key metabolic mechanisms in the lipopeptides synthesis pathway remain unclear, genetic engineering approaches are all ending up with a single or a few gene modifications. The aim of this study is to develop a systematic engineering approach to improve the antifungal activity and biosynthesis of iturin and fengycin in Bacillus amyloliquefaciens. First, blocking the carbon overflow metabolic pathway to increase precursor supply of the branched-chain amino acids by knockout of bdh, disrupting sporulation to extend the stage for producing antifungal lipopeptides by deletion of kinA, blocking of siderophore synthesis to enhance the availability of amino acids and fatty acids by deletion of dhbF, and increasing Spo0A∼P by deletion of rapA, could improve the antifungal activity by 24%, 10%, 13% and 18%, respectively. Second, the double knockout strain ΔbdhΔkinA, triple knockout strain ΔbdhΔkinAΔdhbF and quadruple knockout strain ΔkinAΔbdhΔdhbFΔrapA could improve the antifungal activity by 38%, 44% and 53%, respectively. Finally, overexpression of sfp in ΔkinAΔbdhΔdhbFΔrapA further increased the antifungal activity by 65%. After purifying iturin and fengycin as standards for quantitative analysis of lipopeptides, we found the iturin titer was 17.0 mg/L in the final engineered strain, which was 3.2-fold of the original strain. After fermentation optimization, the titer of iturin and fengycin reached 31.1 mg/L and 175.3 mg/L in flask, and 123.5 mg/L and 1200.8 mg/L in bioreactor. Compared to the original strain, the iturin and fengycin titer in bioreactor increased by 22.8-fold and 15.9-fold in the final engineered strain, respectively. This study may pave the way for the commercial production of green antifungal lipopeptides, and is also favorable for understanding the regulatory and biosynthetic mechanism of iturin and fengycin.

Target Mechanism of Iturinic Lipopeptide on Differential Expression Patterns of Defense-Related Genes against Colletotrichum acutatum in Pepper.

Bacillus subtilis KB21 is an isolate with broad spectrum antifungal activity against plant pathogenic fungi. Our aim was to produce and purify antifungal lipopeptides via fermentation using B. subtilis KB21 and verify their antifungal mechanism against pepper anthracnose. When the KB21 strain was cultured in tryptic soy broth medium, the antifungal activity against pepper anthracnose correlated with biosurfactant production. However, there was no antifungal activity when cultured in Luria-Bertani medium. KB21 filtrates showed the highest degree of inhibition of mycelia (91.1%) and spore germination (98.9%) of Colletotrichum acutatum via increases in the biosurfactant levels. Using liquid chromatography-mass spectrometry (LC-MS) and LC-tandem MS (LC-MS/MS) analyses, the component with antifungal activity in the fermentation medium of the KB21 strain was determined to be the cyclic lipopeptide (CLP) antibiotic, iturin A. When the iturin fractions were applied to pepper fruits inoculated with conidia of C. acutatum, the lesion diameter and hyphal growth on the fruit were significantly suppressed. In addition, iturin CLP elevated the gene expression of PAL, LOX, and GLU in the treatments both with and without following fungal pathogens. Overall, the results of this study show that iturin CLPs from B. subtilis KB21 may be potential biological control agents for plant fungal diseases.

Bacillus Cyclic Lipopeptides Iturin and Fengycin Control Rice Blast Caused by Pyricularia oryzae in Potting and Acid Sulfate Soils by Direct Antagonism and Induced Systemic Resistance.

Rice monoculture in acid sulfate soils (ASSs) is affected by a wide range of abiotic and biotic constraints, including rice blast caused by Pyricularia oryzae. To progress towards a more sustainable agriculture, our research aimed to screen the biocontrol potential of indigenous Bacillus spp. against blast disease by triggering induced systemic resistance (ISR) via root application and direct antagonism. Strains belonging to the B. altitudinis and B. velezensis group could protect rice against blast disease by ISR. UPLC–MS and marker gene replacement methods were used to detect cyclic lipopeptide (CLiP) production and construct CLiPs deficient mutants of B. velezensis, respectively. Here we show that the CLiPs fengycin and iturin are both needed to elicit ISR against rice blast in potting soil and ASS conditions. The CLiPs surfactin, iturin and fengycin completely suppressed P. oryzae spore germination resulting in disease severity reduction when co-applied on rice leaves. In vitro microscopic assays revealed that iturin and fengycin inhibited the mycelial growth of the fungus P. oryzae, while surfactin had no effect. The capacity of indigenous Bacillus spp. to reduce rice blast by direct and indirect antagonism in ASS conditions provides an opportunity to explore their usage for rice blast control in the field.

Effect of zinc oxide nanoparticle supplementation on the enhanced production of surfactin and iturin lipopeptides of endophytic Bacillus sp. Fcl1 and its ameliorated antifungal activity.

Lipopeptides from the Bacillus spp. possess an excellent spectrum of antimicrobial properties which make them suitable candidates to be explored for the food, agricultural, pharmaceutical and biotechnological applications. As the low yield of the lipopeptides limits their applications, methods to enhance their production are highly significant. In this study, extracts prepared from endophytic Bacillus sp. Fcl1 cultured in the presence of various supplements were screened for antifungal activity against Pythium aphanidermatum. From the results, the supplementation of carbon sources and zinc oxide nanoparticles (ZnONPs) was found to have an enhancement effect on the antifungal activity of Bacillus sp. Fcl1. Among these, the highest antifungal activity (73.2%) could be observed for the Fcl1 sample cultured with 5 mg L-1 of ZnONP supplementation. The growth of Fcl1 in the presence of ZnONPs also indicated its compatibility with the nano-supplement in the concentration range used. By liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) analysis, the synthesis of increased numbers of lipopeptide surfactin derivatives could be identified from the extracts of Fcl1 prepared from the carbon sources and ZnONP-supplemented cultures. In addition to the surfactin derivatives, the presence of another lipopeptide iturin was also detected from the extracts of Fcl1 cultured with ZnONPs. ZnONP supplementation was found to enhance antifungal activity and lipopeptide production in the Bacillus sp. Fcl1. The use of nanoparticles to enhance the antifungal mechanisms of Fcl1 as observed in the study provides novel insights to explore its applications for sustainable agricultural productivity.

Recovery of Ag+ by cyclic lipopeptide iturin A and corresponding chain peptide: reaction mechanisms, kinetics, toxicity reduction, and applications

Iturin A, a cyclic lipopeptide produced by Bacillus subtilis, has great potential in removal of Ag+ from water, but the mechanisms and kinetic remain unclear. By comparison with the chain peptide (CP) that has the same amino acid sequence as iturin A, the mechanisms were found as iturin A reduced Ag+ to Ag0 and formed silver nanoparticles (AgNPs) via the groups of Ar-OH, CO, -NH-, O=C-O, and -C(CH).The cycle peptide fraction played an important role for the faster formation of AgNPs by iturin A than by CP. The overall Ag+ removal process by iturin A and CP could be well described by a Freundlich isotherm, with the equilibrium Ag+ removal capacity ranging from 58.41 to 61.03 mg/g within 293.15–333.15 K for iturin A. With the application of iturin A, the overall removal rate of Ag+ reached 91.8% in wastewater, the formed AgNPs could be easily recovered via charging the direct electric current, and the toxicity of Ag+ to paddy growth was greatly reduced.

Identification and characterization of a cyclic lipopeptide iturin A from a marine-derived Bacillus velezensis 11-5 as a fungicidal agent to Magnaporthe oryzae in rice

Magnaporthe oryzae is an important rice pathogen globally. However, plant beneficial microbes and their secondary metabolites to control blast disease in rice are poorly understood. In the present study, a marine-derived Bacillus velezensis 11-5 has been characterized as an antagonist against an isolate of Magnaporthe oryzae, the causal agent of rice blast disease. A cyclic lipopeptide (CLP) iturin A has been identified from the fermentation broth of B. velezensis 11-5 by nuclear magnetic resonance spectroscopy and mass spectrometry. In addition, the in vitro and in planta biocontrol activities of CLP iturin A were evaluated in the further study, respectively. The results revealed that iturin A shows significant activity on the conidia germination and the relative appressoria formation rate of M. oryzae at the concentrations of 10 and 50 µM for 12 h and for 24 h, respectively. In addition, CLP iturin A shows the significant activity to control M. oryzae in rice plants when the concentration of the compound is higher than 10 µM. Taken all results together, this study shows that B. velezensis 11-5 has the possibility to be developed as a biopesticide to control rice blast disease in rice plants, and for the first time, this study shows a CLP iturin A produced by B. velezensis 11-5 is an agrochemical to control rice blast disease both in vitro and in rice. And therefore, the results provide information on the application of B. velezensis 11-5 and its CLP iturin A as potential biopesticides to control blast disease in rice for agricultural practice.

Molecular Docking and Dynamics Simulation of Protein β-Tubulin and Antifungal Cyclic Lipopeptides.

To elucidate interactions between the antifungal cyclic lipopeptides iturin A, fengycin, and surfactin produced by Bacillus bacteria and the microtubular protein β-tubulin in plant pathogenic fungi (Fusarium oxysporum, Colletrotrichum gloeosporioides, Alternaria alternata, and Fusarium solani) in molecular docking and molecular dynamics simulations, we retrieved the structure of tubulin co-crystallized with taxol from the Protein Data Bank (PDB) (ID: 1JFF) and the structure of the cyclic lipopeptides from PubChem (Compound CID: 102287549, 100977820, 10129764). Similarity and homology analyses of the retrieved β-tubulin structure with those of the fungi showed that the conserved domains shared 84% similarity, and the root mean square deviation (RMSD) was less than 2 Å. In the molecular docking studies, within the binding pocket, residues Pro274, Thr276, and Glu27 of β-tubulin were responsible for the interaction with the cyclic lipopeptides. In the molecular dynamics analysis, two groups of ligands were formed based on the number of poses analyzed with respect to the RMSD. Group 1 was made up of 10, 100, and 500 poses with distances 0.080 to 0.092 nm and RMSDs of 0.10 to 0.15 nm. For group 2, consisting of 1000 poses, the initial and final distance was 0.1 nm and the RMSDs were in the range of 0.10 to 0.30 nm. These results suggest that iturin A and fengycin bind with higher affinity than surfactin to β-tubulin. These two lipopeptides may be used as lead compounds to develop new antifungal agents or employed directly as biorational products to control plant pathogenic fungi.

Iturinic Lipopeptide Diversity in the Bacillus subtilis Species Group - Important Antifungals for Plant Disease Biocontrol Applications.

Iturins and closely related lipopeptides constitute a family of antifungal compounds known as iturinic lipopeptides that are produced by species in the Bacillus subtilis group. The compounds that comprise the family are: iturin, bacillomycin D, bacillomycin F, bacillomycin L, mycosubtilin, and mojavensin. These lipopeptides are prominent in many Bacillus strains that have been commercialized as biological control agents against fungal plant pathogens and as plant growth promoters. The compounds are cyclic heptapeptides with a variable length alkyl sidechain, which confers surface activity properties resulting in an affinity for fungal membranes. Above a certain concentration, enough molecules enter the fungal cell membrane to create a pore in the cell wall, which leads to loss of cell contents and cell death. This study identified 330 iturinic lipopeptide clusters in publicly available genomes from the B. subtilis species group. The clusters were subsequently assigned into distinguishable types on the basis of their unique amino acid sequences and then verified by HPLC MS/MS analysis. The results show some lipopeptides are only produced by one species, whereas certain others can produce up to three. In addition, four species previously not known to produce iturinic lipopeptides were identified. The distribution of these compounds among the B. subtilis group species suggests that they play an important role in their speciation and evolution.

Effect of media and fermentation conditions on surfactin and iturin homologues produced by Bacillus natto NT-6: LC\u2013MS analysis

Lipopeptides possess excellent broad spectrum antimicrobial activity. Different lipopeptides have their own unique chemical structures, properties and biological activities. Quantitative analysis of the lipopeptides iturin and surfactin and their homologues produced by Bacillus natto NT–6 subjected to different culture media, shaking speed of rotary shaker, and liquid and solid fermentation methods was conducted using LC–MS. For iturins, liquid-state fermentation in Landy medium at a shaking speed of 160 r min−1 was the most suitable for maximal homologue production. Addition of 0.4% attapulgite powder increased production by 1.92-fold; activated carbon significantly reduced production. For surfactin homologues, solid-state fermentation in potato dextrose broth medium at shaking speed > 160 r min−1 was the best. Addition of 0.4% attapulgite powder increased production by 1.96-fold; activated carbon had no effect. Thus it is clear that fermentation conditions can be manipulated to maximize iturin and surfactin production.

Enhanced production of antifungal lipopeptide iturin A by Bacillus amyloliquefaciens LL3 through metabolic engineering and culture conditions optimization

BackgroundIturins, which belong to antibiotic cyclic lipopeptides mainly produced by Bacillus sp., have the potential for application in biomedicine and biocontrol because of their hemolytic and antifungal properties. Bacillus amyloliquefaciens LL3, isolated previously by our lab, possesses a complete iturin A biosynthetic pathway as shown by genomic analysis. Nevertheless, iturin A could not be synthesized by strain LL3, possibly resulting from low transcription level of the itu operon.ResultsIn this work, enhanced transcription of the iturin A biosynthetic genes was implemented by inserting a strong constitutive promoter C2up into upstream of the itu operon, leading to the production of iturin A with a titer of 37.35 mg l−1. Liquid chromatography-mass spectrometry analyses demonstrated that the strain produced four iturin A homologs with molecular ion peaks at m/z 1044, 1058, 1072 and 1086 corresponding to [C14 + 2H]2+, [C15 + 2H]2+, [C16 + 2H]2+ and [C17 + 2H]2+. The iturin A extract exhibited strong inhibitory activity against several common plant pathogens. The yield of iturin A was improved to 99.73 mg l−1 by the optimization of the fermentation conditions using a response surface methodology. Furthermore, the yield of iturin A was increased to 113.1 mg l−1 by overexpression of a pleiotropic regulator DegQ.ConclusionsTo our knowledge, this is the first report on simultaneous production of four iturin A homologs (C14–C17) by a Bacillus strain. In addition, this study suggests that metabolic engineering in combination with culture conditions optimization may be a feasible method for enhanced production of bacterial secondary metabolites.

Promotion of lipopeptide antibiotic production by Bacillus sp. IA in the presence of rice husk biochar.

The purpose of this study is to isolate the beneficial microorganisms whose growth is promoted in the presence of charcoal materials. We successfully isolated strain IA, whose growth is promoted on an agar plate with charcoal materials, and identified it as a novel strain of the Bacillus sp. The growth of strain IA in the liquid medium was promoted by the addition of both activated charcoal (AC) and rice husk biochar (RHB). Moreover, the sporulation of strain IA in the RHB medium and the antifungal activity of the culture supernatant of the RHB medium were much higher than those with AC. HPLC and MS analyses revealed that strain IA produced an antifungal lipopeptide iturin A, and the yield of iturin A in the RHB medium was 8 times higher than that in the medium without RHB. This is the first paper to describe the positive effect of RHB on microbial metabolisms.

Disease suppression by the cyclic lipopeptides iturin A and surfactin from Bacillus spp. against Fusarium wilt of lettuce

Iturin A and surfactin are antimicrobial cyclic lipopeptides secreted by antagonistic Bacillus strains, and both elicit defence gene expression in host plants. We previously showed that a soil amendment with either of the purified lipopeptides confers suppression against Fusarium yellows of tatsoi (Brassica rapa) caused by the soil-borne pathogen Fusarium oxysporum and that excess amounts of iturin A or surfactin do not confer suppression. Here, we evaluated the impacts of purified iturin A or surfactin against Fusarium wilt of lettuce (Lactuca sativa). As a soil amendment, iturin A and surfactin each conferred suppression of Fusarium wilt of lettuce on diverse lettuce cultivars similar to the results for tatsoi. However, higher concentrations of iturin A (1.88 mg/l of soil or higher) but not of surfactin (up to 7.5 mg/l of soil) negated the suppression. Growth of Fusarium in liquid cultures was suppressed by iturin A but not surfactin.