Osmolyte-producing microbial biostimulants regulate the growth of Arachis hypogaea L. under drought stress.

Globally, drought stress poses a significant threat to crop productivity. Improving the drought tolerance of crops with microbial biostimulants is a sustainable strategy to meet a growing population's demands. This research aimed to elucidate microbial biostimulants' (Plant Growth Promoting Rhizobacteria) role in alleviating drought stress in oil-seed crops. In total, 15 bacterial isolates were selected for drought tolerance and screened for plant growth-promoting (PGP) attributes like phosphate solubilization and production of indole-3-acetic acid, siderophore, hydrogen cyanide, ammonia, and exopolysaccharide. This research describes two PGPR strains: Acinetobacter calcoaceticus AC06 and Bacillus amyloliquefaciens BA01. The present study demonstrated that these strains (AC06 and BA01) produced abundant osmolytes under osmotic stress, including proline (2.21 and 1.75 µg ml- 1), salicylic acid (18.59 and 14.21 µg ml- 1), trehalose (28.35 and 22.74 µg mg- 1 FW) and glycine betaine (11.35 and 7.74 mg g- 1) respectively. AC06 and BA01 strains were further evaluated for their multifunctional performance by inoculating in Arachis hypogaea L. (Groundnut) under mild and severe drought regimes (60 and 40% Field Capacity). Inoculation with microbial biostimulants displayed distinct osmotic-adjustment abilities of the groundnut, such as growth parameters, plant biomass, photosynthetic pigments, relative water content, proline, and soluble sugar in respective to control during drought. On the other hand, plant sensitivity indexes such as electrolyte leakage and malondialdehyde (MDA) contents were decreased as well as cooperatively conferred plant drought tolerance by induced alterations in stress indicators such as catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD). Thus, Acinetobacter sp. AC06 and Bacillus sp. BA01 can be considered as osmolyte producing microbial biostimulants to simultaneously induce osmotic tolerance and metabolic changes in groundnuts under drought stress.

A Novel Bacillus amyloliquefaciens Specifically Improving the Solubility and Antioxidant Activities of Edible Bird's Nest.

Edible bird's nest (EBN), a most highly priced and valuable foodstuff, contains high percentage of proteins and carbohydrates. However, proteins adhering to these carbohydrates make the EBN hard and tough, which need to be boiled as the bird's nest soup to make the Chinese cuisine. To overcome the hard and tough texture of EBN and improve the digestion degrees, the present study screened and identified a probiotic strain Bacillus amyloliquefaciens YZW02 from 5-year stored EBN sample completely solubilizing EBN for the first time. The 24-h B. amyloliquefaciens fermented EBN contained 20.30-21.48 mg/mL of the soluble protein contents with a recovery rate of 98-100%, DPPH radical scavenging rate of 84.76% and ABTS radical scavenging capacity of 41.05%. The mixed fermentation of B. amyloliquefaciens YZW02 and Bacillus natto BN1 were further applied to improve the low-MW peptide percentages and antioxidant activities. The mixed-fermentation of B. natto BN1 with 4-h cultured B. amyloliquefaciens YZW02 had the lowest percentage (82.23%) of >12-kDa proteins/peptides and highest percentages of 3-12 kDa, 1-3 kDa and 0.1-1 kDa peptides of 8.6% ± 0.08, 7.57% ± 0.09, 1.77% ± 0.05 and 0.73% ± 0.05, with the highest DPPH, ABTS and •OH scavenging capacity of 90.23%, 46.45% and 49.12%, respectively. These findings would provide an efficient strategy for improving the solubility and antioxidant activities of EBNs.

Characterization of Bacillus amyloliquefaciens PM415 as a potential bio-preserving probiotic.

Animal feed is vulnerable to fungal infections, and the use of bio-preserving probiotics has received increasing attention. In contrast to Lactobacillus and Bifidobacteria spp., fewer Bacillus spp. have been recognized as antifungal probiotics. Therefore, our objective was to screen antifungal strains and provide more Bacillus candidates to bridge this gap. Here, we screened 56 bacterial strains for cyclic lipopeptide genes and conducted an antifungal assay with Aspergillus niger as a representative fungus. We found that a Bacillus strain Bacillus amyloliquefaciens PM415, isolated from pigeon manure, exhibited the highest fungal inhibition activity as demonstrated by the confrontation assay and morphological observation under scanning electron microscope (SEM). Preliminary safety assessment and probiotic characterization revealed its non-pathogenic feature and stress tolerance capability. Whole genome sequencing of Bacillus amyloliquefaciens PM415 revealed a genome size of 4.16 Mbp and 84 housekeeping genes thereof were used for phylogenetic analysis showing that it is most closely related to Bacillus amyloliquefaciens LFB112. The in silico analysis further supported its non-pathogenic feature at the genomic level and revealed potential biosynthetic gene clusters responsible for its antifungal property. RNA-seq analysis revealed genome-wide changes in transportation, amino acid metabolism, non-ribosomal peptides (NRPs) biosynthesis and glycan degradation during fungal antagonism. Our results suggest that Bacillus amyloliquefaciens PM415 is a safe and effective probiotic strain that can prevent fungal growth in animal feeds.

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.

Modular metabolic engineering of Bacillus amyloliquefaciens for high-level production of green biosurfactant iturin A.

As a kind of biosurfactants, iturin A has attracted people's wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens. Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5'-UTRs of downstream genes (ituA, ituB, and ituC) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH, serC, and introducing ocD. Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. KEY POINTS: • Optimizing 5'-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far.

Bacillus amyloliquefaciens SC06 Relieving Intestinal Inflammation by Modulating Intestinal Stem Cells Proliferation and Differentiation via AhR/STAT3 Pathway in LPS-Challenged Piglets.

Bacillus amyloliquefaciens is a well-accepted probiotic, with many benefits for both humans and animals. The ability of intestinal stem cells (ISCs) to develop into several intestinal epithelial cell types helps accelerate intestinal epithelial regeneration. Limited knowledge exists on how bacteria regulated ISCs proliferation and regeneration. Our study investigated the effects of Bacillus amyloliquefaciens supplementation on ISC proliferation and regeneration and intestinal mucosal barrier functions in piglets exposed to lipopolysaccharide (LPS). Eighteen piglets (male, 21 days old) were randomly split into 3 clusters: CON cluster, LPS cluster, and SC06+LPS cluster. On day 21, 100 µg/kg body weight of LPS was intraperitoneally administered to the SC06+LPS and LPS groups. We found SC06 supplementation maintained the intestinal barrier integrity, enhanced intestinal antioxidant capacity, reduced generation of inflammatory response, and suppressed enterocyte apoptosis against the deleterious effects triggered by LPS. In addition, our research indicated that the SC06 supplementation not only improved the ISC regeneration, but also resulted in upregulation of aryl hydrocarbon receptor (AhR) in LPS-challenge piglets. Further studies showed that SC06 also induced ISC differentiation toward goblet cells and inhibited their differentiation to intestinal absorptive cells and enterocytes. The coculture system of SC06 and ileum organoids revealed that SC06 increased the growth of ISCs and repaired LPS-induced organoid damage through activating the AhR/STAT3 signaling pathway. These findings showed that SC06, possibly through the AhR/STAT3 pathway, accelerated ISC proliferation and promoted epithelial barrier healing, providing a potential clinical treatment for IBD. Our research demonstrated that SC06 is effective in preventing intestinal epithelial damage after pathological injury, restoring intestinal homeostasis, and maintaining intestinal epithelial regeneration.

Metagenomic analysis of the intestinal microbiome reveals the potential mechanism involved in Bacillus amyloliquefaciens in treating schistosomiasis japonica in mice.

Schistosomiasis japonica is one of the neglected tropical diseases characterized by chronic hepatic, intestinal granulomatous inflammation and fibrosis, as well as dysbiosis of intestinal microbiome. Previously, the probiotic Bacillus amyloliquefaciens has been shown to alleviate the pathological injuries in mice infected with Schistosoma japonicum by improving the disturbance of the intestinal microbiota. However, the underlying mechanisms involved in this process remain unclear. In this study, metagenomics sequencing and functional analysis were employed to investigate the differential changes in taxonomic composition and functional genes of the intestinal microbiome in S. japonicum-infected mice treated with B. amyloliquefaciens. The results revealed that intervention with B. amyloliquefaciens altered the taxonomic composition of the intestinal microbiota at the species level in infected mice and significantly increased the abundance of beneficial bacteria. Moreover, the abundance of predicted genes in the intestinal microbiome was also significantly changed, and the abundance of xfp/xpk and genes translated to urease was significantly restored. Further analysis showed that Limosilactobacillus reuteri was positively correlated with several KEGG Orthology (KO) genes and metabolic reactions, which might play important roles in alleviating the pathological symptoms caused by S. japonicum infection, indicating that it has the potential to function as another effective therapeutic agent for schistosomiasis. These data suggested that treatment of murine schistosomiasis japonica by B. amyloliquefaciens might be induced by alterations in the taxonomic composition and functional gene of the intestinal microbiome in mice. We hope this study will provide adjuvant strategies and methods for the early prevention and treatment of schistosomiasis japonica. IMPORTANCE: Targeted interventions of probiotics on gut microbiome were used to explore the mechanism of alleviating schistosomiasis japonica. Through metagenomic analysis, there were significant changes in the composition of gut microbiota in mice infected with Schistosoma japonicum and significant increase in the abundance of beneficial bacteria after the intervention of Bacillus amyloliquefaciens. At the same time, the abundance of functional genes was found to change significantly. The abundance of genes related to urease metabolism and xfp/xpk related to D-erythrose 4-phosphate production was significantly restored, highlighting the importance of Limosilactobacillus reuteri in the recovery and abundance of predicted genes of the gut microbiome. These results indicated potential regulatory mechanism between the gene function of gut microbiome and host immune response. Our research lays the foundation for elucidating the regulatory mechanism of probiotic intervention in alleviating schistosomiasis japonica, and provides potential adjuvant treatment strategies for early prevention and treatment of schistosomiasis japonica.

Bacillus amyloliquefaciens SC06 alleviated intestinal damage induced by inflammatory via modulating intestinal microbiota and intestinal stem cell proliferation and differentiation.

The aim of our research was to investigate the effects of Bacillus amyloliquefaciens SC06 on growth performance, immune status, intestinal stem cells (ISC) proliferation and differentiation, and gut microbiota in weaned piglets. Twelve piglets (male, 21 days old, 6.11 ± 0.12 kg) were randomly allocated to CON and SC06 (1 × 108 cfu/kg to diet) groups. This experiment lasted three weeks. Our results showed that SC06 increased (P < 0.05) growth performance and reduced the diarrhea rate in weaned piglets. In addition, SC06 increased intestinal morphology and interleukin (IL)-10 levels, and decreased (P < 0.01) necrosis factor (TNF-α) levels in jejunum and serum. Moreover, weaning piglets fed SC06 had a better balance of colonic microbiota, with an increase in the abundance of Lactobacillus. Furthermore, SC06 enhanced ISCs proliferation and induced its differentiation to goblet cells via activating wnt/ß-catenin pathway in weaned piglets and intestinal organoid. Taken together, SC06 supplementation improved the growth performance and decreased inflammatory response of piglets by modulating intestinal microbiota, thereby accelerating ISC proliferation and differentiation and promoting epithelial barrier healing.

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.

Inclusion of a Bacillus-based probiotic in non-starch polysaccharides-rich broiler diets.

This study examined the effects of a 3-strain Bacillus-based probiotic (BP; Bacillus amyloliquefaciens and two Bacillus subtilis) in broiler diets with different rye levels on performance, mucus, viscosity, and nutrient digestibility. We distributed 720 one-d-old female broilers into 72 pens and designed nine diets using a 3 × 3 factorial approach, varying BP levels (0, 1.2 × 106, and 1.2 × 107 CFU/g) and rye concentrations (0, 200, 400 g/kg). On d 35, diets with 200 or 400 g/kg rye reduced broiler weight gain (BWG). Diets with 400 g/kg rye had the highest FCR, while rye-free diets had the lowest (p ≤0.05). Adding BP increased feed intake and BWG in weeks two and three (p ≤0.05). It should be noted that the overall performance fell below the goals of the breed. Including rye in diets reduced the coefficient of apparent ileal digestibility (CAID) for protein, ether extract (EE), calcium, phosphorus, and all amino acids (p ≤0.05). Rye-free diets exhibited the highest CAID for all nutrients, except for methionine, EE, and calcium, while diets with 400 g/kg of rye demonstrated the lowest CAID (p ≤0.05). BP in diets decreased phosphorus CAID (p ≤0.05). Diets containing 1.2 × 107 CFU/g (10X) of BP exhibited higher CAID of methionine than the other two diets (p ≤0.05). Diets containing 10X of BP showed higher CAID of cysteine than diets with no BP (p ≤0.05). Ileal viscosity increased as the inclusion level of rye in the diets increased (p ≤0.05). The ileal concentration of glucosamine in chickens fed diets with 400 g/kg of rye was higher than in those fed diets with no rye (p ≤0.05). Furthermore, ileal galactosamine concentrations were elevated in diets with 200 and 400 g/kg of rye when compared to rye-free diets (p ≤0.05). However, BP in diets had no impact on ileal viscosity, galactosamine, or glucosamine (p > 0.05). In conclusion, the applied Bacillus strains appeared to have a limited capacity to produce arabinoxylan-degrading enzymes and were only partially effective in mitigating the negative impacts of rye arabinoxylans on broilers.

Association of Bacillus subtilis and Bacillus amyloliquefaciens: minimizes the adverse effects of necrotic enteritis in the gastrointestinal tract and improves zootechnical performance in broiler chickens.

This study aimed to evaluate the efficiency and capacity of the probiotic composed of Bacillus subtilis and Bacillus amyloliquefaciens, in improving the zootechnical performance of broiler chickens challenged with Eimeria spp. and Clostridium perfringens. The broilers were distributed in a completely randomized design in poultry isolators (12 birds each), resulting in 3 treatments: T1 (control, no challenge and no Bacillus in diet), T2 (challenged with Eimeria spp., followed by Clostridium perfringens infection and no Bacillus in the diet), and T3 (challenged with Eimeria spp., Clostridium perfringens and treated with Bacillus subtilis and Bacillus amyloliquefaciens). They were evaluated for a period of 29 d, divided into preinitial (1-7 d of age), initial (8-21 d), and growth (22-29 d) phases. Assessments of body weight, weight gain, feed consumption, and feed conversion were conducted, along with the classification of the scores and optical microscopy of the tract gastrointestinal. The animals challenged and treated with the probiotic containing Bacillus spp. showed improved indicators of zootechnical performance. Additionally, the animals challenged and treated (T3) had a better score for intestinal lesions compared to the other treatment groups. Therefore, the probiotic consisting of Bacillus subtilis and Bacillus amyloliquefaciens could be considered an effective option for disease prevention and improving the zootechnical performance of broiler chickens.

Bioactivity and genome analysis of Bacillus amyloliquefaciens GL18 isolated from the rhizosphere of Kobresia myosuroides in an alpine meadow.

The unique eco-environment of the Qinghai-Tibet Plateau breeds abundant microbial resources. In this research, Bacillus amyloliquefaciens GL18, isolated from the rhizosphere of Kobresia myosuroides from an alpine meadow, and the antagonistic activity, bacteriostatic hydrolase activity, and low temperature, salt, and drought resistance of it were determined and analysed. The seedlings of Avena sativa were root-irrigated using bacteria suspensions (cell concentration 1 × 107 cfu/mL) of GL18, and the growth-promoting effect of GL18 on it was determined under cold, salt and drought stress, respectively. The whole genome of GL18 was sequenced, and its functional genes were analysed. GL18 presented significant antagonistic activity to Fusarium graminearum, Fusarium acuminatum, Fusarium oxysporum and Aspergillus niger (inhibition zone diameter > 17 mm). Transparent zones formed on four hydrolase detection media, indicating that GL18 secreted cellulase, protease, pectinase and ß-1,3-glucanase. GL18 tolerated conditions of 10 °C, 11% NaCl and 15% PEG-6000, presenting cold, salt and drought resistance. GL18 improved the cold, salt and drought tolerance of A. sativa and it showed significant growth effects under different stress. The total length of the GL18 genome was 3,915,550 bp, and the number of coding DNA sequence was 3726. Compared with the clusters of orthologous groups of proteins, gene ontology and kyoto encyclopedia of genes and genomes databases, 3088, 2869 and 2357 functional genes were annotated, respectively. GL18 contained gene clusters related to antibacterial substances, functional genes related to the synthesis of plant growth-promoting substances, and encoding genes related to stress resistance. This study identified an excellent Bacillus strain and provided a theoretical basis for improving stress resistance and promoting the growth of herbages under abiotic stress.

Potentiation effect of the AI-2 signaling molecule on postharvest disease control of pear and loquat by Bacillus amyloliquefaciens and its mechanism.

An autoinducer-2 (AI-2) signaling molecule from Bacillus was synthesized, and its mechanism on the biofilm formation and biocontrol ability of B. amyloliquefaciens was verified in vitro and in vivo. The 16S/ITS amplicon sequencing was used to analyze the effect of B. amyloliquefaciens B4 with or without AI-2 on the microflora of pears during storage. The results showed that B. amyloliquefaciens B4 secreted AI-2, which promoted biofilm formation. Additionally, AI-2 at a concentration of 40 µmol/L enhanced the biocontrol ability of B. amyloliquefaciens B4 on postharvest pear and loquat fruits. Finally, amplicon sequencing demonstrated that the addition of AI-2 increased the abundance of B. amyloliquefaciens B4 in fruit by stimulating the growth and biofilm formation of this bacterium.

An oxalate decarboxylase-like cupin domain containing protein is involved in imparting acid stress tolerance in Bacillus amyloliquefaciens MBNC.

We report here the structural and functional properties of an oxalate decarboxylase (OxDC)-like cupin domain-containing protein of Bacillus amyloliquefaciens MBNC and its role in imparting tolerance to acid stress conditions. Quantitative real-time PCR (qPCR) analysis revealed 32-fold and 20-fold upregulation of the target gene [(OxDC')cupin] under acetic acid stress and hydrochloric acid stress, respectively, indicating its association with the acid stress response. Bacterial cells with targeted inactivation of the (OxDC')cupin gene using the pMUTIN4 vector system showed decreased growth and survival rate in acidic pH, with drastically reduced exopolysaccharide production. In Silico protein-protein interaction studies revealed seven genes (viz. glmS, nagA, nagB, tuaF, tuaF, gcvT, and ykgA) related to cell wall biosynthesis and biofilm production to interact with OxDC-like cupin domain containing protein. While all these seven genes were upregulated in B. amyloliquefaciens MBNC after 6 h of exposure to pH 4.5, the mutant cells containing the inactivated (OxDC')cupin gene displayed significantly lower expression (RQ: 0.001-0.02) (compared to the wild-type cells) in both neutral and acidic pH. Our results indicate that the OxDC-like cupin domain containing protein is necessary for cell wall biosynthesis and biofilm production in Bacillus amyloliquefaciens MBNC for survival in acid-stress conditions.

Phytoremediation technology for recovery of Ni by Acacia plants in association with Bacillus amyloliquefaciens isolated from E-waste contaminated site.

Electronic waste (e-waste) illegally disposal in Thailand is becoming more widespread. A sustainable metal recovery technology is needed. A phytotechnology called "phytomining" of metals such as nickel (Ni) is a promising technology providing a sustainable solution to the growing e-waste problems. This study investigated the ability of Acacia species in association with e-waste site isolated, plant growth-promoting rhizobacteria (PGPR), Bacillus amyloliquefaciens. Acacia mangium accumulated higher Ni in their tissues when Ni concentrations in soil were lower than 200 mg kg-1. The inoculation of PGPR B. amyloliquefaciens enhanced Ni uptake and accumulation in the leaves, stem, and root. The results showed that the highest Ni concentration was found in the root ash (825.50 mg kg-1) when inoculated plants were grown in soil containing 600 mg kg-1 Ni. Hence, the Ni recovery process and mass balance were performed on root ashes. The highest Ni recovery was 91.3% from the acid (H2SO4) leachate of the ash of inoculated plant treated with 600 mg kg-1 Ni. This demonstrates the feasibility of PGPR-assisted phytomining from Ni-contaminated soil. Phytomining of Ni from any e-waste contaminated sites using Acacia mangium in combination with B. amyloliquefaciens can promote plant growth and improve the uptake of Ni.

Phytomining from electronic waste is an appealing technology that can provide a long-term waste management strategy while valuable trace metals can be recovered. In this study, we evaluated the nickel phytomining ability of Acacia mangium in association with PGPR Bacillus amyloliquefaciens. The results from this study showed that Ni recovery from phytomass using A. mangium with B. amyloliquefaciens can be further improved leading to a sustainable waste management strategy.

Unraveling the role of intra-cellular metabolites in the lactic acid production by novel Bacillus amyloliquefaciens using sugarcane molasses as a substratum.

Lactic acid is a versatile, multi-functional organic monomer in various industries, creating worldwide demand. High titer lactic acid production was achieved by novel Bacillus amyloliquefaciens J2V2AA through sugarcane molasses fermentation up to 178 mg mL-1. A metabolomics approach such as combined GC-MS and LC-MS was applied to elucidate the involvement of key metabolites in lactic acid production. The results revealed the participation of 58 known intra-cellular metabolites at various pathways in lactic acid production. Twenty-eight highly up-regulated and down-regulated metabolites were analyzed, and a schematic diagram of a possible lactic acid production pathway was proposed. The produced lactic acid was analyzed through FTIR, UV-Spectrum, and HPLC analysis.

Unravelling the effect of control agents on Gnomoniopsis smithogilvyi on a chestnut-based medium by proteomics.

BACKGROUND: Gnomoniopsis smithogilvyi is the major chestnut pathogen, responsible for economic losses and recently described as a 3-nitropropionic acid and diplodiatoxin mycotoxin producer. Bacillus amyloliquefaciens QST 713 (Serenade® ASO), B. amyloliquefaciens CIMO-BCA1, and the fungicide Horizon® (tebuconazole) have been shown to reduce the growth of G. smithogilvyi. However, they enhanced mycotoxin production. Proteomics can clarify the mould's physiology and the impact of antifungal agents on the mould's metabolism. Thus, the aim of this study was to assess the impact of Horizon®, Serenade®, and B. amyloliquefaciens CIMO-BCA1 in the proteome of G. smithogilvyi to unveil their modes of action and decipher why the mould responds by increasing the mycotoxin production. For this, the mycelium close to the inhibition zone provoked by antifungals was macroscopically and microscopically observed. Proteins were extracted and analysed using a Q-Exactive plus Orbitrap. RESULTS: The results did not elucidate specific proteins involved in the mycotoxin biosynthesis, but these agents provoked different kinds of stress on the mould, mainly affecting the cell wall structures and antioxidant response, which points to the mycotoxins overproduction as a defence mechanism. The biocontrol agent CIMO-BCA1 acts similar to tebuconazole. The results revealed different responses on the mould's metabolism when co-cultured with the two B. amyloliquefaciens, showing different modes of action of each bacterium, which opens the possibility of combining both biocontrol strategies. CONCLUSION: These results unveil different modes of action of the treatments that could help to reduce the use of toxic chemicals to combat plant pathogens worldwide. © 2023 Society of Chemical Industry.

Identification and investigation of the effects of N-acetylmuramoyl-L-alanine amidase in Bacillus amyloliquefaciens for the cell lysis and heterologous protein production.

Bacillus amyloliquefaciens (BA) is considered as an important industrial strain for heterologous proteins production. However, its severe autolytic behavior leads to reduce the industrial production capacity of the chassis cells. In this study, we aimed to evaluate the autolysis of N-acetylmuranyl-L-alanine amidase in BA TCCC11018, and further slowed down the cell lysis for improved the heterologous protein production by a series of modifications. Firstly, we identified six N-acetylmuramic acid-L-alanines by bioinformatics, and analyzed the transcriptional levels at different culture time points by transcriptome and quantitative real-time PCR. Then, by establishing an efficient CRISPR-nCas9 gene editing method, N-acetylmuramic acid-L-alanine genes were knocked out or overexpressed to verify its effect on cell lysis. Then, by single or tandem knockout N-acetylmuramic acid-L-alanines, it was determined that the reasonable modification of LytH and CwlC1 can slow down cell lysis. After 48 h of culture, the autolysis rate of the mutant strain BA ΔlytH-cwlC1 decreased by 4.83 %, and the amylase activity reached 176 U/mL, which was 76.04 % higher than that of the control strain BA Δupp. The results provide a reference for mining the functional characteristics of autolysin in Bacillus spp., and provide from this study reveal valuable insights delaying the cell lysis and increasing heterologous proteins production.

Bacillus amyloliquefaciens A-1 inhibiting fungal spoilage in agricultural products is improved by metabolic engineering of enhancing surfactin yield.

Fungi and subsequent mycotoxins contamination in agricultural products have caused enormous losses and great harm to human and animal health. Biological control has attracted the attention of researchers due to its advantages, including mild conditions, low cost, high efficiency and low nutrient loss. In this study, a newly isolated strain Bacillus amyloliquefaciens A-1 (A-1), was screened for its ability to inhibit the growth and Aflatoxin B1 (AFB1) production of Aspergillus flavus NRRL 3357. Electron microscopy results revealed that mycelium and conidia of A. flavus were destroyed by A-1, affecting hyphae, cell walls, cell membranes and organelles. RNA-seq analysis indicated disturbance in gene expression profiles of A. flavus, including amino acid degradation and starch and sucrose metabolism pathways. Importantly, the biosynthesis of AFB1 was significantly inhibited by the down-regulation of key regulatory genes, aflR and aflS, and the simultaneous down-regulation of most structural genes. Genome analysis predicted six secondary metabolites biosynthetic gene clusters. Then, four surfactin synthesized by cluster C were identified as the main active substance of A-1 using HPLC-Q-TOF-MS. The addition of alanine, threonine, Fe2+ increased surfactin production. Notably, the overexpression of comX also improved surfactin production. The vivo test results indicated that A-1 could significantly inhibit the decay of pear by Aspergillus westerdijkiae, and the mildew of maize and peanuts. Especially, the overexpression of comX in A-1 could enhance the inhibitory activity. In conclusion, the inhibition mechanism of A-1 was revealed, and comX was found can improve the production of surfactin and subsequent activities, which provides the scientific basis for the development of biocontrol agents to reduce spoilage in agricultural products.

A Probiotic Bacillus amyloliquefaciens D-1 Strain Is Responsible for Zearalenone Detoxifying in Coix Semen.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium spp., which commonly and severely contaminate food/feed. ZEN severely affects food/feed safety and reduces economic losses owing to its carcinogenicity, genotoxicity, reproductive toxicity, endocrine effects, and immunotoxicity. To explore efficient methods to detoxify ZEN, we identified and characterized an efficient ZEN-detoxifying microbiota from the culturable microbiome of Pseudostellaria heterophylla rhizosphere soil, designated Bacillus amyloliquefaciens D-1. Its highest ZEN degradation rate reached 96.13% under the optimal condition. And, D-1 can almost completely remove ZEN (90 µg·g-1) from coix semen in 24 h. Then, the D-1 strain can detoxify ZEN to ZEM, which is a new structural metabolite, through hydrolyzation and decarboxylation at the ester group in the lactone ring and amino acid esterification at C2 and C4 hydroxy. Notably, ZEM has reduced the impact on viability, and the damage of cell membrane and nucleus DNA and can significantly decrease the cell apoptosis in the HepG2 cell and TM4 cell. In addition, it was found that the D-1 strain has no adverse effect on the HepG2 and TM4 cells. Our findings can provide an efficient microbial resource and a reliable reference strategy for the biological detoxification of ZEN.