Enhancement of Green Production of Heme by Deleting Odor-Related Genes from Bacillus amyloliquefaciens Based on CRISPR/Cas9n.

Heme is a crucial component in endowing plant-based meat analogs with flavor and color. This study aimed to develop a green strategy for heme production by reducing fermentation off-odor and accelerating heme synthesis. First, an efficient CRISPR/Cas9n system was constructed in Bacillus amyloliquefaciens to construct the odor-reducing chassis cell HZC9nΔGPSU, and the odor substances including the branched-chain short fatty acids, putrescine, and ammonia were reduced by 62, 70, and 88%, respectively. Meanwhile, the hemA gene was confirmed to be the key gene for enhanced heme synthesis. Various hemA genes were compared to obtain the best gene dhemA, and the catalysis mechanism was explained by molecular docking simulation. After further expression of dhemA in HZC9nΔGPSU, the heme titer of HZC9nΔGPSU/pHY-dhemA reached 11.31 ± 0.51 mg/L, 1.70-fold higher than that of HZC9n/pHY-dhemA. The knockout of off-odor-related genes reduced the odor substances and enhanced the heme synthesis, which is promising for the green production of high-quality heme.

Antifungal efficacy of Bacillus amyloliquefaciens ZK-9 against Fusarium graminearum and analysis of the potential mechanism of its lipopeptides.

Fusarium graminearum is a destructive fungal pathogen that seriously threatens wheat production and quality. In the management of fungal infections, biological control is an environmentally friendly and sustainable approach. Here, the antagonistic strain ZK-9 with a broad antifungal activity was identified as Bacillus amyloliquefaciens. ZK-9 could produce extracellular enzymes such as pectinase, protease, cellulase, and amylase, as well as plant growth-promoting substances including IAA and siderophore. Lipopeptides extracted from strain ZK-9 had the high inhibitory effects on the mycelia of F. graminearum with the minimum inhibitory concentration (MIC) of 0.8 mg/mL. Investigation on the action mechanism of lipopeptides showed they could change the morphology of mycelia, damage the cell membrane, lower the content of ergosterol and increase the relative conductivity of membrane, cause nucleic acid and proteins leaking out from the cells, and disrupt the cell membrane permeability. Furthermore, metabolomic analysis of F. graminearum revealed the significant differences in the expression of 100 metabolites between the lipopeptides treatment group and the control group, which were associated with various metabolic pathways, mainly including amino acid biosynthesis, pentose, glucuronate and glycerophospholipid metabolism. In addition, strain ZK-9 inhibited Fusarium crown rot (FCR) with a biocontrol efficacy of 82.14 % and increased the plant height and root length by 24.23 % and 93.25 %, respectively. Moreover, the field control efficacy of strain ZK-9 on Fusarium head blight (FHB) was 71.76 %, and the DON content in wheat grains was significantly reduced by 69.9 %. This study puts valuable insights into the antifungal mechanism of lipopeptides against F. graminearum, and provides a promising biocontrol agent for controlling F. graminearum.

Biodegradation Characteristics and Mechanism of Aflatoxin B1 by Bacillus amyloliquefaciens from Enzymatic and Multiomics Perspectives.

Aflatoxin B1 (AFB1), a mycotoxin and natural carcinogen, commonly contaminates cereals and animal feeds, posing serious health risks to human and animal. In this study, Bacillus amyloliquefaciens ZG08 isolated from kimchi could effectively remove 80.93% of AFB1 within 72 h at 37 °C and pH 7.0. Metabolome and transcriptome analysis showed that metabolic processes including glycerophospholipid metabolism and amino acid metabolism were most affected in B. amyloliquefaciens ZG08 exposed to AFB1. The adaptation mechanism likely involved activation of the thioredoxin system to restore intracellular redox equilibrium. The key genes, tpx and gldA, overexpressed in Escherichia coli BL21, achieved degradation rates of 60.15% and 47.16% for 100 µg/kg AFB1 under optimal conditions of 37 °C and pH 8.0 and 45 °C and pH 7.0, respectively. The degradation products, identified as AFD1, were less cytotoxic than AFB1 in HepG2 cells. These findings suggest potential strategies for utilizing probiotics and engineered enzymes in AFB1 detoxification.

Peptidomics- inspired discovery and activity evaluation of antioxidant peptides in multiple strains mixed fermentation of Porphyra yezoensis.

This study investigated the production of antioxidant peptides from Porphyra yezoensis through fermentation with three strains of microorganisms: Lactiplantibacillus plantarum L13, Bacillus amyloliquefaciens MMB-02, and Saccharomyces cerevisiae A8. The crude peptides were extracted by aqueous acid precipitation and purified by Sephadex G-25 gel column to produce highly active antioxidant components with molecular weight of <4000 Da. The LC-MS/MS result revealed that the fermentation group contained more hydrophobic amino acids and oligopeptides, which were mainly originated from phycobiliproteins and algal blue proteins. Finally, the antioxidant activity of Porphyra yezoensis was determined with DPPH· and ABTS· scavenging rates of 54.87% and 57.39%, respectively. The ferric ion-reducing power (FRAP) and enzyme activities of SOD and CAT were significantly higher than those of the control group. This study provides a scientific foundation for the deep processing of striped seaweed and contributes to the theoretical understanding of synthetic antioxidant substitutes.

Multiple Perspectives of Study on the Potential of Bacillus amyloliquefaciens JB20221020 for Alleviating Nutrient Stress in Lettuce.

Soil nutrient deficiency has become a key factor limiting crop growth. Plant growth-promoting rhizobacteria (PGPR) are vital in resisting abiotic stress. In this study, we investigated the effects of inoculation with Bacillus amyloliquefaciens JB20221020 on the physiology, biochemistry, rhizosphere microorganisms, and metabolism of lettuce under nutrient stress. Pot experiments showed that inoculation with B. amyloliquefaciens JB20221020 significantly promoted lettuce growth under nutrient deficiency. At the same time, the activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase and the content of proline increased, and the content of Malondialdehyde decreased in the lettuce inoculated with B. amyloliquefaciens JB20221020. Inoculation with B. amyloliquefaciens JB20221020 altered the microbial community of the rhizosphere and increased the relative abundances of Myxococcales, Deltaproteobacteria, Proteobacteria, Devosia, and Verrucomicrobia. Inoculation also altered the rhizosphere metabolism under nutrient deficiency. The folate metabolism pathway was significantly enriched in the Kyoto Encyclopedia of Genes and Genomes enrichment analysis. This study explored the interaction between plants and microorganisms under nutrient deficiency, further explained the critical role of rhizosphere microorganisms in the process of plant nutrient stress, and provided a theoretical basis for the use of microorganisms to improve plant resistance.

Hypocholesterolemic potential of Bacillus amyloliquefaciens KAVK1 modulates lipid accumulation on 3T3-L1 adipose cells and high fat diet-induced obese rat model.

The significance of microorganisms occurring in foods is predominantly targeted due to their application for identifying a novel range of the bacterial spectrum. Diverse microbial species are capable of exhibiting potential pharmacological activities like antimicrobial and anticancer. Microbial strains capable of reducing obesity-related syndromes have also been reported. In the present study, the hypocholesterolemic efficacy of Bacillus amyloliquefaciens isolated from dairy products was scrutinised by in vitro (3T3-L1 adipose cells) and in vivo (high-fat diet-induced obese Wistar albino rats) methods. Potential cholesterol-lowering isolates were screened using a plate assay method and optimised by physical parameters. Molecular identification of the topmost five cholesterol-lowering isolates was acquired by amplification of the 16 S rRNA gene region. Bacillus amyloliquefaciens strain KAVK1, followed by strains KAVK2, KAVK3, KAVK4, and KAVK5 were molecularly determined. Further, cholesterol-lowering strains degraded the spectral patterns determined by the side chain of a cholesterol molecule. The anti-lipase activity was demonstrated using the porcine pancreatic lipase inhibitory method and compared with the reference compound Atorvastatin. Lyophilised strain KAVK1 revealed maximum pancreatic lipase inhibition. Strain KAVK1 attenuated lipid accumulation in 3T3-L1 adipose cell line predicted by Oil Red O staining method. Significant reduction of body weight and change in lipid profile was recognised after the supplement of KAVK1 to obese rats. Histopathological changes in organs were predominantly marked. The result of this study implies that the cholesterol-lowering B. amyloliquefaciens KAVK1 strain was used to treat hypercholesterolemia.

Enhanced Iturin A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and Rap Phosphatase Genes.

Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes (rapC, rapF, and rapH) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens.

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.

Screening signal peptidase based on split-GFP assembly technology to promote the secretion of alkaline protease AprE in Bacillus amyloliquefaciens.

Improving the ability of bacteria to secrete protein is essential for large-scale production of food enzymes. However, due to the lack of effective tracking technology for target proteins, the optimization of the secretory system is facing many problems. In this study, we utilized the split-GFP system to achieve self-assembly into mature GFP in Bacillus amyloliquefaciens and successfully tracked the alkaline protease AprE. The split-GFP system was employed to assess the signal peptidases, a crucial component in the secretory system, and signal peptidase sipA was identified as playing a role in the secretion of AprE. Deletion of sipA resulted in a higher accumulation of the precursor protein of AprE compared to other signal peptidase deletion strains. To explore the mechanism of signal peptidase on signal peptide, molecular docking and calculation of free energy were performed. The action strength of the signal peptidase is determined by its binding affinity with the tripeptides at the C-terminal of the signal peptide. The functions of signal peptides YdbK and NucB rely on sipA, and overexpression of sipA by integrating it into genome of B. amyloliquefaciens increased the activity of extracellular AprE by 19.9 %. These findings provide insights into enhancing the secretion efficiency of chassis strains.

Investigation into the potential mechanism of Bacillus amyloliquefaciens in the fermentation of broad bean paste by metabolomics and transcriptomics.

Pixian broad bean paste is a renowned fermented seasoning. The fermentation of broad bean is the most important process of Pixian broad bean paste. To enhance the flavor of tank-fermented broad bean paste, salt-tolerant Bacillus amyloliquefaciens strain was inoculated, resulting in an increase in total amount of volatile compounds, potentially leading to different flavor characteristics. To investigate the fermentation mechanism, monoculture simulated fermentation systems were designed. Metabolomics and transcriptomics were used to explore Bacillus amyloliquefaciens' transcriptional response to salt stress and potential aroma production mechanisms. The results highlighted different metabolite profiles under salt stress, and the crucial roles of energy metabolism, amino acid metabolism, reaction system, transportation system in Bacillus amyloliquefaciens' hypersaline stress response. This study provides a scientific basis for the industrial application of Bacillus amyloliquefaciens and new insights into addressing the challenges of poor flavor quality in tank fermentation products.

Improvement of branched-chain amino acid production by isolated high-producing protease from Bacillus amyloliquefaciens NY130 on isolated soy/whey proteins and their muscle cell protection.

Branched-chain amino acids (BCAAs) are vital components of human and animal nutrition that contribute to the building blocks of proteins. In this study, 170 protease-producing strains were isolated and screened from soy-fermented foods. Bacillus amyloliquefaciens NY130 was obtained from Cheonggukjang with high production of BCAAs. Optimal production of protease from B. amyloliquefaciens NY130 (protease NY130) was achieved at 42 °C and pH 6.0 for 21 h. It was purified and determined as 27- and 40 kDa. Protease NY130 showed maximum activity at pH 9.0 and 45 °C with Km value of 10.95 mg for ISP and 1.69 mg for WPI. Protease-treated ISP and WPI showed increased sweetness and saltiness via electronic tongue analysis and enhanced the protective effect against oxidative stress in C2C12 myocytes by increasing p-mTOR/mTOR protein expression to 160%. This work possesses potential in producing BCAAs by using protease for utilization in food.

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.

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.

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.

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.

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.

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.

CRISPR-dCpf1 mediated whole genome crRNA inhibition library for high-throughput screening of growth characteristic genes in Bacillus amyloliquefaciens LB1ba02.

Bacillus amyloliquefaciens LB1ba02 is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. However, autolysis affects the growth of bacteria, further affecting the yield of target products. Besides, the restriction-modification system, existed in B. amyloliquefaciens LB1ba02, results in a low transformation efficiency, which further leads to a lack of high-throughput screening tools. Here, we constructed a genome-wide crRNA inhibition library based on the CRISPR/dCpf1 system and high-throughput screening of related genes affecting the cell growth and autolysis using flow cytometry in B. amyloliquefaciens LB1ba02. The whole genome crRNA library was first validated for resistance to the toxic chemical 5-fluorouracil, and then used for validation of essential genes. In addition, seven gene loci (oppD, flil, tuaA, prmA, sigO, hslU, and GE03231) that affect the growth characteristics of LB1ba02 were screened. Among them, the Opp system had the greatest impact on growth. When the expression of operon oppA-oppB-oppC-oppD-oppF was inhibited, the cell growth difference was most significant. Inhibition of other sites could also promote rapid growth of bacteria to varying degrees; however, inhibition of GE03231 site accelerated cell autolysis. Therefore, the whole genome crRNA inhibition library is well suited for B. amyloliquefaciens LB1ba02 and can be further applied to high-throughput mining of other functional genes.

Superoxide dismutase secreting Bacillus amyloliquefaciens spores attenuate pulmonary fibrosis.

Superoxide dismutase (SOD) can convert active oxygen to oxygen or hydrogen peroxide, and recent research has suggested that it can protect against lung damage and fibrosis. Clinical applications based on SOD remain limited however due to costs and low stability. We here investigated a potential new therapeutic delivery system for this enzyme in the form of SOD-overexpressing Bacillus amyloliquefaciens spores which we introduced into a bleomycin-induced pulmonary fibrosis mouse model. This treatment significantly alleviated the disease, as quantified using a hydroxyproline assay, at 107 colony forming unit (CFU) of Bacillus spores per day. Exposure of the mice to the spores was further found to decrease the lung mRNA levels of CTGF, Col1a1, α-SMA, TGF-ß, TNF-α, and IL-6, and the protein levels of TGF-ß, Smad2/3, αSMA and Col1a1, all major indicators of pulmonary fibrosis. Survival benefits, and reduced byproducts of lipid peroxidase such as malondialdehyde and 4-hydroxynen, were also noted in the treated animals. The beneficial effects of these Bacillus spores on pulmonary fibrosis were further found to be greater than the equivalent free SOD concentration. Immunofluorescence staining of primary pulmonary fibroblasts extracted from the bleomycin-induced model showed decreased αSMA expression following the in vivo treatment with SOD-overexpressing Bacillus. Our treatment approach SOD through Bacillus spores shows beneficial effects against pulmonary fibrosis, combined with the suppression of the SMAD/TGF-ß pathway, suggesting that it is an effective novel delivery route for antioxidants.