Combinatorial metabolic engineering of Bacillus subtilis for de novo production of polymyxin B.

Polymyxin is a lipopeptide antibiotic that is effective against multidrug-resistant Gram-negative bacteria. However, its clinical development is limited due to low titer and the presence of homologs. To address this, the polymyxin gene cluster was integrated into Bacillus subtilis, and sfp from Paenibacillus polymyxa was expressed heterologously, enabling recombinant B. subtilis to synthesize polymyxin B. Regulating NRPS domain inhibited formation of polymyxin B2 and B3. The production of polymyxin B increased to 329.7 mg/L by replacing the native promoters of pmxA, pmxB, and pmxE with PfusA, C2up, and PfusA, respectively. Further enhancement in this production, up to 616.1 mg/L, was achieved by improving the synthesis ability of 6-methyloctanoic acid compared to the original strain expressing polymyxin heterologously. Additionally, incorporating an anikasin-derived domain into the hybrid nonribosomal peptide synthase of polymyxin increased the B1 ratio in polymyxin B from 57.5% to 62.2%. Through optimization of peptone supply in the fermentation medium and fermentation in a 5.0-L bioreactor, the final polymyxin B titer reached 962.1 mg/L, with a yield of 19.24 mg/g maltodextrin and a productivity of 10.02 mg/(L·h). This study demonstrates a successful approach for enhancing polymyxin B production and increasing the B1 ratio through combinatorial metabolic engineering.

CRISPR-Cas9-Mediated Genome Editing in Paenibacillus polymyxa.

In recent years, the clustered regularly interspaced palindromic repeats-Cas (CRISPR-Cas) technology has become the method of choice for precision genome editing in many organisms due to its simplicity and efficacy. Multiplex genome editing, point mutations, and large genomic modifications are attractive features of the CRISPR-Cas9 system. These applications facilitate both the ease and velocity of genetic manipulations and the discovery of novel functions. In this protocol chapter, we describe the use of a CRISPR-Cas9 system for multiplex integration and deletion modifications, and deletions of large genomic regions by the use of a single guide RNA (sgRNA), and, finally, targeted point mutation modifications in Paenibacillus polymyxa.

Engineering the carbon and redox metabolism of Paenibacillus polymyxa for efficient isobutanol production.

Paenibacillus polymyxa is a non-pathogenic, Gram-positive bacterium endowed with a rich and versatile metabolism. However interesting, this bacterium has been seldom used for bioproduction thus far. In this study, we engineered P. polymyxa for isobutanol production, a relevant bulk chemical and next-generation biofuel. A CRISPR-Cas9-based genome editing tool facilitated the chromosomal integration of a synthetic operon to establish isobutanol production. The 2,3-butanediol biosynthesis pathway, leading to the main fermentation product of P. polymyxa, was eliminated. A mutant strain harbouring the synthetic isobutanol operon (kdcA from Lactococcus lactis, and the native ilvC, ilvD and adh genes) produced 1 g L-1 isobutanol under microaerobic conditions. Improving NADPH regeneration by overexpression of the malic enzyme subsequently increased the product titre by 50%. Network-wide proteomics provided insights into responses of P. polymyxa to isobutanol and revealed a significant metabolic shift caused by alcohol production. Glucose-6-phosphate 1-dehydrogenase, the key enzyme in the pentose phosphate pathway, was identified as a bottleneck that hindered efficient NADPH regeneration through this pathway. Furthermore, we conducted culture optimization towards cultivating P. polymyxa in a synthetic minimal medium. We identified biotin (B7), pantothenate (B5) and folate (B9) to be mutual essential vitamins for P. polymyxa. Our rational metabolic engineering of P. polymyxa for the production of a heterologous chemical sheds light on the metabolism of this bacterium towards further biotechnological exploitation.

Isolation and Characterization of Paenibacillus polymyxa B7 and Inhibition of Aspergillus tubingensis A1 by Its Antifungal Substances.

Screening of Bacillus with antagonistic effects on paddy mold pathogens to provide strain resources for biological control of mold in Oryza sativa L. screening of Bacillus isolates antagonistic towards Aspergillus tubingensis from rhizosphere soil of healthy paddy; classification and identification of antagonistic strains by biological characteristics and 16S rDNA sequence analysis; transcriptome sequencing after RNA extraction from Bacillus-treated Aspergillus tubingensis; and extraction of inhibitory crude proteins of Bacillus by ammonium sulfate precipitation; inhibitory crude protein and Bacillus spp. were treated separately for A. tubingensis and observed by scanning electron microscopy (SEM). An antagonistic strain of Bacillus, named B7, was identified as Paenibacillus polymyxa by 16S rDNA identification and phylogenetic evolutionary tree comparison analysis. Analysis of the transcriptome results showed that genes related to secondary metabolite biosynthesis such as antifungal protein were significantly downregulated. SEM results showed that the mycelium of A. tubingensis underwent severe rupture after treatment with P. polymyxa and antifungal proteins, respectively. In addition, the sporocarp changed less after treatment with P. polymyxa, and the sporangium stalks had obvious folds. P. polymyxa B7 has a good antagonistic effect against A. tubingensis and has potential for biocontrol applications of paddy mold pathogens.

Mechanisms on salt tolerant of Paenibacillus polymyxa SC2 and its growth-promoting effects on maize seedlings under saline conditions.

Soil salinity negatively affects microbial communities, soil fertility, and agricultural productivity and has become a major agricultural problem worldwide. Plant growth-promoting rhizobacteria (PGPR) with salt tolerance can benefit plant growth under saline conditions and diminish the negative effects of salt stress on plants. In this study, we aimed to understand the salt-tolerance mechanism of Paenibacillus polymyxa at the genetic and metabolic levels and elucidate the mechanism of strain SC2 in promoting maize growth under saline conditions. Under salt stress, we found that strain SC2 promoted maize seedling growth, which was accompanied by a significant upregulation of genes encoding for the biosynthesis of peptidoglycan, polysaccharide, and fatty acid, the metabolism of purine and pyrimidine, and the transport of osmoprotectants such as trehalose, glycine betaine, and K+ in strain SC2. To further enhance the salt resistance of strain SC2, three mutants (SC2-11, SC2-13, and SC2-14) with higher capacities for salt resistance and exopolysaccharide synthesis were obtained via atmospheric and room-temperature plasma mutagenesis. In saline-alkaline soil, the mutants showed better promoting effect on maize seedlings than wild-type SC2. The fresh weight of maize seedlings was increased by 68.10% after treatment with SC2-11 compared with that of the control group. The transcriptome analysis of maize roots demonstrated that SC2 and SC2-11 could induce the upregulation of genes related to the plant hormone signal transduction, starch and sucrose metabolism, reactive oxygen species scavenging, and auxin and ethylene signaling under saline-alkaline stress. In addition, various transcription factors, such as zinc finger proteins, ethylene-responsive-element-binding protein, WRKY, myeloblastosis proteins, basic helix-loop-helix proteins, and NAC proteins, were up-regulated in response to abiotic stress. Moreover, the microbial community composition of maize rhizosphere soil after inoculating with strain SC2 was varied from the one after inoculating with mutant SC2-11. Our results provide new insights into the various genes involved in the salt resistance of strain SC2 and a theoretical basis for utilizing P. polymyxa in saline-alkaline environments.

Exploration of the Native Sucrose Operon Enables the Development of an Inducible T7 Expression System in Paenibacillus polymyxa.

The use of Paenibacillus polymyxa as an industrial producer is limited by the lack of suitable synthetic biology tools. In this study, we identified a native sucrose operon in P. polymyxa. Its structural and functional relationship analysis revealed the presence of multiple regulatory elements, including four ScrR-binding sites and a catabolite-responsive element (CRE). In P. polymyxa, we established a cascade T7 expression system involving an integrated T7 RNA polymerase (T7P) regulated by the sucrose operon and a T7 promoter. It enables controllable gene expression by sucrose and regulatory elements, and a 5-fold increase in expression efficiency compared with the original sucrose operon was achieved. Further deletion of SacB in P. polymyxa resulted in a 38.95% increase in the level of thermophilic lipase (TrLip) production using the cascade T7 induction system. The results highlight the effectiveness of sucrose regulation as a novel synthetic biology tool, which facilitates exploring gene circuits and enables their dynamic regulation.

Response of Paenibacillus polymyxa SC2 to the stress of polymyxin B and a key ABC transporter YwjA involved.

Polymyxins are cationic peptide antibiotics and regarded as the "final line of defense" against multidrug-resistant bacterial infections. Meanwhile, some polymyxin-resistant strains and the corresponding resistance mechanisms have also been reported. However, the response of the polymyxin-producing strain Paenibacillus polymyxa to polymyxin stress remains unclear. The purpose of this study was to investigate the stress response of gram-positive P. polymyxa SC2 to polymyxin B and to identify functional genes involved in the stress response process. Polymyxin B treatment upregulated the expression of genes related to basal metabolism, transcriptional regulation, transport, and flagella formation and increased intracellular ROS levels, flagellar motility, and biofilm formation in P. polymyxa SC2. Adding magnesium, calcium, and iron alleviated the stress of polymyxin B on P. polymyxa SC2, furthermore, magnesium and calcium could improve the resistance of P. polymyxa SC2 to polymyxin B by promoting biofilm formation. Meanwhile, functional identification of differentially expressed genes indicated that an ABC superfamily transporter YwjA was involved in the stress response to polymyxin B of P. polymyxa SC2. This study provides an important reference for improving the resistance of P. polymyxa to polymyxins and increasing the yield of polymyxins. KEY POINTS: • Phenotypic responses of P. polymyxa to polymyxin B was performed and indicated by RNA-seq • Forming biofilm was a key strategy of P. polymyxa to alleviate polymyxin stress • ABC transporter YwjA was involved in the stress resistance of P. polymyxa to polymyxin B.

MALDI-TOF as a powerful tool for identifying and differentiating closely related microorganisms: the strange case of three reference strains of Paenibacillus polymyxa.

Accurate identification and typing of microbes are crucial steps in gaining an awareness of the biological heterogeneity and reliability of microbial material within any proprietary or public collection. Paenibacillus polymyxa is a bacterial species of great agricultural and industrial importance due to its plant growth-promoting activities and production of several relevant secondary metabolites. In recent years, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used as an alternative rapid tool for identifying, typing, and differentiating closely related strains. In this study, we investigated the diversity of three P. polymyxa strains. The mass spectra of ATCC 842T, DSM 292, and DSM 365 were obtained, analysed, and compared to select discriminant peaks using ClinProTools software and generate classification models. MALDI-TOF MS analysis showed inconsistent results in identifying DSM 292 and DSM 365 as belonging to P. polimixa species, and comparative analysis of mass spectra revealed the presence of highly discriminatory biomarkers among the three strains. 16S rRNA sequencing and Average Nucleotide Identity (ANI) confirmed the discrepancies found in the proteomic analysis. The case study presented here suggests the enormous potential of the proteomic-based approach, combined with statistical tools, to predict and explore differences between closely related strains in large microbial datasets.

Abh, AbrB3, and Spo0A play distinct regulatory roles during polymyxin synthesis in Paenibacillus polymyxa SC2.

IMPORTANCE: Polymyxins are considered the last line of defense against multidrug-resistant bacteria. The regulatory mechanism of polymyxin synthesis is poorly studied in Paenibacillus polymyxa. In this study, we found that Abh and AbrB3 negatively regulated, whereas Spo0A positively regulated polymyxin synthesis in P. polymyxa SC2. In addition, a regulatory relationship between Abh, AbrB3, and Spo0A was revealed, which regulate polymyxin synthesis via multiple regulatory mechanisms in P. polymyxa.

Suppression of Strawberry Anthracnose by Paenibacillus polymyxa TP3 In Situ and from a Distance.

Strawberry is a popular fruit with valuable nutrition and an attractive fragrance, but its production and propagation are limited by various diseases, including anthracnose and gray mold. For disease management, biological control measures are environmentally friendly and good alternatives to fungicides to avoid crop losses, reduce carbon emissions, and improve food safety. In this study, Paenibacillus polymyxa TP3, which originated from the strawberry phyllosphere, was shown to antagonize the anthracnose fungal pathogen Colletotrichum siamense and reduce leaf symptoms on strawberry plants. Several mass spectra corresponding to fusaricidin were detected in the confrontation assay of P. polymyxa TP3 and C. siamense by image mass spectrometry. The transcription of fusA and fusG in the fusaricidin biosynthesis gene cluster increased while P. polymyxa TP3 was cultured in the medium containing the culture filtrate of C. siamense, as detected by reverse-transcription polymerase chain reaction, indicating the involvement of fusaricidins in P. polymyxa TP3 antagonism against the anthracnose pathogen. Further disease control assays demonstrated the time frame and spatial mode of P. polymyxa TP3-induced systemic resistance of strawberry against C. siamense. The transcript level of the marker gene FaPDF1.2 of the jasmonic acid pathway increased in strawberry leaves after drenching treatment with P. polymyxa TP3, and the callose deposition was enhanced by further flg22 treatment. In addition, P. polymyxa TP3 treatments of the strawberry mother plants reduced C. siamense infection in the daughter plants, which would be a potent feature for the application of P. polymyxa TP3 in strawberry nurseries and fields to reduce the impact of diseases, especially anthracnose.

Biocontrol Efficacy and Induced Resistance of Paenibacillus polymyxa J2-4 Against Meloidogyne incognita Infection in Cucumber.

Meloidogyne incognita is one of the most destructive agricultural pathogens around the world, resulting in severe damage to yield and quality in agricultural production. Biological control promises to be a great potential alternative to chemical agents against M. incognita. Paenibacillus polymyxa J2-4, isolated from ginger plants injured by M. incognita, has shown excellent biocontrol efficacy against M. incognita in cucumber. In vitro experiments with the strain J2-4 resulted in a correct mortality rate of 88.79% (24 h) and 98.57% (48 h) for second-stage juveniles (J2s) of M. incognita. Strain J2-4 significantly suppressed nematode infection on potted plants, with a 65.94% reduction in galls and a 51.64% reduction in eggs compared with the control. The split-root assay demonstrated that strain J2-4 not only reduced J2s' invasion but also inhibited nematode development through the dependence on salicylic acid and jasmonic acid signaling of strain J2-4 induction of plant resistance in local and systemic roots of cucumbers. Genomic analysis of strain J2-4 indicated biosynthetic gene clusters encoding polymyxin, fusaricidin B, paenilan, and tridecaptin. In addition, genetic analysis showed that none of the genes encoding virulence factors were detected in the genome of J2-4 compared with the pathogenic Bacillus species. Taking all the data together, we conclude that P. polymyxa J2-4 has potential as a biological control agent against M. incognita on cucumbers and can be considered biologically safe when used in agriculture.

Development of PMA-qPCR assay to accurately and reproducible quantify viable bacteria of Paenibacillus polymyxa.

Paenibacillus polymyxa is an important biocontrol bacterium. The combination of propidium monoazide (PMA) and quantitative polymerase chain reactionq (qPCR) has proven effective in quantifying live bacteria from various microorganisms. The objective was to create a PMA-qPCR assay to precisely and consistently measure the number of living bacteria of biocontrol P. polymyxa. The primers were designed for the spo0A gene of P. polymyxa HY96-2. The optimal conditions for treating the target strain with PMA were a PMA concentration of 15 µg/mL, an incubation time of 5 min, and an exposure time of 10 min. The PMA-qPCR method had a limit of quantification (LOQ) of 1.0 × 103 CFU/mL for measuring the amount of viable P. polymyxa bacteria. The PMA-qPCR method is more sensitive than the qPCR method in detecting viable bacteria in the mixtures of viable and dead bacteria. The accuracy and reproducibility of quantifying viable P. polymyxa bacteria using the PMA-qPCR method were higher compared to the plate count method.

Rheological characterization of artificial paenan compositions produced by Paenibacillus polymyxa DSM 365.

Microbial exopolysaccharides offer a sustainable alternative to petroleum-based rheological modifiers. Recent studies revealed that the heteroexopolysaccharide produced by Paenibacillus polymyxa is composed of three distinct biopolymers, referred to as paenan I, II and III. Using CRISPR-Cas9 mediated knock-out variants of glycosyltransferases, defined polysaccharide compositions were produced and rheologically characterized in detail. The high viscosity and gel-like character of the wildtype polymer is proposed to originate from the non-covalent interaction between a pyruvate residue of paenan I and the glucuronic acid found in the backbone of paenan III. Paenan II conveys thermostable properties to the exopolysaccharide mixture. In contrast to the wildtype polymer mixture, knock-out variants demonstrated significantly altered rheological behavior. Using the rheological characterization performed in this study, tailor-made paenan variants and mixtures can be generated to be utilized in a wide range of applications including thickening agents, coatings, or high-value biomedical materials.

Production of highly pure R,R-2,3-butanediol for biological plant growth promoting agent using carbon feeding control of Paenibacillus polymyxa MDBDO.

BACKGROUND: Chemical fertilizers have greatly contributed to the development of agriculture, but alternative fertilizers are needed for the sustainable development of agriculture. 2,3-butanediol (2,3-BDO) is a promising biological plant growth promoter. RESULTS: In this study, we attempted to develop an effective strategy for the biological production of highly pure R,R-2,3-butanediol (R,R-2,3-BDO) by Paenibacillus polymyxa fermentation. First, gamma-ray mutagenesis was performed to obtain P. polymyxa MDBDO, a strain that grew faster than the parent strain and had high production of R,R-2,3-BDO. The activities of R,R-2,3-butanediol dehydrogenase and diacetyl reductase of the mutant strain were increased by 33% and decreased by 60%, respectively. In addition, it was confirmed that the carbon source depletion of the fermentation broth affects the purity of R,R-2,3-BDO through batch fermentation. Fed-batch fermentation using controlled carbon feeding led to production of 77.3 g/L of R,R-2,3-BDO with high optical purity (> 99% of C4 products) at 48 h. Additionally, fed-batch culture using corn steep liquor as an alternative nitrogen source led to production of 70.3 g/L of R,R-2,3-BDO at 60 h. The fed-batch fermentation broth of P. polymyxa MDBDO, which contained highly pure R,R-2,3-BDO, significantly stimulated the growth of soybean and strawberry seedlings. CONCLUSIONS: This study suggests that P. polymyxa MDBDO has potential for use in biological plant growth promoting agent applications. In addition, our fermentation strategy demonstrated that high-purity R,R-2,3-BDO can be produced at high concentrations using P. polymyxa.

Antibacterial activity of epsilon-poly-l-lysine produced by Stenotrophomonas maltophilia HS4 and Paenibacillus polymyxa HS5, alone and in combination with bacteriophages.

Over the past decades, antibiotic resistance has become a major clinical problem, and searching for new therapeutic strategies seems to be necessary. Using novel natural compounds, antimicrobial peptides, and bacteriophages is the most promising solution. In this study, various cationic metabolite-producer bacteria were isolated from different soil samples. Two isolates were identified as Stenotrophomonas maltophilia HS4 (accession number: MW791428) and Paenibacillus polymyxa HS5 (accession number: MW791430) based on biochemical characteristics and phylogenetic analysis using 16S rRNA gene sequences. The cationic compound in the fermentation broth was precipitated and purified with sodium tetraphenylborate salt. The purified cationic peptide was confirmed to be epsilon-poly-l-lysine by structural and molecular analysis using High-Performance Liquid Chromatography, Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, and Fourier-transform infrared spectroscopy. The antibacterial activity of epsilon-poly-l-lysine was evaluated against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Serratia marcescens ATCC 13880, and Klebsiella pneumoniae ATCC 13883 by microdilution method. Furthermore, the antibacterial effects of purified epsilon-poly-l-lysine in combination with two long non-contractile tail bacteriophages against vancomycin-resistant Enterococcus faecalis and colistin-resistant Klebsiella pneumoniae were investigated. The results indicated great antibacterial activity of epsilon-poly-l-lysine which was produced by two novel bacteria. The epsilon-poly-l-lysine as a potent cationic antimicrobial peptide is demonstrated to possess great antimicrobial activity against pathogenic and also antibiotic-resistant bacteria.

Characterization of a newly discovered putative DNA replication initiator from Paenibacillus polymyxa phage phiBP.

The bacteriophage phiBP contains a newly discovered putative replisome organizer, a helicase loader, and a beta clamp, which together may serve to replicate its DNA. Bioinformatics analysis of the phiBP replisome organizer sequence showed that it belongs to a recently identified family of putative initiator proteins. We prepared and isolated a wild type-like recombinant protein, gpRO-HC, and a mutant protein gpRO-HCK8A, containing a lysine to alanine substitution at position 8. gpRO-HC had low ATPase activity regardless of the presence of DNA, while the ATPase activity of the mutant was significantly higher. gpRO-HC bound to both single- and double-stranded DNA substrates. Different methods showed that gpRO-HC forms higher oligomers containing about 12 subunits. This work provides the first information about another group of phage initiator proteins, which trigger DNA replication in phages infecting low GC Gram-positive bacteria.

CRISPR-Cas9 driven structural elucidation of the heteroexopolysaccharides from Paenibacillus polymyxa DSM 365.

Paenibacillus polymyxa is a Gram-positive soil bacterium known for producing a wide range of exopolysaccharides. However, due to the biopolymer's complexity, structural elucidation has so far been inconclusive. Combinatorial knock-outs of glycosyltransferases were generated in order to separate distinct polysaccharides produced by P. polymyxa. Using a complementary analytical approach consisting of carbohydrate fingerprints, sequence analysis, methylation analysis as well as NMR spectroscopy, the structure of the repeating units of two additional heteroexopolysaccharides termed paenan I and paenan III were elucidated. Results for paenan I identified a trisaccharide backbone consisting of 1➔4-ß-d-Glc, 1➔4-ß-d-Man and a 1,3,4-branching ß-d-Gal residue with a sidechain comprising of a terminal ß-d-Gal3,4-Pyr and 1➔3-ß-d-Glc. For paenan III, results indicated a backbone consisting of 1➔3-ß-d-Glc, 1,3,4-linked α-d-Man and 1,3,4-linked α-d-GlcA. NMR analysis indicated monomeric ß-d-Glc and α-d-Man sidechains for the branching Man and GlcA residues respectively.

Genome-wide mapping of GlnR-binding sites reveals the global regulatory role of GlnR in controlling the metabolism of nitrogen and carbon in Paenibacillus polymyxa WLY78.

BACKGROUND: Paenibacillus polymyxa WLY78 is a Gram-positive, endospore-forming and N2-fixing bacterium. Our previous study has demonstrated that GlnR acts as both an activator and a repressor to regulate the transcription of the nif (nitrogen fixation) operon (nifBHDKENXhesAnifV) according to nitrogen availability, which is achieved by binding to the two GlnR-binding sites located in the nif promoter region. However, further study on the GlnR-mediated global regulation in this bacterium is still needed. RESULTS: In this study, global identification of the genes directly under GlnR control is determined by using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assays (EMSA). Our results reveal that GlnR directly regulates the transcription of 17 genes/operons, including a nif operon, 14 nitrogen metabolism genes/operons (glnRA, amtBglnK, glnA1, glnK1, glnQHMP, nasA, nasD1, nasD2EF, gcvH, ansZ, pucR, oppABC, appABCDF and dppABC) and 2 carbon metabolism genes (ldh3 and maeA1). Except for the glnRA and nif operon, the other 15 genes/operons are newly identified targets of GlnR. Furthermore, genome-wide transcription analyses reveal that GlnR not only directly regulates the expression of these 17 genes/operons, but also indirectly controls the expression of some other genes/operons involved in nitrogen fixation and the metabolisms of nitrogen and carbon. CONCLUSION: This study provides a GlnR-mediated regulation network of nitrogen fixation and the metabolisms of nitrogen and carbon.

Paenibacillus polymyxa Antagonism towards Fusarium: Identification and Optimisation of Antibiotic Production.

An antibiotic produced by Paenibacillus polymyxa 7F1 was studied. The 7F1 strain was isolated from the rhizosphere of a wheat field. Response surface methodology was used to optimize the physicochemical parameters. The strain showed broad-spectrum activity against several plant pathogens. Identification of the strain was realized based on 16s rRNA gene and gyrB gene sequencing. The antibiotic was optimized by one-factor-at-a-time (OFAT) and response surface methodology (RSM) approaches. The suitable antibiotic production conditions were optimized using the one-factor-at-a-time method. The individual and interaction effects of three independent variables: culture temperature, initial pH, and culture time, were optimized by Box-Behnken design. The 16SrRNA gene sequence (1239 nucleotides) and gyrB gene (1111 nucleotides) were determined for strain 7F1 and shared the highest identities to those of Paenibacillus polymyxa. The results showed the optimal fermentation conditions for antibiotics produced by Paenibacillus polymyxa 7F1 were a culture temperature of 38 °C, initial pH of 8.0, and culture time of 8 h. The antibiotics produced by Paenibacillus polymyxa 7F1 include lipopeptides such as iturin A and surfactin. The results provide a theoretical basis for the development of bacteriostatic biological agents and the control of mycotoxins.

Characterization of a novel GH26 ß-mannanase from Paenibacillus polymyxa and its application in the production of mannooligosaccharides.

A novel glycoside hydrolase family 26 ß-mannanase gene ppman26a was cloned from Paenibacillus polymyxa KF-1. The full-length enzyme PpMan26A and its truncated products CBM35pp (aa 35-328) and PpMan26A-Δ205 (aa 206-656) were overexpressed in Escherichia coli. PpMan26A hydrolyzed locust bean gum, guar gum, konjac gum and ivory nut mannan, with the highest specific activity toward konjac gum. The Km and kcat values for konjac gum were 2.13 mg/mL and 416.66 s-1, respectively. The oligosaccharides fraction obtained from the hydrolysis of konjac gum by PpMan26A was analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (MALDI-TOF-MS). The degradation products were mainly mannooligosaccharides with a degree of polymerization of 3-8. CBM35pp exerted strong binding activity toward mannans but without ß-mannanase activity. PpMan26A-Δ205, with the deletion of the N-terminal CBM domain, showed lower substrate binding capacity, resulting in reduced enzymatic activity and thermostability. This study complements our understanding of GH26 ß-mannanases and expands the potential industrial application of PpMan26A.