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Considered a "Generally Recognized As Safe" (GRAS) bacterium, the plant growth-promoting rhizobacterium Paenibacillus has been widely applied in: agriculture, medicine, industry, and environmental remediation. Paenibacillus species not only accelerate plant growth and degrade toxic substances in wastewater and soil but also produce industrially-relevant enzymes and antimicrobial peptides. Due to a lack of genetic manipulation tools and methods, exploitation of the bioresources of naturally isolated Paenibacillus species has long been limited. Genetic manipulation tools and methods continue to improve in Paenibacillus , such as shuttle plasmids, promoters, and genetic tools of CRISPR. Furthermore, genetic transformation systems develop gradually, including: penicillin-mediated transformation, electroporation, and magnesium amino acid-mediated transformation. As genetic manipulation methods of homologous recombination and CRISPR-mediated editing system have developed gradually, Paenibacillus has come to be regarded as a promising microbial chassis for biomanufacturing, expanding its application scope, such as: industrial enzymes, bioremediation and bioadsorption, surfactants, and antibacterial agents. In this review, we describe the applications of Paenibacillus bioproducts, and then discuss recent advances and future challenges in the development of genetic manipulation systems in this genus. This work highlights the potential of Paenibacillus as a new microbial chassis for mining bioresources.

Background: The β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low K M value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly., Results: Firstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkat oNPGal /L culture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkat oNPGal /g DCW /h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkat oNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter)., Conclusion: This study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme., (© 2024. The Author(s).)

Two novel Gram-positive strains, chi10 T and PFR10 T , were isolated from the roots of Suaeda japonica Makino and Viola mandshurica W. Becker, respectively. The strains are facultatively aerobic, rod-shaped, motile via peritrichous flagella and endospore-forming. Strains chi10 T and PFR10 T showed the highest 16S rRNA gene sequence similarity to Paenibacillus alvei DSM 29 T (99.0%) and Paenibacillus planticolens LMG 31457 T (99.3%). Furthermore, the phylogenetic and phylogenomic analyses demonstrated that strains chi10 T and PFR10 T were closely related to the genus Paenibacillus . The digital DNA-DNA hybridization (dDDH) values for strains used in the phylogenetic analysis and strain chi10 T ranged from 19.6 to 28.4%, while for strain PFR10 T , the values ranged from 19.8 to 53.5%, according to the Genome-to-Genome Distance Calculator 3.0, which were all less than 70%. Additionally, the dDDH value between strains chi10 T and PFR10 T was 22.0%, confirming that they are different species of the genus Paenibacillus . The average nucleotide identity between chi10 T and PFR10 T and other species of the genus Paenibacillus were 68.4-84.7% and 67.8-93.6%, respectively. In addition, the major polar lipids of chi10 T and PFR10 T consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acid profile of strain chi10 T was iso-C 15 : 0 and anteiso-C 15 : 0 , whereas the major fatty acid profile of strain PFR10 T was iso-C 15 : 0 , anteiso-C 15 : 0 and iso-C 16 : 0 . The sole respiratory quinone in strains chi10 T and PFR10 T was menaquinone-7. Phylogenomic, phenotypic and genome analyses strongly indicated that chi10 T and PFR10 T could be two novel Paenibacillus species for which the names Paenibacillus suaedae sp. nov. (type strain chi10 T = KACC 23258 T = TBRC 17803 T ) and Paenibacillus violae sp. nov. (type strain PFR10 T = KACC 23263 T = TBRC 17804 T ) are proposed, respectively.

Bioproduction of diverse N-acetyl chitooligosaccharides from chitin is of great value. In the study, a novel GH family 18 bifunctional chitinase gene (PsChi82) from Paenibacillus shirakamiensis was identified, expressed and biochemically characterized. PsChi82 was most active at pH 5.0, and 55 °C, and displayed remarkable pH stability with the broad pH range of 3.0-12.0. It showed high chitosanase activity of 10.6 U mg -1 and diverse hydrolysis products of GlcNAc, (GlcNAc) 2 , GlcN-GlcNAc and (GlcN) 2 -GlcNAc, which may facilitate comprehensively understanding of structure-function relationships of N-acetyl COSs. Three engineered variants were then expressed and characterized. Among them, PsChi82-CBM26 possessed specific activity of 25.1 U mg -1 against colloidal chitin, which was 2.1 folds higher than that of PsChi82. The diverse N-acetyl COSs were subsequently produced by PsChi82-CBM26 with a sugar content of 23.2 g L -1 . These excellent properties may make PsChi82-CBM26 potentially useful for N-acetyl COSs production in the food and chemical industries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Lipases play a vital role in various biological processes, from lipid metabolism to industrial applications. However, the ever-evolving challenges and diverse substrates necessitate the continual exploration of novel high-performance lipases. In this study, we employed an in silico mining approach to search for lipases with potential high sn-1,3 selectivity and catalytic activity. The identified novel lipase, PLL, from Paenibacillus larvae subsp. larvae B-3650 exhibited a specific activity of 111.2 ± 5.5 U/mg towards the substrate p-nitrophenyl palmitate (pNPP) and 6.9 ± 0.8 U/mg towards the substrate olive oil when expressed in Escherichia coli (E. coli). Computational design of cysteine mutations was employed to enhance the catalytic performance of PLL. Superior stability was achieved with the mutant K7C/A386C/H159C/K108C (2M3/2M4), showing an increase in melting temperature (T m ) by 1.9°C, a 2.05-fold prolonged half-life at 45°C, and no decrease in enzyme activity. Another mutant, K7C/A386C/A174C/A243C (2M1/2M3), showed a 4.9-fold enhancement in specific activity without compromising stability. Molecular dynamics simulations were conducted to explore the mechanisms of these two mutants. Mutant 2M3/2M4 forms putative disulfide bonds in the loop region, connecting the N- and C-termini of PLL, thus enhancing overall structural rigidity without impacting catalytic activity. The cysteines introduced in mutant 2M1/2M3 not only form new intramolecular hydrogen bonds but also alter the polarity and volume of the substrate-binding pocket, facilitating the entry of large substrate pNPP. These results highlight an efficient in silico exploration approach for novel lipases, offering a rapid and efficient method for enhancing catalytic performance through rational protein design., Competing Interests: Declaration of Competing Interest The authors declare that there are no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This work aimed to isolate and characterize a novel chitin-degrading bacterium from Yok Don National Park, Vietnam, for crop production studies. Among the chitinolytic isolates, strain YSY-4.3 was selected, which grew rapidly and produced a large halo around the colony. 16S rDNA analysis indicated that the strain is a novel species in the genus Paenibacillus, and an in vitro evaluation showed that the strain produced phytohormones (IAA, GA3, and zeatin), biofilms, and siderophores; possessed cellulase; and exerted antifungal activity. The whole genome of the strain was 5,628,400 bp with 49.3% GC content, 5056 coding sequences, 48 tRNA, and 1 rRNA. It shared the highest values of digital DNA-DNA hybridization (67.4%) and average nucleotide identity (89.54%) with those of Paenibacillus woosongensis B2_4 (CP126084.1), suggesting a novel species. Of the coding sequences, 4287 proteins were identified by COG, and 2561 were assigned by KEGG. The genome contained at least 51 genes involved in plant growth and resistance to heavy-metal toxicity and 359 carbohydrate-active enzymes. The chitinolytic system of the strain was composed of 15 enzymes, among them, PsChiC, which contained a GH18 catalytic domain and a GH5 catalytic domain, had not been previously reported. In addition, the genome possessed 15 gene clusters encoding antimicrobial metabolites, 10 of which are possible novel clusters. This study expands knowledge regarding novel chitinolytic bacteria from Yok Don National Park and provides a valuable gene resource for future studies., (© 2024 Wiley‐VCH GmbH.)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A Gram-stain-positive, aerobic, white-coloured, rod-shaped bacteria, designated as a strain dW9 T , was isolated from soil. Strain dW9 T was catalase-positive and oxidase-negative. Strain dW9 T grew at temperature of 20-37°C and at pH of 5.0-7.0. Phylogenetic and 16S rRNA gene analysis indicated that strain dW9 T belonged to the genus Paenibacillus with its closest relative being Paenibacillus filicis S4 T (97.4% sequence similarity). The genome size of dW9 T was 7,787,916 bp with DNA G+C content of 51.3%. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values of dW9 T with its closest relatives were found to be <22.0% and <74.0%, respectively. The only respiratory quinone was MK-7, and the major fatty acids were antiso-C 15:0 and iso-C 16:0 . Overall, the comprehensive taxonomic analysis revealed that strain dW9 T met all the fundamental criteria to be classified as a novel species within the genus Paenibacillus. Accordingly, we propose the name Paenibacillus gyeongsangnamensis sp. nov., with the type strain dW9 T (=KCTC 43431 T =NBRC 116022 T ).

Four Gram-stain-positive and two Gram-stain-negative bacterial strains, designated as W4 T , FW7 T , TW48 T , UW52 T , PT-3 T , and RJY3 T , were isolated from soil samples collected from the Republic of Korea. The 16S rRNA gene sequence analysis showed that strains W4 T and FW7 T belonged to the genus Microbacterium , strains TW48 T and UW52 T were affiliated to the genus Paenibacillus, strain PT-3 T was related to the genus Flavobacterium , and strain RJY3 T was associated with the genus Aquabacterium . The closest phylogenetic taxa to W4 T , FW7 T , TW48 T , UW52 T , PT-3 T , and RJY3 T were Microbacterium bovistercoris NEAU-LLE T (97.7 %), Microbacterium protaetiae DFW100M-13 T (97.9 %), Paenibacillus auburnensis JJ-7 T (99.6 %), Paenibacillus allorhizosphaerae JJ-447 T (95.7 %), Flavobacterium buctense T7 T (97.1 %), and Aquabacterium terrae S2 T (99.5 %), respectively. Average nucleotide identity and digital DNA-DNA hybridization values between the novel strains and related reference type strains were <95.0 % and <70.0 %, respectively. The major cellular fatty acid in strains W4 T , FW7 T TW48 T , and UW52 T was antiso-C 15 : 0 . Similarly, strain PT-3 T revealed iso-C 15 : 0 , iso-C 15 : 1 G, iso-C 17 : 0 3-OH, and iso-C 15 : 0 3-OH as its principal fatty acids. On the other hand, RJY3 T exhibited summed feature 3 (C 16 : 1  ω 7 c and/or C 16 : 1  ω 6 c ), C 16 : 0 , summed feature 8 (C 18 : 1  ω 7 c and/or C 18 : 1  ω 6 c ), and C 12 : 0 as its predominant fatty acids. Overall, the polyphasic taxonomic data indicated that strains W4 T , FW7 T , TW48 T , UW52 T , PT-3 T , and RJY3 T represent novel species within the genera Microbacterium , Paenibacillus , Flavobacterium , and Aquabacterium . Accordingly, we propose the names Microbacterium humicola sp. nov., with the type strain W4 T (=KCTC 49888 T =NBRC 116001 T ), Microbacterium terrisoli sp. nov., with the type strain FW7 T (=KCTC 49859 T =NBRC 116000 T ), Paenibacillus pedocola sp. nov., with the type strain TW48 T (=KCTC 43470 T =NBRC 116017 T ), Paenibacillus silviterrae sp. nov., with the type strain UW52 T (=KCTC 43477 T =NBRC 116018 T ), Flavobacterium terrisoli sp. nov., with the type strain PT-3 T (=KCTC 92106 T =NBRC 116012 T ), and Aquabacterium humicola sp. nov., with the type strain RJY3 T (=KCTC 92105 T =NBRC 115831 T ).

Ascomycetes, basidiomycetes and deuteromycetes can degrade wood, but less attention has been paid to basidiomycetes involved in Esca, a major Grapevine Trunk Disease. Using a wood sawdust microcosm system, we compared the wood degradation of three grapevine cultivars inoculated with Fomitiporia mediterranea M. Fisch, a basidiomycete responsible for white-rot development and involved in Esca disease. The grapevine cultivar Ugni blanc was more susceptible to wood degradation caused by F. mediterranea than the cultivars Cabernet Sauvignon and Merlot. Solid-state Nuclear Magnetic Resonance (NMR) spectroscopy showed that F. mediterranea preferentially degrades lignin and hemicellulose over cellulose (preferential, successive or sequential white-rot). In addition, co-inoculation of sawdust with two cellulolytic and xylanolytic bacterial strains of Paenibacillus (Nakamura) Ash (Paenibacillus sp. (S231-2) and P. amylolyticus (S293)), enhanced F. mediterranea ability to degrade Ugni blanc. The NMR data further showed that the increase in Ugni blanc sawdust degradation products was greater when bacteria and fungi were inoculated together. We also demonstrated that these two bacterial strains could degrade the wood components of Ugni blanc sawdust. Genome analysis of these bacterial strains revealed numerous genes predicted to be involved in cellulose, hemicellulose, and lignin degradation, as well as several other genes related to bacteria-fungi interactions and endophytism inside the plant. The occurrence of this type of bacteria-fungus interaction could explain, at least in part, why necrosis develops extensively in certain grapevine varieties such as Ugni blanc., (© 2024. The Author(s).)

The ecologically and economically vital symbiosis between nitrogen-fixing rhizobia and leguminous plants is often thought of as a bi-partite interaction, yet studies increasingly show the prevalence of non-rhizobial endophytes (NREs) that occupy nodules alongside rhizobia. Yet, what impact these NREs have on plant or rhizobium fitness remains unclear. Here, we investigated four NRE strains found to naturally co-occupy nodules of the legume Medicago truncatula alongside Sinorhizobium meliloti in native soils. Our objectives were to (1) examine the direct and indirect effects of NREs on M. truncatula and S. meliloti fitness, and (2) determine whether NREs can re-colonize root and nodule tissues upon reinoculation. We identified one NRE strain (522) as a novel Paenibacillus species, another strain (717A) as a novel Bacillus species, and the other two (702A and 733B) as novel Pseudomonas species. Additionally, we found that two NREs (Bacillus 717A and Pseudomonas 733B) reduced the fitness benefits obtained from symbiosis for both partners, while the other two (522, 702A) had little effect. Lastly, we found that NREs were able to co-infect host tissues alongside S. meliloti . This study demonstrates that variation of NREs present in natural populations must be considered to better understand legume-rhizobium dynamics in soil communities., Competing Interests: The authors declare there are no competing interests.

GH11 enzyme is known to be specific and efficient for the hydrolysis of xylan. It has been isolated from many microorganisms, and its enzymatic characteristics and thermostability vary between species. In this study, a GH11 enzyme PphXyn11 from a novel xylan-degrading strain of Paenibacillus physcomitrellae XB was characterized, and five mutants were constructed to try to improve the enzyme's thermostability. The results showed that PphXyn11 was an acidophilic endo-β-1,4-xylanase with the optimal reaction pH of 3.0-4.0, and it could deconstruct different kinds of xylan substrates efficiently, such as beechwood xylan, wheat arabinoxylan and xylo-oligosaccharides, to produce xylobiose and xylotriose as the main products at the optimal reaction temperature of 40 °C. Improvement of the thermal stability of PphXyn11 using site-directed mutagenesis revealed that three mutants, W33C/N47C, S127C/N174C and S49E, designed by adding the disulfide bonds at the N-terminal, C-terminal and increasing the charged residues on the surface of PphXyn11 respectively, could increase the enzymatic activity and thermal stablility significantly and make the optimal reaction temperature reach 50 °C. Molecular dynamics simulations as well as computed the numbers of salt bridges and hydrogen bonds indicated that the protein structures of these three mutants were more stable than the wild type, which provided theoretical support for their improved thermal stability. Certainly, further research is necessary to improve the enzymatic characteristics of PphXyn11 to achieve the bioconversion of hemicellulosic biomass on an applicable scale., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Bacterial antimicrobial resistance poses a severe threat to humanity, necessitating the urgent development of new antibiotics. Recent advances in genome sequencing offer new avenues for antibiotic discovery. Paenibacillus genomes encompass a considerable array of antibiotic biosynthetic gene clusters (BGCs), rendering these species as good candidates for genome-driven novel antibiotic exploration. Nevertheless, BGCs within Paenibacillus genomes have not been extensively studied., Results: We conducted an analysis of 554 Paenibacillus genome sequences, sourced from the National Center for Biotechnology Information database, with a focused investigation involving 89 of these genomes via antiSMASH. Our analysis unearthed a total of 848 BGCs, of which 716 (84.4%) were classified as unknown. From the initial pool of 554 Paenibacillus strains, we selected 26 available in culture collections for an in-depth evaluation. Genomic scrutiny of these selected strains unveiled 255 BGCs, encoding non-ribosomal peptide synthetases, polyketide synthases, and bacteriocins, with 221 (86.7%) classified as unknown. Among these strains, 20 exhibited antimicrobial activity against the gram-positive bacterium Micrococcus luteus, yet only six strains displayed activity against the gram-negative bacterium Escherichia coli. We proceeded to focus on Paenibacillus brasilensis, which featured five new BGCs for further investigation. To facilitate detailed characterization, we constructed a mutant in which a single BGC encoding a novel antibiotic was activated while simultaneously inactivating multiple BGCs using a cytosine base editor (CBE). The novel antibiotic was found to be localized to the cell wall and demonstrated activity against both gram-positive bacteria and fungi. The chemical structure of the new antibiotic was elucidated on the basis of ESIMS, 1D and 2D NMR spectroscopic data. The novel compound, with a molecular weight of 926, was named bracidin., Conclusions: This study outcome highlights the potential of Paenibacillus species as valuable sources for novel antibiotics. In addition, CBE-mediated dereplication of antibiotics proved to be a rapid and efficient method for characterizing novel antibiotics from Paenibacillus species, suggesting that it will greatly accelerate the genome-based development of new antibiotics., (© 2024. The Author(s).)

Background: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa., Results: In this study, the cytoplasmic and 120 kDa β-galactosidase of Paenibacillus wynnii (β-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the β-gal-Pw gene led to an increase in extracellular β-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular β-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/L culture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the P AprE promoter. Production of extracellular β-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/L culture with secretion efficiencies of more than 80%., Conclusion: For the first time, the β-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes., (© 2024. The Author(s).)

Commercial β-galactosidases exhibit undesirable kinetic properties regarding substrate affinity (Michaelis-Menten constant [K M ] for lactose) and product inhibition (inhibitor constant [K i ] for galactose). An in silico screening of gene sequences was done and identified a putative β-galactosidase (Paenibacillus wynnii β-galactosidase, BgaPw) from the psychrophilic bacterium Paenibacillus wynnii. The cultivation of the wild-type P. wynnii strain resulted in very low β-galactosidase activities of a maximum of 150 nkat per liter of medium with o-nitrophenyl-β-d-galactopyranoside (oNPGal) as substrate. The recombinant production of BgaPw in Escherichia coli BL21(DE3) increased the yield ∼9,000-fold. Here, a volumetric activity of 1,350.18 ± 11.82 μkat o NPGal /L culture was achieved in a bioreactor cultivation. The partly purified BgaPw showed a pH optimum at 7.0, a temperature maximum at 40°C, and an excellent stability at 8°C with a half-life of 77 d. Kinetic studies with BgaPw were done in milk or in milk-imitating synthetic buffer (Novo buffer), respectively. Remarkably, the K M value of BgaPw with lactose was as low as 0.63 ± 0.045 mM in milk. It was found that the resulting products of lactose hydrolysis, namely galactose and glucose, did not inhibit the β-galactosidase activity of BgaPw, but instead showed a striking activating effect in both cases (up to 144%). In a comparison study in milk, lactose was completely hydrolyzed by BgaPw in 72 h at 8°C, whereas 2 other known β-galactosidases were less powerful and converted only about 90% of lactose in the same time. Finally, the formation of galactooligosaccharides (GOS) was demonstrated with the new BgaPw, starting with pharma-lactose (400 g/L). A GOS production of about 144 g/L was achieved after 24 h (36.0% yield)., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

A Gram-stain-positive, aerobic bacterium, designated as YPD9-1 T , was isolated from the gut contents of a spotty belly greenling, Hexagrammos agrammus , collected near Dokdo island, South Korea. The rod-shaped cells were oxidase-positive, and catalase-negative. The major cellular fatty acids were anteiso-C 15 : 0 , iso-C 15 : 0 , C 16 : 0 , iso-C 16 : 0 and iso-C 17: 0 . The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and two unidentified lipids. The DNA G+C content was 47.6 mol% and the predominant respiratory quinone was menaquinone MK-7. The 16S rRNA gene sequence of YPD9-1 T showed low sequence similarities to species of the genus Paenibacillus , Paenibacillus pocheonensis Gsoil 1138 T (97.21 % of sequence similarity), Paenibacillus aestuarii CJ25 T (97.12 %) and Paenibacillus allorhizoplanae JJ-42 T (96.89 %). The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that YPD9-1 T formed a distinct branch among other species of the genus Paenibacillus . The digital DNA-DNA hybridisation, average nucleotide identity, and average amino acid identity values between YPD9-1 T and the related species were in the ranges of 15.3-16.2 %, 74.1-78.4 %, and 71.1-71.9 %, respectively, which are below the species cutoff values. On the basis of the results of the polyphasic analysis, we conclude that strain YPD9-1 T represents a novel species of the genus Paenibacillus , for which the name Paenibacillus hexagrammi sp. nov. is proposed. The type strain of Paenibacillus hexagrammi is YPD9-1 T (=KCTC 43424 T =LMG 32988 T ).

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The K m and V max of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2 ) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

As a primary nutrient in agricultural soils, phosphorus plays a crucial but growth-limiting role for plants due to its complex interactions with various soil elements. This often results in excessive phosphorus fertilizer application, posing concerns for the environment. Agri-research has therefore shifted focus to increase fertilizer-use efficiency and minimize environmental impact by leveraging plant growth-promoting rhizobacteria. This study aimed to evaluate the in-field incremental effect of inorganic phosphate concentration (up to 50 kg/ha/P) on the ability of two rhizobacterial isolates, Lysinibacillus sphaericus (T19), Paenibacillus alvei (T29), from the previous Breedt et al. (Ann Appl Biol 171:229-236, 2017) study on maize in enhancing the yield of commercially grown Duzi® cultivar wheat. Results obtained from three seasons of field trials revealed a significant relationship between soil phosphate concentration and the isolates' effectiveness in improving wheat yield. Rhizospheric samples collected at flowering during the third season, specifically to assess phosphatase enzyme activity at the different soil phosphate levels, demonstrated a significant decrease in soil phosphatase activity when the phosphorus rate reached 75% for both isolates. Furthermore, in vitro assessments of inorganic phosphate solubilization by both isolates at five increments of tricalcium phosphate-amended Pikovskaya media found that only isolate T19 was capable of solubilizing tricalcium at concentrations exceeding 3 mg/ml. The current study demonstrates the substantial influence of inorganic phosphate on the performance of individual rhizobacterial isolates, highlighting that this is an essential consideration when optimizing these isolates to increase wheat yield in commercial cultivation., (© 2024. The Author(s).)

Dendrobium is a rich source of high-value natural components. Endophytic fungi are well studied, yet bacteria research is limited. In this study, endophytic bacteria from Dendrobium nobile were isolated using an improved method, showing inhibition of pathogens and growth promotion. JC-3jx, identified as Paenibacillus peoriae , exhibited significant inhibitory activity against tested fungi and bacteria, including Escherichia coli . JC-3jx also promoted corn seed rooting and Dendrobium growth, highlighting its excellent biocontrol and growth-promoting potential.

β-1,3-Galactanases selectively degrade β-1,3-galactan, thus it is an attractive enzyme technique to map high-galactan structure and prepare galactooligosaccharides. In this work, a gene encoding exo-β-1,3-galactanase (PxGal43) was screened form Paenibacillus xylanexedens, consisting of a GH43 domain, a CBM32 domain and α-L-arabinofuranosidase B (AbfB) domain. Using β-1,3-galactan (AG-II-P) as substrate, the recombined enzyme expressed in Escherichia coli BL21 (DE3) exhibited an optimal activity at pH 7.0 and 30 °C. The enzyme was thermostable, retaining >70 % activity after incubating at 50 °C for 2 h. In addition, it showed high tolerance to various metal ions, denaturants and detergents. Substrate specificity indicated that PxGal43 hydrolysis only β-1,3-linked galactosyl oligosaccharides and polysaccharides, releasing galactose as an exo-acting manner. The function of the CBM32 and AbfB domain was revealed by their sequential deletion and suggested that their connection to the catalytic domain was crucial for the oligomerization, catalytic activity, substrate binding and thermal stability of PxGal43. The substrate docking and site-directed mutagenesis proposed that Glu191, Gln244, Asp138 and Glu81 served as the catalytic acid, catalytic base, pKa modulator, and substrate identifier in PxGal43, respectively. These results provide a better understanding and optimization of multi-domain bacterial GH43 β-1,3-galactanase for the degradation of arabinogalactan., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. This article does not contain any studies involving human or animal subjects., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bacterial α-1,3-glucanase, classified as glycoside hydrolase (GH) family 87, has been divided into 3 subgroups based on differences in gene sequences in the catalytic domain. The enzymatic properties of subgroups 1 and 3 of several bacteria have been previously investigated and reported; however, the chemical characterization of subgroup 2 enzymes has not been previously conducted. The α-1,3-glucanase gene from Paenibacillus alginolyticus NBRC15375 (PaAgl) belonging to subgroup 2 of GH family 87 was cloned and expressed in Escherichia coli. PgAgl-N1 (subgroup 3) and PgAgl-N2 (subgroup 1) from P. glycanilyticus NBRC16188 were expressed in E. coli, and their enzymatic characteristics were compared. The amino acid sequence of PaAgl demonstrated that the homology was significantly lower in other subgroups when only the catalytic domain was compared. The oligosaccharide products of the mutan-degrading reaction seemed to have different characteristics among subgroups 1, 2, and 3 in GH family 87., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Two Gram-positive, rod-shaped, endospore-forming strains, YIM B05601 and YIM B05602 T , were isolated from soil sampled at Hamazui hot spring, Tengchong City, Yunnan Province, PR China. Phylogenetic analysis based on 16S rRNA gene sequences suggested that the two strains fell within the genus Paenibacillus , appearing most closely related to Paenibacillus alkalitolerans YIM B00362 T (96.9 % sequence similarity). Genome-based phylogenetic analysis confirmed that strains YIM B05601 and YIM B05602 T formed a distinct phylogenetic cluster within the genus Paenibacillus . The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of strains YIM B05601 and YIM B05602 T with the related species P. alkalitolerans YIM B00362 T were within the ranges of 74.43-74.57 % and 12.1-18.5 %, respectively, which clearly indicated that strains YIM B05601, YIM B05602 T represented a novel species. Strains YIM B05601 and YIM B05602 T exhibited 99.6 % 16S rRNA gene sequence similarity. The ANI and dDDH values between the two strains were 99.8 and 100 %, respectively, suggesting that they belong to the same species. Optimum growth for both strains occurred at pH 7.0 and 45 °C. The diagnostic diamino acid in the cell-wall peptidoglycan of strains YIM B05601 and YIM B05602 T was meso -diaminopimelic acid. MK-7 was the predominant menaquinone. The polar lipids of strain YIM B05602 T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, four unidentified glycolipids, an unidentified polarlipid and phosphatidylinositol mannoside. The major fatty acids of the two stains were iso-C 15 : 0 and anteiso-C 15 : 0 . Based on phylogenomic and phylogenetic analyses coupled with phenotypic and chemotaxonomic characterizations, strains YIM B05601 and YIM B05602 T could be classified as a novel species of the genus Paenibacillus , for which the name Paenibacillus thermotolerans sp. nov. is proposed. The type strain is YIM B05602 T (=CGMCC 1.60051 T =KCTC 43460 T =NBRC 115924 T ).

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius., (© 2024. The Author(s).)

Aims: In this work, we aimed to isolate marine bacteria that produce metabolites with antifungal properties., Methods and Results: Paenibacillus polymyxa 188 was isolated from a marine sediment sample, and it showed excellent antifungal activity against many fungi pathogenic to plants (Fusarium tricinctum, Pestalotiopsis clavispora, Fusarium oxysporum, F. oxysporum f. sp. Cubense (Foc), Curvularia plantarum, and Talaromyces pinophilus) and to humans (Aspergillus terreus, Penicillium oxalicum, and Microsphaeropsis arundinis). The antifungal compounds produced by P. polymyxa 188 were extracted and analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The complete genome sequence and biosynthetic gene clusters of P. polymyxa 188 were characterized and compared with those of other strains. A total of 238 carbohydrate-active enzymes (CAZymes) were identified in P. polymyxa 188. Two antibiotic gene clusters, fusaricidin and tridecaptin, exist in P. polymyxa 188, which is different from other strains that typically have multiple antibiotic gene clusters., Conclusions: Paenibacilluspolymyxa 188 was identified with numerous biosynthetic gene clusters, and its antifungal ability against pathogenic fungi was verified., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Paenibacillus sonchi SBR5 T is a Gram-positive, endospore-forming facultative aerobic diazotrophic bacterium that can fix nitrogen via an alternative Fe-only nitrogenase (AnfHDGK). In several bacteria, this alternative system is expressed under molybdenum (Mo)-limiting conditions when the conventional Mo-dependent nitrogenase (NifHDK) production is impaired. The regulatory mechanisms, metabolic processes, and cellular functions of N 2 fixation by alternative and/or conventional systems are poorly understood in the Paenibacillus genus. We conducted a comparative proteomic profiling study of P. sonchi SBR5T grown under N 2 -fixing conditions with and without Mo supply through an LC-MS/MS and label-free quantification analysis to address this gap. Protein abundances revealed overrepresented processes related to anaerobiosis growth adaption, Fe-S cluster biosynthesis, ammonia assimilation, electron transfer, and sporulation under N 2 -fixing conditions compared to non-fixing control. Under Mo limitation, the Fe-only nitrogenase components were overrepresented together with the Mo-transporter system, while the dinitrogenase component (NifDK) of Mo‑nitrogenase was underrepresented. The dinitrogenase reductase component (NifH) and accessory proteins encoded by the nif operon had no significant differential expression, suggesting post-transcriptional regulation of nif gene products in this strain. Overall, this was the first comprehensive proteomic analysis of a diazotrophic strain from the Paenibacillaceae family, and it provided insights related to alternative N 2 -fixation by Fe-only nitrogenase. SIGNIFICANCE: In this work, we try to understand how the alternative nitrogen fixation system, presented by some diazotrophic bacteria, works. For this, we used the SBR5 lineage of P. sonchi, which presents the alternative system in which the nitrogenase cofactor is composed only of iron. In addition, we tried to unravel the proteome of this strain in different situations of nitrogen fixation, since, for Gram-positive bacteria, these systems are little known. The results achieved, through LC-MS/MS and label-free quantitative analysis, showed an overrepresentation of proteins related to different processes involved with growth under stressful conditions in situations of nitrogen deficiency, in addition to suggesting that some encoded proteins by the nif operon may be regulated at post-transcriptional levels. Our findings represent important steps toward the elucidation of nitrogen fixation systems in Gram-positive diazotrophic bacteria., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Plant diseases caused by pathogenic fungi are devastating. However, commonly used fungicides are harmful to the environment, and some are becoming ineffective due to fungal resistance. Therefore, eco-friendly biological methods to control pathogenic fungi are urgently needed., Results: In this study, a strain, Paenibacillus sp. lzh-N1, that could inhibit the growth of the pathogenic fungus Mycosphaerella sentina (Fr) Schrorter was isolated from the rhizosphere soil of pear trees, and the complete genome sequence of the strain was obtained, annotated, and analyzed to reveal the genetic foundation of its antagonistic ability. The entire genome of this strain contained a circular chromosome of 5,641,488 bp with a GC content of 45.50%. The results of species identification show that the strain belongs to the same species as P. polymyxa Sb3-1 and P. polymyxa CJX518. Sixteen secondary metabolic biosynthetic gene clusters were predicted by antiSMASH, including those of the antifungal peptides fusaricidin B and paenilarvins. In addition, biofilm formation-related genes containing two potential gene clusters for cyclic lactone autoinducer, a gene encoding S-ribosylhomocysteine lyase (LuxS), and three genes encoding exopolysaccharide biosynthesis protein were identified., Conclusions: Antifungal peptides and glucanase biosynthesized by Paenibacillus sp. lzh-N1 may be responsible for its antagonistic effect. Moreover, quorum sensing systems may influence the biocontrol activity of this strain directly or indirectly., (© 2024. The Author(s).)

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., (© 2024 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd.)

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.

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 ., Competing Interests: The authors declare no conflict of interest.

2,3-Butanediol is an essential renewable fuel. The synthesis of 2,3-butanediol using Paenibacillus polymyxa has attracted increasing attention. In this study, the glucose-derived 2,3-butanediol pathway and its related genes were identified in P. polymyxa using combined transcriptome and metabolome analyses. The functions of two distinct genes ldh1 and ldh3 encoding lactate dehydrogenase, the gene bdh encoding butanediol dehydrogenase, and the spore-forming genes spo0A and spoIIE were studied and directly knocked out or overexpressed in the genome sequence to improve the production of 2,3-butanediol. A raw hydrolysate of poplar wood containing 27 g/L glucose and 15 g/L xylose was used to produce 2,3-butanediol with a maximum yield of 0.465 g/g and 93 % of the maximum theoretical value, and the total production of 2,3-butanediol and ethanol reached 21.7 g/L. This study provides a new scheme for engineered P. polymyxa to produce renewable fuels using raw poplar wood hydrolysates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Currently, alginate oligosaccharides (AOS) become attractive due to their excellent physiological effects. AOS has been widely used in food, pharmaceutical, and cosmetic industries. Generally, AOS can be produced from alginate using alginate lyase (ALyase) as the biocatalyst. However, most ALyase display poor thermostability. In this study, a thermostable ALyase from Paenibacillus sp. YN15 (Payn ALyase) was characterized. It belonged to the polysaccharide lyase (PL) 31 family and displayed poly β-D-mannuronate (Poly M) preference. Under the optimum condition (pH 8.0, 55 °C, 50 mM NaCl), it exhibited maximum activity of 90.3 U/mg and efficiently degraded alginate into monosaccharides and AOS with polymerization (DP) of 2-4. Payn ALyase was relatively stable at 55 °C, but the thermostability dropped rapidly at higher temperatures. To further improve its thermostability, rational design mutagenesis was carried out based on a combination of FireProt, Consensus Finder, and PROSS analysis. Finally, a triple-point mutant K71P/Y129G/S213G was constructed. The optimum temperature was increased from 55 to 70 °C, and the T m was increased from 62.7 to 64.1 °C. The residual activity after 30 min incubation at 65 °C was enhanced from 36.0 % to 83.3 %. This study provided a promising ALyase mutant for AOS industrial production., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

During the study of microbial ecology of forest soil, two circular, white-colored bacterial colonies were isolated and labeled as strains TW38 T and TW40 T . Both strains were catalase positive and oxidase negative. Strains TW38 T and TW40 T demonstrated growth within a temperature range of 10-37 °C and 18-37 °C, respectively, and thrived within a pH range of 5.5-9.0 and 6.0-8.0, respectively. Both strains grew at 0-2.0% (w/v) NaCl concentrations. The phylogenetic analysis indicated that strains TW38 T and TW40 T affiliated to the genus Paenibacillus, with the closest neighbors being Paenibacillus montanisoli RA17 T (98.6%) and Paenibacillus arachidis E3 T (95.4%), respectively. In both strains, the sole respiratory quinone was MK-7, the signature fatty acid was antiso-C 15:0 , and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. The digital DNA-DNA hybridization and the average nucleotide identity values between TW38 T , TW40 T , and closest reference strains were < 29.0% and < 85.0%, respectively. The DNA G+C content of TW38 T and TW40 T was 54.5% and 57.1%, respectively. In general, the phylogenetic, genomics, chemotaxonomic, and phenotypic data support the differentiation of TW38 T and TW40 T from other closest members of the genus Paenibacillus. Thus, we conclude both strains TW38 T and TW40 T represent novel species of the genus Paenibacillus, for which the name Paenibacillus silvisoli sp. nov. and Paenibacillus humicola sp. nov. are proposed, respectively. The type strain of Paenibacillus silvisoli is TW38 T (= KCTC 43468 T  = NBRC 116015 T ) and type strain of Paenibacillus humicola is TW40 T (= KCTC 43469 T  = NBRC 116016 T )., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Drought has become the most important limiting factor to crop productions. Research thus far has been devoted to identifying drought-responsive genes (DRGs) via breeding and engineering approaches. Still, these efforts have not resulted in a solution to combat drought's effects because the ectopic expression of most DRGs causes adverse effects that reduce plant growth and yields. Lately, we discovered that two DRGs, Response to Desiccation (RD)29A and RD29B, induced by Paenibacillus polymyxa CR1, a plant growth-promoting rhizobacterium capable of priming drought tolerance and concurrently stimulating plant growth, play pivotal roles in defense responses against drought. In this study, we employ the ChlP and qRT-PCR analyses and further clarify that P. polymyxa CR1 reformats the chromatin/transcriptional memory of RD29s, positioned as upstream controllers that fine-tune the temporal dynamic of stress-regulating transcription factors (TFs) in elaborating induced systemic drought tolerance without growth penalties. Two genes coordinate the upregulation of NAC TFs, while feedback inhibiting CBF TFs, which regulate downstream DRG expressions. This supports that RD29s are unique, feasible transgene candidates for improving plants' survival capacity in both optimal and drought conditions. However, the mode of action of RD29A and RD29B are partly independent, exerting distinct roles in disparate ecological states. When subjected to increasing NaCl concentrations, the KO mutant of RD29A (rd29a) displayed enhanced tolerance compared to WT and rd29b plants, proposing that RD29B, but not RD29A, a key player in conferring WT-like tolerance to salinity stress; further studies will be needed to optimize/maximize their applications in engineering for-profit drought and/or broad-spectrum stress tolerant crops., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Mangrove soil is a reliable source for screening cellulose-degrading bacteria due to the high diversity of microbes. To effectively utilize crop straw resources, a cellulolytic bacterium, Paenibacillus silvae strain CH2 was isolated from mangrove soil. We determined the carboxymethyl cellulose (CMC) and filter paper assay (FPA) activities of CH2 at different incubation times, NaCl concentrations, pH and temperatures, estimated the degradation efficiencies of rice and maize straw by CH2, sequenced and analyzed the whole genome of CH2. The results showed that along with the increases of incubation time, NaCl concentration, pH and temperature, the CMC and FPA activities increased first and then decreased . The highest CMC and FPA activities were observed at incubation time of 72-84 h, NaCl concentration of 6.0 g·L -1 , pH of 7 and temperature of 36 ℃. Degradation of straw assays revealed that CH2 could effectively degrade rice and maize straw. At 0 g·L -1 NaCl (the control), the 10-day degradation rates of rice and maize straw were 30.4% and 47.0%, respectively. In the presence of 15 g·L -1 NaCl, the degradation rates were not significantly different from the control, indicating that CH2 had a high tolerance to salts. The whole genome of P. silvae CH2 was 6797325 bp, containing 6312 coding genes. P. silvae CH2 contained multiple genes encoding cellulose and hemicellulose degrading enzymes. These enzymes mainly belonged to the GH family, including endo-1,4-β-xylanase, Xylan 1,4-β-xylosidase, β-glucosidase, and endoglucanase. The results indicated that the bacterium had the potential to be used in crop straw degradation.

D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The K m and k cat / K m values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s·mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35 ℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.

Glycosidases are essential for the industrial production of functional oligosaccharides and many biotech applications. A novel β-galactosidase/α-L-arabinopyranosidase (PpBGal42A) of the glycoside hydrolase family 42 (GH42) from Paenibacillus polymyxa KF-1 was identified and functionally characterized. Using p NPG as a substrate, the recombinant PpBGal42A (77.16 kD) was shown to have an optimal temperature and pH of 30 °C and 6.0. Using p NPαArap as a substrate, the optimal temperature and pH were 40 °C and 7.0. PpBGal42A has good temperature and pH stability. Furthermore, Na + , K + , Li + , and Ca 2+ (5 mmol/L) enhanced the enzymatic activity, whereas Mn 2+ , Cu 2+ , Zn 2+ , and Hg 2+ significantly reduced the enzymatic activity. PpBGal42A hydrolyzed p NP-β-D-galactoside and p NP-α-L-arabinopyranoside. PpBGal42A liberated galactose from β-1,3/4/6-galactobiose and galactan. PpBGal42A hydrolyzed arabinopyranose at C20 of ginsenoside Rb2, but could not cleave arabinofuranose at C20 of ginsenoside Rc. Meanwhile, the molecular docking results revealed that PpBGal42A efficiently recognized and catalyzed lactose. PpBGal42A hydrolyzes lactose to galactose and glucose. PpBGal42A exhibits significant degradative activity towards citrus pectin when combined with pectinase. Our findings suggest that PpBGal42A is a novel bifunctional enzyme that is active as a β-galactosidase and α-L-arabinopyranosidase. This study expands on the diversity of bifunctional enzymes and provides a potentially effective tool for the food industry.

With the advent of long-term human habitation in space and on the moon, understanding how the built environment microbiome of space habitats differs from Earth habitats, and how microbes survive, proliferate and spread in space conditions, is becoming more important. The microbial tracking mission series has been monitoring the microbiome of the International Space Station (ISS) for almost a decade. During this mission series, six unique strains of Gram-stain-positive bacteria, including two spore-forming and three non-spore-forming species, were isolated from the environmental surfaces of the ISS. The analysis of their 16S rRNA gene sequences revealed > 99% similarities with previously described bacterial species. To further explore their phylogenetic affiliation, whole genome sequencing was undertaken. For all strains, the gyrB gene exhibited < 93% similarity with closely related species, which proved effective in categorizing these ISS strains as novel species. Average nucleotide identity and digital DNA-DNA hybridization values, when compared to any known bacterial species, were < 94% and <50% respectively for all species described here. Traditional biochemical tests, fatty acid profiling, polar lipid, and cell wall composition analyses were performed to generate phenotypic characterization of these ISS strains. A study of the shotgun metagenomic reads from the ISS samples, from which the novel species were isolated, showed that only 0.1% of the total reads mapped to the novel species, supporting the idea that these novel species are rare in the ISS environments. In-depth annotation of the genomes unveiled a variety of genes linked to amino acid and derivative synthesis, carbohydrate metabolism, cofactors, vitamins, prosthetic groups, pigments, and protein metabolism. Further analysis of these ISS-isolated organisms revealed that, on average, they contain 46 genes associated with virulence, disease, and defense. The main predicted functions of these genes are: conferring resistance to antibiotics and toxic compounds, and enabling invasion and intracellular resistance. After conducting antiSMASH analysis, it was found that there are roughly 16 cluster types across the six strains, including β-lactone and type III polyketide synthase (T3PKS) clusters. Based on these multi-faceted taxonomic methods, it was concluded that these six ISS strains represent five novel species, which we propose to name as follows: Arthrobacter burdickii IIF3SC-B10 T (= NRRL B-65660 T = DSM 115933 T ), Leifsonia virtsii F6&#95;8S&#95;P&#95;1A T (= NRRL B-65661 T = DSM 115931 T ), Leifsonia williamsii F6&#95;8S&#95;P&#95;1B T (= NRRL B-65662 T = DSM 115932 T ), Paenibacillus vandeheii F6&#95;3S&#95;P&#95;1C T (= NRRL B-65663 T = DSM 115940 T ), and Sporosarcina highlanderae F6&#95;3S&#95;P&#95;2 T (= NRRL B-65664 T = DSM 115943 T ). Identifying and characterizing the genomes and phenotypes of novel microbes found in space habitats, like those explored in this study, is integral for expanding our genomic databases of space-relevant microbes. This approach offers the only reliable method to determine species composition, track microbial dispersion, and anticipate potential threats to human health from monitoring microbes on the surfaces and equipment within space habitats. By unraveling these microbial mysteries, we take a crucial step towards ensuring the safety and success of future space missions., (© 2023. The Author(s).)

A novel bacterial strain, designated as PHS-Z3 T , was isolated from a marine sponge belonging to the genus Theonella on the Puerto Galera Deep Monkey, Philippines. Cells of PHS-Z3 T were Gram-stain-positive, motile, oxidase- and catalase-positive, white-pigmented, spore-forming, short rods that could grow at 10-40 °C (optimum, 20 °C), pH 6.0-9.5 (optimum, pH 7.5) and with 2-16 % (w/v) NaCl (optimum, 7 %). The 16S rRNA gene sequence of PHS-Z3 T showed 97.9 %, 96.7 %, and 96.2 % identities to Paenibacillus mendelii C/2 T , Paenibacillus oenotherae DT7-4 T and Paenibacillus aurantiacus RC11 T , respectively. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that PHS-Z3 T formed an independent cluster with Paenibacillus mendelii C/2 T . The total genome of PHS-Z3 T was approximately 7 613 364 bp in size with a DNA G+C content of 51.6 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between PHS-Z3 T and other type strains of species of the genus Paenibacillus were 68.0-81.4 % [ANI by blast (ANIb)], 83.0-88.0 % [ANI by MUMmer (ANIm)] and 12.7-32.1 % (dDDH). The dDDH and ANI values were below the standard cut-off criteria for delineation of bacterial species. The percentage of conserved proteins (POCP) values between the genome of PHS-Z3 T and those of members of the genus Paenibacillus were 39.7-75.7 %, while the average amino acid identity (AAI) values were 55.9-83.7 %. The sole respiratory quinone in the strain was MK-7, and the predominant fatty acids were anteiso-C 15 : 0 and C 16 : 0 . The major polar lipids of PHS-Z3 T consisted of diphosphatidylglycerol, phospholipid and phosphatidylglycerol. The characteristic amino acid in the cell wall of PHS-Z3 T was diamino heptanoic acid ( meso -DAP). On the basis of the molecular, physiological, biochemical and chemotaxonomic features, strain PHS-Z3 T represents a novel species of the genus Paenibacillus , for which the name Paenibacillus spongiae sp. nov. is proposed, with the type strain PHS-Z3 T (=MCCC 1K07848 T =KCTC 43443 T ).

A Gram-stain-positive, aerobic, rod-shaped, non-motile, yellowish and glossy strain, C31 T , was isolated from a wetland plant Polygonum lapathifolium L. located south of Poyang Lake, Jiangxi Province, PR China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain C31 T showed similarity values of lower than 97.0 % to other type species belonging to the genus Paenibacillus . The genomic average nucleotide identity values between strain C31 T and its reference type species ranged from 68.9-70.9 % and the digital DNA-DNA hybridization values were lower than 27.8 %. The genomic DNA G+C content of strain C31 T was 41.9 mol%. The optimal growth temperature, pH and NaCl concentration were 37 °C, pH 7 and 0.5 %, respectively. The major cellular fatty acids (>5.0 %) of strain C31 T were anteiso-C 15 : 0 (73.7 %), anteiso-C 17 : 0 (8.4 %) and iso-C 15 : 0 (5.2 %). The polar lipids of strain C31 T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and unidentified phospholipids. The respiratory quinone was MK-7. Based on these phylogenetic and phenotypic characterizations, strain C31 T represents a novel species within the genus Paenibacillus . Therefore, the proposed name for this newly identified species is Paenibacillus polygoni sp. nov. and the type strain is C31 T (=CCTCC AB 2022349 T =KCTC 43565 T ).

Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic β-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor). CsaB protein structure modelling was done using Phyre2 and I-Tasser based on the partial crystal structure of the Schizosaccharomyces pombe pyruvyltransferase Pvg1p and by AlphaFold. The models informed the construction of twelve CsaB mutants targeted at plausible PEP and acceptor binding sites and K M and k cat values were determined to evaluate the mutants, indicating the importance of a loop region for catalysis. R148, H308 and K328 were found to be critical to PEP binding and insight into acceptor binding was obtained from an analysis of Y14 and F16 mutants, confirming the modelled binding sites and interactions predicted using Molecular Operating Environment. These data lay the basis for future mechanistic studies of saccharide pyruvylation as a novel target for interference with bacterial cell wall assembly., (© 2023. Springer Nature Limited.)

Background: Paenibacillus thiaminolyticus is a cause of postinfectious hydrocephalus among Ugandan infants. To determine whether Paenibacillus spp is a pathogen in neonatal sepsis, meningitis, and postinfectious hydrocephalus, we aimed to complete three separate studies of Ugandan infants. The first study was on peripartum prevalence of Paenibacillus in mother-newborn pairs. The second study assessed Paenibacillus in blood and cerebrospinal fluid (CSF) from neonates with sepsis. The third study assessed Paenibacillus in CSF from infants with hydrocephalus., Methods: In this observational study, we recruited mother-newborn pairs with and without maternal fever (mother-newborn cohort), neonates (aged ≤28 days) with sepsis (sepsis cohort), and infants (aged ≤90 days) with hydrocephalus with and without a history of neonatal sepsis and meningitis (hydrocephalus cohort) from three hospitals in Uganda between Jan 13, 2016 and Oct 2, 2019. We collected maternal blood, vaginal swabs, and placental samples and the cord from the mother-newborn pairs, and blood and CSF from neonates and infants. Bacterial content of infant CSF was characterised by 16S rDNA sequencing. We analysed all samples using quantitative PCR (qPCR) targeting either the Paenibacillus genus or Paenibacillus thiaminolyticus spp. We collected cranial ultrasound and computed tomography images in the subset of participants represented in more than one cohort., Findings: No Paenibacillus spp were detected in vaginal, maternal blood, placental, or cord blood specimens from the mother-newborn cohort by qPCR. Paenibacillus spp was detected in 6% (37 of 631 neonates) in the sepsis cohort and, of these, 14% (5 of 37 neonates) developed postinfectious hydrocephalus. Paenibacillus was the most enriched bacterial genera in postinfectious hydrocephalus CSF (91 [44%] of 209 patients) from the hydrocephalus cohort, with 16S showing 94% accuracy when validated by qPCR. Imaging showed progression from Paenibacillus spp-related meningitis to postinfectious hydrocephalus over 1-3 months. Patients with postinfectious hydrocephalus with Paenibacillus spp infections were geographically clustered., Interpretation: Paenibacillus spp causes neonatal sepsis and meningitis in Uganda and is the dominant cause of subsequent postinfectious hydrocephalus. There was no evidence of transplacental transmission, and geographical evidence was consistent with an environmental source of neonatal infection. Further work is needed to identify routes of infection and optimise treatment of neonatal Paenibacillus spp infection to lessen the burden of morbidity and mortality., Funding: National Institutes of Health and Boston Children's Hospital Office of Faculty Development., Competing Interests: Declaration of interests KB is Principal Investigator on a Joint Global Health Trials grant from the UK Medical Research Council and has received travel grants and honoraria for speaking at Hot Topics in Neonatology. JEE has received honoraria for participation on the Data Safety Monitoring Board of AbbVie. BCW has received honoraria for participation on the Data Safety Monitoring Board for the Endoscopic versus Shunt Treatment of Hydrocephalus in Infants trial of the Hydrocephalus Clinical Research Network and is the Chairman of Neurokids. JNP has been employed by Genentech and N-Power Medicine; has held stocks and holds and is planning patents at Genentech and N-Power Medicine; has received honoraria for speaking at the International Human Microbiome Consortia meeting; and has received travel support from Genentech. All other authors declare no competing interests. The National Institutes of Health (NIH) Director's Pioneer Award 5DP1HD086071 and NIH Director's Transformative Award 1R01AI145057 has been given to SJS, and the Boston Children's Hospital Office of Faculty Development Career Development Award to SUM. No authors are employed by the NIH., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Members of Paenibacillus genus from diverse habitats have attracted great attention due to their multifarious properties. Considering that members of this genus are mostly free-living in soil, we characterized the genome of a halotolerant environmental isolate belonging to the genus Paenibacillus. The genome mining unravelled the presence of CAZymes, probiotic, and stress-protected genes that suggested strain S-12 for industrial and agricultural purposes., Results: Molecular identification by 16 S rRNA gene sequencing showed its closest match to other Paenibacillus species. The complete genome size of S-12 was 5.69 Mb, with a GC-content 46.5%. The genome analysis of S-12 unravelled the presence of an open reading frame (ORF) encoding the functions related to environmental stress tolerance, adhesion processes, multidrug efflux systems, and heavy metal resistance. Genome annotation identified the various genes for chemotaxis, flagellar motility, and biofilm production, illustrating its strong colonization ability., Conclusion: The current findings provides the in-depth investigation of a probiotic Paenibacillus bacterium that possessed various genome features that enable the bacterium to survive under diverse conditions. The strain shows the strong ability for probiotic application purposes., (© 2023. The Author(s).)

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 C 4 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., (© 2023. The Author(s).)

Xylan is the most abundant hemicellulose in hardwood and graminaceous plants. It is a heteropolysaccharide comprising different moieties appended to the xylose units. Complete degradation of xylan requires an arsenal of xylanolytic enzymes that can remove the substitutions and mediate internal hydrolysis of the xylan backbone. Here, we describe the xylan degradation potential and underlying enzyme machinery of the strain, Paenibacillus sp. LS1. The strain LS1 was able to utilize both beechwood and corncob xylan as the sole source of carbon, with the former being the preferred substrate. Genome analysis revealed an extensive xylan-active CAZyme repertoire capable of mediating efficient degradation of the complex polymer. In addition to this, a putative xylooligosaccharide ABC transporter and homologues of the enzymes involved in the xylose isomerase pathway were identified. Further, we have validated the expression of selected xylan-active CAZymes, transporters, and metabolic enzymes during growth of the LS1 on xylan substrates using qRT-PCR. The genome comparison and genomic index (average nucleotide identity [ANI] and digital DNA-DNA hybridization) values revealed that strain LS1 is a novel species of the genus Paenibacillus . Lastly, comparative genome analysis of 238 genomes revealed the prevalence of xylan-active CAZymes over cellulose across the Paenibacillus genus. Taken together, our results indicate that Paenibacillus sp. LS1 is an efficient degrader of xylan polymers, with potential implications in the production of biofuels and other beneficial by-products from lignocellulosic biomass. IMPORTANCE Xylan is the most abundant hemicellulose in the lignocellulosic (plant) biomass that requires cooperative deconstruction by an arsenal of different xylanolytic enzymes to produce xylose and xylooligosaccharides. Microbial (particularly, bacterial) candidates that encode such enzymes are an asset to the biorefineries to mediate efficient and eco-friendly deconstruction of xylan to generate products of value. Although xylan degradation by a few Paenibacillus spp. is reported, a complete genus-wide understanding of the said trait is unavailable till date. Through comparative genome analysis, we showed the prevalence of xylan-active CAZymes across Paenibacillus spp., therefore making them an attractive option towards efficient xylan degradation. Additionally, we deciphered the xylan degradation potential of the strain Paenibacillus sp. LS1 through genome analysis, expression profiling, and biochemical studies. The ability of Paenibacillus sp. LS1 to degrade different xylan types obtained from different plant species, emphasizes its potential implication in lignocellulosic biorefineries., Competing Interests: The authors declare no conflict of interest.

Background: Icaritin is an aglycone of flavonoid glycosides from Herba Epimedii. It has good performance in the treatment of hepatocellular carcinoma in clinical trials. However, the natural icaritin content of Herba Epimedii is very low. At present, the icaritin is mainly prepared from flavonoid glycosides by α-L-rhamnosidases and β-glucosidases in two-step catalysis process. However, one-pot icaritin production required reported enzymes to be immobilized or bifunctional enzymes to hydrolyze substrate with long reaction time, which caused complicated operations and high costs. To improve the production efficiency and reduce costs, we explored α-L-rhamnosidase SPRHA2 and β-glucosidase PBGL to directly hydrolyze icariin to icaritin in one-pot, and developed the whole-cell catalytic method for efficient icaritin production., Results: The SPRHA2 and PBGL were expressed in Escherichia coli, respectively. One-pot production of icaritin was achieved by co-catalysis of SPRHA2 and PBGL. Moreover, whole-cell catalysis was developed for icariin hydrolysis. The mixture of SPRHA2 cells and PBGL cells transformed 200 g/L icariin into 103.69 g/L icaritin (yield 95.23%) in 4 h in whole-cell catalysis under the optimized reaction conditions. In order to further increase the production efficiency and simplify operations, we also constructed recombinant E. coli strains that co-expressed SPRHA2 and PBGL. Crude icariin extracts were also efficiently hydrolyzed by the whole-cell catalytic system., Conclusions: Compared to previous reports on icaritin production, in this study, whole-cell catalysis showed higher production efficiency of icaritin. This study provides promising approach for industrial production of icaritin in the future., (© 2023. The Author(s).)

PsEst3, a psychrophilic esterase obtained from Paenibacillus sp. R4, which was isolated from the permafrost of Alaska, exhibits relatively high activity at low temperatures. Here, crystal structures of PsEst3 complexed with various ligands were generated and studied at atomic resolution, and biochemical studies were performed to analyze the structure-function relationship of PsEst3. Certain unique characteristics of PsEst3 distinct from those of other classes of lipases/esterases were identified. Firstly, PsEst3 contains a conserved GHSRA/G pentapeptide sequence in the GxSxG motif around the nucleophilic serine. Additionally, it contains a conserved HGFR/K consensus sequence in the oxyanion hole, which is distinct from that in other lipase/esterase families, as well as a specific domain composition (for example a helix-turn-helix motif) and a degenerative lid domain that exposes the active site to the solvent. Secondly, the electrostatic potential of the active site in PsEst3 is positive, which may cause unintended binding of negatively charged chemicals in the active site. Thirdly, the last residue of the oxyanion hole-forming sequence, Arg44, separates the active site from the solvent by sealing the acyl-binding pocket, suggesting that PsEst3 is an enzyme that is customized to sense an unidentified substrate that is distinct from those of classical lipases/esterases. Collectively, this evidence strongly suggests that PsEst3 belongs to a distinct family of esterases., (open access.)

Paenibacillus mucilaginosus has widely been reported as a plant growth-promoting rhizobacteria (PGPR). However, the important genomic insights into plant growth promotion in this species remain undescribed. In this study, the genome of P. mucilaginosus G78 was sequenced using Illumina NovaSeq PE150. It contains 8,576,872 bp with a GC content of 58.5%, and was taxonomically characterized. Additionally, a total of 7337 genes with 143 tRNAs, 41 rRNAs, and 5 ncRNAs were identified. This strain can prohibit the growth of the plant pathogen, but also has the capability to form biofilm, solubilize phosphate, and produce IAA. Twenty-six gene clusters encoding secondary metabolites were identified, and the genotypic characterization indirectly proved its resistant ability to ampicillin, bacitracin, polymyxin and chloramphenicol. The putative exopolysaccharide biosynthesis and biofilm formation gene clusters were explored. According to the genetic features, the potential monosaccharides of its exopolysaccharides for P. mucilaginosus G78 may include glucose, mannose, galactose, fucose, that can probably be acetylated and pyruvated. Conservation of the pelADEFG compared with other 40 Paenibacillus species suggests that Pel may be specific biofilm matrix component in P. mucilaginosus . Several genes relevant to plant growth-promoting traits, i.e., IAA production and phosphate solubilization are well conserved compared with other 40 other Paenibacillus strains. The current study can benefit for understanding the plant growth-promoting traits of P. mucilaginosus as well as its potential application in agriculture as PGPR.

In this study, site saturation mutagenesis was performed on the - 3 (R44, D86, S90, and D192) and - 6 subsite (Y163, G175, G176, and N189) of Bacillus stearothermophilus NO2 cyclodextrin glucosyltransferase to enhance its specificity for the donor substrate maltodextrin for 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) preparation. The AA-2G yields produced by the mutants S90D, G176H, and S90D/G176H were 181, 171, and 185 g/L, respectively. Our previous study found that the mutant K228R/M230L also increased the AA-2G yield. Therefore, the mutants S90D, G176H, S90D/G176H, and K228R/M230L were further used to generate combinatorial mutants. Among these mutants, the highest AA-2G yield (217 g/L) was produced by S90D/K228R/M230L with 500 g/L maltodextrin as the glucosyl donor, which was 56 g/L higher than that produced by wild-type CGTase. In addition, AA-2G was prepared by adding isoamylase to hydrolyze α-1,6 glucosidic linkages in maltodextrin that could not be utilized by CGTase to improve the utilization rate of maltodextrin. The addition of isoamylase reduced the concentration of maltodextrin from 500 to 350 g/L, while the AA-2G yield remained high (208 g/L). The preparation of AA-2G by complexing isoamylase with mutant S90D/K228R/M230L reduced the maltodextrin concentration by 150 g/L, while the AA-2G yield increased by 47 g/L than preparation with wild-type CGTase alone, which laid a foundation for the large-scale preparation of AA-2G. KEY POINTS: • Mutants exhibited improved maltodextrin specificity. • Mutant S90D/K228R/M230L produced high yield of AA-2G with maltodextrin as substrate. • AA-2G was first synthesized by a combination of isoamylase and CGTase., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)