Pseudomonas putida PCL1760 is a well-studied bacterium that can be used for a wide range of biotechnological applications. In our study we investigated the effect of deletion of the genes involved in alginate accumulation (algA), flagellar export (flhA), and pili formation pilQ on the behavior of the strain in bioreactors. We obtained the knockout mutant P. putida LN6160 with the deletion of these genes and showed that the absence of these genes reduces mobility and biofilm formation (40% lower after 72 h) in the mutant. At the same time, we noted the positive effect of these deletions on the growth of the mutant strain on rich medium (1.39 × 10¹⁰ CFU/mL in the mutant and 6.4 × 10⁹ CFU/mL in the wild type) and on mineral medium (6.11 × 10⁹ CFU/mL in the mutant and 1.36 × 10⁹ CFU/mL in the wild type) by growing them in small-volume bioreactors. A significant decrease in the biofilm and the foam formation was also observed for LN6160 in a small-volume bioreactor. Most probably, the rapid growth of the deletion strain occurs due to a decrease in the energy load on the bacterial apparatus.

We present the cloning and heterologous expression of the α-amylase gene amyBL159 from a thermophilic strain Bacillus licheniformis MGMM159, which was isolated from wastewater sediments self-heated to 70 °C. The gene was successfully cloned into the pET22b and pHT01 vectors, expressed and AmyBL159_Ec and AmyBL159_Bs recombinant α-amylases were purified from Escherichia coli BL21(DE3)pLys and Bacillus subtilis 168 strains, respectively. The AmyBL159_Ec enzyme was most active in the range of 75–95 °C, while AmyBL159_Bs showed maximum activity at temperatures from 45 to 75 °C. AmyBL159_Bs was shown to be more thermostable. Both enzymes were active over a broad pH range of 4.0–12.0. It was shown that Mn²⁺ ions enhanced the activity of both enzymes (up to 163% for AmyBL159_Ec and 142% for AmyBL159_Bs). These results highlight the importance of choosing an expression system for modulating the functional characteristics of recombinant α-amylase. The obtained AmyBL159_Ec and AmyBL159_Bs enzymes are promising for biotechnological applications under extreme conditions. The structure of the α-amylase was generated using the AlphaFold 3 web service. A structure–function analysis of this enzyme and previously studied α-amylases from B. licheniformis identified significant amino acid substitutions at positions 134(133) and 210(209) of the amino acid chain which may contribute to enhanced enzyme thermostability.

Lignin is a complex aromatic polymer that constitutes a major fraction of plant biomass and represents a valuable renewable carbon resource. Naturally decaying wood serves as an environmental reservoir of microorganisms capable of degrading lignin. In this study, we isolated and characterized sixteen bacterial strains from decaying Tilia cordata wood using an enrichment culture technique with lignin as the sole carbon source. Taxonomic identification via 16S rRNA gene sequencing revealed microbial diversity spanning the genera Bacillus, Pseudomonas, Stenotrophomonas, and several members of the Enterobacteriaceae family, including Raoultella terrigena isolates. Metagenomic sequencing of the wood substrate revealed an exceptionally rich and balanced bacterial community (Shannon index H′ = 5.07), dominated by Streptomyces, Bradyrhizobium, Bacillus, and Pseudomonas, likely reflecting a specialized consortium adapted to lignin rich late-stage decay. Functional phenotyping demonstrated that all isolates possess ligninolytic potential, evidenced by peroxidase/laccase-type activity through methylene blue decolorization. Dynamic Light Scattering (DLS) and HPLC analyses showed that some isolates, such as Raoultella terrigena MGMM806, effectively depolymerized lignosulfonate into low molecular weight fragments (1.23 nm), while others accumulated intermediate metabolites or completely mineralized the substrate. Growth profiling on monolignol substrates revealed a broad spectrum of catabolic specialization in lignin monomer degradation. The results demonstrate a complex system of metabolic partitioning within a natural bacterial consortium. This collection represents a foundational genetic resource for developing engineered biocatalysts and synthetic microbial communities aimed at the efficient conversion of lignin into valuable aromatic compounds.

The phylum Armatimonadota represents one of the least characterized bacterial lineages, with only three formally described species despite its widespread distribution in various environments. Deep subsurface thermal environments harbor significant microbial diversity and represent promising sources for novel enzyme discovery through metagenomic approaches. This study reports the identification, cloning, and biochemical characterization of Cel7465, a novel endoglucanase from an uncultured GBS-DC family within the order Fimbriimonadales. The enzyme was identified through metagenomic analysis of microbial mats from the Biragzang deep thermal well (North Ossetia, Russia, 48 °C) and successfully expressed in cells of Escherichia coli strain ArcticExpress (DE3). Phylogenetic analysis assigned Cel7465 to glycoside hydrolase family 5 subfamily 36. The purified recombinant enzyme exhibited optimal activity at 55 °C and pH 8.0, with high specific activity of 4347 U/mg. The enzyme demonstrated broad pH tolerance (50% activity retained from pH 4.0 to 10.0) and notable thermal stability, retaining 60% activity after one hour at 80 °C and 20% after four hours. Remarkably, the presence of Mn²⁺ ions enhanced enzyme activity more than 7-fold, while Mg²⁺ and Ca²⁺ ions provided moderate activation. Cel7465 represents the first biochemically characterized glycoside hydrolase from the order Fimbriimonadales, expanding our understanding of enzymatic capabilities within the understudied phylum Armatimonadota and demonstrating the continued potential of extreme environments as sources of novel industrial biocatalysts.

Objective: Bacterial biofilms, as a lifestyle strategy for surviving hostile environments, are influenced by their architecture, which is mainly composed of exopolysaccharides. Alginate ensures the structure and biofilm stability, and its biosynthesis is governed by multiple genes, including algA, which encodes enzymes for the alginate precursor. In this study, we evaluated the impact of algA gene deletion on functional biocontrol properties of Pseudomonas putida, including extracellular polymeric substance (EPS), alginate exopolysaccharide production, plant colonization ability, and biocontrol efficacy against Fusarium oxysporum f.sp. radicis-lycopersici (Forl) ZUM2407 in tomato plants. Methods: The extracellular polymeric substance (EPS) was evaluated after precipitation from a 48-h culture grown statically in LB medium covered with cellophane. Alginate content was quantified calorimetrically at 485 nm using a dissolved EPS dissolved in milli-Q water, filtered, and reacted with 80% phenol and H2SO4. The colonization ability was assessed in competitive and non-competitive environments using tomato plants in a gnotobiotic system. The biocontrol efficiency against Forl ZUM2407 was assayed under laboratory conditions. Results: The results obtained demonstrated that algA deletion significantly reduced EPS and alginate production by up to 12.8 ± 1.4% and 5.8 ± 0.2%, respectively. The root colonization ability evaluated in competitive and non-competitive environments demonstrated substantially reduced ability of P. putida PCL1760-ΔalgA, with 69.0 ± 0.4% and 82.5 ± 0.1% decreases in basal and apical tomato part-plants, respectively. Most notably, P. putida PCL1760-ΔalgA was up to 47.01 ± 7.51% less effective in suppressing disease caused by Forl ZUM2407 compared to its wild-type strain, which was up 49.52 ± 8.2% effective. Conclusion: The algA as a critical gene for alginate-mediated biofilm architecture, contributes to root colonization and biocontrol ability in P. putida. These findings extended the critical role for algA in maintaining the plants colonization capacity of PCL1760 and consequent biocontrol performance against Fusarium wilt.

Fungal laccases oxidize a wide range of substrates with a diverse spectrum of subsequent non-specific free radical reactions, leading to the production of unwanted byproducts. This work describes a unique recombinant alkaliphilic laccase from Paramyrothecium roridum VKM F-3565 capable of performing specific oligomerization of phenylpropanoids (precursors of natural lignin and lignans) in a neutral environment, thus preventing the reverse reaction of depolymerization which occurs in an acidic environment. The recombinant alkaliphilic laccase from P. roridum VKM F-3565 with a specific enzyme activity of about 154.0 U/mg (in the reaction with 1 mM ABTS) was obtained using a Komagataella phaffii transformant with a yield of 20 ± 1.5 mg/L. The recombinant laccase had an increased degree of N-glycosylation (MW = 97 kDa), higher pH optimum in reaction with phenylpropanoids and a decreased temperature optimum, compared to the wild-type laccase. The enzyme exhibited great resistance to surfactants and the EDTA in the neutral conditions rather than the acidic ones, whereas its tolerance to mono- and divalent-metal ions was high at acidic conditions. This work demonstrates the important role of N-glycosylation of the alkaliphilic laccase of P. roridum VKM F-3565 in its functional activity. The presence of pH-dependent reactions makes the studied laccase attractive for the phenylpropanoid oligomerization with the production of novel oligomeric phenylpropanoid derivatives for industrial and pharmacological purposes.