Applied Microbiology and Biotechnology最新文献

Intestinal bacteria can convert indole to a range of compounds; however, the precise enzymatic processes facilitating this conversion have not been fully elucidated. Certain Bifidobacterium strains convert exogenous indole to indole-3-lactic acid (ILA) via tryptophan synthase beta chain and aromatic lactate dehydrogenase. Moreover, the metabolism of indole is enhanced when the strain is combined with non-digestible oligosaccharides, forming synbiotics. However, the mechanism by which synbiotics enhance indole metabolism remains unclear. Here, the conversion of indole to ILA was investigated in the context of synbiotic-mediated enhancement. The combination of Bifidobacterium bifidum YIT 10347 and galactooligosaccharides (non-digestible oligosaccharides) in human fecal suspension medium synergistically increased this conversion. Screening Tn5 transposon-mutated B. bifidum YIT 10347 clones revealed that phosphoserine phosphatase B (PSP), encoded by serB, and tryptophan synthase alpha chain (TrpA), encoded by trpA, play crucial roles in indole metabolism. Mutant strains lacking either PSP or TrpA showed a significant reduction in ILA production, confirming the roles of PSP and TrpA in this pathway. Additionally, serine supplementation restored ILA production in PSP-deficient strains, further supporting the role of serine biosynthesis in indole metabolism. These results suggest that PSP and TrpA play a vital role in the metabolism of indole to ILA. This study provides novel insights into microbial indole metabolism and suggests potential applications of synbiotics in improving health.

• PSP and TrpA are essential for indole metabolism

• Bifidobacterium bifidum YIT 10347 and GOS synergistically metabolize indole to ILA

• This pathway offers potential therapeutic benefits for gut and kidney health

Bacillus strains are widely used in food fermentation and plant disease control. An efficient transformation method is crucial for genetic manipulation in these organisms. To enhance the transformation efficiency of three Bacillus strains—Bacillus amyloliquefaciens YN-J3 (B.a YN-J3), Bacillus velezensis JN-Y2 (B.v JN-Y2) and Bacillus subtilis S-16 (B.s S-16), we optimized transformation conditions using orthogonal experiments combined with response surface analysis. Additionally, we tested various cell wall agents to improve competence. Our results showed that the optimal transformation parameters for B.a YN-J3 and B.v JN-Y2 had an OD₆₀₀ of 0.70, a competent cell volume of 91 μL, a plasmid concentration of 1040 ng·μL⁻¹, and a field strength of 18.1 kV·cm⁻¹. For B.s S-16, the optimal conditions were an OD₆₀₀ of 0.71, a competent cell volume of 92 μL, a plasmid concentration of 1052 ng·μL⁻¹, and a field strength of 18.2 kV·cm⁻¹. Under these optimal conditions, the transformation efficiencies for B.a YN-J3, B.v JN-Y2, and B.s S-16 were 22,198.33 CFU·μg⁻¹ DNA, 24,498.67 CFU·μg⁻¹ DNA, and 23,305.00 CFU·μg⁻¹ DNA, respectively. Screening of cell wall agents revealed that 50 mg/mL glycine significantly boosted transformation efficiency by 40, 36, and 24 times for B.a YN-J3, B.v JN-Y2, and B.s S-16, respectively. These findings demonstrate that combining glycine treatment with optimized transformation conditions provides an efficient approach for the genetic manipulation of Bacillus strains.

• The electroporation transformation parameters of three Bacillus were optimized by combining orthogonal experiments with response surface methodology

• A stable and efficient electroporation transformation system suitable for three types of Bacillus was established.

• 3. 50 mg/mL glycine solution can increase the transformation efficiency by 40, 36 and 24 times, respectively.

The glutamate decarboxylase (GAD) system catalyzed the conversion of L-glutamate to gamma-aminobutyric acid (GABA) in a proton-consuming reaction, which played a critical role to maintain intracellular pH homeostasis. However, the genetic organization and functional roles of GAD system in Lactiplantibacillus plantarum remain incompletely understood. In this study, L. plantarum ZR79, a GABA-producing strain, was successfully screened from 120 L. plantarum strains based on gas production and pH increase after 48-h fermentation. Comparative genomic analysis revealed that only L. plantarum ZR79 harbors two glutamate decarboxylases encoded by gadA and gadB, respectively. Insertional inactivation of the gadB gene abolished the ability to synthesize GABA, suggesting that the gadB gene plays a critical role in GABA biosynthesis in L. plantarum ZR79. The complete genome sequencing analysis combined with RT-PCR revealed that the gadB gene was located on the plasmid pZR79, which was co-transcribed with gadR and gadC. Plasmid stability assays revealed that pZR79 was stably maintained in ZR79 over 200 generations. Furthermore, acid stress survival assays confirmed that gadB-mediated GABA production contributes to the acid tolerance of L. plantarum ZR79. This study provides the first evidence of a plasmid-encoded gadRCB operon in L. plantarum, offering new insights into the strain-specific genetic basis of GABA biosynthesis and its physiological role in acid stress resistance.

• L. plantarum ZR79 harbors two distinct glutamate decarboxylase-encoding genes, gadA and gadB, with the gadB gene cluster located on its plasmid pZR79.

• Insertional inactivation of the gadB gene abolished the ability of L. plantarum ZR79 to synthesize GABA.

• The gadB gene increased the survival ability in L. plantarum ZR79 under acidic conditions.

Antimicrobial resistance (AMR) is problematic for the management of chronic wound infection, where biofilms confer increased tolerance to treatments. A wealth of research describes the antimicrobial activity of essential oils, but none have been formulated for clinical use. We screened ten commercially available essential oils from the Lamiaceae plant family (thyme, rosemary, basil, oregano, clary sage) for bacteriostatic, bactericidal, and anti-biofilm activity. TD-GCMS was used to identify highly abundant compounds which were mapped to efficacy data. Thyme essential oils were antimicrobial against both Pseudomonas aeruginosa and Staphylococcus aureus and had the most potent anti-biofilm activity. Three compounds were common and highly abundant in these oils: o-cymene, 2-isopropyl-4-methyl-phenol (o-thymol/carvacrol), and thymol. The most efficacious red and white thyme oils were formulated into Laponite-based hydrogel emulsions capable of inhibiting both P. aeruginosa and S. aureus in static and dynamic biofilm models. Notably, the efficacy of both gels diverged from that predicted by MIC, MBC, and MBIC values, highlighting the limitations of reductionist analyses in predicting real-world antimicrobial performance.

• Thyme oils are the most efficacious of the Lamiaceae plant oils tested

• Thymol isomers and o-cymene are abundant in thyme oils, but minor components also play a role in antimicrobial activity

• Hydrogel efficacy arises from interactions between formulation and wound microenvironment

Drought poses a major threat to global food security, reducing yields of staple crops such as wheat. Sustainable solutions to improve plant resilience are urgently needed. Tobacco is widely cultivated for cigarette production, but its dried leaves can also harbor thermotolerant bacterial strains with potential plant growth-promoting properties. This study aimed to evaluate the biostimulatory potential of endophytic bacteria isolated from dried tobacco leaves in enhancing drought tolerance in winter wheat (Triticum aestivum L.). Five bacterial strains were characterized for key PGP (plant growth-promoting) traits, including auxin production, phosphate solubilization, siderophore production, and salt tolerance. In greenhouse experiments under controlled drought stress (100%, 75%, and 50% field capacity), selected strains were applied to wheat seedlings, and their effects on plant growth, biomass accumulation, and oxidative stress markers (malondialdehyde and hydrogen peroxide) were assessed. The effects of bacterial inoculation varied by strain and irrigation level, with the most beneficial responses generally observed under moderate drought stress. Certain strains significantly reduced oxidative damage and enhanced shoot biomass compared to noninoculated controls These findings demonstrate the untapped potential of tobacco leaf-derived PGP bacteria as sustainable bioinoculants to improve drought resilience in wheat, contributing to climate-resilient agriculture and reduced reliance on chemical inputs.

• Tobacco leaf bacteria improve wheat growth under drought stress

• Isolated strains show biostimulant and drought tolerance effects

• Bacterial application may support sustainable wheat farming in dry conditions

Recent studies on yeast-insect associations demonstrated that social wasps of the genera Polistes and Vespula act as a reservoir for the conservation of yeasts and as vectors capable of transferring such yeasts on the grapes. This work aimed to assess yeast species associated with social wasps and to obtain new strains to be used as bio-protection agents in winemaking. The wine yeast communities present on the exoskeleton, intestine of social wasps, and on the surface of grapes sampled in the vineyards of three Tuscan wineries were determined. Regardless of the wasp species, yeasts were mostly associated with female workers and found mainly in their intestine (up to 7 × 10⁵ CFU/mL). The identification revealed 20 species belonging to 10 genera; Aureobasidium pullulans and Metschnikowia pulcherrima were isolated from wasps of all the wineries, the latter occurring at the highest frequency. Strain-level characterization highlighted that three strains present on grapes were also present in the gut of wasps from the same vineyard. All the isolated M. pulcherrima strains underwent in vitro tests to select the most suitable for use as bio-protective cultures. Three strains showed good inhibitory activity against Kloeckera apiculata and Brettanomyces bruxellensis; hence, they were selected for bio-protection trials on artificially contaminated grapes. Results highlighted the reduction of non-Saccharomyces populations, suggesting the effectiveness of M. pulcherrima as a biocontrol agent. The study confirmed the role of social wasps as yeast vectors in the vineyard and as a reservoir of yeast strains to be exploited for biotechnological applications in oenology.

• Social wasps can carry yeast strains that are also present on the surface of grapes

• Metschnikowia pulcherrima was found within the wasp gut as the prevalent yeast species

• M. pulcherrima proved to be a suitable biocontrol agent on grapes

Extremophilic environments are rich reservoirs for discovering microorganisms with vast biotechnological potential. Among these, microalgae stand out for their pivotal role in sustainable wastewater treatment and nutrient recycling. This study introduces Coelastrella thermophile D14, a microalga isolated from a solar panel, identified through morphological studies and genomic sequencing. The genus Coelastrella has been characterized and classified as highly productive strains valuable for biofuel and bioproduct generation as well as for their ability to produce significant amounts of carotenoids. Experiments revealed the extraordinary resilience of this strain to prolonged desiccation and high-strength piggery wastewater. Notably, D14 cultivated in 10% pig effluent exhibited biostimulant properties, achieving a germination index 23% higher than the control on Lepidium sativum. In a groundbreaking development, we have successfully established an Agrobacterium-mediated transformation protocol for C. thermophila D14, optimizing key parameters for effective T-DNA transfer. This marks a pioneering achievement within the genus Coelastrella. These findings highlight the significant potential of D14 as a robust platform for future biotechnological applications, opening new opportunities for innovative solutions, especially in environmental protection and sustainable agriculture.

• First microalga from solar panel biofilm: Coelastrella sp. D14 isolated and characterized.

• Strain D14 tolerates prolonged desiccation and grows well in piggery wastewater.

• Stable Agrobacterium-mediated transformation enables future metabolic engineering.

Coenzyme Q10 biosynthesis in Escherichia coli is constrained by kinetic mismatches between precursor synthesis and methylation, alongside bioenergetic uncoupling. We implemented an optogenetic phase-control strategy integrating dynamic light induction, ribosome binding site (RBS) engineering, and real-time membrane potential (ΔΨ) feedback. Temporal coordination of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and UbiG methyltransferase (UbiG) via a 6-h phase delay reduced methylglyoxal shunt flux by 41 ± 3% (p < 0.01) through enhanced precursor channeling. Membrane hyperpolarization to − 90 ± 2 mV (relative to − 70 mV in controls) triggered voltage-gated UbiG membrane localization (62 ± 3%) and ATP-driven S-adenosylmethionine regeneration, increasing methylation efficiency 2.3-fold. Multivariate modeling identified ΔΨ and acetate as critical control parameters, enabling optimized fermentation (dissolved oxygen (DO) 15–20%, pH 6.7–6.9). The engineered strain achieved 0.63 ± 0.07 g/L CoQ10 in 5-L bioreactors—a 4.3-fold improvement over the static control strain (0.15 ± 0.02 g/L)—with 82.5% carbon efficiency and 25.8% glycerol-to-product yield. This work establishes bioenergetically coupled temporal control as a scalable paradigm for membrane-bound isoprenoid biomanufacturing.

• Phase-driven enzyme synchronization via optogenetics resolves kinetic mismatch.

• Membrane hyperpolarization gates enzyme localization and ATP regeneration.

• Model-integrated bioenergetic-process control enhances CoQ10 production efficiency.