Applied Microbiology and Biotechnology最新文献

Gram-negative bacteria are emerging as an important source of natural products with pharmaceutical potential. However, the limited availability of genetic tools for drug discovery and sustainable production of secondary metabolites remains a challenge. Burkholderia spp. serve as a promising source for such tools, as these bacteria produce diverse natural products and are amenable to genetic modification. We sequenced the genome of Burkholderia stagnalis TBRC 18363 and performed transcriptomic analysis to identify genes highly expressed in early to late exponential cultures. We hypothesized that the sequences upstream of the most highly expressed genes contain strong and constitutive promoters active in heterologous Gram-negative hosts. Twenty-six B. stagnalis TBRC 18363 promoters were evaluated in Escherichia coli and Pseudomonas putida reporter systems. Two promoters, p2035 and p5642, exhibited superior performance in both systems. Promoter exchange experiments at biosynthetic gene clusters showed that these promoters can enhance the production titers of icosalide in B. stagnalis TBRC 18363 and FR900359, a G_q/11 protein inhibitor depsipeptide, in Chromobacterium vaccinii. Therefore, the p2035 and p5642 promoters are applicable for target gene overexpression in Gram-negative bacteria and can serve as tools for unlocking the potential of cryptic biosynthetic genes.

• Strong constitutive promoters of Burkholderia stagnalis TBRC 18363 were identified.

• Efficiencies of the selected promoters were evaluated in two heterologous hosts.

• p2035 and p5642 promoters boosted BGC expression in Burkholderia and Chromobacterium.

Haloferax mediterranei has shown strong potential for bioremediating brines contaminated with nitrogenous compounds, oxychlorides, and metals, making it a promising candidate for treating saline wastewater. Therefore, this study aims to evaluate its capacity to bioremediate industrial wastewaters (IWWs) from desalination and textile industries. After characterising IWWs and formulating suitable media, growth was compared with two control media: an optimal medium (NH₄⁺, 20% salts) and a control medium providing basic nutrients and ionic strength (NO₃⁻, 15% salts). Raw IWWs were heterogeneous and unsuitable per se for optimal growth, requiring supplementation with minimal essential nutrients (C, N, P, and Fe). H. mediterranei grown in IWWs media exhibited reduced growth and increased cell pigmentation, indicating stress. Particularly, textile residues induced tenfold higher carotenoid production than the optimal medium and only fourfold less than the carotenoid production medium. Carotenoid extract composition showed variations within different IWWs, but bacterioruberin revealed as the main natural pigment in all cases. Elemental biomass analysis showed Fe and Zn accumulation, as metalloprotein cofactors for stress tolerance, and Ca, related to exopolysaccharide production. Despite the stress, H. mediterranei effectively removed 60–90% of the NO₃⁻ in the wastes—among the higher removal rate values reported for matrices under comparable salinities and initial NO₃⁻ loads. While further optimisation of waste mixtures is needed to improve nutrient balance and eliminate the need for supplementation, our findings suggest that IWWs from both industries can support H. mediterranei growth, enabling a cost-effective, industrially viable bioremediating strategy with the added value from pigment production.

The current study provides the first detailed characterization of two novel bacteriophages, DG23 and RG24, that infect Pseudomonas marginalis, a causative agent of soft rot in potato and other vegetable crops. The phages were assessed for environmental stability, genetic characteristics, and biocontrol efficacy. Both DG23 and RG24 showed broad tolerance throughout a wide pH range (3–9), with RG24 still viable at pH 11, while DG23 was more sensitive to extreme pH conditions. Thermal stability assay demonstrated that both phages remained infectious up to 45 °C, but activity decreased dramatically at higher temperatures, with total inactivation at 75 °C. Phage viability reduced considerably under UV irradiation (254 nm), with DG23 demonstrating better resistance than RG24. Whole-genome sequencing revealed that both phages are lytic, with no integrase, pathogenicity, or antibiotic resistance genes, ensuring biosafety for prospective agricultural uses. Comparative genomic analysis indicated a 99% average nucleotide identity (ANI) between DG23 and RG24, showing they are the same species, but both were genetically distinct from their nearest relative, Pseudomonas phage XD2 (ANI 92%). In addition, comparative proteomic and phylogenetic analyses revealed that DG23 and RG24 form a distinct clade within the class Caudoviricetes, separate from other related phages. Biocontrol experiments showed that both phages efficiently inhibited potato soft rot when used individually, but when combined, disease severity was decreased by more than 80%, demonstrating the higher efficiency of phage cocktails. These data suggest that DG23 and RG24 are promising, safe, and effective candidates for phage-based biocontrol of soft rot caused by P. marginalis.

• Novel phages DG23 and RG24 lyse Pseudomonas marginalis and lack virulence genes.

• Phages show stability under broad pH, temperature, and UV conditions.

• Cocktail treatment reduces potato soft rot severity by more than 80%.

The fermentation of nitrogen-containing compounds by biological nitrogen fixation is a sustainable strategy that is independent of the Haber–Bosch process. We previously reported that the nitrogen-fixing bacterium Klebsiella pasteurii (formerly K. oxytoca) NG13 synthesized and excreted large amounts of ʟ-glutamate using gaseous nitrogen when citrate synthase (CS) and citrate transporter (CitS) were overproduced; however, the majority of carbon atoms in ʟ-glutamate were derived from citrate, not glucose, in the glucose and citrate-containing medium. To examine biased carbon flux to ʟ-glutamate, K. pasteurii overproducing CS and a 2-oxoglutarate (2-OG) transporter (KgtP) was constructed, and its carbon origin was investigated. This strain produced 2-OG-derived ʟ-glutamate in a culture medium containing glucose and 2-OG as the carbon sources. Since CS was inhibited by 2-OG competitively with oxaloacetate, a cognate substrate of CS, the deviated carbon flux from citrate/2-OG to ʟ-glutamate was attributed to the suppression of CS by 2-OG. Based on the structural model of CS from K. pasteurii (KpCS), H227 and V362 were selected as candidates to detect 2-OG binding, and KpCS variants (KpCS*) with H227L, H227Q, and V362L substitutions were confirmed to have inhibition constants that increased by 2.5- to 12.5-fold. As expected, the strains co-overproducing each of the KpCS variants and CitS generated larger amounts of ʟ-glutamate from glucose than the wild-type KpCS + CitS strain. When the KpCS(H227Q) + CitS strain was cultured under continuous glucose-fed conditions, maximum ʟ-glutamate production reached 2.35 g L⁻¹. These results suggest the potential of the Haber–Bosch process-independent strategy as a technological basis for the sustainable and eco-friendly utilization of nitrogen.

• CS was inhibited by 2-OG in K. pasteurii

• CS variants with increased K_i^2−OG allowed glucose-derived ʟ-glutamate production

• Under glucose-fed culture, ʟ-glutamate production finally reached 2.35 g L⁻¹

Whey permeate (WP), the main waste stream of the dairy industry, was used as a raw material to produce fully bio-based non-ionic surfactants. Specifically, on the one hand, WP was submitted to a transglycosylation reaction catalyzed by the immobilized β-galactosidase from Aspergillus oryzae in 1-BuOH, affording 1-butyl β-D-galactopyranoside (yield 40%), which was used as the polar “head” of the surfactant. On the other hand, a WP-based fermentation process by the yeast Cutaneotrichosporon oleaginosus ATCC 20509 was employed to produce single cell oil (45% w/w_{cell dry weight}). The microbial lipids were recovered from the freeze-dried cells and derivatized in a one-pot system by acid-catalysis to yield the corresponding ethyl esters as apolar “tails” (75% w/w yield, based on lipid content). These bio-based building blocks were converted into the sugar fatty acid esters (SFAE) n-butyl 6-O-acyl-β-D-galactopyranosides by a lipase-catalyzed transesterification reaction (yield 40%). The hydrophilic–lipophilic balance and solubility parameters of the synthesized SFAE mixture were calculated. Additionally, its ability to reduce the interfacial tension was examined, including the effect of fatty tail unsaturation. The interfacial performance of the SFAE mixture, containing both palmitic (45%) and oleic (40%) acid residues, was lower (6.3 mN m⁻¹) compared to the SFAE containing only palmitic acid as the fatty acid tail (0.1 mN m⁻¹) at the same concentration, but still highly promising.

• Whey permeate (WP) is the main waste stream of dairy industry.

• WP was upcycled by coupling fermentation and biocatalysis.

• Bio-based surfactants (sugar fatty acid esters) were obtained using WP as biomass.

Struvite is a phosphate-based mineral commonly formed in ammonia-rich settings such as soils, aquatic environments, wastewater systems, and the urinary tract. Its distinct morphology and mineral properties have drawn interest across disciplines, including microbiology, geology, medicine, and materials science, especially for its ammonia capturing ability. Microbial metabolic activities have been recently recorded to play a critical role in its formation. However, the influence of microbial characteristics, such as enzymatic activity, biofilm, and extracellular polymeric substances (EPS), on struvite morphology and precipitation mechanisms remains underexplored. This study investigates microbial struvite precipitation mediated by three distinct microbes, Sporosarcina pasteurii, Bacillus subtilis, and Pseudomonas fluorescens to investigate the role of different enzymes and EPS on struvite mineralisation. The bacterial cultures selected for the study have varying ureolytic and alkaline phosphatase activity, as well as EPS and biofilm production potential. Struvite precipitation kinetics under different microbial conditions were studied along with its morphology and biofilm-mineral interactions employing microstructural and mineralogical analyses (via field emission scanning electron microscopy, confocal laser scanning microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy). This study reports that urease and phosphatase activities (between 7 and 7.5 U/mL) along with moderate EPS production (~6.5 g/L), lead to the generation of the highest quantities of struvite crystals with moderate size (~103 ± 5 µm), contrary to the larger crystals (~180 ± 5 µm) produced with a higher urease activity (8.4 U/mL) and lower EPS (2 g/L). The experimental findings suggest a complex interplay between enzymatic activity and EPS in shaping crystal structure and its features, which have a significant impact on mineral characteristics. Additionally, moderate biofilm formation (OD₅₇₀ ~1.2) resulted in maximal struvite precipitation, indicating that structured biofilms support efficient mineral nucleation and accumulation. Overall, this study has unravelled key insights into microbial struvite precipitation mechanisms and can pave the way for tailored engineering and biotechnological applications utilising this phosphate mineral.

The growing demand for sustainable agricultural pest control solutions has increased the use of microbial bioinput. This study characterized an industrial bioinput produced by Saccharopolyspora spinosa, focusing on identifying metabolites and quantifying spinosyns A and D by HPLC-MS. The concentrations of these compounds were 29.4 ± 3.6 mg L⁻¹ and 13.3 ± 1.5 mg L⁻¹, respectively, for a total of 42.7 mg L⁻¹. The limit of quantification (LOQ) and limit of detection (LOD) values obtained were 13.7 ng L⁻¹ and 4.16 ng L⁻¹ for spinosyn A and 7.63 ng L⁻¹ and 2.31 ng L⁻¹ for spinosyn D, respectively. The bioinput contained essential amino acids (leucine, phenylalanine, and tryptophan), plant cell death inducers (sphinganine), diketopiperazines with insecticidal potential (Cyclo(Met-Val)), larvicides (N-stearoyl tryptophan), antimicrobials (Penicitrinol D), and phospholipids associated with cell defense and stress responses (phosphatidylethanolamine, phosphatidylserine, ceramides, and mycolactones). In vitro trials demonstrated mortalities of 82.5% for Spodoptera frugiperda, 100.0% for Dalbulus maidis, and 64.1% for Ceratitis capitata. These results are statistically equivalent to those obtained using commercial products, which cost up to five times more. This bioinput’s chemical diversity suggests multifunctional action, probable synergism, and a lower risk of resistance, which reinforces its potential in biological agricultural management.

• Identification of a diverse set of bioactive compounds, including spinosyns A and D.

• Bioinput achieves statistical efficacy equal to commercial products in in vitro tests.

• A sustainable, cost-effective alternative for large-scale production and application.

Thiol peroxidases have been identified as antioxidant enzymes in different organisms. However, there have been only a few reports on deleting or overexpressing its gene to characterize its function. Recently, the ability of the enzyme thiol peroxidase belonging to the Micrococcus sp. IITD107 on asphaltene biotransformation has been reported. Here, we report the construction of deletion and overexpression strains of tpx and compared them with the wild-type strain of Micrococcus sp. IITD107. For this purpose, whole-genome sequencing of the strain was carried out. The genome sequence revealed the presence of only one copy of the tpx gene and several other genes which might play a role in stress response, polyaromatic hydrocarbon (PAH) degradation, etc. The tpx deletion mutant was constructed by homologous recombination. For overexpression, a pRC4 replicon from Rhodococcus was used, and the gene for tpx was expressed under the control of an IPTG-inducible tac promoter. The replicon was found to be stable in Micrococcus. The deletion mutant showed coccoid morphology, and the doubling time was found to be 7.2 h as compared to the doubling times of 3.5 h for the wild-type strain and 4.5 h for the overexpression strain in LB medium. The deletion strain was unable to grow in the presence of high salt or hydrogen peroxide concentrations, whereas the overexpression strain showed less growth as compared to the wild type. Further, about a 50% decrease and only a 5% increase in the rate of asphaltene biotransformation in the deletion and overexpression strains, respectively, were observed. Overall, our results suggest that thiol peroxidase helps not only in asphaltene biotransformation but also aids the bacterium to survive in the presence of stress agents such as salt and hydrogen peroxide.

• Gene for thiol peroxidase confirmed to enhance asphaltene biotransformation

• Change in growth and morphology

• Alteration in halotolerance and stress response

In this study, the composition of the postmortem mycobiome and microbiome of a cadaver in an advanced stage of decomposition, had been deposited outdoors and showed extensive mycelial growth, was characterized using culture methods. This approach allowed for the identification of a total of 26 fungal and 16 bacterial species. The dominant fungal species were Penicillium polonicum, Debaryomyces hansenii, and Penicillium commune. Sensitivity tests for voriconazole and amphotericin B were also performed, to which several isolates were resistant. In the case of bacteria, the distribution of dominant species differed between samples taken from outside the body and samples taken from inside the body. Sensitivity tests for 16 antibiotics showed that 23.08% of isolates were resistant to the tested drugs. Importantly, to the best of our knowledge, we detected several species that have not been previously associated with cadavers: Botryotrichum domesticum, Chaetomium subaffine, Penicillium allii, Scopulariopsis crassa, Scopulariopsis fusca and Yarrowia deformans. These results not only expand our understanding of the ecological roles of fungi in cadaver decomposition but also highlight their potential forensic value. First and foremost, it broadens our understanding of local fungal communities associated with human remains, which in the future may provide valuable information about the location or environmental conditions of body deposition, while specific taxa could assist in estimating the postmortem interval. Moreover, the identification of drug-resistant strains underscores the importance of biosafety in forensic practice and raises awareness of the potential for pathogen dissemination from decomposing remains.

• Twenty-six fungi and sixteen bacterial species were identified from the cadaver

• New fungal records isolated from human remains, expanding forensic mycology knowledge

• Antimicrobial susceptibility testing revealed the presence of drug-resistant fungal and bacterial isolates

Parageobacillus thermoglucosidasius is a carboxydotrophic microorganism that produces H₂ through the water-gas shift (WGS) reaction, using carbon monoxide (CO) as the main substrate. CO is a common constituent of syngas, alongside CO₂, H₂, O₂, and other gases. The facultatively anaerobic nature of P. thermoglucosidasius is particularly pertinent for hydrogenogenesis from O₂-containing syngas. Here, we evaluated the effects of different syngas compositions (5, 12, and 20% of H₂ gas, with constant CO and CO₂; 10, 30, and 50% CO gas with constant CO₂ and H₂) on hydrogenogenesis at the bioreactor scale. Electron balance analysis showed that 88–91% of electrons coming from CO were converted into H₂, regardless of the gas composition. The presence of H₂ in different compositions had no inhibitory effect on hydrogen production rate (HPR), and the maximum HPR corresponded to 13.65 L H₂ L⁻¹ day⁻¹ in fermentations containing 30% CO. A carbon source, other than CO, is needed for biomass formation of P. thermoglucosidasius. Acetate was shown to be the primary intermediate metabolite of glucose metabolism, but could also be used as an initial carbon source for biomass generation. When this carbon source was used, most electrons from CO were converted to H₂, demonstrating that this organic acid can be used as an effective alternative to glucose for H₂ production with P. thermoglucosidasius.

• Evaluation of lab-defined syngas at different compositions for H2 production with P. thermoglucosidasius at the bioreactor scale.

• Hydrogen presence in the headspace was not inhibiting for subsequent H2 production.

• Acetate can replace glucose to generate biomass when growing P. thermoglucosidasius.