Applied Microbiology and Biotechnology最新文献

Xanthan gum, a natural heteropolysaccharide produced by Xanthomonas species, has many biotechnological applications across industries due to its unique rheological properties. Expanding its utility requires specific enzymes capable of targeted xanthan modification or degradation. In this study, a novel bacterial strain, isolated from a spoiled xanthan sample and identified as Paenibacillus taichungensis I5, was shown to degrade xanthan using a plate screening assay with Congo red. Activity tests of crude enzyme in culture supernatant demonstrated the secretion of xanthan-degrading enzymes. Genome and proteome analyses suggest a chromosomal xanthan utilization locus encoding a suite of enzymes, including a xanthanase (Pt_XanGH9), two xanthan lyases (Pt_XanPL8a and Pt_XanPL8b), two unsaturated glucuronidases, two α-mannosidases, as well as transport and regulator proteins. Functional characterization through recombinant protein expression and enzyme assays confirmed the functions of Pt_XanGH9, Pt_XanPL8a and Pt_XanPL8b on native xanthan and xanthan-derived oligosaccharides. The polysaccharide degradation products released by these enzymes were identified via LC–MS analysis and suggested two xanthan lyases with divergent cleavage preferences. In contrast to Pt_XanPL8a, Pt_XanPL8b is synthesized with an N-terminal signal peptide, yet both lyases were detected in cell-free supernatant during growth on xanthan. Based on the composition of the xanthan utilization gene cluster and preliminary enzyme characteristics, a working model for xanthan utilization by P. taichungensis I5 is proposed. Reaching a better understanding of bacterial xanthan degrading pathways and the enzymes involved may help to develop modified xanthan derivatives and xanthan degrading enzymes that align with the specific demands of various industrial process.

• The genome of P. taichungensis I5 encodes a xanthan utilization locus.

• P. taichungensis I5 employs a twin lyase-dependent xanthan utilization system.

• The two xanthan lyases differ in cellular localization and in cleavage specificity.

In recent years, the biotechnological production of carotenoids and lipids by yeasts emerges as a valuable strategy at the industrial level, also fitting the circular economy pillars when agri-food waste can be used as the main components of the culture media. In this study, bioprocesses employing red yeasts were developed using cost-effective agri-industrial residues, such as soy okara (a soybean industry byproduct). This low-cost substrate was investigated as a source of carbohydrates and essential nutrients, with its enzymatic pre-treatment optimized to create a balanced and efficient fermentation medium. The screening of a collection of red oleaginous yeasts identified Rhodotorula paludigena CBS 6565 and Rhodotorula diobovata CBS 324 as promising strains capable of efficiently producing both lipids and uncommon carotenoids. These two strains were cultivated in a lignocellulose hydrolysate-based medium supplemented with urea, validating the promising results of the screening. Urea, a cost-effective nitrogen source, was found to enhance carotenoid production compared to ammonium sulfate. Finally, soy okara was used as the fermentation medium for Rhodotorula paludigena CBS 6565. Soy okara underwent optimized enzymatic hydrolysis to maximize fermentable sugar release, while the addition of waste cooking oil and syrup from candied fruit processing significantly boosted carotenoid production, reaching 262.4 mg/L in 90 h. Among these, β-carotene and torularhodin contributed 140 mg/L and 72.5 mg/L, respectively. Furthermore, the yeast cells accumulated lipids, constituting 56% of their dry weight, with a final concentration of 18 g/L. Overall, this study underscores the synergy between agri-food waste valorization and the sustainable production of yeast biomass enriched in carotenoids and lipids, offering a versatile and high-value resource for various industrial applications.

• β-carotene, torulene, and torularhodin levels are highly strain-dependent in yeasts

• R. paludigena CBS 6565 achieved high carotenoid–lipid co-production yields

• Fed-batch with multiple agri-food residues enabled a sustainable bioprocess design

The outbreak of certain bacteria can trigger severe diseases, even posing a threat to public safety, leading to significant social panic and economic losses. Thus, tracing the origin of bacteria is of great significance. Stable isotope analysis technology offers a new way to determine the geographical information of bacteria, yet related research still fails to meet the application requirements of this technology in practical cases of bacterial traceability. In this study, a systematic stable isotope analysis method for bacteria and their culture conditions, based on practical geographical environments, was established for the first time. Escherichia coli and Staphylococcus aureus were cultured with water from five regions and different culture media, and the stable isotope ratios of H/O/C/N in the two bacteria and the culture media were measured to explore the relationship between bacteria and their cultivation site. The results showed that there were linear relationships between the hydrogen and oxygen stable isotopes of the two bacteria and the culture water. The combined discriminant model constructed using multi-isotope (H/O/C/N) characteristics achieved a 100% accuracy rate in identifying the types of culture media. These results indicate that research on the isotope association between bacteria and their culture water can be used to infer the cultivation region, and the specific source of bacteria can be further inferred through the multi-isotope combination discriminant model. This study can provide a relatively complete research idea for bacterial geographical traceability research, and improve the efficiency and accuracy of bacterial traceability work in practical investigations.

Noxious odors from swine farms pose significant health and environmental concerns. Hydrogen sulfide (H₂S) is one of the major compounds causing noxious odors. Using biological inoculants offers a promising, efficient, and cost-effective method to reduce H₂S emissions from these farms. Several studies have found that Priestia megaterium (formerly Bacillus megaterium) can help mitigate the production of H₂S. However, detailed knowledge regarding its mechanisms of action is still limited. In this study, the potential of novel strain of P. megaterium, strain S188, for the reduction of H₂S in swine manure has been demonstrated. The artificial manure-based assay revealed that P. megaterium S188 significantly reduced the headspace concentration of H₂S. Moreover, transcriptomic analysis revealed that exposure to H₂S upregulated the expression of several cysteine and methionine biosynthesis-related genes in P. megaterium S188. Furthermore, treatment of swine manure with P. megaterium S188 was associated with significant modulation of the microbiota in the manure. Overall, this study provides insights into the putative mechanisms underlying the reduction in H₂S levels mediated by P. megaterium S188 and its potential as a biological inoculant for H₂S mitigation in swine farms.

• Priestia megaterium S188 has shown the ability to lower H₂S from swine manure.

• Transcriptomic analysis revealed upregulation of H₂S-related biosynthesis genes.

• P. megaterium S188 treatment is associated with changes in the manure microbiota.

Improving Escherichia coli’s thermotolerance through rational engineering is hindered by limited knowledge of the molecular mechanisms involved in supraoptimal thermal adaptation. To address this issue, we applied a reverse metabolic engineering strategy to develop a d-homolactic E. coli strain under non-aerated conditions. We first characterized the thermal reaction norms of the kinetic and stoichiometric parameters in strains evolved through continuous adaptive laboratory evolution, identifying those that maintained parental volumetric productivity and the lactate/glucose yield at high temperatures. Then, the genomic analysis of two thermally adapted strains was performed to determine the mutations acquired during thermal adaptation. Thermally adapted strains revealed convergent mutations in regulatory genes, notably metJ, lrp, and components of the RNA polymerase complex. Introducing these point mutations into the parental strain demonstrated that individual mutations in rpoB and metJ significantly improved growth at high temperatures, despite the presence of complex epistatic interactions in combinatorial analyses.

• ALE significantly enhanced the thermotolerance of homolactic Escherichia coli

• Mutations in rpoB and metJ notably enhance growth at high temperatures

• Lactate productivity and yield stayed similar to the parental strain

Lactiplantibacillus plantarum strains are increasingly recognized for their combined probiotic and antimicrobial activities, offering potential applications in gut health management and pathogen control. This study characterized the intracellular (Met-Int) and extracellular (Met-Ext) metabolomic profiles of L. plantarum UTNGt2 (Gt2), UTNGt3 (Gt3), and UTNGt28L (Gt28L) isolated from tropical fruits and evaluated their probiotic, antimicrobial, cytotoxic, and immunomodulatory properties in vitro. Metabolomic profiling was performed using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with a SWATH (Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra) acquisition method. Cytotoxicity and cell viability were assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and LDH (lactate dehydrogenase) release assays in colon epithelial cells, while cytokine responses (IL-10, IL-1β) were quantified to determine immunomodulatory effects. Antimicrobial mechanisms were examined by scanning and transmission electron microscopy (SEM/TEM) on Staphylococcus aureus ATCC1026. LC–MS/MS identified 117 Met-Int and 32 Met-Ext across the three strains, revealing shared metabolites (e.g., l-tryptophan, adenosine) and distinct strain-specific compounds (e.g., harmine, lincomycin, baicalein) associated with bioactivity. Pathway enrichment analysis indicated four enriched pathways in Gt2, eight in Gt3, and ten in Gt28L, reflecting differential specialization in amino-acid, carbohydrate, and cofactor metabolism. Gt3 exhibited the most diverse antimicrobial metabolite repertoire, whereas Gt28L showed the strongest anti-inflammatory effect, increasing IL-10 secretion by ~ 6.5-fold and reducing IL-1β by ~ 50% compared with control cells. All strains maintained > 85% cell viability with minimal LDH release. SEM/TEM analysis confirmed that Met-Ext fractions caused membrane disruption and intracellular damage in S. aureus. Overall, these results demonstrate strain-specific probiotic, antimicrobial, and immunomodulatory signatures, identifying Gt3 and Gt28L as promising candidates for therapeutic or food-grade applications.

Mounting evidence supports population-specific variation in the microbiome. This study applied PacBio HiFi 16S rRNA amplicon sequencing to profile the oral microbiome of 62 individuals (aged 18–55 years) from the four major South African population groups (Black, Coloured, Indian and White). Bioinformatics analysis was performed using QIIME2 with taxonomic classification based on the SILVA database. Across all groups, Firmicutes was the most dominant phylum (80.92%), followed by Proteobacteria (8.94%) and Bacteroidota (4.22%). A total of 236 genera and 376 species were identified. Veillonella was the most abundant genus (27.57%), followed by Streptococcus (25.08%) and Granulicatella (15.54%). Streptococcus (30.40%) and Veillonella (41.82%) were the most abundant in the Indian and Coloured populations, respectively. Significant microbiome variation was observed between groups (β-diversity, p < 0.001), highlighting distinct population-specific microbial profiles. The Coloured population exhibited the highest microbiome diversity, likely due to complex genetic makeup and diverse cultural influences. The White population displayed the lowest microbiome diversity, likely due to more uniform lifestyle patterns. Despite these differences, three bacterial species, namely, Streptococcus salivarius, Veillonella atypica, and Prevotella melaninogenica were present in the majority of individuals across the populations, suggesting a core component of the South African oral microbiome. No significant associations were observed between factors such as sex, age, diet antibiotic use, lifestyle and oral microbiome variation. Several undetermined factors, such as psychological factors, stress, level of exercise, host genetics and immunity, could have contributed to the observed diversity. Our results present the first report of oral microbiome analysis of healthy South African populations by PacBio HiFi sequencing and warrant further research to provide insight into how these differences influence health disparities and potential application in forensics.

• First report of the oral microbiome of healthy South Africans by PacBio HiFi sequencing

• S. salivarius, P. melaninogenica and V. atypica define South Africa’s core oral microbiome

• Population-specific microbiome signatures can guide forensics and health research

Bacterial blight (causal agent Pseudomonas syringae complex, Psc) is an endemic and economically important disease of northern highbush blueberry production in Canada and the Pacific Northwest of the USA. To date, there is no comprehensive survey of the disease in the region and detailed characterization of associated pathogens from Pacific western Canada. Therefore, we did comprehensive disease survey and characterization of associated pseudomonads population using pathogen morphology, biochemical tests, and molecular characterization. We isolated 380 strains of pseudomonads from symptomatic plants from 32 research and commercial fields in 10 diverse geographic locations in British Columbia. We used P. syringae specific (Psy) primers and identified 197 Psy-PCR positive isolates out of 380. We further sequenced Psy-PCR positive isolates of pseudomonads using four housekeeping genes and identified four phylogenomic species: P. syringae (40%), Pseudomonas avellanae (29%), Pseudomonas viridiflava (20%), and phylogenomic species A (7%). P. avellanae and P. viridiflava are new phylogenomic species of Psc causing bacterial blight in highbush blueberry. We found some patterns among geographical locations and highbush blueberry varieties in the frequency distribution of isolates of these phylogenomic species. Genetic fingerprinting with rep-PCR assays identified a very high genetic diversity of pseudomonads populations among geographical locations, varieties, and phylogenomic species. Biochemical characterization (LOPAT- levan, oxidase, pectolytic activity, arginine dihydrolase, and tobacco hypersensitivity) revealed that the vast majority of isolates were Pseudomonas Group Ia. Findings of this study provide insight into the population biology of pseudomonads infecting highbush blueberry, provide information for disease diagnosis, and exploit disease management options, including identifying sources of disease resistance.

• High prevalence of bacterial blight caused by P. syringae complex (Psc) in highbush blueberry in Pacific western Canada

• We report two new phylogenomic species of Psc, P. viridiflava and P. avellanae, that cause bacterial blight and canker disease in highbush blueberry

• The genetic diversity of the population of Psc was very high

Palm oil is the world’s most widely used vegetable oil, with a sizeable impact on the environment. As an alternative, microalgae are considered oil producers since they produce a variety of fatty acids (FA) depending on growth conditions. A collection of ten microalgae strains naturally producing oils similar in composition to palm oil was selected, and the effects of cultivation regime and varying light intensity on their growth and FA production and composition were analysed. To achieve high biomass density as well as total fatty acid (TFA) content, the optimum irradiance of 400 µmol photons m⁻² s⁻¹ in a photoautotrophic regime was determined for most of the strains. The growth rates of Scenedesmus and Desmodesmus strains in general were approximately twice as high as Chlamydomonas. The highest TFA content was found in S. obliquus CCALA 455 and D. subspicatus CCALA 467, grown photoautotrophically, reaching the values of about 66% and 58% of their dry weight, respectively. Moreover, the content of palmitic (PA), oleic (OA) and linoleic acid (LA) of about 39%, 30% and 14% of TFA, respectively, determined in D. subspicatus CCALA 467 was closest to that in palm oil (44% of PA, 39% of OA and 10% of LA). Eight of the ten microalgae strains were capable of heterotrophic growth, although their production under this regime has not been considered suitable in terms of TFA and individual FA content.

• The optimum irradiance of 400 µmol photons m⁻² s⁻¹ was determined

• CCALA 467 produces selected FAs in amounts close to those in palm oil

• TFA content (% of dry weight) in CCALA 467 is 1.6-fold higher than in the palm