Applied Microbiology and Biotechnology最新文献

Aspergillus carbonarius is the main producer of ochratoxin A (OTA) in grape and grape products. Good agricultural practices and preventive measures such as the use of biological control agents are key to decreasing OTA concentration in the final product. This study evaluated non-ochratoxigenic strains from Aspergillus section Nigri isolated from grape environments for their ability to control OTA production. In vitro interactions between an ochratoxigenic strain of A. carbonarius and 22 non-ochratoxigenic strains were evaluated using microtiter plates. Biocontrol potential was determined by measuring OTA reduction, while competitiveness was quantified through a specific qPCR assay. Results showed that both OTA reduction and competitive ability were strain-dependent. Co-inoculation experiments revealed that most non-OTA-producing strains reduced OTA levels, with Aspergillus uvarum showing the strongest inhibition. Aspergillus japonicus and Aspergillus trinidadensis also reduced OTA, whereas biseriate species such as Aspergillus niger, Aspergillus welwitschiae, and Aspergillus brasiliensis had minimal impact. The qPCR competitiveness assays revealed that A. carbonarius typically dominated mixed cultures, except when co-cultured with highly competitive A. uvarum strains. Notably, strain A-6760 reduced A. carbonarius abundance to below 6%. This strong competitiveness aligned with significant OTA suppression, suggesting competitive exclusion as the main biocontrol mechanism. Overall, the developed qPCR assay provides a rapid, precise method for fungal interaction evaluation. A. uvarum strains showed great promise for mitigating OTA contamination in grapes and wine through its combined dominance and toxin reduction capacity. Future research should evaluate their effectiveness under field conditions.

• Atoxigenic black aspergilli were tested as biocontrol agents vs. A. carbonarius.

• Competitiveness and OTA reduction varied by strain; uniseriates performed best.

• A. uvarum A-6760 shows promise as a biocontrol agent to reduce OTA in grapes.

Microalgae play a crucial role in the biogenesis of polyunsaturated fatty acids (PUFAs), essential bioactive molecules widely used in the pharmaceutical industry for their therapeutic efficacy and health-enhancing benefits. These microorganisms, due to their rapid growth and resilient nature, make an efficient source for PUFA production. The biogenesis of PUFAs in microalgae involves a series of enzymatic reactions catalyzed by elongases and desaturases that convert precursor fatty acids into PUFAs, including omega-6 and omega-3. This study explores cost-effective, and environmentally friendly strategies to enhance PUFA production in microalgae for use in the food and feed industries. The present studyevaluates the influences of salinity, pH, and low temperature on the nutraceutical properties of Chlorococcum oleofaciens and Leptolyngbya sp., with a focus on PUFA production. Among the conditions tested, 10 mM NaCl yielded in the highest specific growth rates for Chlorococcum oleofaciens and Leptolyngbya sp., reaching 0.142 ± 0.0010, 0.122 ± 0.0016 day⁻¹, respectively. Under the same conditions, biomass yields peaked at 6.89 ± 0.22, 7.06 ± 0.35 g/L. The highest PUFA content of linolenic acid (29, 7.7%), eicosapentaenoic acid (47, 41%), and docosahexaenoic acid (56, 47%) was recorded at 15 °C. The activities of superoxide dismutase and malondialdehyde increased in response to elevated salinity, low temperature, and pH, indicating oxidative stress adaptation. In conclusion, low temperature had a significant effect on the enhancement of PUFA content in Chlorococcum oleofaciens and Leptolyngbya sp., making these microalgae potential strains for PUFA production. These findings offer valuable insights for optimizing cultivation practices and enhancing the productivity of these economically important microalgae.

• Chlorococcum oleofaciens and Leptolyngbya sp. were studied for PUFA production.

• Salinity, pH, and low temperature have a marked effects on the PUFA profile of microalgae.

• The highest specific growth rate occurred at 10 mM NaCl.

• The highest PUFA content was observed at 15 °C.

Here we report the first β-fructofuranosidase from the Trichoderma genus producing fructooligosaccharides (FOS). The novel enzyme from Trichoderma atroviride (TaINV) here characterized was heterologously expressed, purified, and biochemically analyzed. TaINV exhibited hydrolytic activity mainly toward sucrose and other substrates containing β-(2 → 1) linkages, with minor activity toward β-(2 → 6) bonds. In addition to hydrolysis, it catalyzed the synthesis of FOS of all three structural series (¹F-FOS, ⁶F-FOS, and ⁶G-FOS). At the maximal production point, TaINV synthesized 252 g/L of total FOS, representing 50.3% (w/w) of the total sugars in the reaction mixture, with 1-kestose as the major product, representing ~ 85% of the total products synthesized. Structural analysis based on AlphaFold-predicted TaINV model and comparative superimposition with GH32-substrate complexes revealed conserved catalytic motifs and residues located in positions associated with substrate binding and specificity in characterized GH32 enzymes. Site-directed mutagenesis confirmed the essential role of the catalytic triad (Asp63, Asp201, Glu277) and identified additional residues shaping transfructosylation specificity. Variants including substitutions W60Y and N62S increased total FOS production, reaching 62.7% and 57.4% (w/w) of total sugars, respectively, which are comparable to yields obtained with commercial enzymes. Overall, TaINV represents a distinct intracellular fungal β-fructofuranosidase with strong transfructosylation capacity and preference for short-chain FOS. These findings expand the current knowledge of GH32 enzyme diversity and highlight TaINV as a promising biocatalyst for the efficient production of low-degree polymerization FOS with potential prebiotic applications.

• Novel Trichoderma β-fructofuranosidase with high transfructosylation activity.

• Catalytic residues defining FOS synthesis identified by structure–function analysis.

• Engineered variant boosting FOS yield to 62% of total sugars.

Members of the genus Lysinibacillus are increasingly explored as probiotic candidates, yet thorough screening and safety assessment remain essential due to the toxin-producing potential of related species. In this study, a soil-derived isolate, Lysinibacillus sp. MK212927 demonstrated strong antagonistic activity against multiple human enteropathogens and was selected for comprehensive characterization. The strain exhibited high resilience under physiologically relevant stress conditions, including low pH, simulated gastric and intestinal fluids, bile salts, and thermal exposure. It also displayed desirable probiotic attributes such as antioxidant capacity and bile salt hydrolase activity. Safety evaluation revealed the absence of hemolytic activity, minimal cytotoxicity toward Caco-2 cells, and susceptibility to vancomycin, levofloxacin, sulfamethoxazole, and doxycycline, with intermediate susceptibility to azithromycin and amoxicillin, suggesting a lack of plasmids or mobile genetic elements. To enhance its industrial applicability, response surface methodology (RSM) was applied to optimize biomass and spore production. Optimal conditions (pH 6.1, 33.5 °C, 200 rpm, and 0.21 vvm aeration) resulted in a 3.1-fold increase in biomass and a 5.4-fold increase in spore yield. In vivo assessment further showed that administration of Lysinibacillus sp. MK212927 improved body weight gain in rats, supporting its functional benefits as a feed supplement. Overall, the comprehensive phenotypic and safety evaluations highlight Lysinibacillus sp. MK212927 as a robust probiotic candidate with significant potential for controlling enteropathogens and for use in animal and human nutrition, warranting further preclinical and functional development.

• Lysinibacillus sp. MK212927 exhibits strong inhibitory activity against human enteropathogens.

• The strain tolerates low pH, bile salts, gastric and intestinal fluids, and heat, while displaying antioxidant and bile salt hydrolase activity.

• Optimization of biomass and spore production, along with improved body weight in rats, supports its potential as a feed supplement.

Ornithine lipids (OLs) are phosphorus-free membrane lipids present in many bacteria, but absent from eukaryotes and archaea. Three pathways for OL synthesis have been reported to date. Conditions that induce OL synthesis include elevated temperature, low pH, low phosphate concentration, and low salt concentration. OLs can be modified by different hydroxylations, N-methylation, or taurine transfer. These modifications can be expected to alter the biophysical properties of individual lipid molecules and the membrane as a whole, with potential applications in synthetic biology. The presence and synthesis of OLs are frequently associated with increased stress resistance, and bacterial mutants of some species deficient in OL synthesis show increased susceptibility to elevated temperatures or reduced pH. OLs have been shown to be important for bacteria-host interactions and, recently, to interact with Toll-like receptor 4 (TLR4). We present a comprehensive analysis of the taxonomic distribution of genes encoding putative OL synthases, enabling predictions of which bacteria are expected to have the capacity to synthesize OL at least under specific growth conditions. Lipids structurally analogous to OLs in which other amino acids replace ornithine have also been described and are synthesized by enzymes homologous to OL synthases. In recent years, a wide range of studies and observations related to OLs have been published, including the identification of genes encoding novel OL synthases, novel OL-modifying enzymes, and novel OL structures; the sensing of OLs and other aminolipids by eukaryotic organisms; and their possible use in synthetic biology. In the present review, we discuss these recent advances.

Microbes that generate copious amounts of hydrogen (H₂) via dark fermentation are a promising means to evolve and improve renewable biofuels. Many anaerobic hyperthermophilic archaea, such as the fast-growing, genetically tractable, heterotroph Thermococcus kodakarensis, produce generous quantities of H₂ and provide an idealized platform to further optimize naturally high levels of biohydrogen reduction. Precise genetic manipulations and modifications to growth conditions have already resulted in substantial increases to H₂ output but additional improvements are desired. An unexamined and potentially valuable route towards increased H₂ production is to tether select electron donor and acceptor proteins together to reroute and maximize the flow of electrons towards H₂ production. Such strategies have shown promise in Bacteria and Eukarya but have not yet been investigated in thermophilic Archaea. Here, we generate and evaluate twelve novel T. kodakarensis strains wherein a proteinaceous electron carrier (a ferredoxin, Fd) is physically tethered to the membrane-bound-hydrogenase (MBH), the sole H₂ producing enzyme, to direct electron flux towards biohydrogen generation. Growth assessments and H₂ output measurements demonstrate that strains encoding protein-fusions evolve up to ~ 40% more H₂ per cell than the host strain. Eliminating H₂ consumption and alternative routes of electron sinks in concert with protein tethering further increased H₂ output per cell for a maximum increase of ~ 66% over the host strain. Our results demonstrate that rerouting electron flux via protein tethering coupled with the elimination of reductant sinks is a promising means towards improved biohydrogen production in T. kodakarensis.

Clostridium species have garnered increasing attention for their ability to convert lignocellulosic biomass into renewable fuels and platform chemicals. Among the enzymes involved in lignin degradation, feruloyl esterases (FAEs) cleave ester bonds between ferulic acid and polysaccharide side chains, thereby facilitating the disruption of lignin-carbohydrate complexes. However, the biochemical and structural properties of Clostridium FAEs remain poorly characterized, with activity studies largely limited to model substrates rather than native lignin-derived compounds. Here, we report the functional and crystallographic characterization of a novel FAE (CaFaeA) from Clostridium acetobutylicum. CaFaeA exhibits broad catalytic activity toward a range of hydroxycinnamate esters as well as bis(2-hydroxyethyl) terephthalate (BHET), distinguishing it from typical carboxyl esterases. Furthermore, the 2.45 Å crystal structure of CaFaeA reveals a canonical α/β-hydrolase fold with a unique lid domain of three α-helices and two antiparallel β-strands partially covering the active site. Mutagenesis identified two gatekeeper residues, Y151 and E168, that regulate substrate access and catalytic performance. Remarkably, CaFaeA demonstrates exceptional tolerance to organic solvents, retaining or even enhancing activity in the presence of 25% dimethyl sulfoxide and n-hexane. With insoluble wheat arabinoxylan (I-WAX) as substrate, its unique lid architecture enabled efficient cleavage of ferulic acid–arabinose ester linkages, resulting in a release of free ferulic acid by 5.39 mg·μmol⁻¹·h⁻¹, representing a high activity within the range reported for FAEs. These findings not only provide mechanistic insights into microbial FAE function but also highlight CaFaeA as a promising candidate for lignocellulosic biomass utilization.

Candida tropicalis, the most prevalent non-Candida albicans Candida species, is an emerging pathogen forming robust biofilms on medical devices, contributing to biofouling, virulence, and antifungal resistance. In this study, growth conditions for six C. tropicalis clinical isolates (C4, U873, U951, U1179, U1309, U1360) and a standard strain (MTCC-184) were optimized on polypropylene using central composite design-based response surface methodology. The parameters tested included temperature, pH, shaker speed, inoculum size, and incubation time, with biofilm formation quantified by crystal violet, cell viability by MTT, biomass by calcofluor white, and wet/dry weight measurements. Notably, C. tropicalis forms biofilm on polypropylene surfaces, resembling extracellular polymeric substance-rich matrices. Among the isolates, C4, U873, U951, and U1179 fit the CCD model, whereas for MTCC-184, U1309, and U1360, the Johnson Transformation was required to obtain unified optimal conditions. Temperature and pH were the major factors influencing biofilm formation in C4 and U1179, while temperature and incubation time were significant for U873 and U951. A direct correlation was observed between cell viability and biofilm formation, though biomass varied, indicating strain-specific virulence. This high-throughput optimization strategy establishes a platform for antifungal screening, biofilm–material interaction studies, and the development of medical devices resistant to fungal colonization.

• Optimized growth conditions of Candida tropicalis biofilm on polypropylene material by RSM

• Four C. tropicalis isolates fit the CCD model; the other three isolates were modelled using CCD–JT

• A direct correlation was observed between cell viability and biofilm with variations in cell mass.