Applied Microbiology and Biotechnology最新文献

The increasing spread of antimicrobial resistance has prompted the search for innovative alternatives to conventional antibiotics. Chitosan, a biopolymer derived from chitin, is known for its broad-spectrum antimicrobial activity. This study evaluated both direct and indirect antimicrobial activity of chitosan obtained from Hermetia illucens, a novel and sustainable source compared to the traditionally crustacean-derived biopolymer. Chitosan produced from H. illucens larvae, pupal exuviae and adults, through heterogeneous and homogeneous deacetylation, was tested for both its indirect and direct antimicrobial effects. The indirect effect was evaluated by measuring the induction of Human Beta-Defensin-2 (HBD-2) expression in HaCaT keratinocytes stimulated with lipopolysaccharide of Salmonella typhimurium, a Gram-negative bacterium. The direct antimicrobial activity was assessed against Gram-positive pathogens (Enterococcus faecalis, Staphylococcus epidermidis, and Streptococcus agalactiae), using a microdilution assay and plate colony count. Results demonstrated significant bacteriostatic effects at 0.5 mg/mL, with some samples, particularly the homogeneous unbleached pupal exuviae chitosan and the heterogeneous unbleached larvae chitosan, comparable to or even superior to commercial chitosan in terms of biological activity. Furthermore, insect-chitosan significantly up-regulated HBD-2 expression, suggesting immunomodulatory activity. These findings validated H. illucens as a promising alternative source of chitosan with dual antimicrobial activity, and supported its potential use in clinical, pharmaceutical and biomedical applications.

• Insect-chitosan activates innate immunity via strong HBD-2 induction

• Chitosan samples showed notable growth-inhibition toward key Gram-positive strains

• Hermetia illucens chitosans provide efficacy comparable or superior to the commercial biopolymer

The escalating challenge of antibiotic resistance in aquaculture critically threatens global fish health and food security, underscoring an urgent need for novel antimicrobial strategies. This study explored the bioactive potential of metabolites from the marine actinomycete Streptomyces zaomyceticus, isolated from a deep-sea sediment sample off Sharm El-Sheikh, Egypt. Bioactivity-guided fractionation led to the isolation and structural elucidation of SPIROST-8-EN-11-ONE, 3-HYDROXY- (SEOH), identified as a novel spirostenoid. SEOH exhibited significant broad-spectrum in vitro growth inhibition against a diverse panel of aquaculture-relevant pathogens, including Gram-positive and Gram-negative bacteria, and opportunistic fungi. It demonstrated potent minimum inhibitory concentrations (MICs) ranging from 0.25 to 1.0 µg/mL, notably effective against multidrug-resistant (MDR) Klebsiella pneumoniae (0.25 µg/mL) and Enterococcus faecalis (0.5 µg/mL). Scanning electron microscopy (SEM) revealed that SEOH treatment (2× MIC) induced significant morphological alterations, including visible cell surface modifications and reduced cell numbers, in both bacterial (E. faecalis, K. pneumoniae, P. aeruginosa) and fungal (C. albicans) pathogens. Preliminary cytotoxicity assessment using the MTT assay on HepG2 cells yielded a promising IC₅₀ value of 71.76 ± 0.62 µg/ml, indicating a favorable in vitro safety profile. The novel structure of SEOH coupled with its potent, broad-spectrum in vitro antimicrobial activity against crucial aquaculture pathogens positions it as a highly promising candidate. These compelling in vitro findings strongly warrant comprehensive in vivo efficacy and safety studies to fully establish SEOH’s potential as a novel therapeutic agent or feed additive for advancing aquaculture sustainability and animal health.

• Novel Spirostenoid Discovery: SEOH, a new spirostenoid from Streptomyces zaomyceticus, was identified

• Potent Broad-Spectrum Activity: It shows strong inhibition against MDR aquaculture pathogens (MICs = 1.0 µg/mL)

• Warrants Further Study: Its promising safety profile and potency merit in vivo testing for aquaculture use

Porins govern nutrient uptake and antibiotic influx in Gram-negative bacteria, making their characterization critical for understanding permeability, resistance mechanisms, and structure-function relationships. From a biotechnological point of view, they are effective tools for modulating the transport of substances across the outer bacterial membrane or for building catalytically active nanoreactors and biosensors. Quantitative data on mass transport through membranes is of great interest, but not trivial to obtain, as in vivo analyses are confounded by cellular complexity and variability. Here, we present a synthetic bottom-up approach, based on polymersomes containing reconstituted purified porins, enabling direct, quantitative measurement of substrate translocation, while minimizing interferences from native processes. Encapsulation of Gaussia luciferase allowed real-time monitoring of coelenterazine (CLZ) translocation across the polymeric membrane in the absence and presence of porins. The typically flash-type luciferase kinetics adapts a glow-type light emission profile, whose signal increases over time. This allows conclusions to be drawn about the substrate concentration accessible to the enzyme, enabling quantitative calculations of the transport rates. The novel approach was exemplarily used to compare the transport characteristics of three Escherichia coli porins: Outer membrane protein F (OmpF), a deletion variant selected for larger pore size OmpF∆, and Phosphoporin E (PhoE). OmpF∆ exhibited the highest transport rate of 78 molecules s⁻¹ per porin trimer, exceeding OmpF (10.8 molecules s⁻¹) more than sevenfold, whereas PhoE showed a lower rate of 2.8 molecules s⁻¹ for the neutral CLZ substrate. Analysis of two CLZ derivatives of slightly higher molecular mass and notably greater hydrophobicity revealed that transport through OmpF and OmpF∆ was reduced by half, whereas PhoE exhibited lower selectivity for the selected substrates.

• Synthetic polymersomes enable direct, quantitative analysis of porin transport

• OmpF∆ exhibits a sevenfold higher molecular flux than wildtype OmpF porins

• The assay provides a versatile platform to study porin selectivity and permeability

Solventogenic Clostridium species can efficiently produce n-butanol and other valuable chemicals via acetone–butanol–ethanol (ABE) fermentation from plant-based feedstocks. For economic and ecological sustainability, cheap and abundant substrates such as lignocellulosic and hemicellulosic residues from agricultural or forestry side streams are preferable. Cereal brans, rich in hemicellulose, represent a promising substrate. However, for direct fermentation of this material, only low product titers are reported. In this study, we characterized the utilization of arabinoxylan, the main polysaccharide component of cereal bran, by the industrial ABE producer Clostridium saccharobutylicum DSM 13864^T and report inefficient degradation of the substrate. Supplementation with hemicellulolytic enzyme mixtures derived from the thermophilic organism Thermoclostridium stercorarium subsp. stercorarium DSM 8532^T significantly enhanced substrate utilization. The best improvement was achieved by the addition of the arabinofuranosidase Axh43A, which reduced the residual sugar content in the fermentation broth from 48.2 to 17.8%. Analysis of the remaining oligosaccharides after growth on arabinoxylan showed that C. saccharobutylicum cannot remove O-2 and O-3 α-L-arabinofuranosyl groups from double-substituted xyloses, creating a key bottleneck in arabinoxylan degradation that is overcome by Axh43A addition. Plasmid-based expression of Axh43A in C. saccharobutylicum DSM 13864^T replicated the enzymatic supplementation effects, confirming the enzyme’s role in overcoming this limitation. This underscores the potential of genetic engineering to enhance the valorization of lignocellulosic biomass in biotechnological fermentation processes.

Benzyl alcohol (BA) and two BA esters were examined for their ability to support growth of Burkholderia vietnamiensis G4 and expression of toluene-2-monooxygenase (T2MO) activity. Resting cell assays and an activity-based labeling (ABL) approach were used to quantify T2MO activity and levels of T2MO hydroxylase subunit in cells grown either on substrates already known to support T2MO expression (toluene, phenol); novel aromatic substrates (BA, benzyl acetate (BAc); benzyl butyrate (BBu)); and non-aromatics (lactate, acetate, and butyrate). Specific rates of TCE oxidation and levels of activity-based fluorescent labeling of T2MO hydroxylase α-subunits in cells grown on BA or BA esters were comparable to or greater than those of cells grown on toluene or phenol. In contrast, levels of activity and protein labeling of cells grown on acetate or butyrate were like those of lactate-grown cells. Cells grown on BA also degraded 1,2-cis-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC). While toluene-grown cells generally exhibited higher specific rates of chloroethene transformation, BA-grown cells had consistently higher transformation capacities for these compounds. Stable accumulation of BA in batch cultures grown on BBu in the presence of propyne and the inhibitory effects of BA on TCE transformation both suggest BA is a T2MO substrate. Substrate-specific O₂ uptake studies also suggest 2-hydroxybenzyl alcohol is a likely product of T2MO-dependent BA oxidation. Our results suggest that BA and benzyl esters could be useful for promoting cometabolic transformations, because unlike toluene or phenol, these are “generally regarded as safe” (GRAS) compounds with little or no human toxicity.

• Burkholderia vietnamiensis G4 cometabolizes TCE when grown on benzyl alcohol (BA).

• Activity-based labeling showed BA and BA producing esters support T2MO expression.

• BA and BA producing esters are safe compounds to promote aerobic TCE transformation.

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.