Applied Microbiology and Biotechnology最新文献

Cytochrome P450 enzymes capable of performing iterative oxidation at the same substrate site contribute to compound diversification; however, reaction control is also necessary for the efficient production of the desired compound. RosC, a cytochrome P450 enzyme involved in the biosynthesis of the 16-membered ring macrolide antibiotic rosamicin, catalyzes stepwise oxidation of the ethyl group at C-20 via hydroxylation to an alcohol, followed by successive oxidation to the corresponding aldehyde and carboxylic acid. The P107S/L176Q mutant produces the hydroxylated intermediate in the first oxidation step, but the efficiency of the subsequent conversion to aldehyde and carboxylic acid is significantly reduced. To elucidate the factors responsible for the reduced efficiency of the second and subsequent oxidation steps in the P107S/L176Q mutant and to understand how RosC facilitates multistep oxidative modification, we compared the reaction time courses and substrate-binding affinities of RosC and the P107S/L176Q mutant. The mutant exhibited a reduced reaction rate for the initial hydroxylation and showed reduced substrate-binding affinities for both the hydroxylated and aldehydic intermediates.

Furthermore, crystallographic analysis revealed that Leu-176 played a key role in binding to the desosamine moiety of the substrate, and its mutation resulted in the loss of this function. Ser-248 was presumed to play a role in re-anchoring the modification site to the active center through hydrogen bonding with the hydroxyl and aldehyde groups generated in the first and second reactions. These findings are expected to contribute to the multifunctionalization of cytochrome P450 enzymes and the regulation of their reactivity.

• RosC-catalyzed three-step oxidation is limited to one step in the P107S/L176Q mutant.

• P107S/L176Q mutations impair substrate binding by increasing structural fluctuations.

• Leu-176 and Ser-248 position the substrate for RosC-catalyzed iterative oxidation.

Bacillus species have demonstrated beneficial effects on intestinal health, production parameters, and immune function in poultry under both standard and disease-challenged conditions. Previously, we found that several novel ingredients (beta-glucan, vegetable protein hydrolysate, and liquid seaweed extract) demonstrated growth stimulation effects on Bifidobacterium lactis and Lactobacillus plantarum, Here, we extended this approach to examine in vitro synbiotic combinations of five novel marine-derived candidate probiotic Bacillus strains to assess their potential for in ovo applications. Beta-glucan enhanced the growth of all candidate Bacillus probiotic strains compared to a glucose control (p ≤ 0.05), suggesting a broad-spectrum modulatory role over a 24-h period, with variable magnitudes of response observed between strains. Species specificity was also observed, with lentinus stimulating the Bacillus pumilus but not the Bacillus altitudinis strains. A seaweed extract consistently stimulated the growth of one of the B. altitudinis strains (p ≤ 0.05), which, like all of the strains evaluated here, is seaweed-derived. This suggests potential ecological adaptation in substrate utilization. The shared environmental origin may influence substrate specificity and metabolic complementarity between strains and prebiotic candidates. Both B. altitudinis strains also exhibited enhanced growth at almost all time points (p ≤ 0.05) when cultured with vegetable protein hydrolysate. Based on these findings, we evaluated the effect of a potential synbiotic formulation comprising one of the B. altitudinis strains and vegetable protein hydrolysate in chickens, in ovo. The components were administered intra-amniotically at embryonic development day 18.5, utilizing a standard vaccination protocol. The hatchability of the chickens was not affected, thereby demonstrating the established dose as safe and applicable for further investigation.

• Shared origin of bioactive compounds may enhance probiotic-prebiotic compatibility in vitro

• Protein hydrolysate offers a novel alternative to carbohydrate prebiotics

• In ovo delivery of Bacillus-based synbiotic formulations offers potential as an early microbiome programming strategy

Poly-γ-glutamic acid (γ-PGA) has diverse applications from cosmetic to drug delivery. The production of γ-PGA primarily relies on microbial fermentation using Bacillus spp. supplemented with l-glutamate supplementation. However, the high cost of l-glutamate supplementation limits industrial production. This study aimed to achieve direct γ-PGA production from glucose using a Corynebacterium glutamicum-Bacillus licheniformis coculture system. To create such a coculture system, we utilized B. licheniformis ATCC 9945a, a natural l-glutamate-dependent γ-PGA producing strain, and C. glutamicum F343, which exhibited an excellent capacity to produce l-glutamate from glucose. B. licheniformis ATCC 9945a grew well and produced small amounts of γ-PGA in the medium of C. glutamicum F343. Subsequently, B. licheniformis ATCC 9945a was cultured using the supernatant collected from the C. glutamicum F343 fermentation broth to investigate its effect on the fermentation profile. It was found that B. licheniformis ATCC 9945a produced more γ-PGA in the supernatant compared to when exogenously supplemented with l-glutamate. Moreover, nine intracellular metabolites were discovered to be strongly connected to γ-PGA synthesis by UPLC-MS. Finally, the coculture of C. glutamicum F343 and B. licheniformis ATCC 9945a to produce γ-PGA was conducted. We successfully achieved direct γ-PGA production from glucose under optimal conditions, including an inoculation time of 4 h for B. licheniformis after C. glutamicum inoculation, a 75% inoculum ratio of C. glutamicum, and a total inoculum size of 10% culture volume. The coculture system produced 12.49 g/L of γ-PGA in a shake flask and 22.7 g/L in a 5-L fermentor.

• C. glutamicum F343 could produce L-glutamate from glucose as a precursor for PGA synthesis by B. licheniformis ATCC 9945a.

• The C. glutamicum-B. licheniformis coculture system could produce γ-PGA up to 22.7 g/L.

• Nine intracellular metabolites demonstrated a remarkable influence on γ-PGA synesis by UPLC-MS and metabolite profiling.

Streptomyces species are prolific producers of bioactive natural products, yet their genetic manipulation remains constrained by inefficient DNA delivery methods in many strains. Conjugation from methylation-deficient Escherichia coli has become the preferred approach for introducing plasmids into Streptomyces, relying on the presence of the oriT sequence within the mobilizable plasmid and the conjugation machinery (tra genes) encoded on the non-mobilizable helper plasmid pUZ8002. Among these, traJ encodes an essential component of the relaxosome. An additional copy of traJ is present downstream of oriT in some mobilizable plasmids, whereas many other commonly used plasmids lack traJ. Here, we investigated the impact of including traJ in mobilizable plasmids on conjugation efficiency by engineering two oriT-containing plasmids that initially lacked traJ: the ΦC31 integrative vector pRASK-SP44 and the non-replicative transposon delivery vector pHL734. We also examined the effect of introducing a second copy of traJ into the recombination-based chromosomal end-removal vector pCER. Incorporation of traJ into pRASK-SP44 and pHL734 resulted in tenfold and 100-fold increases in transconjugant numbers, respectively. Furthermore, introducing a second copy of traJ into pCER led to a fivefold improvement in plasmid transfer. Our data suggest that the inclusion of traJ improves transfer efficiency and may help overcome limiting steps in conjugation from E. coli to Streptomyces. Modulating the presence and copy number of traJ could represent a simple yet effective strategy to enhance genetic accessibility in Streptomyces. These findings have broad implications for the optimisation of genetic tools used in Streptomyces genome engineering and natural product discovery.

• traj in mobilizable plasmids enhances conjugation to S. coelicolor.

• traj increases plasmid transfer efficiency up to 100-fold in S. coelicolor.

• traj may aid development of genetic tools for genome engineering in Streptomyces.

Bovicin HC5, a bacteriocin produced by Streptococcus equinus HC5, demonstrates inhibitory activity against pathogenic and spoilage microorganisms. However, low production yields hinder its widespread application. This study investigated the impact of temperature on S. equinus HC5 growth and employed adaptive laboratory evolution (ALE) under heat stress to obtain variants with improved bovicin HC5 production. The optimal growth temperature for the wild-type strain was determined to be 42 °C, with growth ceasing above 49 °C. Following 400 generations of ALE at 47 °C and 48 °C, eight variants were selected. Two of these variants exhibited significantly enhanced bovicin HC5 production, reaching up to a 140% increase (P < 0.05). The variant with the highest bacteriocin yield showed increased expression of bvcA, the gene encoding the bovicin HC5 precursor peptide. This high-producing variant also displayed enhanced thermal resistance, a higher growth rate (μ = 1.33 ± 0.02 h⁻¹), and increased biomass accumulation (OD_600nm = 4.03 ± 0.06) at 48 °C compared to the wild-type strain (μ = 0.98 ± 0.04 h⁻¹; OD_600nm = 1.96 ± 0.12) (P < 0.05). Furthermore, the selected variants exhibited alterations in membrane composition, characterized by an increased concentration of saturated fatty acids and a reduced Zeta potential (P < 0.05). Genomic analysis of these variants identified mutations in genes involved in protein modification, transcriptional regulation, and cellular transport, including a lantibiotic permease. These results demonstrate the effectiveness of ALE for generating S. equinus HC5 variants with improved bovicin HC5 production and provide valuable insights for optimizing bacteriocin biosynthesis strategies.

• The optimal growth temperature for the Streptococcus equinus HC5 strain was determined to be 42 °C, with growth ceasing above 49 °C

• The variant Streptococcus equinus HC5 40048 with the highest bacteriocin yield showed increased expression of bvcA, the gene encoding the bovicin HC5 precursor peptide

• ALE is an efficient metabolic engineering strategy to increase bacteriocin production in Streptococcus equinus HC5

Acinetobacter baumannii and Staphylococcus aureus are major multidrug-resistant (MDR) pathogens frequently associated with healthcare-acquired infections. The emergence of antimicrobial resistance underscores the urgent need for alternative therapeutics. This study explores the antimicrobial potential of fatty acids (FAs) extracted from Hermetia illucens (HI) larvae fat (AWME3) against MDR strains A. baumannii ATCC 19606 and S. aureus ATCC 55804. AWME3 exhibited potent inhibitory effects, with minimum inhibitory concentrations (MICs) of 0.38 mg/mL for A. baumannii and 0.19 mg/mL for S. aureus. Bactericidal activity occurred within 5–10 min at 0.75 mg/mL. Broth microdilution and propidium iodide uptake assays manifested FA-induced membrane permeabilization (55–70%) within 5 min, supporting a rapid membrane-targeting mechanism. Disruption of membrane integrity was accompanied by significant intracellular ATP depletion, cytoplasmic protein leakage, and altered cellular ultrastructure. AFM imaging showed significant morphological damage, with increased cell surface roughness in both bacterial strains. A. baumannii showed a significant height reduction (51–80%), while S. aureus had a reduction of 26–38% after exposure to 1 × MIC and 2 × MIC. AFM visualizations indicated severe cell envelope damage, including pore formation, blebbing, and surface collapse, consistent with membrane lysis. These findings reveal the swift and membrane-disrupting effects of AWME3 fatty acids on MDR nosocomial pathogens, underscoring their potential as a natural antimicrobial agent.

• Fatty acids from H. illucens fat show strong activity against MDR pathogens.

• Rapid bactericidal effect via membrane disruption and cytoplasmic leakage.

• AFM reveals nanoscale cell damage confirming membranolytic action.

Ergothioneine (EGT), which exhibits strong antioxidant properties, is an amino acid derivative with a betaine structure. Currently, studies have examined EGT import systems and its physiological roles in various organisms. Despite the broad applicability of EGT, industrial production with high productivity has not yet been achieved. In this study, we aimed to develop fermentative production methods for EGT using Corynebacterium glutamicum as a host and successfully achieved the highest yield of EGT (459 mg L⁻¹) reported to date. A cysteine-producing strain C. glutamicum CYS-2, which was constructed in a previous study, was engineered to enhance the biosynthesis of histidine and S-adenosylmethionine, both of which, along with cysteine, are required for EGT production. Additionally, heterologous metabolic pathways for EGT biosynthesis from Mycolicibacterium smegmatis and Methylobacterium pseudosasicola were introduced into the engineered strain, which was designated CHS2. In batch cultivation, the CHS2 strain produced more EGT than the CYS-2 strain harboring the same EGT biosynthesis pathway. Interestingly, batch cultivation of the CHS2 strain under high osmotic pressure conditions prolonged the time for EGT production and increased the intracellular accumulation of EGT. These results suggest that increasing osmotic pressure together with engineering the biosynthesis of cysteine, histidine, and S-adenosylmethionine is an effective strategy for enhancing EGT production in recombinant C. glutamicum harboring heterologous EGT biosynthesis pathways.

• Ergothioneine production in C. glutamicum was enhanced by metabolic engineering.

• Osmotic pressure affects ergothioneine production in engineered C. glutamicum.

• Ergothioneine may function as a compatible solute in C. glutamicum.

Biopharmaceuticals, such as antibody therapeutics, are produced by culturing mammalian cells with chemically defined media that consist of more than 50 synthesized components. The screening of medium components related to culture performance and the subsequent optimization of the composition are required in the development of new modalities, host cells, and culture methods. Screening the components to be optimized is typically labor-intensive. The easiest approach is media blending, which creates variations in the concentrations of the components with only liquid mixing. However, a workflow for systematically determining experimental conditions (i.e., how to blend media) has not been established. Therefore, we reassessed media blending from a mathematical perspective and proposed a workflow for the first time. In the workflow, we evaluated the use of a commercially available chemically defined media to maximize simplicity and applicability. From a mathematical perspective, we clarified that multicollinearity is an inevitable challenge in both experimental design and its analysis. Under the constraint, we showed that one of the most appropriate experimental conditions could be systematically calculated and selected by applying D-optimal design focusing on the principal components. We performed a case study of cell culture to screen medium components under 120 experimental conditions using 11 chemically defined media designed for Chinese Hamster ovary cells. The case study provided a reasonable set of components that explained the variance in viable cell concentrations, which range from 5.8 to 19.4 (× 10⁶) cells/mL. Finally, our mathematical redefinition also enabled the design of a dedicated media set for media blending.

• The constraints in media blending were clearly explained.

• A systematic workflow from blending design to analysis was proposed.

• The workflow also enabled the design of a dedicated media set for media blending.

This study presents a sustainable one-step process for converting raw sugarcane bagasse (SB) into bioethanol, highlighting the innovative use of cerium-doped iron oxide nanoparticles (CeFe₃O₄NPs). Initially, these nanoparticles facilitated simultaneous pretreatment and hydrolysis of the raw SB biomass under ambient conditions (50 °C), demonstrating direct catalytic activity by producing 6.55 ± 0.112 g/L glucose and 4.73 ± 0.143 g/L xylose within 24 h. The scalability of this approach was confirmed with similar results achieved in a larger 7.5 L-scale fermentation. A key novelty of this research lies in demonstrating the synergistic effect of CeFe₃O₄NPs with enzymatic hydrolysis. By incorporating a minimal amount of in-house generated cellulase enzymes alongside CeFe₃O₄NPs, the sugar yields dramatically increased to 23.1 ± 1.12 g/L of glucose and 13.9 ± 0.88 g/L of xylose. This indicates that CeFe₃O₄NPs are not merely catalysts but function effectively as promoters, significantly enhancing the efficiency of enzymatic process. The subsequent fermentation using Saccharomyces cerevisiae efficiently converted these sugars, including xylose, into 17.3 ± 0.98 g/L of bioethanol with a productivity of 1.44 g/L/h. Further gene expression studies using quantitative real-time PCR (qRT-PCR) analysis revealed that CeFe₃O₄NPs played a role in upregulating xylose-utilizing genes within yeast strain, leading to near-complete utilization of xylose. This stimulation of xylose metabolism is a crucial finding that significantly aids in improving the overall economics of the biomass conversion process. This integrated approach, combining magnetic CeFe₃O₄NPs with enzymatic activity and xylose metabolism, represents a significant step towards more cost-effective and scalable bioethanol production from lignocellulosic biomass.

• Eco-friendly bioethanol production from sugarcane bagasse using nanobiotechnology

• Delignification and hydrolysis of biomass by enzyme-mimicking CeFe3O4 nanoparticles

• Xylose utilization by S. cerevisiae noticed due to CeFe3O4 nanoparticles

Microbial fuel cells (MFCs) offer a promising alternative for sustainable wastewater treatment and energy recovery. However, the mechanisms underpinning electrogenic biofilm formation remain poorly understood. This study investigates the spatial and temporal dynamics of microbial community assembly using a novel multi-electrode MFC design under two substrate conditions: acetate and starch. Pre-inoculation of three designated electrodes led to successful current generation within 110 h in both MFCs, while a dispersed inoculation strategy failed to establish electrogenic biofilms despite equivalent inoculum volume. Electrode positioning significantly influenced start-up, with vertical alignment above inoculated electrodes facilitating faster colonisation and current generation than lateral spacing. Notably, starch-fed MFCs exhibited more rapid and widespread biofilm proliferation, suggesting that complex microbial consortia may disperse more efficiently than single-function electrogens. Community sequencing revealed spatial heterogeneity and a shift from diverse to more optimised anodic communities over time. Geobacter initially dominated, but community succession was shaped by substrate complexity, competition, and spatial structure. Interestingly, non-inoculated electrodes often outperformed inoculated ones, indicating that deterministic selection pressures favoured more efficient biofilms. However, long-term current production declined, particularly under batch conditions, suggesting that population drift and limited microbial renewal limited sustained performance. This study is the first to characterise electrogenic biofilm assembly in a multi-electrode MFC, highlighting the interplay between stochastic dispersal and deterministic selection. These findings underscore the importance of inoculation strategy, substrate selection, and continuous microbial replenishment for optimising MFC performance and real-world applicability.

• Substrate complexity shaped colonisation and distinct microbial communities.

• Vertical electrode positioning enhanced colonisation and start-up efficiency.

• Temporal succession led to specialised but less diverse electrogenic biofilms.