Biotechnology Letters最新文献

Purpose: Bacillus subtilis spores, known for their durability and well-developed genetic manipulation tools, show promise for oral vaccine delivery. Staphylococcus aureus is a major global health concern due to multidrug resistance and lack of a vaccine. This study explored the potential of B. subtilis spores engineered to express IsdB, a key S. aureus iron acquisition protein, on their surface and evaluated the immunogenic effect of recombinant spores through oral administration in mice.

Method: B. subtilis spores were engineered to display a key S. aureus protein, IsdB, fused with spore coat protein, CotB, and confirmed by PCR. Western blotting, sporeELISA, and immunofluorescence microscopy verified the surface expression of IsdB. The engineered BsHT2377 spores were orally administered to Swiss mice at two different doses, and antibody levels in both serum and feces were measured using ELISA.

Result: PCR confirmed the targeted integration of the isdB gene into B. subtilis, generating the strain BsHT2377. Western blotting, sporeELISA, and immunofluorescence microscopy validated IsdB surface display on BsHT2377 spores. Oral administration triggered a gut-localised immune response in mice, with significantly elevated fecal IgA but no substantial increase in serum IgG.

Conclusion: This study engineered a novel B. subtilis strain, BsHT2377, that displays the S. aureus IsdB protein on its spore surface. Oral administration in mice significantly increased fecal IgA, indicating a mucosal immune response. These findings highlight the potential of B. subtilis spores as oral vaccine carriers and offer insights to support the optimization of future vaccine designs.

The macrolide antibiotics spinosad, synthesized by Saccharopolyspora spinosa, is a highly effective, environmentally-friendly insecticide. However, due to poor fermentation performance and difficulties in engineering S. spinosa strains, the production cost of spinosad is still high, which restricts its industrial application. The industrial strains used for antibiotic production primarily originate from mutagenic screening, whereas traditional screening methods are time-consuming and laborious. Here, an in vitro detection method for spinosad was established to accelerate the breeding of mutated strains of S. spinosa. Firstly, a broad substrate promiscuity glycosyltransferase OleD from Streptomyces antibioticus was selected and employed for the detection of pseudoaglycone (PSA), which serves as the precursor compound for spinosad, through the utilization of colorimetric reactions coupled with glycosylation. Subsequently, the in vitro PSA detection system was optimized and applied for S. spinosa high throughput screening. The final selected mutant strain DUA15 was obtained and showed a 0.80-fold and 0.66-fold increase in spinosad and PSA production compared to the original strain, respectively. Furthermore, genetic engineering technology was combined to obtain the engineered strain D15-102, which showed a 2.9-fold increase in spinosad production compared to the original strain.

Dark septate endophytes (DSE) are widely used in ecological restoration. In this study, we first discovered that the metabolic products of the DSE strain Alternaria sp. 17463 contain extracellular polymeric substances (EPS). This study aimed to optimize the culture conditions, characterize the structural composition of EPS, and evaluate its effects on soil improvement. The ethanol precipitation method was used to extract EPS from the metabolites of Alternaria sp. 17463, increasing the yield 4.48-fold to 6.96 g L⁻¹ through optimized cultivation conditions. The molecular mass of EPS was 533.754 kDa, with an exopolysaccharide content of up to 77.8% and a protein content of 8.4%. The EPS were mainly composed of glucan units T)-Glc p-(1 → and → 4)-Glc p-(1 → , with minor fractions of mannose and galactose. By acting as a cementing agent, EPS strengthened the linkages between soil particles. At an optimal concentration, it significantly enhanced the activity of extracellular enzymes involved in carbon, nitrogen, and phosphorus metabolism, thereby improving soil nutrient transformation and cycling. This also promoted changes in the metabolism of small molecules in the soil. This research reveals why this strain can accelerate ecological restoration and lays the foundation for further exploration of its metabolic products and potential applications in this field, particularly in open-pit dump reclamation and agricultural soil improvement.

Chlamydomonas reinhardtii CW15, a cell-wall-deficient strain, is a widely used chassis in microalgal genetic engineering. However, its low cell density under autotrophic and mixotrophic cultivation restricts industrial scalability. This study aimed to establish a cost-effective, high-density heterotrophic cultivation process for C. reinhardtii CW15. Initially, an optimized AP medium (Tris-free TAP with K₂HPO₄ as the sole phosphorus source) significantly reduced medium costs while achieving a cell density of 0.446 g/L. Subsequently, a fed-batch process in a 5 L fermentor yielded a record cell density of 22.1 g/L (growth rate: 94.6 mg/L·h) at 233 h by optimizing nutrient supplementation. Scaling up to a 50 L fermentor revealed challenges in ammonia volatilization and salinity accumulation during sterilization. These were resolved by substituting the nitrogen source with ammonia water and supplementing ammonium acetate as a dual nitrogen and partial carbon source, ultimately achieving 30.2 g/L (140 mg/L·h) after 215 h-the highest reported density for cell-wall-deficient C. reinhardtii. This work provides an efficient and scalable cultivation strategy, facilitating its industrial and biotechnological applications.

Lepidoptera has caused huge economic losses to a variety of agricultural and forestry plants. The prevention and control measures of pests and diseases include agricultural, physical, chemical, and biological control. Microbial pesticides (including bacteria, fungi and viruses) were more and more widely used in the prevention and control of lepidopteran pests. They represent the development trend of the pesticide industry in the future and realize the sustainable control of major pests and diseases. As a novel strategy for pest control, a large number of microbial insecticides have been used in the actual production process. Although it has many advantages compared with chemical pesticides, it also has its own drawbacks. In the future, due to the search for new strategies, a variety of techniques are used in combination to form a large-scale application of microbial pesticide formulations, and continue to strengthen the application of microbial pesticides in the future lepidoptera pest control.

Chitosan beads (CB) and calcium alginate beads (CAB) are widely used for immobilizing Saccharomyces cerevisiae in fermentation, but their low mechanical strength and limited surface area reduce ethanol yield. To overcome these limitations, ferric oxide (Fe₂O₃) nanoparticles were incorporated into CB and CAB to enhance both mechanical strength and surface area. The modified carriers were employed for Saccharomyces cerevisiae immobilization in a bubble column bioreactor under semi-batch fermentation conditions at 35 °C, an air flow rate of 0.01 L/min, and pH 4.0 for 15 h. The Fe₂O₃ nanoparticles incorporation significantly improved the rupture forces of CB and CAB, increasing from 2 ± 0.05 N to 8 ± 0.5 N and 2 ± 0.05 N to 9 ± 0.07 N, respectively. The surface area of CB increased from 18 ± 0.3 m²/g to 48 ± 0.2 m²/g, while CAB increased from 2 ± 0.2 m²/g to 50 ± 0.1 m²/g, leading to enhanced cell adsorption from 1.13 × 10⁸ to 1.10 × 10⁹ cells/mL Consequently, ethanol yield improved from 37 ± 0.28% to 45 ± 1.23%. Unlike unmodified CB and CAB, which exhibited significant rupture after five reuse cycles, the modified beads retained their structural integrity and activity, demonstrating their durability for yeast immobilization and reuse. This approach offers a promising strategy for enhancing fermentation efficiency and carrier stability in ethanol production.

Pseudomonas aeruginosa (P. aeruginosa) is a significant opportunistic pathogen associated with nosocomial infections, particularly in pediatric populations, where it can lead to severe clinical manifestations, including P. aeruginosa-associated meningitis. To meet the critical need for highly sensitive detection of P. aeruginosa, a novel fluorescent biosensor utilizing a three-way junction (TWJ) probe has been developed. This biosensor capitalizes on the specific binding interaction between P. aeruginosa and a tailored aptamer embedded within a DNA TWJ structure. Upon target binding, the double-stranded DNA branches of the TWJ undergo a conformational rearrangement, resulting in the formation of two distinct DNA "Y" junction structures. These structures are subsequently linked by a designed sequence, initiating a DNA polymerase/endonuclease-mediated strand displacement amplification process. The TWJ-based biosensor offers several key advantages: (i) the integration of the aptamer sequence within the TWJ probe ensures high specificity for target recognition, and (ii) the subsequent enzymatic amplification significantly enhances the sensitivity of detection. Under optimized experimental conditions, the biosensor demonstrated a broad linear detection range from 10 to 10 to 10⁵ cfu/mL, with an exceptionally low limit of detection of 4.12 cfu/mL. Recovery studies further confirmed the reliability and robustness, highlighting its potential for clinical implementation. This innovative bio-sensing strategy represents a significant advancement in diagnostic technology, offering a promising tool for the early and accurate detection of infectious diseases in pediatric patients, with potential applications in improving clinical outcomes.

Objectives: Expressing cellulase systems in C. glycerinogenes with extracellular secretion ability, and using fermentation with residue, enabled the recombinant strain to degrade lignocellulosic waste for efficient glycerol production, offering new option for agricultural waste transformation.

Results: Candida glycerinogenes is employed as the host strain, various cellulases were screened. Signal peptide mining and screening, and semi-rational design strategy was adopted in the host strain. The recombinant strain Cg4 had a total cellulase activity of 13.6 U/mL. Cg4 was applied to 110 g/L sugarcane bagasse hydrolysate with 40 g/L of pretreated bagasse as the substrate. The glycerol yield reached 50 g/L, with a 43.3% bagasse utilization rate, the amount of cellulase used in the degradation of lignocellulose was reduced by 12.1%.

Conclusion: Opened up a route for efficient degradation of agricultural waste of lignocellulose by C. glycerinogenes for glycerol production, the emission of lignocellulose waste had been reduced, and decreasing the need for cellulases in hydrolysis.

The cost-effective and high-yield production of hyaluronic acid (HA) by microbial means remains challenging, necessitating the optimization of existing processes through the implementation of novel approaches. The present study investigates the production of HA under varying temperature and pH conditions utilizing independent, fully controlled fermenters. The synthesis of HA was evaluated at four temperatures (32 °C, 35 °C, 37 °C, 40 °C) and four pH levels (6.5, 7.0, 7.5, and 8.0), with optimal parameters identified through the Taguchi design methodology. The Taguchi optimization method effectively identified 37 °C and pH 6.5 as the optimal conditions for HA production corresponding to the highest signal-to-noise (S/N) ratios of 58.18 and 57.55, respectively. These conditions resulted in a maximum yield of 1.08 g.L^-1, demonstrating the efficacy of this parameter combination in maximizing production efficiency. The carbazole method was utilized to quantify the production of HA following a five-hour fermentation period, with the culture conditions subjected to a statistical comparison. A temperature of 37 °C yielded significantly higher HA levels than 32 °C and 40 °C (Dunn test, respectively p = 0.019, p = 0.001). The lowest HA production was observed at 40 °C, while the 32 °C group exhibited relatively low variation in HA production. Furthermore, the hourly bacterial counts demonstrated a direct correlation between bacterial proliferation and HA synthesis, with the highest bacterial growth observed at 37 °C and pH 7.0. This study highlights the trends in HA concentration under different temperature and pH conditions during the pre-fermentation phase, offering critical insights for optimizing HA production.

The enzymatic conversion of lactose to galactooligosaccharides (GOS) within raw milk offers a promising avenue for reducing lactose content while enhancing its prebiotic benefits. This process hinges on utilizing enzymes with high transglycosylation activity compatible with current milk processing methods. In the present studies, Bacillus circulans β-galactosidase (BglD) was identified as an effective enzyme, achieving a lactose conversion rate of 73.61% in milk. However, BglD's resistance to pasteurization underscored the need for enzyme modifications. Through molecular directed evolution, a mutant T473L/R484P was engineered. It exhibited improved catalytic performance at lower temperatures, with optimal activity at pH 6.5 and 55 °C. In contrary to the wild-type, mutant T473L/R484P exhibited reduced thermostability, and its activity diminished rapidly above 50 °C. Remarkably, in simulated dairy environments, mutant T473L/R484P converted over 78% to 82% of lactose at low temperatures (5 to 10 °C), reducing lactose to around 10.4 to13.0 g/L while generating approximately 30 to 34 g/L of GOS. These findings highlight the mutant's potential in producing low-lactose, GOS-enriched milk, and pave the way for innovative in situ lactose-to-GOS conversion processes in raw milk.