Biotechnology Letters最新文献

The engineered Aspergillus niger strain AnCALB005 was selected as the research strain, which is a high-yield strain of Candida antarctica B lipase constructed in our laboratory. CRISPR/Cas9-mediated gene knockout was employed to construct the multiple protease-deficient strains targeting five genes (pepA, pepB, pepC, pepE and pepF) in the A. niger AnCALB005. Among the engineered variants, a triple-knockout strain lacking pepA, pepB, and pepF demonstrated 56% enhanced hydrolytic lipase activity relative to the parental strain. Fermentation culture conditions were initially screened through single-factor experiments. Building on these results, critical parameters were statistically determined via Plackett-Burman (PB) design. This was followed by a steepest ascent method combined with Box-Behnken (BB) response surface methodology. Key factors influencing lipase production (identified as maltose concentration, corn steep concentration, and shaking speed) were optimized. The final optimized fermentation conditions comprised: maltose (52 g/L), corn steep (52 g/L), K₂HPO₄ (5 g/L), soybean cake flour (30 g/L), initial pH 6.5, inoculation amount 10% (v/v), and shaking speed 220 rpm. Under the optimized fermentation conditions, Shake-flask validation of the engineered A. niger yielded a lipase activity of 46.66 U/mL, representing an increase of 92.01%. Scale-up fermentation in a 5 L bioreactor applying these optimized conditions over 120 h of cultivation achieved a lipase activity of 79.31 U/mL.

For the reconstitution of artificial cell membranes, silica microbeads are often selected as scaffolds to reinforce fragile lipid membranes; these bilayer-covered beads are often called spherical supported lipid bilayer membranes (SS-BLMs). SS-BLMs were made through the adsorption of small unilamellar vesicles (SUVs) preformed from a mixture of phosphatidylcholine (PC) and phosphatidylserine (PS). Fluorescence microscopic observation showed that budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) labeled with octadecyl rhodamine B chloride (R18) could fuse and be optimally distributed homogeneously over the SS-BLMs when using a combination of PC and PS. We also demonstrated that when BVs expressing the recombinant transmembrane protein, G-protein coupled receptor (GPCR), like β2 adrenergic receptor and corticotropin-releasing hormone receptor 1, the protein could be specifically visualized on SS-BLMs by immunofluorescence microscopy. This indicates that recombinant proteins were incorporated into the spherical supported lipid bilayers through virion-lipid bilayer membrane fusion.

Purpose: Phosphoinositide-specific phospholipase C plays a crucial role in the trans-membrane and lipid signal transduction. Previous studies have elucidated the biological functions of PI-PLC. However, its function mechanism has not been fully understood.

Results: We present a novel approach to analyze the in vitro activity of phospholipase C from Populus simonii x P. nigra. The results indicate that the optimal condition for PsnPLC activity is pH 7.4/10 μM Ca²⁺. The increase of Ca²⁺ and pH, as well as the deletion of the EF-hand domain or C2 domain, will lead to the reduced PsnPLC activity. Electrophoresis migration shift assays confirm that PsnPLC could bind Ca²⁺ in vitro, while conditions of Ca²⁺ binding and pH 8.8 might lead to its degradation.

Conclusion: The findings identify Ca²⁺ and pH as key factors modulating PsnPLC function, which will provide evidence linking PI-PLC and the Ca²⁺ signaling network.

As demand for rare earth elements (REEs) increases, biotechnological solutions to their extraction and purification are becoming more critical. Identification of chassis organisms capable of surviving in high REE concentrations is a crucial step towards development of sustainable biotechnologies. In this study, we show that growth of mesophilic Escherichia coli is not significantly inhibited by < 300 µM REEs in complex media of, but in phosphate-depleted minimal media a half-maximum inhibitory concentration (IC₅₀) for REEs is 80 ± 100 µM. REE-utilizing mesophile Pseudomonas alloputida KT2440 is growth-impaired in presence of REEs, with an IC₅₀ of 100 ± 20 µM. In contrast, we demonstrate that extreme acidophiles survive in conditions of 500 µM REEs without apparent inhibition of growth. Indeed, we observe that hyperacidophilic bacteria Acidithiobacillus ferrooxidans, A. thiooxidans, and A. caldus appear to enter log phase earlier in presence of REEs and grow to significantly higher densities. Acidophilic archaeon Sulfulobus acidocaldarius, a hyperthermophile, is similarly not significantly inhibited by 500 µM REEs. These data indicate that acidophiles have broad-range stress tolerance mechanisms that apply to REE stress. Importance: Rare earth elements (REEs) are essential for a diverse and expanding range of high-technology applications, which are an important part of the industrial economy. Application of biomining technologies could introduce "greener" extraction and processing steps, however, many fundamental challenges must be addressed before a biological approach to REEs recovery and separation can be fully adopted at scale. Fundamentally, any chassis organism used for REE biomining must be able to tolerate high concentrations of REEs, and existing literature along with this study demonstrate that neutrophilic mesophiles are highly sensitive to REEs. In contrast, we demonstrate that mesophilic extreme acidophiles and thermophilic acidophiles exhibit inherent REE tolerance. This phenomenon highlights their potential for bioprocessing and corroborates existing evidence that the extremophile response to REE presence may allow for broader adaptation behavior in comparison to other model chassis strains.

Objective: This study aimed to isolate an efficient bacterial cellulose (BC)-producing strain from rotten mango, optimize BC production though single-factor tests followed by Box-Behnken design (BBD) under agitated culture conditions using tobacco waste extract as the medium, and conduct the characterization of BC via Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and X-ray diffraction (XRD).

Results: Komagataeibacter sp. XMZ1 with a high-efficiency BC biosynthesis capacity was successfully isolated from rotten mango. Subsequently, BBD was utilized to optimize the co-supplementation of lactic acid, ethanol, and yeast extract, demonstrating this statistical approach to be a reliable tool for both optimization and predictive modeling of BC yield. The optimized medium containing 2.1 g/L lactic acid, 1% (v/v) ethanol, and 2 g/L yeast extract resulted in a 2.1-fold enhancement of BC production, increasing the yield from 4.20 g/L to 8.85 g/L. The characterization of BC through FTIR, SEM and XRD revealed that BC produced under agitated culture conditions maintained similar chemical functional groups as the BC produced under static culture. However, the agitated culture products exhibited a more porous fibrillar network and a reduced crystallinity index.

Conclusion: This present study provides valuable insights into BC production under agitated culture conditions by making use of tobacco waste. Additionally, it offers BC structural characterizations that are relevant to potential industrial applications.

Thermostable α-amylase from Geobacillus sp. DS3, isolated from the Sikidang Crater, Dieng Plateau, Indonesia, was previously purified and characterized. However, production from thermophilic bacteria requires high-temperature cultivation. This study aimed to clone and express the amy gene encoding α-amylase in Escherichia coli BL21(DE3) for easier enzyme production. The amy gene (1638 bp) was amplified via PCR, TA-cloned, and inserted into the pET-SUMO expression vector, which includes an N-terminal His-tag and SUMO-tag to enhance expression and solubility. The recombinant plasmid (pET-SUMO-amy) was transformed into E. coli BL21(DE3) for protein expression. Homology modelling using MOE software and template PDB ID 1HVX (91.5% identity) revealed a reliable 3D structure. Structural analysis showed altered calcium and sodium ion binding compared to the template, with calcium ions interacting with more residues. Docking studies revealed that maltotetraose binding is stabilized by five key residues: Asp268, His272, Trp300, Asn363, and Asp365. The enzyme displayed optimal activity at 70 °C and retained 60% activity at 90 °C. Kinetic parameters showed a low K_m (6.77 mM) and V_max (0.20 U/mL), indicating high substrate affinity. In conclusion, the recombinant α-amylase exhibited thermostability and substrate affinity suitable for industrial applications such as starch liquefaction and porous starch production at elevated temperatures.

Purpose: Several scientific studies have revealed the role of probiotic lactic acid bacteria and their metabolites, including antioxidant activity, which is required for reducing oxidative stress, and have implications for cancer treatment. This study evaluated the ability of isolates to exert antioxidative and in vitro anti-tumor effects against the HCT-116 colon cancer cell line, with fewer side effects compared to chemotherapy.

Methods: The in vitro antioxidative capacity of LAB isolates was quantified against DPPH free radicals. The cytotoxicity effects against the HCT-116 cells were analyzed using the MTT assay, microscopic fluorescence, flow cytometry, and qRT-PCR analysis of immunomodulatory cytokines and cancer-related gene expression.

Results: Seven LAB isolates exhibited potent antioxidant activity, scavenging 51.20 to 78.29% of DPPH free radicals. Lactobacillus delbrueckii subsp. bulgaricus (RE 254) demonstrated superior activity, with 78.29% scavenging and a ferric-reducing activity power of 38.04%. Conversely, Lactiplantibacillus plantarum (RE 298) showed 53.67% and 16.08% activity, respectively. At a concentration of 25 × 10³ CFU/ml, both strains induced pronounced cytotoxic effects on HCT-116 cells after 72 h (71.1% for RE 254 and 62.5% for RE 298). This cytotoxicity was mediated by apoptosis, driven by the upregulation of IL-2 and the concomitant downregulation of key genes, including BCL-2, PARK, TARC, LIF, IL-4, IL-6, CD1A, and CD1B.

Conclusion: The findings strongly suggest that RE 254 and RE 298 are compelling candidates for use as functional supplements and adjunctive therapeutics in colon cancer management. Their efficacy, demonstrated through multifaceted anti-tumor mechanisms, warrants further validation through in vivo studies to assess their clinical potential.

Diseases are a global threat to rice production. Among them, rice blast and bacterial blight (BB) are most significant. To enhance targeted disease resistance, the marker-assisted backcross breeding (MABB) approach was employed to pyramid two blast (Pi9 and Pb1) and three BB resistant (R) genes (Xa4, xa13, and Xa21) into a high-quality rice cultivar BRRI dhan100. The donor parents Pi9-US2 (Pi9), Pb1-US2 (Pb1), and IRBB58 (Xa4, xa13, and Xa21) contributed these five genes. We followed a triple-cross breeding strategy, followed by backcrossing, selfing, and foreground selection to produce BC₃F₅ progenies. Chi-square analysis of 420 BC₃F₂ individuals confirmed the inheritance patterns of blast and BB resistance were controlled by a simple Mendelian fashion. Finally, we selected 38 advanced lines (ALs), and among them, twelve lines possessed all 5 candidate R genes, while fifteen consisted of 4 genes in different combinations. The disease rating scale of these ALs varied from 0 to 3 for both blast and BB diseases, while BRRI dhan100 ranging from 7 to 9. The G23, G13, G5, G10, G15, and G29 ALs exhibited higher yields ranging from 7.22-7.34 ton ha^-1 compared to the check, BRRI dhan100. Marker-trait association analysis displayed molecular markers negatively associated with disease susceptibility. Therefore, gene introgression by MABB could effectively identify and functionally validate the candidate R genes with high accuracy to develop a resistant variety to multiple diseases in rice breeding programs.