Journal of biotechnology最新文献

Heavy metal pollution is a worldwide problem that threaten agricultural production and human health. Methyl jasmonate (MeJA) is a phytohormone that could enhance plant resistance against various stresses. However, the mechanism of MeJA in cadmium (Cd) uptake, distribution, and translocation in rice plants remains elusive. In this study, we found that the Cd induced-growth inhibition was ameliorated by MeJA. Upon MeJA application, Cd content in root and shoot was decreased by 10.15% and 36.39%, which paralleled with less Cd²⁺ influx of rice roots and depressed expression of the cation transporters (OsNramp1 and OsNramp5). The subcellular distribution revealed that MeJA enriched Cd distribution in cell wall, which was accompanied by increased cell wall thickness and altered cell wall polysaccharide (pectin, cellulose, hemicellulose) content, meanwhile, the Cd content in pectin, cellulose, hemicellulose was increased, the FTIR analysis implied that functional groups (especially -OH and COO-) on cell wall were involved in Cd fixation. The root to shoot translocation of Cd was hindered by exogenous MeJA, this was validated by the decreased expression of OsHMA2 in root and declined Cd level in xylem sap. Overall, our results revealed that MeJA could act as a foliar resistance control substance to reduce Cd accumulation in rice plants. The detailed molecular mechanisms of MeJA in Cd detoxification in plants still need further investigation.

The Pichia pastoris expression system is a favorable platform for production of pharmaceutical proteins. Treatment of strains with N-acetyl-L-cysteine (NAC) has been shown to enhance the yield of recombinant proteins, thereby contributing to a reduction in production costs. However, the specific mechanism of action of NAC remains unclear. Previous research has indicated that glutathione (GSH) and autophagy are involved in the increased production of human serum albumin and porcine follicle-stimulating hormone β (HSA-pFSHβ) by NAC. This study investigated the potential interaction between GSH and autophagy in the production of HSA-pFSHβ. The findings indicated that sulfhydryl-free antioxidants such as melatonin, vitamin C, or vitamin E did not exhibit similar effects to NAC in enhancing HSA-pFSHβ yield. Moreover, NAC was found to enhance HSA-pFSHβ production by modulating GSH metabolism to reduce GSH consumption, increase total GSH levels, as well as glutathione peroxidase (GSH-Px) and glutathione reductase (GR) activities. Additionally, inhibition of autophagy through disruption of autophagy scaffolding proteins Atg1 or Atg11 led to an increase in recombinant HSA-pFSHβ production. Furthermore, NAC significantly decreased the phosphorylation of Slt2, and the absence of the SLT2 gene influenced the effect of NAC on HSA-pFSHβ secretion by modulating mitophagy and GSH metabolism. In conclusion, these results suggest a complex interplay between GSH metabolism and autophagy in the regulation of NAC-induced HSA-pFSHβ secretion.

This study investigates the crucial role of transfection methods in the manufacturability and potency of recombinant adeno-associated virus (rAAV) gene therapies. By employing a novel analytical approach, multiplex digital PCR (dPCR), we evaluated the impact of different transfection reagents and conditions on the scalability and quality of rAAV. Our research demonstrates that the selection of transfection approach significantly influences not only the yield and ease of scale-up but also the potency of the final product. Importantly, later changes to transfection parameters established in the early stages of development can be challenging, potentially compromising product quality and leading to comparability issues. Leveraging multiplex dPCR has proven instrumental in guiding these early-stage decisions, ensuring a reliable manufacturing process that consistently delivers high-quality therapeutic products. Our findings highlight the importance of optimizing transfection strategies early in development to guarantee the successful production of potent and cost-effective gene therapies, ultimately impacting patient accessibility.

Cold-active pullulanases with good catalytic performance possess promising applications in cold hydrolysis of starch. Adopting bioinformatics-assisted mining strategies, 7 candidate cold-active pullulanases were initially screened out from IMG/MER database. Among the candidates, PulBs exhibited good thermostability and the highest specific activity of 147.4 U/mg. The half-life of PulBs was about 200 h at 35 °C. Employing PulBs as the initial enzyme, the active-site design of FuncLib was implemented to enhance the activity. The design PulBs-20 exhibited an enhanced specific activity of 209.9 U/mg, which was 1.4 times that of PulBs. Furthermore, the thermostability of PulBs-20 was augmented, with a half-life of 250 h at 35 °C. When applied in the cold hydrolysis of starch, PulBs-20 can effectively enhance the hydrolysis effect of raw starch. Supplemented with the raw starch-hydrolyzing α-amylase AmyZ1 and PulBs-20, the hydrolysis rate of raw corn starch increased to 53.5 %, which was 1.3 times that of using AmyZ1 alone. Due to its high hydrolysis activity and good thermostability, PulBs-20 can serve as an efficient accessory enzyme in starch cold hydrolysis.

In order to improve the production and quality of lipid of D. intermedius Z₈ under the furfural stress environment, different exogenous γ-aminobutyric acid (GABA) addition strategies and their reasons for improving the fermentation performance were investigated. For this purpose, the effect of different concentrations of furfural on the production of biomass and lipid in D. intermedius Z₈ was researched. The result shows that the growth of D. intermedius Z₈ nearly stopped when 1.0 g/L furfural was adopted. However, when 1.5 mmol/L GABA was used, the highest biomass and lipid production of 4.56 g/L and 1.83 g/L were obtained, respectively, which were 36.53 % and 61.95 % higher compared to the control group (the normal development BG11 medium). Additionally, the changes in microalgal cell morphology were analyzed using the scanning electron microscope (SEM) technology, and the results suggest that GABA addition could significantly mitigate the toxic effects of excessive furfural by maintaining the integrity of the cell. Simultaneously, the activities of key enzymes involved in fatty acid synthesis, such as nitrate reductase (NR), phosphatidic acid phosphatase (PAP), acetyl CoA carboxylase (ACC), and fatty acid synthase (FAS), were enhanced under the optimal condition, and thus improving lipid production. According to the results in this study, the exogenous GABA addition strategy is a simple and effective approach to improve microbial tolerance and enhance its fermentation performance.

Surface display technology has garnered significant attention for preparing efficient whole cell catalysts, while reported carrier proteins still cannot meet the demand to display various passenger domains, especially for those with high molecular weight. This study demonstrates that the autotransporter of esterase Est7 (E7AT) from Stenotrophomonas maltophilia played a decisive role in its efficient surface display. Guided by the original signal peptide, the surface display ratio of Est7 was determined as 89.67% with the total enzymatic activity of 16.67U/mL, which was much higher than 4.60U/mL and 5.70U/mL for the signal peptides derived from pectolase B (pelB) and cholera toxin B (ctxB), respectively. Then, the E7AT unit was successfully developed to surface display proteins with varying molecular weight from 19.3kDa to 117.9kDa, showing a more effective autodisplay ability than adhesin involved in diffuse adherence (AIDA-I), ice nucleation proteins (InaK and InaP), and outer membrane proteins (lipoprotein/ompA, MltA-interacting protein A, and yiaT) systems. Additionally, a galactosidase (GAL) displayed by E7AT was employed to hydrolyze lactose, achieving a promising hydrolysis rate of 31.63% in 2h. The displayed GAL retained 63.24% and 41.41% activity in third and sixth batch, respectively, indicating the considerable potential of E7AT in developing efficient whole cell catalysts.

Spermidine has broad application potential in food, medicine and other fields. In this study, a novel Bacillus amyloliquefaciens cell factory was constructed for production of spermidine from renewablebiomass resources. Firstly, the speB gene was found to be optimal for synthesis of spermidine, and the function of SpeB was explained by amino acid sequence analysis and molecular docking. By replacing the native promoter of the speEB operon with the P43, the synthesis of spermidine was significantly enhanced in B. amyloliquefaciens HSPM1-P43speEB. After knockout of the genes yobN and bltD associated with spermidine degradation, the spermidine titer of the strain HSPM2 was further improved to 115.96 mg/L, increased by 108 % compared to HSPM1-P43speEB. Subsequently, the titer of spermidine was further increased to 277.47 mg/L through enhancing the supply of the precursor methionine by overexpression of speD. Finally, the renewable biomass resources, xylose and feather meal were optimized to produce spermidine, and the maximum titer is up to 588.10 mg/L after optimization. In conclusion, an efficient spermidine producing B. amyloliquefaciens was constructed through combinatorial metabolic engineering strategies, and the sustainable production of spermidine was achieved using the biomass resources of xylose and feather meal.

RNA (Ribonucleic Acid) is an essential component of organisms and is widely used in the food and pharmaceutical industries. Saccharomyces cerevisiae, recognized as a safe strain, is widely used for RNA production. In this study, the S. cerevisiae W303-1a was used as a starting strain and molecular modifications were made to the functional ncRNA-SRG1 to evaluate the effect on RNA production. At the same time, its transcriptionally associated helper genes (Spt2, Spt6 and Cha4) were overexpressed and the culture medium was supplemented with serine to induce SRG1 transcription, to increase SRG1 transcription levels and investigate its effect on intracellular RNA levels. The results showed that the intracellular RNA content of the recombinant strain W303-1a-SRG1 was 10.27 %, an increase of 11.15 % compared to the starting strain (W303-1a, with an intracellular RNA content of 9.24 %). On this basis, a gene co-overexpression strain-W303-1a-SRG1-Spt6 was constructed. Simultaneously, the addition of 2 % serine strategy was used to increase the transcription level of SRG1 and RNA content of the recombinant strain. The intracellular RNA of the recombinant strain reached 11.41 %, an increase of 23.38 % compared to the starting strain (W303-1a, without serine supplementation). In addition, the growth performance of the strain was assessed by measuring the SRG1 transcription level in the strain and plotting the growth curve. Therefore, we found that improving the transcription level of ncRNA can be used as a new idea to construct S. cerevisiae with high RNA content, which provides a strong help for subsequent research in related fields. This work provides a new strategy for increasing the nucleic acid content of S. cerevisiae.