Biotechnology Letters最新文献

The pernicious nature of low-quality sequencing data warrants improvement in the bioinformatics workflow for profiling microbial diversity. The conventional merging approach, which drops a copious amount of sequencing reads when processing low-quality amplicon data, requires alternative methods. In this study, a computational workflow, a combination of merging and direct-joining where the paired-end reads lacking overlaps are concatenated and pooled with the merged sequences, is proposed to handle the low-quality amplicon data. The proposed computational strategy was compared with two workflows; the merging approach where the paired-end reads are merged, and the direct-joining approach where the reads are concatenated. The results showed that the merging approach generates a significantly low number of amplicon sequences, limits the microbiome inference, and obscures some microbial associations. In comparison to other workflows, the combination of merging and direct-joining strategy reduces the loss of amplicon data, improves the taxonomy classification, and importantly, abates the misleading results associated with the merging approach when analysing the low-quality amplicon data. The mock community analysis also supports the findings. In summary, the researchers are suggested to follow the merging and direct-joining workflow to avoid problems associated with low-quality data while profiling the microbial community structure.

Pentachlorophenol (PCP) was once used as a pesticide, germicide, and preservative due to its stable properties and resistance to degradation. This study aimed to design a biosensor for the quantitative and prompt detection of capable of PCP. A cell-free fluorescence biosensor was developed while employing NalC, an allosteric Transcription Factor responsive to PCP and In Vitro Transcription. By adding a DNA template and PCP and employing Electrophoretic Mobility Shift Assay while monitoring the dynamic fluorescence changes in RNA, this study offers evidence of NalC's potential applicability in sensor systems developed for the specific detection of PCP. The biosensor showed the capability for the quantitative detection of PCP, with a Limit of Detection (LOD) of 0.21 μM. Following the addition of Nucleic Acid Sequence-Based Amplification, the fluorescence intensity of RNA revealed an excellent linear relationship with the concentration of PCP, showing a correlation coefficient (R²) of 0.9595. The final LOD was determined to be 0.002 μM. This study has successfully translated the determination of PCP into a fluorescent RNA output, thereby presenting a novel approach for detecting PCP within environmental settings.

Objectives: This study aimed to discuss the essential amino acid residues and catalytic mechanism of trans-epoxysuccinate hydrolase from Pseudomonas koreensis for the production of meso-tartaric acid.

Results: The optimum conditions of the enzyme were 45 °C and pH 9.0, respectively. It was strongly inhibited by Zn²⁺, Mn²⁺ and SDS. Michaelis-Menten enzyme kinetics analysis gave a K_m value of 3.50 mM and a k_cat of 99.75 s^-1, with an exceptional EE value exceeding 99.9%. Multiple sequence alignment and homology modeling revealed that the enzyme belonged to MhpC superfamily and possessed a typical α/β hydrolase folding structure. Site-directed mutagenesis indicated H34, D104, R105, R108, D128, Y147, H149, W150, Y211, and H272 were important catalytic residues. The ¹⁸O-labeling study suggested the enzyme acted via two-step catalytic mechanism.

Conclusions: The structure and catalytic mechanism of trans-epoxysuccinate hydrolase were first reported. Ten residues were critical for its catalysis and a two-step mechanism by an Asp-His-Asp catalytic triad was proposed.

Purpose: Fed-batch cultures have rarely been used in single cell protein (SCP) research. This work evaluated multiple yeast species for suitability as SCP cultivated using glucose- and sucrose-based substrate and performed in-depth studies of fed-batch SCP cultivation kinetics for selected yeasts, including determination of specific crude nitrogen-to-protein conversion factors.

Methods: SCP was cultivated using fully synthetic media in flask batch or bioreactor fed-batch cultures. Crude nitrogen and nucleic acid content were determined using the Dumas method and fluorescence assay kits, respectively.

Results: C. utilis compared favorably to other yeasts in flask batch cultures in terms of process yield (0.52 ± 0.01 g_x g_s^-1) and crude nitrogen content (10.0 ± 0.5 and 9.9 ± 0.5%_CDW for glucose and sucrose, respectively). This is the first time biomass composition data was reported for SCP cultivated in fed-batch mode. C. utilis crude nitrogen content was consistent across the tested conditions (protein content stabilized around 50%_CDW in fed-batch), while that of the benchmark yeast S. cerevisiae was higher in batch cultures and at the beginning of fed-batch relative to the end (protein content decreased over time and stabilized around 43%_CDW). Total nucleic acid content of the yeasts was similar (6.8%_CDW and 6.3%_CDW, for C. utilis and S. cerevisiae, respectively), with crude nitrogen-to-protein conversion factors of 4.97 and 5.80.

Conclusion: This study demonstrated the suitability of C. utilis as SCP, notably the robustness of its crude nitrogen content (as an indicator of protein content) across batch and fed-batch conditions, compared to that of the benchmark yeast S. cerevisiae.

Pearl millet (Cenchrus americanus) is a cereal crop that can tolerate high temperatures, drought, and low-fertility conditions where other crops lose productivity. However, genes regulating this ability are largely unknown. Transcription factors (TFs) regulate transcription of their target genes, regulate downstream biological processes, and thus are candidates for regulators of such tolerance of pearl millet. PgWRKY74 encodes a group IIc WRKY TF in pearl millet and is downregulated by drought. PgWRKY74 may have a role in drought tolerance. The objective of this study was to gain insights into the physiological and biochemical functions of PgWRKY74. Yeast one-hybrid and gel shift assays were performed to examine transcriptional activation potential and deoxyribonucleic acid (DNA)-binding ability, respectively. Transgenic Arabidopsis thaliana plants overexpressing PgWRKY74-green fluorescent protein (GFP) fusion gene were generated and tested for growth and stress-responsive gene expression under mannitol and NaCl-stressed conditions. A construct with PgWRKY74 enabled yeast reporter cells to survive on test media in the yeast one-hybrid assays. The electrophoretic mobility of DNA with putative WRKY TF-binding motifs was lower in the presence of a recombinant PgWRKY74 protein than its absence. The PgWRKY74-GFP-overexpressing Arabidopsis plants exhibited smaller rosette areas than did wild-type plants under mannitol-stressed and NaCl-stressed conditions, and exhibited weaker expression of RD29B, which is induced by the stress-related phytohormone abscisic acid (ABA), under the mannitol-stressed condition. PgWRKY74 have transcriptional activation potential and DNA-binding ability, and can negatively regulate plant responses to mannitol and NaCl stresses, possibly by decreasing ABA levels or ABA sensitivity.

Objectives To screen high active volatile organic compounds (VOCs)-producing Trichoderma isolates against strawberry gray mold caused by Botrytis cinerea, and to explore their antagonistic mode of action against the pathogen. VOCs produced by nine Trichoderma isolates (Trichoderma atroviride T1 and T3; Trichoderma harzianum T2, T4 and T5; T6, T7, T8 and T9 identified as Trichoderma asperellum in this work) significantly inhibited the mycelial growth (13.9-63.0% reduction) and conidial germination (17.6-96.3% reduction) of B. cinerea, the highest inhibition percentage belonged to VOCs of T7; in a closed space, VOCs of T7 shared 76.9% and 100% biocontrol efficacy against gray mold on strawberry fruits and detached leaves, respectively, prolonged the fruit shelf-life by 3 days in presence of B. cinerea, completely protected the leaves from B. cinerea infecting; volatile metabolites of T7 damaged the cell membrane permeability and integrity of B. cinerea, thereby inhibiting the mycelial growth and conidial germination. Gas chromatography-mass spectrometry (GC-MS) analysis revealed the VOCs contain 23 potential compounds, and the majority of these compounds were categorised as alkenes, alcohols, and esters, including PEA and 6PP, which have been reported as substances produced by Trichoderma spp. T. asperellum T7 showed high biofumigant activity against mycelial growth especially conidial germination of B. cinerea and thus protected strawberry fruits and leaves from gray mold, which acted by damaging the pathogen's plasma membrane and resulting in cytoplasm leakage, was a potential biofumigant for controlling pre- and post-harvest strawberry gray mold.

Purpose: Cardiac tissue engineering is suggested as a promising approach to overcome problems associated with impaired myocardium. This is the first study to investigate the use of BC and gelatin for cardiomyocyte adhesion and growth.

Methods: Bacterial cellulose (BC) membranes were produced by Komagataeibacter xylinus and coated or mixed with gelatin to make gelatin-coated BC (BCG) or gelatin-mixed BC (mBCG) scaffolds, respectively. BC based-scaffolds were characterized via SEM, FTIR, XRD, and AFM. Neonatal rat-ventricular cardiomyocytes (nr-vCMCs) were cultured on the scaffolds to check the capability of the composites for cardiomyocyte attachment, growth and expansion.

Results: The average nanofibrils diameter in all scaffolds was suitable (~ 30-65 nm) for nr-vCMCs culture. Pore diameter (≥ 10 µm), surface roughness (~ 182 nm), elastic modulus (0.075 ± 0.015 MPa) in mBCG were in accordance with cardiomyocyte requirements, so that mBCG could better support attachment of nr-vCMCs with high concentration of gelatin, and appropriate surface roughness. Also, it could better support growth and expansion of nr-vCMCs due to submicron scale of nanofibrils and proper elasticity (~ 0.075 MPa). The viability of nr-vCMCs on BC and BCG scaffolds was very low even at day 2 of culture (~ ≤ 40%), but, mBCG could promote a metabolic active state of nr-vCMCs until day 7 (~ ≥ 50%).

Conclusion: According to our results, mBCG scaffold was the most suitable composite for cardiomyocyte culture, regarding its physicochemical and cell characteristics. It is suggested that improvement in mBCG stability and cell attachment features may provide a convenient scaffold for cardiac tissue engineering.

Mesenchymal stem/stromal cells (MSC) play a pivotal role in regenerative therapies. Recent studies show that factors secreted by MSC can replicate their biological activity, driving the emergence of cell-free therapy, likely to surpass stem cell therapy. Patents are an objective measure of R&D and innovation activities, and patent mapping allows us to verify the state of the art and technology, anticipate trends, and identify emerging lines of research. This review performed a search on Derwent World Patents Index™ and retrieved 269 patent families related to the MSC-derived cell-free products. Analysis reveals an exponential increase in patents from the mid-2010s, primarily focusing on exosomes. The patent's contents offer a great diversity of applications and associated technologies by using the products as medicinal agents or drug delivery systems. Nevertheless, numerous application branches remain unexplored, suggesting vast potential for cell-free technologies alone or combined with other approaches.

Objective: Evaluation of Nepeta cataria as a host with specific endogenous metabolite background for transient expression and metabolic engineering of secondary biosynthetic sequences.

Results: The reporter gene gfp::licBM3 as well as three biosynthetic genes leading to the formation of the cannabinoid precursor olivetolic acid were adopted to the modular cloning standard GoldenBraid, transiently expressed in two chemotypes of N. cataria and compared to Nicotiana benthamiana. To estimate the expression efficiency in both hosts, quantification of the reporter activity was carried out with a sensitive and specific lichenase assay. While N. benthamiana exhibited lichenase activity of 676 ± 94 μmol g^-1 s^-1, N. cataria cultivar '1000', and the cultivar 'Citriodora' showed an activity of 37 ± 8 μmol g^-1 s^-1 and 18 ± 4 μmol g^-1 s^-1, respectively. Further, combinatorial expression of genes involved in cannabinoid biosynthetic pathway acyl-activating enzyme 1 (aae1), olivetol synthase (ols) and olivetolic acid cyclase (oac) in N. cataria cv. resulted presumably in the in vivo production of olivetolic acid glycosides.

Conclusion: Nepeta cataria is amenable to Agrobacterium-mediated transient expression and could serve as a novel chassis for the engineering of secondary metabolic pathways and transient evaluation of heterologous genes.

Objectives: In order to investigate the impact of L-cysteine (L-Cys) on starch and protein degradation during barley germination. The amylase activities, degradation of macromolecules during germination were determined in this study.

Methods: Barley was germinated in petri dish for 0 to 5 days with different levels of L-Cys (0 mM, 2.5 mM, 5 mM, 10 mM).

Results: L-Cys addition increased the total limit dextrinase (LD) activities and decreased the LD inhibitor activities during whole germination stage. The activities of α-amylase, β-amylase and free LD were increased with the addition of 2.5, 5 mM L-Cys at germination days 1 to 4. Due to higher amylase in malt with the addition of L-Cys, the non-fermentable sugars were reduced and the glucose, maltotriose were improved. Furthermore, the protein degradation analysis showed that low molecular weight protein increased and middle molecular weight protein decreased obviously in wort from the malt germinated with L-Cys, demonstrating that the L-Cys promote the protein degradation. Lastly, the filtration performance of malt with the addition of L-Cys during malting was better than the control.

Conclusion: In conclusion, L-Cys can promote the degradation of storage material (starch, protein) during barley germination, leading to a better green malt quality.