Journal of bioscience and bioengineering最新文献

d-Amino acid oxidase from the thermophilic fungus Rasamsonia emersonii strain YA (ReDAAO) exhibits high thermostability. To understand the structural basis for this high stability, we isolated thermolabile variants of ReDAAO with a single amino acid substitution (L134P, K203E, C230S, V275G, and V305L), whose T₅₀ (the temperature at which 50 % of the initial enzyme activity was retained) values were 12-18 °C lower than that of the wild-type. The L134P substitution in a flexible protein surface loop caused the most severe destabilization, likely due to increased loop flexibility through hydrogen bond disruption. The other substitutions affected stability by impairing distinct structural elements: K203E might disrupt an amino acid interaction network involved in both flavin adenine dinucleotide binding and subunit interactions, C230S might eliminate the unique disulfide bond that likely fixes a long α-helix involved in subunit interactions, and V275G and V305L might perturb critical interactions at subunit interfaces, with V305L also potentially affecting the subunit structure. Notably, the thermostabilization conferred by the disulfide bond and the interaction network involving K203 were unique to thermophilic fungal DAAOs. These findings revealed multiple distinct mechanisms of thermostabilization in ReDAAO, providing valuable insights for engineering flavoenzymes with improved thermostability.

Ethanol production using the model cyanobacterium Synechocystis sp. PCC 6803 (PCC6803) has garnered considerable attention. A heterologous pyruvate decarboxylase (PDC) is essential for synthesizing ethanol in PCC6803. Although many organisms possess PDCs, no systematic search for suitable PDCs has been reported. This study employed a two-step approach to identify promising PDCs. First, nine diverse natural PDCs with confirmed activity in BRENDA were evaluated for ethanol production in PCC6803. Ethanol production was observed only with PDCs from Zymomonas mobilis (Zm PDC) and Gluconobacter diazotrophicus, suggesting that bacterial PDCs are suitable. In the second step, the search focused on bacterial PDCs, not only natural PDCs but also artificial sequences designed via the Protein Repair One-Stop Shop or ancestral sequence reconstruction. A PDC from Gluconobacter oxydans showed higher ethanol productivity (88.9 mg/L/5 days) than Zm PDC. Although productivity did not surpass that of Zm PDC, ethanol production was achieved with previously unconfirmed or engineered PDCs, expanding the range of useable sequences. This stepwise strategy demonstrates a robust approach for identifying and designing useful enzymes across sequence spaces.

Alkaline phosphatase (ALP) is an essential enzyme that is involved in various metabolic processes. Abnormal ALP levels are linked to diseases and pathological conditions. Herein, a simple and sensitive assay is reported for ALP detection by using glutathione-conjugated gold nanoclusters (GSH-AuNCs) and p-nitrophenyl phosphate (pNPP), based on the fluorescence quenching mechanism. In the underlying mechanism of this assay, the fluorescence of GSH-AuNCs is initially quenched by pNPP, followed by further quenching caused by p-nitrophenol (pNP), a product of ALP activity. To investigate this mechanism for the diagnostic ALP detection, UV-Vis spectrophotometry, transmission electron microscopy (TEM), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis were employed, and this method was tested in real samples. The prepared GSH-AuNCs exhibited an absorption peak at 600 nm under excitation at 365 nm. TEM analysis revealed that GSH-AuNCs were spherical in shape and exhibited uniform particle size and distribution. Furthermore, the gradual reduction in fluorescence intensity of GSH-AuNCs was observed with increasing concentration of pNPP increased (0.03 mM-2.7 mM), suggesting the quenching of the fluorescence by pNPP. SDS-PAGE analysis further confirmed the quenching effect of pNPP on GSH-AuNCs. In addition, fluorescence intensity was decreased by the increasing amounts of ALP. The relationship curve revealed a detectable concentration range of 1.95-1000 U/L and the correlation coefficient of 0.976. The developed method was successfully applied to human osteosarcoma MG-63 cell lysates, culture medium, and extracts from root plants for detection of ALP. Therefore, this assay will be beneficial for the diagnosis of ALP activity in clinical medicine.

Yeast ethanol fermentation and growth are affected by environmental factors such as nutrients, pH, and temperature. Ethanol fermentation in yeast is typically accompanied by cell proliferation. Thus, the specific nutrients required for fermentation remain unclear. To determine nutrients required for fermentation, first a semi-synthetic medium with a small inoculum size (OD₆₀₀ = 2) was used, and CO₂ gas emissions were monitored. The addition of sodium aspartate (Asp) and MgSO₄ to a medium containing 0.2 % yeast extract and 9.1 % glucose efficiently increased gas emissions. Further addition of KH₂PO₄ and myo-inositol to the medium increased the fermentation rate. Next, to obtain no-growth conditions during fermentation, the glucose and cell amounts were increased. Cell growth was repressed but fermentation proceeded in 28.6 % glucose with a large inoculum size (OD₆₀₀ = 16 or 32). The elimination of medium components under these conditions revealed that Asp was sufficient to increase gas emissions without cell growth. Further analysis indicated that pH maintenance and aspartic acid are required to enhance fermentation under no-growth conditions.

Shewanella oneidensis MR-1 possesses an extracellular electron transfer (EET) pathway that enables bidirectional electron exchange with electrodes, making it a promising host for electro-fermentation (EF). However, the intracellular redox reactions driven by MR-1 during electron uptake from the electrodes remain poorly characterized. This study investigated the metabolic fate of pyruvate, a key fermentation intermediate, during inward electron transfer from a low-potential cathode. To examine this, an MR-1 derivative lacking formate dehydrogenase (ΔFDH), which is unable to utilize formate as an electron donor for pyruvate reduction, was incubated under open-circuit (OC) conditions and closed-circuit (CC) conditions with an electrode poised at -0.36 V (vs. the standard hydrogen electrode). A comparative analysis of pyruvate-derived metabolites under these conditions revealed that ΔFDH produced significantly higher amounts of d-lactate under CC conditions, indicating cathode-derived electron utilization for pyruvate reduction to d-lactate. Further gene knockout experiments in the ΔFDH background showed that two d-lactate dehydrogenases (D-LDHs) in MR-1, Dld (a quinone-dependent inner membrane D-LDH) and LdhA (an NADH-dependent D-LDH), contributed almost equally to cathode-dependent d-lactate production. These results indicate that electron transfer from electrodes to pyruvate in MR-1 cells involves both inner membrane quinone-mediated and NADH-mediated redox reactions, highlighting the potential applicability of MR-1 in diverse EF processes.

The construction of small proteins by removing amino acid subsequences that are not involved in function, activity, or structure is crucial for bioprocessing and drug development. Traditional design methods often focus on reconstructing functional motifs, but they face challenges in stabilizing structure and reproducing function. In this study, we aimed to develop a design method for small proteins using ProtGPT2, a model that generates protein sequences based on function and structure. First, amino acid sequence data of malate dehydrogenase (MDH) was collected, and ProtGPT2 was fine-tuned (ProtGPT2 for MDH). The chain length and perplexity (ppl) of the generated sequences were evaluated, producing shorter sequences than the natural ones. The validity of the generated sequences was assessed using both population and individual analyses. Population analysis, including multiple sequence alignment (MSA) and t-distributed stochastic neighbor embedding (tSNE), revealed that ProtGPT2 for MDH identified functional motifs of MDH and incorporated them into the generated sequences. Additionally, tSNE showed that the generated sequences were highly similar to natural MDH sequences. In individual analysis, 10 randomly selected sequences were evaluated using BLAST, AlphaFold2, and InterPro. BLAST indicated that 9 sequences were novel MDH variants. AlphaFold2 confirmed that their 3D structures were highly similar to known MDH structures. InterPro identified domains and active sites in 2 sequences, suggesting that they were novel, small MDH variants. In conclusion, ProtGPT2 for MDH has the potential to design amino acid sequence candidates for small MDHs. The validity and utility of the model will be established through future experimental efforts.

The vascular and neuronal networks are structurally complex and highly branched. These networks play an important role in supplying oxygen and sending signals to the tissues and organs in the body. This study proposes procedures to prepare in vitro neurovascular models on glass substrates and collagen gels for a better understanding of neurovascular interactions. Human umbilical vein endothelial cells (HUVECs) elongated the neurites of neuronal model cells (PC12 cells) on a glass substrate. In collagen gel cultures, the timing of supplementing nerve growth factor (NGF) was crucial for the formation of vascular and neural networks. Thus, neurite outgrowth was more effectively promoted by initially culturing the two cell types in the medium for vascular endothelial cells for 5 days to induce vascular organization and then adding NGF, rather than culturing the cells in the presence of NGF from the beginning. This simple sequential method may be useful for preparing 3-dimensional neurovascular models.

A phosphite (Pt)-dependent biological containment strategy, achieved by introducing a Pt-metabolic pathway and disrupting endogenous phosphate transporters, renders Escherichia coli growth strictly dependent on Pt, a compound rarely detected in natural environments, thereby preventing unintended environmental spread. In this study, we demonstrated that expression of phosphate regulon (Pho regulon) genes was markedly upregulated in a Pt-dependent E. coli strain due to the elimination of phoU, a negative regulator of the Pho regulon, along with the high-affinity phosphate transporter pstSCAB. However, further genetic modification of this strain for detailed analysis was hindered by the presence of multiple antibiotic resistance markers. To overcome this limitation, we reconstructed a Pt-dependent E. coli strain using CRISPR-Cas12a-mediated genome editing, enabling the removal of the antibiotic resistance markers and facilitating subsequent genetic manipulation. Using this strain, we disrupted the PhoBR two-component regulatory genes and found that deletion of phoBR alleviated the constitutive overexpression of Pho regulon genes and partially restored growth of the Pt-dependent strain. These findings provide mechanistic insights and technical advances for the refinement and practical application of Pt-dependent biocontainment strategy.