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[学术文献 ] A cysteine-less and ultra-fast split intein rationally engineered from being aggregation-prone to highly efficient in protein trans-splicing 进入全文
Nature Communications
Split inteins catalyze protein trans-splicing by ligating their extein sequences while undergoing self-excision, enabling diverse protein modification applications. However, many purified split intein precursors exhibit partial or no splicing activity for unknown reasons. The Aes123 PolB1 intein, a representative of the rare cysteine-less split inteins, is of particular interest due to its resistance to oxidative conditions and orthogonality to thiol chemistries. In this work, we identify β-sheet-dominated aggregation of its N-terminal intein fragment as the origin of its low (~30%) splicing efficiency. Using computational, biochemical, and biophysical analyses, we characterize the fully active monomeric fraction and pinpoint aggregation-prone regions. Supported by a crystal structure, we design stably monomeric mutants with nearly complete splicing activity. The optimized CLm intein (Cysteine-Less and monomeric) retains the wild-type’s ultra-fast reaction rate and serves as an efficient, thiol-independent protein modification tool. We find that other benchmark split inteins show similar precursor aggregation, suggesting that this general phenomenon arises from the intrinsic challenge to maintain the precursor in a partially disordered state while promoting stable folding upon fragment association.
[学术文献 ] Rational design of DAHP synthase and prephenate dehydrogenase for metabolic engineering of Bacillus amyloliquefaciens to produce L-tyrosine 进入全文
International Journal of Biological Macromolecules
The rational design of enzymes represents a critical strategy for achieving efficient and sustainable biocatalysis. In this study, enzyme evolution guided by rational design was utilized to engineer two key enzymes, DAHP synthase (AroA) and prephenate dehydrogenase (TyrA), within the biosynthetic pathway of L-tyrosine. The beneficial mutants AroAR27A/K38A and TyrAI309A/E330V were identified, leading to a 102 % and 105 % increase in L-tyrosine yield, respectively. Molecular dynamics simulations further explained the possible mechanism underlying their improved catalytic efficiency. Co-expression of these two mutant genes resulted in a significant increase in L-tyrosine yield. Additionally, modifications in the branching metabolic pathways, which altered both material and energy flux, further enhanced L-tyrosine production. Ultimately, the L-tyrosine yield (0.14 g/g) from xylose was much higher than that from glucose, and the final L-tyrosine titer (9.39 g/L) and productivity (0.26 g/(L·h)) were achieved through fermentation optimization in shake flasks. This represents the highest reported yield in shake flasks. The strategies described here will contribute to the development of microbial strains for the efficient production of L-tyrosine from sustainable biomass resources.
[学术文献 ] Modulating the electronic configuration of single-atom nanozymes using cobalt nanoclusters for enhanced mycotoxin degradation 进入全文
Food Chemistry
Herein, Co- and Fe-based single-atom nanozymes (M/N-PC, M = Co or Fe) were successfully fabricated and their catalytic performances for patulin degradation were evaluated systematically. Co/N-PC, consisting of Co–N4 and nanoclusters sites, achieved a higher patulin degradation efficiency (99.4 %, within 60 min) than Fe/N-PC (only consisting of Fe–N5 sites). Synergistic interactions between Co–N4 and Co nanoclusters greatly enhanced electron density near the Fermi level in Co/N-PC, enabling its high catalytic performance. The degradation products of patulin exhibited negligible cytotoxicity. The M/N-PCs demonstrated good reusability, broad pH adaptability and high practical application potential for patulin degradation in apple juice. M/N-PC also exhibited high efficiency in degrading aflatoxin B1, deoxynivalenol and zearalenone (∼100 %, 10–40 min). This study provides in-depth insights into the relationship between metal active site structures in M/N-PCs and their catalytic properties for mycotoxin detoxification, offering guidance for the design of highly efficient single-atom nanozymes.
[学术文献 ] Mechanistic investigation of repurposed photoenzymes with new-to-nature reactivity 进入全文
Current Opinion in Green and Sustainable Chemistry
Biocatalysis is widely renowned for its remarkable efficiency, selectivity, and known for operating under mild conditions. While most enzymatic reactions progress without light irradiation, recent studies have identified light as a crucial factor in the activation of certain naturally occurring enzymes. These findings have spurred the rapid advancement of photoenzymatic catalysis in the past few years, where enzymes are not typically known for light activation perform excited-state chemistry with or without the presence of external photocatalysts to facilitate new-to-nature transformations that are challenging for traditional chemical synthesis. In this review, we summarize the experimental and computational methods used to investigate the catalytic mechanisms of repurposed photoenzymes with new-to-nature reactivity and discuss how these insights can inform the design of new photoenzymatic catalytic systems.
[前沿资讯 ] The evolution of low-temperature adapted enzymes 进入全文
EurekAlert
A research team led by Professor Satoshi Akanuma from Waseda University, Japan, in collaboration with Assistant Professor Sota Yagi from Waseda University, Dr. Subrata Dasgupta, and Dr. Shunsuke Tagami from the RIKEN Center for Biosystems Dynamics Research, investigated the evolutionary improvement of IPMDH activity at low temperatures. They traced its evolution from the enzyme of the most ancient thermophilic common ancestor to the mesophilic bacterium Escherichia coli using ASR. Their study was published online in the journal Protein Science on February 19, 2025.“We reconstructed 11 intermediate ancestral enzymes along the evolutionary trajectory connecting the last common bacterial ancestor and E. coli IPMDH (EcIPMDH),” explains Akanuma. “After that, we analyzed changes in enzyme activity at each evolutionary stage, especially improvements in catalytic activity at low temperatures.” They observed a notable increase in catalytic activity at 25 °C, which did not follow a gradual, linear pattern. Instead, a dramatic improvement occurred between the fifth (Anc05) and sixth (Anc06) intermediate ancestors. What caused this sudden boost in enzymatic efficiency? To find the underlying molecular mechanisms, the researchers compared the amino acid sequences of the ancestral enzymes and used site-directed mutagenesis, a technique that allows precise alterations to DNA and protein sequences. They identified three key amino acid substitutions that significantly enhanced catalytic activity at 25 °C. Surprisingly, these mutations occurred far from the active site, challenging the previous belief that temperature adaptation is primarily driven by active-site modifications. Molecular dynamics simulations revealed a key structural shift between Anc05 and Anc06. While Anc05 remained in an open conformation, Anc06 could adopt a partially closed conformation, reducing activation energy and enhancing enzymatic efficiency at low temperatures. This transition occurred 2.5–2.1 billion years ago, coinciding with the Great Oxidation Event, which led to a sharp decline in atmospheric methane and global cooling. The researchers suggest that this climate shift may have driven the adaptation of enzymes to lower temperatures. By identifying key mutations that enhance enzyme efficiency, ASR provides valuable insights into how life evolved in response to Earth's changing environment. “Applying this approach to various enzymes is expected to reveal how organisms and their enzymes have evolved in response to Earth's environmental changes over the past four billion years,” Akanuma concluded.
[学术文献 ] Synthetic Biology in Natural Product Biosynthesis 进入全文
Chemical Reviews
Synthetic biology has played an important role in the renaissance of natural products research during the post-genomics era. The development and integration of new tools have transformed the workflow of natural product discovery and engineering, generating multidisciplinary interest in the field. In this review, we summarize recent developments in natural product biosynthesis from three different aspects. First, advances in bioinformatics, experimental, and analytical tools to identify natural products associated with predicted biosynthetic gene clusters (BGCs) will be covered. This will be followed by an extensive review on the heterologous expression of natural products in bacterial, fungal and plant organisms. The native host-independent paradigm to natural product identification, pathway characterization, and enzyme discovery is where synthetic biology has played the most prominent role. Lastly, strategies to engineer biosynthetic pathways for structural diversification and complexity generation will be discussed, including recent advances in assembly-line megasynthase engineering, precursor-directed structural modification, and combinatorial biosynthesis.
