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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 ] Atomic context-conditioned protein sequence design using LigandMPNN 进入全文
Nature Methods
Protein sequence design in the context of small molecules, nucleotides and metals is critical to enzyme and small-molecule binder and sensor design, but current state-of-the-art deep-learning-based sequence design methods are unable to model nonprotein atoms and molecules. Here we describe a deep-learning-based protein sequence design method called LigandMPNN that explicitly models all nonprotein components of biomolecular systems. LigandMPNN significantly outperforms Rosetta and ProteinMPNN on native backbone sequence recovery for residues interacting with small molecules (63.3% versus 50.4% and 50.5%), nucleotides (50.5% versus 35.2% and 34.0%) and metals (77.5% versus 36.0% and 40.6%). LigandMPNN generates not only sequences but also sidechain conformations to allow detailed evaluation of binding interactions. LigandMPNN has been used to design over 100 experimentally validated small-molecule and DNA-binding proteins with high affinity and high structural accuracy (as indicated by four X-ray crystal structures), and redesign of Rosetta small-molecule binder designs has increased binding affinity by as much as 100-fold. We anticipate that LigandMPNN will be widely useful for designing new binding proteins, sensors and enzymes.
[学术文献 ] Comprehensive evaluation of the capacities of microbial cell factories 进入全文
Nature Communications
Systems metabolic engineering is facilitating the development of high-performing microbial cell factories for producing chemicals and materials. However, constructing an efficient microbial cell factory still requires exploring and selecting various host strains, as well as identifying the best-suited metabolic engineering strategies, which demand significant time, effort, and costs. Here, we comprehensively evaluate the capacities of various microbial cell factories and propose strategies for systems metabolic engineering steps, including host strain selection, metabolic pathway reconstruction, and metabolic flux optimization. We analyze the metabolic capacities of five representative industrial microorganisms as cell factories for the production of 235 different bio-based chemicals and suggest the most suitable host strain for the corresponding chemical production. To improve the innate metabolic capacity by constructing more efficient metabolic pathways, heterologous metabolic reactions, and cofactor exchanges are systematically analyzed. Additionally, we present metabolic engineering strategies, which include up- and down-regulation target reactions, for the improved production of chemicals. Altogether, this study will serve as a comprehensive resource for the systems metabolic engineering of microorganisms in the bio-based production of chemicals.
[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.
