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[学术文献 ] RiboJ-assisted non-repeated sgRNA arrays for enhanced CRISPR multiplex genome engineering in Escherichia coli 进入全文
Chemical Engineering Journal
CRISPR-based systems have revolutionized genome editing by enabling precise and efficient genetic modifications. However, achieving multiplex genome editing remains challenging due to limitations in encoding, transcribing, and processing multiple single-guide RNAs (sgRNAs) in repetitive DNA arrays. In this study, we present the RiboJ-Aided Multiplexed Base Editing (RAMBE) system and its advanced iteration, the Non-Repetitive RAMBE (NR-RAMBE) system, designed for efficient and scalable multiplex genome engineering in Escherichia coli. The RAMBE system leverages RiboJ insulators to autonomously process sgRNA arrays, enhancing sgRNA maturation and enabling simultaneous multi-gene editing. We demonstrate editing of up to six endogenous genes in E. coli Nissle 1917 (EcN) in a single step, achieving high target-specific efficiencies of up to 100%, depending on the target and context. This multiplex editing enabled robust butyrate production and improved acetate utilization in engineered EcN strains. Building on this, the NR-RAMBE system incorporates diverse ribozymes and engineered non-repetitive sgRNA handles to minimize sequence repetition. This design reduced synthesis complexity and enabled simultaneous editing of six genomic loci with efficiencies comparable to those of the RAMBE system. The NR-RAMBE system broadens the scope of CRISPR multiplexing by allowing precise and scalable genome editing without labor-intensive sgRNA array assembly, paving the way for diverse large-scale genomic applications.
[前沿资讯 ] 中科院天津工业生物技术研究所实现淀粉低碳微生物制造 进入全文
中科院天津工业生物技术研究所
“粮食安全是国之大者”，淀粉是为人类生命活动提供能量的关键粮食组分，也是重要的工业原料。近日，中国科学院天津工业生物技术研究所人工淀粉研发团队发展了一条以乙酸为原料的低碳酵母细胞淀粉合成路线。通过优化异源淀粉合成途径的表达强度、精确调控糖异生途径及淀粉分解代谢通路，并结合形态工程扩大胞内淀粉储存空间等工程化策略，系统解决了酵母天然代谢网络淀粉合成能力不足的瓶颈，实现了淀粉的低碳微生物高效合成，以电还原二氧化碳合成的乙酸为原料，淀粉含量达到细胞干重的47.18%，时空生产效率243.7 g/m²/d，生产强度达到160.9 mg/L/h，比其他微生物高一个数量级，为已报道工程细胞合成淀粉的最高水平。系统揭示了重塑细胞资源分配网络支撑高水平淀粉合成的新机制，并通过代谢调控与路径优化相结合的菌株工程，成功实现了人工微籽粒酵母细胞中的淀粉组成及淀粉-蛋白比例调控，展示了应用工程生物技术按需定制功能营养原料组成、创制理想食品和饲料的巨大潜力。相关成果近日发表在国际学术期刊Nature Communications上。
[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.
