Sheng wu gong cheng xue bao = Chinese journal of biotechnology最新文献

Phytase (EC 3.1.3.8) is an enzyme that specifically hydrolyzes phytic acid, yielding products such as orthophosphate, inositol phosphate isomers, and free inositol. It is a promising enzyme for food applications. This study aims to enhance the thermostability of the phytase derived from Aspergillus tubingensis. The FireProt platform was employed to predict the impacts of mutation sites on protein stability, which was complemented by FoldX and Rosetta-based calculations of mutation free energy changes for mutant screening. Four distinct mutants were subsequently constructed. The elite mutant T273K was screened out for enzymatic characterization. The half-life of T273K was increased by 67.56% at 60 ℃ compared with that of the wild type (WT), and by 92.86% at 80 ℃. The catalytic efficiency (k_cat/K_m) of T273K was 196.11 L/(μmol·min), representing a 50.56% increase relative to that of WT. These results collectively demonstrated improved thermostability and activity of T273K. Kinetic simulation analysis revealed a significant increase in the number of salt bridges in the distal fragments (205-210 and 380-400) of T273K, which led to improved overall protein rigidity, thereby enhancing the thermostability. This study successfully identified a mutant T273K, which effectively enhanced the phytase thermostability. Furthermore, kinetic simulations revealed that a local mutation remodeled salt bridges in distal fragments to increase overall protein rigidity and improve the protein thermostability. This provides an effective strategy for future modifications aimed at enhancing enzyme thermostability.

Living systems often rely on dynamically assembled multi-enzyme complexes to achieve precise control over metabolic reactions. In recent years, liquid-liquid phase separation (LLPS) has been recognized as one of the important mechanism by which living systems organbize and regulate enzymatic reactions, and has gradually emerged as a powerful tool for constructing artificial catalytic systems. Owing to the reversible, self-organizing, and programmable properties, LLPS-driven biomolecular condensates can modulate the local concentration, spatial arrangement, and microenvironmental features of enzymes, thereby influencing catalytic efficiency and substrate selectivity. To date, a variety of enzymatic systems, from intracellular multi-enzyme pathways to in vitro artificial reactors, have been reconstructed and enhanced within condensates. This review provides a concise overview of the physiological context of LLPS and, together with the authors' recent work on LLPS-based enzymatic systems, highlights the major mechanisms by which LLPS modulates enzymatic activity. We further summarize advances in the applications of LLPS in metabolic pathway engineering and in vitro biocatalysis and discuss the potential of programmable condensates for dynamic regulation of catalytic processes. This review proides a systematic framework for understanding the mechanisms by which LLPS regulates enzymatic catalysis and offers a theoretical basis for the rational design of efficient and controllable multi-enzyme catalytic systems.

Global salinized land greatly affects the ecology and agriculture. Biological remediation is an important green approach for managing salinized soil. Enhancing the salt tolerance of plant growth-promoting rhizobacteria (PGPR) is key to improving the effectiveness of biological remediation. Glycine betaine (GB), an important osmotic regulator, can enhance the stress resistance of microorganisms and plants. In order to improve the survival rate of PGPR in salinized soil, GB was synthesized by biological method. In this study, we used a novel PGPR strain Bacillus paralicheniformis Bp1 as the host to reconstruct the GB synthesis pathway. First, the genes maeA, aceB, iclR, and gcvP in the glycine competition/consumption pathway were knocked out, and the glyoxylate cycle was strengthened by introduction of aceAK from Escherichia coli. Additionally, the exogenous high-efficiency transaminase gene agx1 was introduced. The highest yield of GB precursor, glycine, reached 34.27 mg/L within 30 h of fermentation, representing a 293.9% increase compared with the highest yield of the wild-type at the time point of 12 h. Meanwhile, to ensure the stability of the strain, aceAK was integrated into the genome to obtain strain Bp1Z11. Next, the methyl transferase genes gsmt and sdmt from Aphanothece halophytica were introduced into Bp1Z11 to construct the engineered strain Bp1Z12, which achieved a GB yield of 2.56 mg/L (a 326.7% increase compared with the wild type) after 36 h of fermentation. Under 0.3 mol/L NaCl stress (simulating moderately salinized soil conditions), the engineered strain achieved a GB yield of 4.94 mg/L (a 93.7% increase compared with the salt-free control) after 36 h of fermentation, with the biomass (OD₆₀₀) increasing to 16.22 (a 20.9% increase compared with the salt-free control). Additionally, Bp1Z12 effectively alleviated salt stress of tomato plants and enhanced their growth in salinized soil. The root length of tomato plants in the Bp1Z12 treatment increased significantly by 75.0% and 27.3% compared with that in the water and Bp1 treatments, respectively. The plant height of the Bp1Z12 treatment increased by 76.9% and 21.1% compared with that in the water and Bp1 treatments, respectively. The leaf area of this treatment increased by 77.8% and 45.0% compared with that in the water and Bp1 treatments, respectively. The engineered strain Bp1Z12 can efficiently utilize glucose to synthesize GB, while exhibiting good salt tolerance and plant growth-promoting ability in salinized soils. This study provides new ideas for application of this strain in the development of stress-tolerant microbial fertilizers or the remediation of salinized soils in the future.

Chiral reticuline serves as the core skeleton molecule in the biosynthetic pathway of benzylisoquinoline alkaloids (BIAs). Its efficient and highly selective biosynthesis is crucial for overcoming the limitations of natural extraction and enabling the large-scale production of BIAs. Recent advances in synthetic biology and metabolic engineering have led to significant breakthroughs in the heterologous production of chiral reticuline and BIAs in microbial hosts. However, the biosynthesis of (R)-reticuline remains constrained by the stereoselectivity of key enzymes, necessitating resolution through strategies such as enzyme engineering and pathway reconstitution. Studies on Nelumbo nucifera revealing an (R)-stereochemical preference offer novel pathways for synthesizing (R)-configured BIAs. This review systematically summarizes the application value of chiral reticuline as a key scaffold of BIAs, its synthetic pathways, and the latest research progress. We critically analyze current challenges and propose future research directions, aiming to provide both theoretical support and practical guidance for developing next-generation high-efficiency, high-selectivity BIAs.

Monensin, a polyether ionophore antibiotic produced by Streptomyces cinnamonensis, is widely used in the livestock industry. To address the yield bottleneck caused by insufficient intracellular reducing power (NADPH/NADH) during industrial fermentation, we employed a multi-pathway collaborative metabolic engineering strategy. We fused the genes zwf (encoding glucose-6-phosphate dehydrogenase) and gnd (encoding 6-phosphogluconate dehydrogenase) from the pentose phosphate pathway with fadB (encoding 3-hydroxyacyl-ACP dehydratase) from the fatty acid β-oxidation pathway to construct a synthetic expression cassette. This cassette was cloned into the integrative vector pSET152 under the control of the strong ermE promoter and fd terminator and then introduced into S. cinnamonensis SDSL6002 (WT) via intergeneric conjugation, yielding the recombinant strain S-zwf-gnd-fadB. Shake-flask fermentation demonstrated that the engineered strain achieved the intracellular NADPH/NADH level 1.8 folds of that in the wild type, with a monensin titer of 22.6 g/L (a 51.7% increase). The yield remained stable after five generations of antibiotic-free subculturing. Further scale-up validation in 50 L and 5 m³ industrial fermenters demonstrated that the engineered strain achieved monensin titers of 37.2 g/L and 40.4 g/L, which represented 16.3% and 18.8% improvements, respectively, over that of the control strain. These results highlight the exceptional scalability and industrial production potential of the engineered strain. This study establishes a multi-gene co-expression system to reinforce the reducing power network, providing a high yield engineered strain and a feasible metabolic engineering approach for industrial biosynthesis of monensin.

In 2025, synthetic biology and biomanufacturing have demonstrated remarkable progress characterized by intelligent integration, systematic optimization, and diversified applications. Artificial intelligence has evolved from an auxiliary tool into a core driving force, deeply embedded throughout the entire pipeline from biomolecular design to fermentation process control, catalyzing a fundamental shift from "experience-driven" to "data- and model-driven" research paradigms. Breakthroughs in enzyme engineering and protein design continue to expand the functional boundaries of biocatalysis, while cell factory construction is advancing from single-target modification toward global systematic optimization. The synergistic development of traditional and emerging chassis hosts enables efficient biosynthesis of high-value chemicals and novel proteins. Environmental bioremediation is transitioning from functional strain engineering to the rational design of synthetic microbial consortia, marking a new era of predictable and controllable remediation strategies. Meanwhile, advances in interdisciplinary frontiers, such as artificial cells, are continuously expanding the horizons of biomanufacturing. Looking forward, the deep integration of artificial intelligence with automation platforms, a deeper understanding of biological complexity, and industrial applications aligned with carbon neutrality goals and human health demands will define the future trajectory of the field. Synthetic biomanufacturing is evolving from "understanding life by creating life and creating life for applications" toward "creating life for good and creating life for the future", offering indispensable solutions to address global challenges in resources, environment, and health.

The efficient production of target chemicals by microbial cell factories, characterized by renewable raw materials, clean and efficient production processes, and mild reaction conditions, has been established as a significant direction for sustainable industrial development. Currently, microbial cell factories primarily use glucose and glycerol as carbon sources, which compete with humans for grain and compete with grain for land, restricting the rapid development of the microbial manufacturing industry. As a non-grain carbon source, acetate can avoid the aforementioned issues and is widely sourced from industrial waste and biomass conversion, and thus it has become an ideal alternative carbon source for the microbial synthesis of chemicals. This paper systematically evaluates the potential of acetate as a microbial carbon source for the synthesis of chemicals from four dimensions: microorganisms capable of utilizing acetate, metabolic engineering strategies to enhance the utilization of acetate, methods to improve tolerance to high concentrations of acetate, and the chemicals that can be synthesized from acetate. Furthermore, this paper analyzes the main challenges faced by microbial cell factories in utilizing acetate and envisions the future development directions, providing theoretical guidance for the in-depth research and industrial application of acetate-based biomanufacturing technologies.

Currently, organoids emerges as novel ex vivo models for biomedical research and biopharmaceutical development. However, the recombinant growth factors used for organoid culture faces challenges such as high costs, low batch consistency, and poor stability, which limit the standardization and scaling of organoid applications. This study aims to develop a membrane-anchored bifunctional growth factor mimetic based on functional nucleic acids to replace recombinant growth factors in organoid culture. Using nucleic acid aptamers as functional mimic units and cholesterol (Chol) as the membrane anchoring module, we constructed two bifunctional growth factor mimetics through base-complement pairing principles. The first was a dual-specificity aptamer (ApE+F-22-Chol) targeting the dimerization activation of both epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1), simulating the synergistic function of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). The second was a dual-specificity aptamer (ApF+M-Chol) designed to target and induce the dimerization activation of mesenchymal-epithelial transition factor (Met) and FGFR1, mimicking the function of hepatocyte growth factor (HGF) and bFGF. The results demonstrated that ApE+F-22-Chol successfully anchored to cell membranes while efficiently activating both EGFR and FGFR1 signaling pathways, synergistically promoting cell proliferation and migration. In a patient-derived prostate cancer organoid culture system, ApE+F-22-Chol can fully replace EGF and bFGF in the culture medium, effectively supporting organoid formation and growth. Further experiments confirmed that the ApF+M-Chol constructed with the same strategy also exhibited significant growth-promoting activity in organoid culture, validating the broad applicability of this membrane-anchored bifunctional growth factor mimetic strategy. This study provides a novel tool with well-defined components, high stability, and high efficiency for organoid culture systems, showing promising applications in disease modeling, drug screening, and regenerative medicine.

Spermidine is a natural polyamine with autophagy-inducing properties, capable of extending lifespan, inhibiting tumors, protecting cardiovascular health, regulating neural functions, and exerting anti-inflammatory effects. It holds significant applications in agriculture, industry, and healthcare. This study focuses on the microbial synthesis of spermidine by tracing the biosynthetic pathways of its precursors. Through the overexpression of key enzymes involved in precursor synthesis, spermidine was successfully synthesized in vitro with amino acids as substrates under multi-enzyme cascade conditions, which significantly reduced the production costs. The phylogenetic analysis identified an efficient S-adenosylmethionine decarboxylase from Bacillus subtilis, and its activity was enhanced by 2.68 folds through mutagenesis. Subsequently, the superiority of whole-cell catalysis for spermidine synthesis was confirmed, and the continuous and efficient synthesis of spermidine was achieved by optimizing gene tandem sequences and introducing an ATP regeneration system. Further, the reaction conditions, including cell density, substrate concentration, reaction temperature, pH, and metal ions, were optimized. Under optimal conditions, the recombinant Escherichia coli EC12 achieved a maximum spermidine yield of 17.83 g/L in a 5 L bioreactor after 20 hours of transformation. The recombinant E. coli constructed in this study demonstrates potential for industrial-scale spermidine production, laying a theoretical and practical foundation for the green and efficient synthesis of spermidine.

Chondroitinase ABC (ChABC) holds significant value in biomedical applications and polysaccharide structure analysis. However, the poor thermostability of this enzyme severely limits its industrial production and utilization. Focusing on the multidomain characteristics of PvChABC derived from Proteus vulgaris and targeting the loop region connecting the N-terminal domain and the catalytic domain, we obtained an optimized mutant M3 with significantly improved thermostability through a computer-aided rational design strategy. M3 exhibited a half-life of (48.12±1.85) h at 45 ℃, which represented a 13.7-fold increase over that of the wild type. It showed the specific activity 110.5% of that in the wild type, which indicated no loss of enzymatic activity. Molecular dynamics simulations revealed that the mutation in the loop region introduced hydrophobic interaction and hydrogen bond network, thereby enhancing the inter-domain binding and stabilizing the overall protein conformation. Notably, the enhanced thermostability enabled efficient heterologous expression at 37 ℃, with M3 showing a 14.84-fold higher yield than the wild type under industrial fermentation conditions. This work not only establishes a foundation for the industrial production of PvChABC but also provides a generalizable strategy for engineering other multidomain proteins.