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

We had engineered Caldicellulosiruptor morganii CE (CmCE) to construct the enzyme CmCE/D226G for catalyzing the isomerization of lactose to lactulose. However, the poor thermostability and catalytic efficiency of CmCE/D226G limits its application under high substrate concentrations. To address these issues, we employed directed evolution to improve the catalysis performance of CmCE/D226G. First, we used error-prone PCR to construct a variant library and identified the positive variants through cysteine-tryptophan chromogenic high-throughput screening. Then, aiming the crucial residue positions, we conducted saturation mutagenesis to obtain the variants with improved thermostability and catalysis. Accordingly, we successfully obtained a variant named CmCE/D226G/S180T. Its half-life and catalysis efficiency (k_cat/K_m) at 70 ℃ were 184.2 min and 36.5 L/(mmol·s), respectively, which were higher than those (117.7 min and 14.96 L/(mmol·s), respectively) of CmCE/D226G. The improvement of isomerization efficiency resulted from the shortening distance between His377 and the O1 of substrate enol intermediate. The improvement of thermostability was attributed to the rigidness enhancement of a flexible loop. With 350 g/L lactose as the substrate, CmCE/D226G/S180T achieved a lactulose yield of 54.8% and an epilactose yield of 9.2%, while CmCE/D226G only reached a lactulose yield of 50% and an epilactose yield of 10.3%. Taken together, this work provides an elite enzyme for catalyzing the transformation of lactose to lactulose, which especially facilitates the innovation of eco-friendly synthesis of lactulose.

Chiral amines as pivotal intermediates in organic synthesis are widely utilized in the production of pharmaceuticals, fine chemicals, and bioactive molecules, holding significant industrial value. The asymmetric synthesis of chiral amines has always gained great attention. NAD(P)H-dependent imine reductases (IREDs) with wide substrate ranges, high activity, and high enantiomeric selectivity can be used for asymmetric reduction of imines to chiral amines. Our study aims to develop an efficient and stable immobilized dual-enzyme system through the fusion expression of imine reductase and formate dehydrogenase, in order to address the challenge of coenzyme regeneration and enhance catalytic efficiency, thereby providing a novel strategy for the green synthesis of chiral amines. The constructed system was applied to catalyze the asymmetric reduction of 1-methyl-3,4-dihydroisoquinoline to synthesize (S)-1-methyl-1,2,3,4-tetrahydroisoquinoline. Comparative analysis indicated that the catalytic efficiency of this fusion expression system exceeded that of both co-expression and standalone dual-enzyme systems. Furthermore, mesoporous silica nanoflowers were utilized as carriers to immobilize the fusion-expressed dual enzymes through a covalent method. In the case of covalent binding duration of 1.5 h and an initial enzyme concentration of 2.5 mg/mL, the protein loading achieved 193.2 mg/g. The immobilized enzymes demonstrated excellent pH, thermal, and storage stability. When the immobilized enzymes were employed to catalyze asymmetric reduction reactions of other cyclic imines, such as 1-ethyl-3,4-dihydroisoquinoline, 5-phenyl-3,4-dihydro-2H-pyrrole, 2,3,3-trimethyl-3H-indole, 2,3,3,5-tetramethylindole, and myosmine, the conversion rates exceeded 95%, and the values of e.e. surpassed 96%. The data confirm the application potential of the immobilized fusion enzymes in the green and efficient synthesis of chiral amines. Our study provides a novel strategy for the industrial biosynthesis of chiral amines, and the developed fused-enzyme immobilization approach holds significant theoretical and practical value for addressing common technical challenges in cofactor-dependent biocatalytic processes.

To serve the national "dual carbon" goals and strengthen the role of higher education in cultivating talents for carbon sequestration and emission reduction, we focused on the main problems in current teaching practice and constructed a new teaching model for "dual carbon" education in Microbiology. Leveraging the online course resources, we systematically sorted out the knowledge system related to the "dual carbon" goals in Microbiology and reorganized the teaching contents through the dual approaches of embedding real-world production and daily life cases and dynamically tracking cutting-edge technologies. By designing a problem chain to guide the involvement of students and deeply integrating the "dual carbon" concept with course knowledge, this teaching model strengthens both the cognitive cultivation of "dual carbon" goals and the ability of microbial technology application. Teaching practice showed that this model effectively improved students' literacy of "dual carbon" goals and innovative ability, as well as teachers' teaching and research levels. The blended learning model developed in this study offers a referenceable paradigm for the teaching reform incorporating "dual carbon" concept in university courses and has demonstrative significance in fostering "profession+green" composite talents needed in new era.

O-acetyl- l- homoserine (OAH) is a key intermediate in the synthesis of various high-value compounds such as l-methionine and has a high market value. However, the production of OAH still faces problems such as low yields and long fermentation periods. In this study, the wild type of the probiotic Escherichia coli Nissle 1917 (EcN) was used as the starting strain. Firstly, we obtained the mutant MetX^{K185R-G210S-N204T} by modifying the key enzyme l-homoserine acetyltransferase (MetX). Further, ppc, asd, and metL were overexpressed to strengthen the synthetic pathways of the precursors l-aspartic acid and l-homoserine. Strategies such as dynamic regulation were adopted to weaken the synthesis of the by-product amino acids. Different intensities of 5{L-End} ' UTR were screened to downregulate the expression level of gltA to balance the supply of another precursor acetyl-CoA, and finally an efficient OAH-producing strain was constructed. The fermentation yield of OAH in the shake flask was 11.18 g/L, and that in the 5 L fermenter reached 63.54 g/L at the time point of 52 h. The strain engineering improved the yield and productivity of OAH and laid a research foundation for the subsequent biological production of l-methionine.

China's agriculture is progressing towards more intensive, information-driven, smart, and environmentally friendly practices. Consumers are increasingly prioritizing safety, sustainability, and ecological health when selecting agricultural products. Accordingly, the safety, low residue, and environmental friendliness of chemical fertilizers have become crucial. Biostimulants derived from natural fermentation have attracted considerable attention due to their eco-friendly nature, safety, efficacy, and ability to reduce fertilizer application while enhancing efficiency. Among these, γ-polyglutamic acid (γ-PGA), a microbially synthesized polyamino acid, is non-toxic, edible, moisturizing, readily chelatable, and highly dispersible. It is extensively used in the medical, food, and agricultural sectors, making it an ideal novel biofertilizer and biostimulant. This review summarizes the latest research on biosynthetic pathways, production conditions, process optimization, and applications of γ-PGA. This review provides an overview and analysis of the fermentation process for γ-PGA production by Bacillus strains, with a focus on optimizing the medium composition. Furthermore, this article discusses various analytical methods for quantifying γ-PGA in agricultural applications and examines its effects on soil improvement, fertilizer efficiency enhancement, and crop growth regulation. This review aims to provide valuable insights into the microbial synthesis of γ-PGA and propose new directions for its development in the field of new fertilizers.

This study addresses the demand for green biosynthesis of chiral amine drugs by developing a novel R-selective amine transaminase (PamAT) derived from the marine actinomycete Pseudonocardia ammonioxydans. Following recombinant expression in Escherichia coli and purification via nickel-affinity chromatography, PamAT exhibited remarkable catalytic properties: strict stereoselectivity; a broad substrate spectrum; enhanced solvent tolerance (retaining>80% activity in 10% methanol or acetonitrile). Enzyme activity assays revealed that PamAT achieved the activity of (7.57±0.42) U/mg under mild conditions. Molecular docking and structural simulations elucidated the substrate recognition mechanism and key amino acid residues controlling stereoselectivity. The results indicate that PamAT holds significant potential for industrial applications, offering not only a new tool for the green synthesis of chiral amine drugs but also novel insights for the development of marine microbial enzymes.

Spores are specialized dormant structures formed by bacteria of the order Bacillales under adverse conditions such as nutrient deprivation, exhibiting extremely strong resistance to environmental stresses. Investigating the regulatory mechanisms of sporulation is of great significance for advancing theoretical understanding in biology and guiding practical applications. Sporulation relies on the coordinated action of environmental signals and intracellular signaling pathways. When external stimuli initiate a kinase-catalyzed phosphorylation cascade, the transcription factor Spo0A is activated, subsequently triggering a sigma factor cascade regulatory system, ultimately initiating the sporulation program. Spo0A is the central hub governing sporulation, and its dynamic phosphorylation state persists throughout the entire sporulation process and orchestrates the sporulation pathway at transcriptional, translational, and post-translational levels. This review systematically elucidates the key environmental inducers of sporulation, the Spo0A-mediated regulatory network, and its multi-level molecular regulatory mechanisms, aiming to provide a theoretical foundation for designing Spo0A pathway-based spore application strategies.

Several critical challenges exist in the graduate course Genetic Engineering at Beijing Institute of Technology, including conceptual confusion in core topics such as CRISPR-Cas9, difficulties in transferring theoretical knowledge to experimental design, fragmentation of teaching resources across multiple platforms, and the lack of process-oriented assessment for complex reasoning and research competence. In response, we propose a new paradigm that deeply integrates artificial intelligence (AI) and outcome-based education (OBE) in course design and implementation. Specifically, we construct a triadic framework of knowledge graph-ability matrix-intelligent learning companion, in which 421 core knowledge points are systematically mapped to the graduate program's ability indicators, forming a dynamic knowledge-ability matrix that supports personalized learning paths and traceable competence development. A research task-oriented AI companion is further designed for the graduate context. By leveraging retrieval-augmented generation (RAG) and a course-specific large language model (LLM) around comprehensive tasks such as the vector design for gene therapy, the system analyzes students' decision-making trajectories and task outputs to enable process-based evaluation of complex thinking and research skills. In addition, a data-driven teaching analytics cockpit aggregates learning behaviors, knowledge mastery, and ability performance in real time, helping instructors accurately identify conceptual bottlenecks and competence gaps and refine instructional interventions. Teaching practice conducted in the Fall 2025 semester suggests, within the context of this study, that students in the experimental class using the platform achieved higher final examination scores than those in two parallel control classes (P<0.05). The platform was also associated with high learning engagement, with a daily activity rate of 87% and an average of 5.3 questions per student per day. Most students reported observed improvements within this study in self-directed learning efficiency and understanding of advanced technologies. Overall, the findings indicate that compared with conventional smart courses primarily focusing on resource integration and content recommendation, this AI- and OBE-integrated course-centered on authentic research tasks and competence attainment-partially addresses challenges related to knowledge transfer and competence evaluation in graduate education, providing a referenceable practical pathway for smart-course reform in life science graduate programs.

Biomanufacturing, as an emerging industry, is profoundly reshaping the global industrial landscape due to its green, low-carbon, efficient, and sustainable characteristics. Driven by artificial intelligence, this field urgently demands interdisciplinary applied talents proficient in both biological and computational sciences. In response, establishing the course Computational Biology to aligns with the national major development plans and cultivate the required talents is a significant measure. This paper elaborates on the urgency and practical significance of developing this course, as well as the challenges involved. Basing on the development direction of biomanufacturing, disciplinary trends, and students' learning needs in Computational Biology, a development framework aimed at cultivating applied talents is proposed. Furthermore, the implementation plan for the course is detailed from three perspectives: designing the teaching contents, integrating diverse pedagogical approaches, and improving the teaching evaluation system. The implementation of this course will help to cultivate professionals skilled in applying computational biology tools to the biomanufacturing sector, while also providing valuable experience and reference for other universities offering similar courses.

In the special marine environment characterized by high salinity, high osmotic pressure, and oligotrophy, marine actinomycetes have evolved unique survival strategies. Some of their secondary metabolites, with novel structures and potent biological activities, hold significant research and application value in pharmaceutical development. Under conventional laboratory culture conditions, only 20% of the biosynthetic gene clusters (BGCs) in marine actinomycetes are expressed, and a large number of silent BGCs urgently need to be explored. This paper systematically reviews the silencing mechanisms and activation strategies of silent BGCs in marine actinomycetes, comprehensively summarizes recent research advances in physical activation, chemical activation, and bioengineering technologies, and deeply analyzes the advantages and limitations of various activation methods. The silencing of BGCs often resulted from the combined effects of multiple factors. Therefore, this paper proposes a multi-technology combination strategy for activating silent BGCs. This strategy simulates the original survival environment of microorganisms from multiple aspects or constructs specific expression environments for gene clusters to promote and induce the expression of silent BGCs at multiple levels, thereby obtaining novel secondary metabolites. This strategy provides theoretical references and technical pathways for exploring the metabolic potential of marine actinomycetes and promoting the discovery of new natural products.