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

The 8-10 repeat of fibronectin (FN) type III domain (FNIII8-10) contains multiple integrin-binding sites and serves as the core module mediating cell adhesion. Currently, recombinant FNIII8-10 is expressed only in prokaryotic microorganisms. This study aims to establish a Pichia pastoris expression system for the high-level production of recombinant FNIII8-10. Although the recombinant FNIII8-10 was successfully expressed in Pichia pastoris, the secretion efficiency was low. Through rational signal peptides design and fermentation optimization, secretion efficiency was significantly improved. Screening of multiple signal peptides identified the α-factor signal peptide as the most efficient. Site-directed mutagenesis of its hydrophobic core generated the high-efficiency mutant α-V50A, which increased secreted protein yield by 44.8%. Using the response surface methodology, fermentation conditions were optimized to 117.46 h, pH 6.91, and 1.91% (V/V) methanol supplementation. Meanwhile, with sorbitol as an auxiliary carbon source to alleviate methanol stress, the yield of the recombinant FNIII8-10 reached 65.49 mg/L. Finally, high-cell-density fermentation achieved a yield of 532.82 mg/L, representing a 7-fold increase compared with the highest yield in shake flasks. This study lays a technical foundation for the industrial-scale production of recombinant FNIII8-10.

To address the fragmented knowledge, lagging case updates, and disconnect between theory and practice in the Metabolic Engineering course for undergraduates, we established a teaching reform model centered on research-integrated pedagogy. By translating the latest research outputs into instructional resources, the model constructs a progressive sequence from phenotype perception to mechanism elucidation and regulatory optimization. Instruction is organized around key themes such as pathway reconstruction and dynamic regulation, and is extended beyond the classroom through laboratory practices, participation in innovation projects, and academic competitions. In parallel, a multidimensional assessment framework combining formative and summative components is designed, quantifying knowledge mastery, data interpretation, design capability, and practical operation. Through coordinated endeavor in content updating, localization of case studies, and engineering-oriented training, the model alleviates textbook latency and the theory-practice divide and strengthens students' systems analysis and engineering design abilities, providing a replicable implementation pathway for the continuous optimization and dissemination of biotechnology courses.

Selenium is an essential trace element for the human body, enhancing the body resistance to a variety of diseases induced by oxidative stress. Selenium nanoparticles (SeNPs) possess advantages such as low toxicity and broad biocompatibility. In order to screen for a strain with high efficiency in synthesizing SeNPs, we investigated the selenium tolerance and SeNPs synthesis capacities of three selenium-tolerant yeast strains and the in vitro antioxidant activities and antimicrobial activities of the synthesized SeNPs. Furthermore, we characterized SeNPs synthesized by strain M16-28 with the best performance. The results indicated that strain M16-28 was capable of tolerating 300 mmol/L Na₂SeO₃, with a SeNPs synthesis capacity of 1 018.92 μg/mL, which was 2.86 times that of strain M1. The in vitro antioxidant experiments indicated that the synthesized SeNPs exhibited the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rate of 65.7%, the hydroxyl radical scavenging rate of 57.5%, and the reducing power of 0.77. The antimicrobial experiments indicated that the synthesized SeNPs displayed significantly stronger inhibitory effects against Gram-positive bacteria than against Gram-negative bacteria, and they had a significant inhibitory effect on the pathogenic microorganisms causing stem rot of Lycium barbarum. The SeNPs synthesized by the three yeast strains exhibited an inhibition rate higher than 90% against Fusarium spp., and a lower inhibition rate against Alternaria alternata.The SeNPs produced by strain M16-28 exhibited the highest antimicrobial activity, with an inhibition rate of (66.53±1.37)%. Characterization results indicated that SeNPs were closely arranged, encapsulated by polysaccharides, and negatively charged, with a size ranging from 190 nm to 390 nm and the zeta potential of -7.87 mV. In conclusion, strain M16-28 demonstrates ideal performance of synthesizing SeNPs, and the capacity for selenium enrichment is positively correlated with the ability to synthesize SeNPs. This study provides a potential microbial resource for the efficient synthesis of SeNPs.

Leveraging a solid foundation in engineering education, we have built a bioengineering talent development system tailored to intelligent biomanufacturing and biomedicine in response to the intelligent transformation of biomedicine and the national initiative for emerging engineering education. This paper introduces a digital-intelligence driven, competency-oriented educational philosophy, a "trinity" evaluation framework, a "Three-Integration & Three-Articulation" curriculum, and a comprehensive "Holistic Education" system. A distinctive laboratory teaching model featuring "Three-Coupling, Four-Infusion, and Five-Enhancement" has been established. This model was integrated with practical activities such as innovation projects, academic competitions, and industry internships to form a progressive "Bio-Maker" practice platform. These outcomes strongly support the development of the A+ bioengineering discipline, serve industrial needs in Shanghai and the Yangtze River Delta, and have generated broad influence nationally, promoting progress in related fields at peer institutions.

Cytidine 5{L-End} '-monophosphate (5{L-End} '-CMP), a fundamental component of RNA and a key intermediate for nucleotide derivatives, has broad applications in the medical, food, and agricultural industries. However, the biosynthesis of 5{L-End} '-CMP faces challenges such as low enzyme catalytic efficiency, low substrate conversion rates, and high production costs. To address these limitations, we first screened and identified a cytidine kinase (MmUCK) with high product tolerance and strong stability. After that, an AMP/ATP regeneration system was introduced to reduce ATP consumption. With cytidine, sodium hexametaphosphate, and adenosine monophosphate (AMP) as substrates, 5{L-End} '-CMP was efficiently synthesized via a one-pot, dual-enzyme biocatalytic system. That is, (76.94±3.26) mmol/L 5{L-End} '-CMP was produced when 100 mmol/L cytidine was used as the substrate. Furthermore, the cytidine deaminase gene (cdd) and the pyrimidine-specific ribonucleoside hydrolase gene (rihC) in the cytidine branch were knocked out, which increased the molar conversion rate to 98.1%. Finally, in a 10 L bioreactor, (563.93±8.84) mmol/L 5{L-End} '-CMP was synthesized after 7 h of enzymatic reaction with 600 mmol/L cytidine, 150 mmol/L sodium hexametaphosphate, and 5 mmol/L AMP as substrates, and a molar conversion rate of 94.2% was achieved. Our study significantly improves catalytic efficiency and conversion rates by using a highly tolerant MmUCK enzyme, constructing an AMP/ATP regeneration system, and knocking out genes related to branch metabolic pathways, providing an economically efficient and feasible route for the industrial biomanufacturing of 5{L-End} '-CMP.

Nerve growth factor (NGF) is one of the most critical neurotrophic factors in the nervous system and plays an important role in regulating neuronal growth, development, differentiation, and survival, as well as the regeneration and repair of damaged nerves. Currently, the main active component of pharmaceutical NGF products in China is mouse-derived nerve growth factor (mNGF). However, mNGF may cause allergic reactions and carries the risk of animal virus contamination. This study used the tobacco BY-2 cell expression platform to produce bioactive recombinant human nerve growth factor (rhNGF), aiming to overcome the limitations associated with mNGF. The human NGF gene was introduced into BY-2 cells via genetic engineering, and PCR analysis confirmed its integration into the cell genome. Western blotting results demonstrated that rhNGF was successfully expressed at 166 μg/L in BY-2 cells, and the recombinant protein induced PC12 cell differentiation and promote TF1 cell proliferation, confirming its favorable biological activity. The results of this study paves the way for the use of tobacco BY2 cells as an effective production system for active human nerve growth factor. Our work not only provides a feasible plant-based expression strategy for obtaining safe and low-immunogenicity human NGF, but also lays a technical foundation for utilizing plant cell platforms to produce other complex therapeutic proteins.

The rapid development of the biomanufacturing industry has put an increasing demand for interdisciplinary talents equipped with strong bioengineering design capabilities. However, the conventional teaching model of the AutoCAD Design course fails to effectively support the cultivation of high-quality bioengineering professionals, as students commonly face challenges such as weak engineering application ability, limited innovation and practical skills, and poor alignment with industry needs. To address these issues and enhance students' integrated design and engineering innovation capabilities, we developed a systematic teaching reform framework known as the "one core and five dimensions" model according to the student-centered and competence-oriented educational philosophy. This framework drives comprehensive reforms in teaching contents and teaching methods through five dimensions: curriculum system optimization, online and offline teaching integration, production-education collaboration, incorporation of ideological and political elements, and the integration of competitions with teaching. Since the reform measures were implemented, students have exhibited significantly improvements in operational skills, practical ability, and innovation awareness, which indicate that the overall teaching effectiveness of this course has continued to strengthen. This study offers an effective path for reforming the AutoCAD Design course in bioengineering programs and provides valuable insights for optimizing the talent competency structure in the biomanufacturing field.

With the advance of gene sequencing technology, genome-scale metabolic models have become favored tools for systematically analyzing microbial metabolic networks. In this study, a genome-scale metabolic model of Bifidobacterium animalis subsp. lactis BLa80 was constructed via a semi-automated modeling approach and subsequently validated, providing a tool for subsequent research into its metabolic mechanisms and probiotic functions. In terms of structural quality, the model achieved a Memote score of 82%. For wet-lab validation, the model's simulations of growth on fourteen single carbon sources and amino acid requirements were fully consistent with experimental findings. The maximum specific growth rate and the corresponding specific production rates of two major organic acids predicted by the model based on the existing synthetic medium and modified MRS medium showed errors within 5% and 10%, respectively, compared to the experimental results. The final model, designated iMJ519, comprises 1 005 reactions, 1 047 metabolites, and 519 genes, providing a powerful tool for in-depth research on the growth and metabolic regulation of B. lactis BLa80.

Hyaluronic acid (HA) is a glycosaminoglycan with significant biological activities, which render it widely applicable in the cosmetics and pharmaceutical industries. The development of safe and efficient chassis cells to enhance HA synthesis efficiency has thus emerged as a key factor in HA production. Our study aims to construct a high-performance HA biosynthesis system using Streptomyces as the chassis cell, thereby providing technical support for the efficient microbial production of HA. Thus, our study focused on the metabolic engineering of Streptomyces for strengthening the HA synthesis pathway and then optimized the culture conditions for efficient HA synthesis. First, the HA-synthesizing capabilities of four hyaluronate synthases from different sources were evaluated in two host strains: Streptomyces coelicolor M1146 and Streptomyces albus J1074. The results indicated that the hyaluronate synthases derived from Streptococcus pyogenes exhibited the strongest HA synthesis capability. Notably, the HA yield in S. albus J1074 was higher. Building on this finding, S. albus J1074 was selected as the starting strain to construct a chassis strain tailored for HA synthesis: key genes in the competitive metabolic pathway of HA synthesis were knocked out, while the expression levels of genes associated with the bypass pathway were down-regulated. Furthermore, different combinations of key genes involved in the HA precursor synthesis pathway were designed, and their expression levels were enhanced via a constitutive strong promoter. The recombinant strain obtained therefrom achieved a maximum HA yield of 2.62 g/L. Finally, the synthetic capacity of this high-yield engineered strain was further unleased through the optimization of culture conditions, leading to a final HA yield of 4.63 g/L. The recombinant strain constructed in this study not only lays a foundation for the development of engineered Streptomyces but also provides an excellent chassis strain for the microbial production of HA and other related bioproducts.

Pectinases play a crucial role in the enzymatic degumming process of ramie. However, existing pectinases often suffer from issues such as low degumming efficiency and long processing time. Therefore, it is essential to discover novel efficient pectinases for the ramie degumming process. In this study, we performed carbohydrate-active enzymes annotation on the coding sequence (CDS) and protein sequence of Bacillus licheniformis ATCC 14580, identifying a pectate lyase BlicPL1 whose function has not yet been verified. An expression vector for this gene was constructed and expressed in Escherichia coli BL21(DE3), followed by protein purification. This study marks the first experimental confirmation that BlicPL1 functions as a pectate lyase, with a specific enzyme activity of 3 952.54 U/mg, which is higher than those of most reported pectate lyases from the polysaccharide lyase 1 (PL1) family. Enzymatic characterization revealed that the optimal pH and temperature for BlicPL1 activity were 10.0 and 60 ℃, respectively. Notably, BlicPL1 maintained high activity (over 80% relative activity) within a broad range of 50-80 °C and pH 10.0-12.0. This thermostable and alkali-tolerant profile aligns well with the high-temperature, high-pH conditions typical of ramie degumming processes. Unlike the conventional pectate lyases which are activated by Ca²⁺, BlicPL1 showcased the activity not promoted by Ca²⁺, or even inhibited by Ca²⁺, which highlighted its uniqueness in the Ca²⁺ response mechanism and suggested that BlicPL1 was a novel pectate lyase. Furthermore, BlicPL1 was employed for ramie degumming, resulting in a weight loss rate of 16.17% and a degumming rate of 44.09% for ramie fibers. Scanning electron microscopy results indicated that the treatment with BlicPL1 helped remove residual middle lamella tissue from the ramie fiber surface and reduced the adhesion of free gummy substances, resulting in smoother and more separated fibers. X-ray diffraction analysis confirmed that the characteristic cellulose peaks of the ramie fibers remained unchanged after degumming by BlicPL1, while the crystallinity index increased from 74.59% to 79.69%, indicating an increased proportion of cellulose and the successful removal of non-cellulosic components. The pectate lyase BlicPL1 identified in this study, with high activity and suitability for medium-high temperatures and alkaline environments, demonstrates significant application potential in the ramie degumming process. This work lays a foundation for advancing the industrial application of pectinases in ramie degumming.