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[前沿资讯 ] 天津工业生物技术研究所实现大肠杆菌实时动态调控葡萄糖摄取率及中心途径代谢 进入全文
中科院天津工业生物技术研究所
近日，中国科学院天津工业生物技术研究所张大伟研究员带领的蛋白表达系统与微生物代谢研究团队开发了实时动态监测大肠杆菌葡萄糖吸收速率的方法及其遗传回路，能够动态调节葡萄糖摄取速率及相关代谢途径的碳通量。在大肠杆菌摄取葡萄糖时，会经历一系列复杂的过程，包括跨膜转运、磷酸化、去磷酸化、辅助蛋白招募，以及相关因子的表达或抑制等。基于此调控机制，研究团队开发出了能够实时响应葡萄糖摄取速率的生物传感器（GURBs）（图1），并建立了对葡萄糖摄取速率和中央代谢流进行正负调节的遗传回路。GURBs的性能和灵敏度在不同条件下得到了验证。在线荧光和离线葡萄糖检测技术表明，GURBs可以直接测量葡萄糖摄取速率。GURBs被应用于氨基酸，维生素，有机酸等产品合成（图2），通过调控中央代谢途径代谢流，或调控遗传回路的激活或抑制，有效的提高了其产量。这些结果表明，GURBs可以根据葡萄糖摄取速率动态调节葡萄糖摄取率，及中央代谢和相关途径的碳通量，从而提高目标产品产量。葡萄糖作为细胞摄取碳源的第一步，建立其实时监测及动态调控技术十分重要，通过基因回路优化代谢流分配，不仅能很好地适应培养环境变化，还能有效平衡细胞生长与产物合成之间的代谢竞争，合理分配和利用碳资源，为合成生物设计与细胞工厂的构建提供了重要工具和更多选择。
[前沿资讯 ] 中国科学院深圳先进技术研究院综述肠道微生物群落建模研究进展 进入全文
https://www.siat.ac.cn/kyjz2016/202501/t20250120_7520256.html
近日，中国科学院深圳先进技术研究院陈禹课题组与查尔姆斯理工大学Jens Nielsen教授合作，在Current Opinion in Biotechnology期刊发表综述文章“Personalized gut microbial community modeling by leveraging genome-scale metabolic models and metagenomics”。陈禹研究员和Jens Nielsen教授为文章的共同通讯作者，研究助理李龙涛为第一作者。该工作获得了国家重点研发计划及深圳合成生物学创新研究院的支持。 文章首先回顾了近些年GEM相关资源与建模工具（如AGORA2，CarveMe等）及其在肠道微生物研究中的应用，然后介绍了构建个性化人类肠道Co-GEM的两种主流策略 （图1）：一是通过宏基因组中获得的微生物分类信息与已有的多个菌株的GEM资源整合构建Co-GEM；二是直接利用宏基因组测序数据构建GEM并结合环境中微生物分类信息来构建Co-GEM。最后，文章总结了该领域的挑战与展望。首要挑战便是不同数据库与GEM资源之间的标准化，目前不同GEM和数据库之间的代谢物、反应等关键信息存在多种不同的格式和命名规则。单一模型的性能是群落建模的基础，基于先验知识对GEM进行多约束（比如酶动力学参数，蛋白限制等）的整合至关重要。例如，最新的GECKO 3.0工具通过构建酶约束模型显著提升了模型的预测能力，有望运用于肠道微生物模型构建。此外，新的“泛”模型构建方法，比如MIGRENE和Pan-draft等，使得构建个性化肠道Co-GEM成为了可能；而多组学数据的整合以及机器学习和神经网络方法也能够进一步提升模型性能。随着新方法的不断涌现并应用在提高Co-GEM的性能上，相信在不久的将来，将能从肠道微生物的角度为人类健康与疾病提供更深入的见解。
[学术文献 ] Insights into the zearalenone degradation performance and pathway by Gordonia hydrophobica HAU421 and characterization of a novel lactonohydrolase involved 进入全文
International Journal of Biological Macromolecules
Zearalenone (ZEN) is a harmful macrolide mycotoxin, posing a serious hazard to human health. In this study, a highly efficient ZEN-degrading bacterium Gordonia hydrophobica HAU421 was isolated from soil by using spiramycin (SPM)-containing selective medium. Mass spectrometry analysis revealed that strain HAU421 could transform ZEN into hydrolyzed zearalenone (HZEN), zearalenol (ZEL), and hydrolyzed zearalenol (HZEL). A novel lactonohydrolase GhZH capable of hydrolyzing ZEN was mined from the genome of strain HAU421 and heterologously expressed in Escherichia coli. The recombinant GhZH exhibited peak activity at pH 7.0 and 42 °C. The catalytic triad of GhZH was identified as S122-D147-H297 via sequence comparison, molecular docking and site-directed mutagenesis. Moreover, toxicological analysis suggested that GhZH-catalyzed ZEN hydrolyzation resulted in the detoxification of its hepatotoxicity. To meet the industrial demands, GhZH was immobilized onto chitosan microspheres using the crosslinker glutaraldehyde. The stability of immobilized GhZH at harsh acidic pH and high temperature was enhanced in comparison with free GhZH. The immobilized GhZH achieved a ZEN removal rate of 53.2 % in beer and 74.0 % in corn steep liquor. These findings offer new insights into microbial ZEN degradation and support the advancement of enzyme-catalyzed ZEN detoxification.
[学术文献 ] Protein Engineering of Substrate Specificity toward Nitrilases: Strategies and Challenges 进入全文
Journal of Agricultural and Food Chemistry
Nitrilase is extensively applied across diverse sectors owing to its unique catalytic properties. Nevertheless, in industrial production, nitrilases often face issues such as low catalytic efficiency, limited substrate range, suboptimal selectivity, and side reaction products, which have garnered heightened attention. With the widespread recognition that the structure of enzymes has a direct impact on their catalytic properties, an increasing number of researchers are beginning to optimize the functional characteristics of nitrilases by modifying their structures, in order to meet specific industrial or biotechnology application needs. Particularly in the artificial intelligence era, the innovative application of computer-aided design in enzyme engineering offers remarkable opportunities to tailor nitrilases for the widespread production of high-value products. In this discussion, we will briefly examine the structural mechanism of nitrilase. An overview of the protein engineering strategies of substrate preference, regioselectivity and stereoselectivity are explored combined with some representative examples recently in terms of the substrate specificity of enzyme. The future research trends in this field are also prospected.
[学术文献 ] Metabolic Engineering of Escherichia coli for De Novo Biosynthesis of the Platform Chemical Pelletierin 进入全文
ACS Sustainable Chemistry & Engineering
Pelletierine is a versatile plant alkaloid having a C5N–C3 structure from which numerous chemicals can be derived. One notable derivative is huperzine A (HupA) which may alleviate the symptoms of Alzheimer’s disease. Currently, industrial production of pelletierine relies primarily on chemical synthesis and plant extraction. However, chemical synthesis leads to analogues that complicate product separation, and plant extraction is constrained by limited resources. Herein, we report that pelletierine can be produced by recombinant Escherichia coli in which the engineered pelletierine biosynthesis pathway comprises four modules involving seven key genes native to E. coli, three genes from other bacteria, and three genes from plants. To overproduce pelletierine, the intrinsic l-lysine biosynthesis pathway in E. coli was simplified, and a clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) system was engineered to minimize the byproducts. Moreover, the transporter MatC was overexpressed to enhance the intracellular concentration of 3-oxoglutaryl ketide, which is another precursor of pelletierine. Based on the aforementioned manipulations, the resulting recombinant E. coli harboring the pelletierine biosynthesis pathway and CRISPRi system produced 3.40 and 8.23 mg/L pelletierine in a shake-flask and a 5 L bioreactor, respectively. This is the first report of microbial production of pelletierine, which represents a sustainable route to produce the precursor of HupA and beyond.
[学术文献 ] Efficient Expression and Activity Optimization of Manganese Peroxidase for the Simultaneous Degradation of Aflatoxins AFB1, AFB2, AFG1, and AFG2 进入全文
Journal of Agricultural and Food Chemistry
Aflatoxins (AFs), notorious mycotoxins that pose significant risks to human and animal health, make biodegradation extremely crucial as they offer a promising approach to managing and reducing their harmful impacts. In this study, we identified a manganese peroxidase from Punctularia strigosozonata (PsMnp) through protein similarity analysis, which has the capability to degrade four AFs (AFB1, AFB2, AFG1, and AFG2) simultaneously. The gene encoding this enzyme was subject to codon optimization, followed by cold shock induction expression using the pColdII vector, leading to the soluble expression of manganese peroxidase (Mnp) in Escherichia coli. This study tackled the problem of inclusion body formation that often occurs during Mnp expression in E. coli. After optimizing the degradation conditions, the degradation rates for AFB1, AFB2, AFG1, and AFG2 were 87.9, 72.8, 77.3, and 85.6%, respectively. Molecular docking and molecular dynamics simulations indicated that PsMnp facilitated the degradation of AFs through hydrophobic and polar interactions among various amino acid residues. This research offers novel insights into the rapid discovery of enzymes capable of degrading AFs and establishes a theoretical foundation for the efficient expression of mycotoxin detoxification enzymes.
