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[学术文献 ] Semirationally Engineering an Efficient P450 Peroxygenase for Regio- and Enantioselective Hydroxylation of Steroids 进入全文
ACS Catalysis
Enzymatic direct hydroxylation of unactivated C–H bonds in steroids provides a promising approach to enrich their structural and functional diversity, together with higher physiological and pharmacological activity. Here, we construct an efficient peroxide-driven P450 hydroxylase for the regio- and enantioselective hydroxylation of steroids. The NADH-dependent CYP154C5 monooxygenase is smoothly transformed into its peroxygenase mode by combining the strategies of H2O2 tunnel engineering and the introduction of a catalytic aspartate residue, which avoids the use of expensive nicotinamide cofactors and redox partner proteins. The variant F92A/R114A/E282A/T248D (AAA/T248D) quantitatively converted testosterone and nandrolone into the corresponding 16α-hydroxylation products, showing the best catalytic efficiency (kcat/Km) for testosterone hydroxylation among all known natural and engineered P450 peroxygenases to date. Crystal structural analysis and molecular dynamics simulations suggest that H2O2 tunnel engineering plays a crucial role in promoting the flow of H2O2 into active centers, and the introduced aspartate residue may participate in the activation of H2O2. Moreover, the milligram-scale preparation of 16α-hydroxytestosterone by AAA/T248D gave a substrate conversion rate (>98%) and an isolated yield (90%), suggesting potential for synthetic application. This work not only establishes a feasible semirational approach to engineered non-natural P450 peroxygenases but also provides a potentially practical approach for the enzymatic synthesis of hydroxylated steroid compounds.
[学术文献 ] Patulin-degrading enzymes sources, structures, and mechanisms: A review 进入全文
International Journal of Biological Macromolecules
Patulin (PAT), a fungal secondary metabolite with multiple toxicities, is an unavoidable contaminant in fruit and vegetable processing, posing potential health risks to consumers and causing significant economic losses to the global food industry. Traditional control strategies, such as physical and chemical methods, face several challenges, including low efficiency, high costs, and unverified safety. In contrast, microbial degradation of patulin is considered a more efficient and environmentally friendly approach, which has become a popular research focus. However, there is still insufficient research on the key degradation enzymes involved in microorganisms. Therefore, this review comprehensively summarizes recent research progress on the biological degradation of patulin, with a focus on microbial species capable of degrading patulin, the degradation enzymes they express, potential degradation mechanisms, and the toxicity of degradation products, while providing prospects for future research. It offers valuable insights for controlling patulin in food and stimulates further investigation. Ultimately, this review aims to promote the development of efficient and eco-friendly methods to mitigate patulin contamination in fruits and vegetables.
[前沿资讯 ] 中国科学院深圳先进技术研究院合成生物学研究所研究基于对比学习的酶促反应分类AI模型 进入全文
中国科学院深圳先进技术研究院
中国科学院深圳先进技术研究院的罗小舟领衔的研究团队，近日在Journal of Cheminformatics期刊发表重要研究成果"CLAIRE: A Contrastive Learning-based Predictor for EC Number of Chemical Reactions"。在该研究成果中，团队利用对比学习，数据扩增，以及基于化学反应预训练模型的特征提取（embedding）策略，构建了一个用于预测EC分类编号的高效人工智能模型（CLAIRE）。作者将CLAIRE与当前最领先的Theia模型进行了对比。Theia是2023年由瑞士洛桑联邦理工学院的科学家Daniel Probst发表在Journal of Cheminformatics期刊上的基于常规深度学习的模型——然而常规深度学习方法不能有效解决数据不平衡的问题。借助对比学习和数据扩增的策略，CLAIRE展现出了优异的性能——在测试集上，CLAIRE比Theia有数倍的准确率提升，且在三级EC分类编号预测之间的一致性也显著高于Theia。此外，作者利用酵母菌的代谢模型构建了另一个大型独立测试集。在该数据集中，CLAIRE的表现也显著高于Theia。 通过一系列严格的评估，研究人员展示了CLAIRE的强大能力：在酵母代谢模型中，它成功区分了真实的酶-反应配对与错误配对。代谢模型是生物体内代谢反应的定量化表示，涵盖基因、酶、代谢物及其细胞内分布，广泛应用于代谢工程和通量平衡分析等领域。CLAIRE的加入使得研究人员能够更高效地分析和注释反应网络，为代谢研究提供了全新可能。此外，CLAIRE在逆合成路径规划和药物代谢预测等关键领域展示出巨大应用潜力。逆合成预测旨在推断生成目标化合物所需的原料及反应路径。在这一过程中，多个中间产物可能生成大量候选反应。通过CLAIRE预测的EC编号，可为这些反应分配相关酶，大幅提升最终目标化合物成功合成的可能性。另外，药物在人体内的代谢转化及路径是评估其安全性和有效性的重要环节。通过对潜在反应注释EC编号，CLAIRE能够清晰描绘可能的药物代谢路径，为毒性评估及药物开发提供有力支持。总而言之，该项成果在代谢工程和合成生物学领域中有着广泛的应用。
[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.
