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[学术文献 ] A Widespread Radical-Mediated Glycolysis Pathway 进入全文
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Glycyl radical enzymes (GREs) catalyze mechanistically diverse radical-mediated reactions, playing important roles in the metabolism of anaerobic bacteria. The model bacterium Escherichia coli MG1655 contains two GREs of unknown function, YbiW and PflD, which are widespread among human intestinal bacteria. Here, we report that YbiW and PflD catalyze ring-opening C–O cleavage of 1,5-anhydroglucitol-6-phosphate (AG6P) and 1,5-anhydromannitol-6-phosphate (AM6P), respectively. The product of both enzymes, 1-deoxy-fructose-6-phosphate (DF6P), is then cleaved by the aldolases FsaA or FsaB to form glyceraldehyde-3-phosphate (G3P) and hydroxyacetone (HA), which are then reduced by the NADH-dependent dehydrogenase GldA to form 1,2-propanediol (1,2-PDO). Crystal structures of YbiW and PflD in complex with their substrates provided insights into the mechanism of radical-mediated C–O cleavage. This “anhydroglycolysis” pathway enables anaerobic growth of E. coli on 1,5-anhydroglucitol (AG) and 1,5-anhydromannitol (AM), and we probe the feasibility of harnessing this pathway for the production of 1,2-PDO, a highly demanded chiral chemical feedstock, from inexpensive starch. Discovery of the anhydroglycolysis pathway expands the known catalytic repertoire of GREs, clarifies the hitherto unknown physiological functions of the well-studied enzymes FsaA, FsaB, and GldA, and demonstrates how enzyme discovery efforts can cast light on prevalent yet overlooked metabolites in the microbiome.
[学术文献 ] Progress in Enzyme-Catalyzed C(sp3)-H Amination 进入全文
ACS CATALYSIS
Amine structures are widely present in various biologically active natural products, drug molecules, and material structures. Among the various strategies of amine synthesis, C(sp3)–H amination has become a powerful strategy due to its atom economy and multiple potential reaction sites compared with conventional amine synthesis methods. Due to the advantages of high catalytic efficiency, high selectivity, environmental friendliness, and high modifiability of the enzyme, the enzymatic C(sp3)–H amination is of great research significance. However, it was not until recent years that natural enzymes capable of catalyzing the amination of C(sp3)–H bonds were discovered. Modifying enzymes to confer unnatural C(sp3)–H amination activity holds great potential. In the past decade, a series of protocols for the amination of C(sp3)–H bonds using engineered enzymes have been developed, several of which showed comparable properties to those of natural enzymes.
[前沿资讯 ] 学者构建超分子光-酶催化体系应用于水体净化 进入全文
科学网
暨南大学环境与气候学院环境健康系（筹）副教授江瑞芬团队联合中山大学副教授陈国胜、副教授沈勇以及教授欧阳钢锋，创新性地构建了一种基于直接电子转移的超分子光-酶催化体系，并将其应用于水体有机污染物的高效去除。相关成果近日发表于《科学进展》（Science Advances）。 研究团队创新性地构建了一种基于直接电子转移的超分子光-酶催化体系，并成功应用于水体净化。研究中，氧化还原酶—漆酶被封装在多孔的光活性氢键有机框架结构的缺陷区域内，形成了促进光活性氢键有机框架与漆酶之间电子转移的稳定生物界面，避免了使用昂贵的辅因子或辅助介质。同时，这一光-酶体系不产生强破坏性的活性物种，如超氧自由基和羟基自由基，成功地保护了酶的活性，从而解决了光催化与生物催化难以兼容的难题。 此外，与传统无孔纳米结构相比，该超分子体系的长程有序介孔结构有效促进了反应物分子的传质，大幅提升了光-酶偶联催化的效率。以双酚A（BPA）为例进行验证实验，该体系在可见光条件下的催化转化率和周转频率比游离酶高出两个数量级，且无需使用任何牺牲剂。在每克催化剂每小时的污染物转化量方面，其净化效率远超现有报道的催化剂。
[前沿资讯 ] 天津工业生物所等在人工辅酶合成应用领域取得进展 进入全文
中科院天津工业生物技术研究所
手性胺类化合物在药物、农用化学品和精细化工合成中扮演了重要角色。亚胺还原酶（IREDs）作为一种能够催化还原胺化反应生成高立体选择性手性胺的酶，因其绿色环保的优势备受关注。然而，IREDs在催化反应中依赖于还原型辅酶Ⅱ（NADPH）作为电子供体，而NADPH成本高、稳定性差，极大限制了其在工业规模中的广泛应用。因此，寻找一种能够替代NADPH的高效、低成本人工辅因子成为了研究的重点。尽管人工辅因子研究在多种氧化还原酶中取得了积极效果，其在IRED催化反应中的应用尚未被充分挖掘。 近日，中国科学院天津工业生物技术研究所崔成森研究员团队和中国海洋大学的王长云教授团队合作，通过使用人工合成的烟酰胺辅因子替代NADPH，成功实现了IRED催化的高效还原胺化反应。该团队合成了16个不同取代基的小分子人工辅酶，通过分子对接实验深入分析了人工辅因子与IRED酶活性位点的相互作用，揭示了不同取代基在还原胺化反应中的催化机制。本研究为理解小分子人工辅因子的作用机制提供了理论基础，而葡萄糖脱氢酶再生系统则进一步增强了反应的可操作性。下一步团队将着重于对这些人工辅因子进行进一步优化，扩展其在其他酶促反应中的应用潜力，并推动其在工业中的实际应用。
[学术文献 ] Multilevel Systematic Optimization To Achieve Efficient Integrated Expression of Escherichia coli 进入全文
ACS Synthetic Biology
Genomic integration of heterologous genes is the preferred approach in industrial fermentation-related strains due to the drawbacks associated with plasmid-mediated microbial fermentation, including additional growth burden, genetic instability, and antibiotic contamination. Synthetic biology and genome editing advancements have made gene integration convenient. Integrated expression is extensively used in the field of biomanufacturing and is anticipated to become the prevailing method for expressing recombinant proteins. Therefore, it is pivotal to strengthen the expression of exogenous genes at the genome level. Here, we systematically optimized the integrated expression system of Escherichia coli from 3 aspects. First, the integration site slmA with the highest expression activity was screened out of 18 sites in the ORI region of the E. coli BL21 (DE3) genome. Second, we characterized 16 endogenous promoters in E. coli and combined them with the T7 promoter. A constitutive promoter, Plpp-T7, exhibited significantly higher expression strength than the T7 promoter, achieving a 3.3-fold increase in expression levels. Finally, to further enhance the T7 expression system, we proceeded with overexpression of T7 RNA polymerase at the chassis cell level. The resulting constitutive efficient integrated expression system (CEIES_Ecoli) showed a 2-fold increase in GFP expression compared to the pET3b recombinant plasmid. Therefore, CEIES_Ecoli was applied to the integrated expression of nitrilase and hyaluronidase, achieving stable and efficient enzyme expression, with enzyme activities of 22.87 and 12,195 U·mL–1, respectively, comparable to plasmid levels. Overall, CEIES_Ecoli provides a stable and efficient method of gene expression without the need for antibiotics or inducers, making it a robust tool for synthetic biology, enzyme engineering, and related applications.
[学术文献 ] Integrating Deep Learning and Synthetic Biology: A Co-Design Approach for Enhancing Gene Expression via N-Terminal Coding Sequences 进入全文
ACS Synthetic Biology
N-terminal coding sequence (NCS) influences gene expression by impacting the translation initiation rate. The NCS optimization problem is to find an NCS that maximizes gene expression. The problem is important in genetic engineering. However, current methods for NCS optimization such as rational design and statistics-guided approaches are labor-intensive yield only relatively small improvements. This paper introduces a deep learning/synthetic biology codesigned few-shot training workflow for NCS optimization. Our method utilizes k-nearest encoding followed by word2vec to encode the NCS, then performs feature extraction using attention mechanisms, before constructing a time-series network for predicting gene expression intensity, and finally a direct search algorithm identifies the optimal NCS with limited training data. We took green fluorescent protein (GFP) expressed by Bacillus subtilis as a reporting protein of NCSs, and employed the fluorescence enhancement factor as the metric of NCS optimization. Within just six iterative experiments, our model generated an NCS (MLD62) that increased average GFP expression by 5.41-fold, outperforming the state-of-the-art NCS designs. Extending our findings beyond GFP, we showed that our engineered NCS (MLD62) can effectively boost the production of N-acetylneuraminic acid by enhancing the expression of the crucial rate-limiting GNA1 gene, demonstrating its practical utility. We have open-sourced our NCS expression database and experimental procedures for public use.
