共检索到62条,权限内显示50条;
[学术文献 ] Engineering Peroxygenase Activity into Cytochrome P450 Monooxygenases through Modification of the Oxygen Binding Region 进入全文
ACS Catalysis
Cytochrome P450 enzymes (CYPs) are biocatalysts for the generation of fine chemicals including natural products, drug metabolites, and flavor and fragrance compounds. However, both the high cost of the required nicotinamide cofactors and their need for additional electron transfer proteins limit their use. Here, we investigate whether CYPs can be converted into more efficient peroxygenases through protein engineering of the enzyme's oxygen activation machinery. We improve the peroxygenase activity by modifying selected residues within the I-helix to more closely resemble those of a natural peroxygenase. We produced mutants containing two, four, and six mutations, within this region of the I-helix. In our model CYP system, the double mutant in which glutamine and glutamate residues replaced aspartate and threonine, respectively, was found to have significantly higher peroxygenase activity for the O-demethylation of 4-methoxybenzoic acid than a single glutamate mutant prototype. Importantly, it functioned better at lower H2O2 concentrations and could convert all the added substrate to product. All the mutants maintained the stereoselectivity of the CYP enzyme for the epoxidation of 4-vinylbenzoic acid. The X-ray crystal structures of these enzymes showed significant structural changes at the oxygen-binding groove in the I-helix. In crystallo reactions with 4-methylbenzoic acid exhibit electron density corresponding to the 4-(hydroxymethyl)benzoic acid metabolite. We extended this mutagenesis strategy to a bacterial steroid-hydroxylating CYP and an uncharacterized CYP from a thermophilic bacterium. In these instances, we generate peroxygenases, which catalyze the regio- and stereoselective hydroxylation of progesterone and the hydroxylation of fatty acids at low hydrogen peroxide concentrations.
[学术文献 ] Practical Machine Learning-Assisted Design Protocol for Protein Engineering: Transaminase Engineering for the Conversion of Bulky Substrates 进入全文
ACS Catalysis
Protein engineering is essential for improving the catalytic performance of enzymes for applications in biocatalysis, in which machine learning provides an emerging approach for variant design. Transaminases are powerful biocatalysts for the stereoselective synthesis of chiral amines but one major challenge is their limited substrate scope. We present a general and practical variant design protocol for protein engineering to combine the advantages of three strategies, including directed evolution, rational design, and machine learning, and demonstrate the application of the protocol in the protein engineering of transaminases with higher activity toward bulky substrates. A high-quality data set was obtained by rational design of selected key positions, which was then applied to create a machine learning model for transaminase activity. This model was applied for the data-assisted design of optimized variants, which showed improved activity (up to 3-fold over parent) for three bulky substrates, maintaining enantioselectivity of the starting enzyme scaffold as well as improving the enantiomeric excess (up to >99%ee).
[学术文献 ] CRISPR–dCas12a-mediated genetic circuit cascades for multiplexed pathway optimization 进入全文
Nature Chemical Biology
The production efficiency of microbial cell factories is sometimes limited by the lack of effective methods to regulate multiple targets in a coordinated manner. Here taking the biosynthesis of glucosamine-6-phosphate (GlcN6P) in Bacillus subtilis as an example, a 'design-build-test-learn' framework was proposed to achieve efficient multiplexed optimization of metabolic pathways. A platform strain was built to carry biosensor signal-amplifying circuits and two genetic regulation circuits. Then, a synthetic CRISPR RNA array blend for boosting and leading (ScrABBLE) device was integrated into the platform strain, which generated 5,184 combinatorial assemblies targeting three genes. The best GlcN6P producer was screened and engineered for the synthesis of valuable pharmaceuticals N-acetylglucosamine and N-acetylmannosamine. The N-acetylglucosamine titer reached 183.9 g liter(-1) in a 15-liter bioreactor. In addition, the potential generic application of the ScrABBLE device was also verified using three fluorescent proteins as a case study.
[前沿资讯 ] 中科院天津工业生物所发布REME平台 推动非天然反应酶挖掘与评估 进入全文
中科院天津工业生物所
近日，中国科学院天津工业生物技术研究所生物设计中心发布了首个集成的反应酶挖掘与评估平台——REME（https://reme.biodesign.ac.cn）。该平台结合底物原子到产物原子映射、原子类型变化识别和反应相似性计算，实现了相似反应的计算、快速排序和可视化。用户可以根据功能基团筛选相似反应，并进一步通过酶号或序列同源性筛选或扩展候选酶。REME平台结合多种人工智能方法对候选酶进行多角度评估（如kcat、Km、最佳温度和pH），帮助科研人员迅速识别潜在酶。REME平台的推出为非天然反应的酶挖掘和评估提供了新的解决方案。
[前沿资讯 ] Bioengineered enzyme creates natural vanillin from plants in one step 进入全文
Eurek Alert
Professor Toshiki Furuya from the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, and his graduate students Shizuka Fujimaki and Satsuki Sakamoto, successfully developed an enzyme that generates vanillin from plant-derived ferulic acid. “Ferulic acid, the raw material, is a compound that can be obtained in abundance from agricultural waste such as rice bran and wheat bran. Vanillin is generated simply by mixing ferulic acid with the developed enzyme at room temperature. So, the established technology can provide a simple and environmentally friendly method for producing flavor compounds,” explains Prof. Furuya. Their study was published on May 10, 2024 in Applied and Environmental Microbiology. The researchers used genetic engineering approaches to modify the molecular structure of an enzyme – ‘Ado.’ Ado is originally an oxidase enzyme that adds an oxygen atom to the substrate – isoeugenol. In its native state, it does not have the ability to convert ferulic acid into vanillin. Using structural modeling analysis, the researchers were able to predict amino acid changes in Ado which would enable its interaction with ferulic acid. On these lines, they conducted a series of experiments by replacing phenylalanine and valine amino acid residues at specific positions in the structure of Ado, with various other amino acids. They went on to examine the ferulic acid conversion ability of the various engineered mutant proteins.
[学术文献 ] Advances and prospects in microbial production of biotin 进入全文
Microbial Cell Factories
Biotin, serving as a coenzyme in carboxylation reactions, is a vital nutrient crucial for the natural growth, development, and overall well-being of both humans and animals. Consequently, biotin is widely utilized in various industries, including feed, food, and pharmaceuticals. Despite its potential advantages, the chemical synthesis of biotin for commercial production encounters environmental and safety challenges. The burgeoning field of synthetic biology now allows for the creation of microbial cell factories producing bio-based products, offering a cost-effective alternative to chemical synthesis for biotin production. This review outlines the pathway and regulatory mechanism involved in biotin biosynthesis. Then, the strategies to enhance biotin production through both traditional chemical mutagenesis and advanced metabolic engineering are discussed. Finally, the article explores the limitations and future prospects of microbial biotin production. This comprehensive review not only discusses strategies for biotin enhancement but also provides in-depth insights into systematic metabolic engineering approaches aimed at boosting biotin production.
