Halogenases in Biocatalysis: Advances in Mechanism Elucidation， Directed Evolution， and Green Manufacturing Synthetic Biology Journal    DOI: 10.12211/2096-8280.2024-091

Fig. 4 Workflow of genome mining for halogenases and their application examples^[34]

Extracts from the Article

Fisher等［34］对黄素依赖型卤化酶进行全家族范围的基因组挖掘，使用已知卤化酶RebH的保守区域作为查询序列，在UniProt序列数据库中进行BLAST搜索，共获得3975个匹配结果，从中挑选并合成了128个假定FDH基因，高通量筛选了其中87种可溶性表达的FDHs对62种底物及2种卤素的大于2万个催化反应；发现39种新的活性卤化酶可选择性地进行后期C-H官能化。挖掘得到的卤化酶1-F08和1-F11比之前报道的FDHs更具优势，成功实现几种大分子化合物的位点选择性卤化（图4）。尽管1-F11源自拟南芥根际微生物组中的一种中温型Sphingomonas菌种，但其热稳定性（T_m = 66.5 ± 0.2°C）与经过三轮定向进化后的RebH突变体3-LSR（T_m = 69.5 ± 0.4°C）相当。这些结果显著扩展了可用于生物催化的FDH范围，并为不同应用的持续迭代基因组挖掘提供了指导。

Other Images/Table from this Article

• Fig. 1  Halogens are the most common and important modification groups in pesticides and pharmaceutical products
• Fig. 2  Examples of halogenated natural products and the role of halogenases in molecular synthesis^[4,12,15-16]
• Fig. 3  Cofactors and catalytic mechanisms of different types of halogenase^[1,4,17-25]
• Table 1  Examples of halogenase discovered by genome mining
• Table 2  The uniqueness and superiority of the CLEAN algorithm framework
• Fig. 5  Overview of chloride depletion workflow for revealing cryptic halogenation^[59]
• Fig. 6  The typical structure and catalytic elements of flavin-dependent halogenases
• Fig. 7  The typical structure of single-component flavin-dependent halogenases
• Fig. 8  The proposed catalytic mechanism of PrnA by calculation and simulation^[69]
• Fig. 9  The typical structure of NHFe/αKGHs^[25,70-76]
• Fig. 10  The proposed catalytic mechanism of BesD by calculation and simulation^[80]
• Fig. 11  The action mode and engineering strategies of novel nucleotide halogenase ^[46]
• Fig.12  The structural, function and catalytic mechanism of copper-dependent halogenase ApnU^[81]
• Table 3  Typical engineering cases of natural halogenase in recent years
• Fig. 13  The typical structure SAM-dependent halogenase ^[84-91]The red lines indicate the residues that are crucial for fluorination activity; the yellow lines indicate the residues that can form hydrogen bonds with the modification groups at the C2′ position of adenine
• Fig. 14  Tunnel engineering improves the catalytic performance of Thal halogenase^[96]
• Fig. 15  Directed evolution enhances the fluorination activity of non-heme iron oxidase^[108-109]
• Fig. 16  Biocatalytic synthesis platform of halogenated tryptophan derivatives^[113]
• Tab. 4  Examples of FDHs catalyzing enantioselective reactions^[125-127]
• Tab. 5  Recent cases and key data related to the potential industrial application of halogenases
• Fig. 17  Representative research progress in the field of halogenase research
• Fig. 18  AI/ML drives the intelligent evolution of halogenase^[138-156]