Novel transaminases from thermophiles: from discovery to application.
Novel transaminases from thermophiles: from discovery to application.
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DOI：
10.1111/1751-7915.13940
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发表时间：
2022-01
影响因子：
5.7
通讯作者：
Ward JM
中科院分区：
文献类型：
--
作者：
Cárdenas-Fernández M;Sinclair O;Ward JM
氨基转移酶（TA）是一种很有前途的手性胺合成生物催化剂，然而，迄今为止，只有少数嗜热TA已被描述。在这项工作中，基因组挖掘的方法被用来寻找新的TA从9个嗜热微生物。从它们各自的基因组序列中鉴定TA序列，并预测它们的Pfam，证实I-II类TA最丰富（50%），其次是III类（26%）、V类（16%）、IV类（8%）和VI类（1%）。开放阅读框（ORF）是TA的百分比范围从滨海热球菌中的0.689%到硫磺硫化叶菌中的0.424%。总共有94个推定的TA被成功克隆并表达到大肠杆菌中，当使用含有d-山梨醇的化学伴侣培养基时，显示出大多数良好的表达水平。使用不同氨基供体-受体的动力学和终点比色测定证实了TA的活性，允许对底物范围进行初步探索。选择立体选择性和非立体选择性丝氨酸-TA用于羟基丙酮酸（HPA）的合成。用四种非立体选择性丝氨酸-TA观察到低HPA反应产率，而两种立体选择性丝氨酸-TA显示出显著更高的产率。将丝氨酸-TA反应与转酮醇酶偶联以产生L-藜芦酮糖（Ery）显著增加了丝氨酸转化为HPA。使用廉价的外消旋D/L-丝氨酸组合立体选择性丝氨酸-TA和转酮醇酶导致高Ery产率（82%）。立体选择性丝氨酸-TA的热表征证实它们具有高达60°C的优异热稳定性和高的最佳温度。氨基转移酶（TA）是一种很有前途的手性胺合成生物催化剂，但迄今为止，只有少数嗜热TA已被描述。我们已经从9种嗜热细菌中克隆并鉴定了94种TA。选择立体选择性和非立体选择性丝氨酸-TA用于合成羟基丙酮酸（HPA），其是酮醇转移酶C-C键形成的重要供体底物。用4种非立体选择性丝氨酸TA观察到低HPA产率，而两种立体选择性丝氨酸TA显示出显著更高的产率。将丝氨酸-TA反应与转酮醇酶偶联以产生L-藜芦酮糖（Ery）显著增加了丝氨酸转化为HPA。使用廉价的外消旋D/L-丝氨酸组合立体选择性丝氨酸-TA和转酮醇酶导致高Ery产率（82%）。
Transaminases (TAs) are promising biocatalysts for chiral amine synthesis; however, only few thermophilic TAs have been described to date. In this work, a genome mining approach was taken to seek novel TAs from nine thermophilic microorganisms. TA sequences were identified from their respective genome sequences and their Pfam were predicted confirming that TAs class I–II are the most abundant (50%), followed by class III (26%), V (16%), IV (8%) and VI (1%). The percentage of open reading frames (ORFs) that are TAs ranges from 0.689% in Thermococcus litoralis to 0.424% in Sulfolobus solfataricus. A total of 94 putative TAs were successfully cloned and expressed into E. coli, showing mostly good expression levels when using a chemical chaperone media containing d‐sorbitol. Kinetic and end‐point colorimetric assays with different amino donors–acceptors confirmed TAs activity allowing for initial exploration of the substrate scope. Stereoselective and non‐stereoselective serine‐TAs were selected for the synthesis of hydroxypyruvate (HPA). Low HPA reaction yields were observed with four non‐stereoselective serine‐TAs, whilst two stereoselective serine‐TAs showed significantly higher yields. Coupling serine‐TA reactions to a transketolase to yield l‐erythrulose (Ery) substantially increased serine conversion into HPA. Combining both stereoselective serine‐TAs and transketolase using the inexpensive racemic D/L‐serine led to high Ery yield (82%). Thermal characterization of stereoselective serine‐TAs confirmed they have excellent thermostability up to 60°C and high optimum temperatures. Transaminases (TAs) are promising biocatalysts for chiral amine synthesis, however only few thermophilic TAs have been described to date. We have cloned and characterised 94 TAs from nine thermophilic bacteria. Stereoselective and non‐stereoselective serine‐TAs were selected for the synthesis of hydroxypyruvate (HPA) which is a vital donor substrate for transkeolase C‐C bond formation. Low HPA yields were observed with 4 non‐stereoselective serine‐TAs, whilst two stereoselective serine‐TAs showed significant higher yields. Coupling serine‐TA reactions to a transketolase to yield l‐erythrulose (Ery) substantially increased serine conversion into HPA. Combining both stereoselective serine‐TAs and transketolase using the inexpensive racemic D/L‐serine led to high Ery yield (82%).
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