Department of Biochemical Engineering, University College London, Gower St, WC1E 6BT, London, UK.
School of Biosciences, University of Kent, CT2 7NJ, Kent, UK.
Microb Biotechnol. 2022 Jan;15(1):305-317. doi: 10.1111/1751-7915.13940. Epub 2021 Oct 29.
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.
转氨酶（TAs）是手性胺合成有前途的生物催化剂；然而，迄今为止仅描述了少数嗜热 TAs。在这项工作中，采用了基因组挖掘方法从九种嗜热微生物中寻找新的 TAs。从各自的基因组序列中鉴定出 TA 序列，并预测了它们的 Pfam，证实 TA 类 I-II 是最丰富的（50%），其次是类 III（26%）、V（16%）、IV（8%）和 VI（1%）。TA 的开放阅读框（ORFs）的百分比范围从 Thermococcus litoralis 的 0.689%到 Sulfolobus solfataricus 的 0.424%。成功克隆了 94 个推定的 TA 并在 E. coli 中表达，当使用含有 d-山梨糖醇的化学伴侣培养基时，大多数表现出良好的表达水平。使用不同的氨基供体-受体进行动力学和终点比色测定证实了 TA 的活性，从而可以初步探索底物范围。立体选择性和非立体选择性丝氨酸-TA 被用于合成羟基丙酮酸（HPA）。用四种非立体选择性丝氨酸-TA 观察到 HPA 反应产率低，而两种立体选择性丝氨酸-TA 显示出明显更高的产率。将丝氨酸-TA 反应与转酮醇酶偶联以生成 l-赤藓糖（Ery）可大大提高丝氨酸转化为 HPA 的效率。使用廉价的外消旋 D/L-丝氨酸结合两种立体选择性丝氨酸-TA 和转酮醇酶可导致高 Ery 产率（82%）。立体选择性丝氨酸-TA 的热特性分析证实它们具有出色的热稳定性，最高可达 60°C 和较高的最佳温度。
