(R)-1-[3,5-bis(trifluoromethyl)phenyl]ethanamine, a key chiral intermediate of selective tetrodotoxin-sensitive blockers, was efficiently synthesized by a bienzyme cascade system formed by with R-ω-transaminase (ATA117) and an alcohol dehydrogenase (ADH) co-expression system. Herein, we report that the use of ATA117 as the biocatalyst for the amination of 3,5-bistrifluoromethylacetophenone led to the highest efficiency in product performance (enantiomeric excess > 99.9%). Moreover, to further improve the product yield, ADH was introduced into the reaction system to promote an equilibrium shift. Additionally, bienzyme cascade system was constructed by five different expression systems, including two tandem expression recombinant plasmids (pETDuet-ATA117-ADH and pACYCDuet-ATA117-ADH) and three co-expressed dual-plasmids (pETDuet-ATA117/pET28a-ADH, pACYCDuet-ATA117/pET28a-ADH, and pACYCDuet-ATA117/pETDuet-ADH), utilizing recombinant engineered bacteria. Subsequent studies revealed that as compared with ATA117 single enzyme, the substrate handling capacity of BL21(DE3)/pETDuet-ATA117-ADH (0.25 g wet weight) developed for bienzyme cascade system was increased by 1.50 folds under the condition of 40 °C, 180 rpm, 0.1 M pH9 Tris-HCl for 24 h. To the best of our knowledge, ours is the first report demonstrating the production of (R)-1-[3,5-bis(trifluoromethyl)phenyl]ethanamine using a bienzyme cascade system, thus providing valuable insights into the biosynthesis of chiral amines.

中文翻译：

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具有 R-ω-转氨酶和乙醇脱氢酶功能的双酶级联系统高效合成止痛药关键手性中间体 (R)-1-[3,5-双(三氟甲基)苯基]乙胺

(R)-1-[3,5-双(三氟甲基)苯基]乙胺是选择性河豚毒素敏感阻断剂的关键手性中间体，通过与 R-ω-转氨酶 (ATA117) 和乙醇脱氢酶 (ADH) 共表达系统。在此，我们报道了使用 ATA117 作为 3,5-双三氟甲基苯乙酮胺化的生物催化剂导致产品性能效率最高（对映体过量 > 99.9%）。此外，为了进一步提高产品收率，将ADH引入反应体系以促进平衡移动。此外，双酶级联系统由五个不同的表达系统构建，包括两个串联表达重组质粒（pETDuet-ATA117-ADH 和 pACYCDuet-ATA117-ADH）和三个共表达双质粒（pETDuet-ATA117/pET28a-ADH，pACYCDuet-ATA117/pET28a-ADH 和 pACYCDuet-ATA117/pETDuet-ADH)，利用重组工程细菌。后续研究表明，与ATA117单酶相比，为双酶级联系统开发的BL21(DE3)/pETDuet-ATA117-ADH(0.25 g湿重)的底物处理能力在40 °C条件下提高了1.50倍， 180 rpm，0.1 M pH9 Tris-HCl 24 小时。据我们所知，我们的报告是第一份证明使用双酶级联系统生产 (R)-1-[3,5-双(三氟甲基)苯基]乙胺的报告，从而为手性胺的生物合成提供了宝贵的见解. 为双酶级联系统开发的 BL21(DE3)/pETDuet-ATA117-ADH（0.25 g 湿重）的底物处理能力在 40 °C、180 rpm、0.1 M pH9 Tris-HCl 条件下提高了 1.50 倍，用于 24 H。据我们所知，我们的报告是第一份证明使用双酶级联系统生产 (R)-1-[3,5-双(三氟甲基)苯基]乙胺的报告，从而为手性胺的生物合成提供了宝贵的见解. 为双酶级联系统开发的 BL21(DE3)/pETDuet-ATA117-ADH（0.25 g 湿重）的底物处理能力在 40 °C、180 rpm、0.1 M pH9 Tris-HCl 条件下提高了 1.50 倍，用于 24 H。据我们所知，我们的报告是第一份证明使用双酶级联系统生产 (R)-1-[3,5-双(三氟甲基)苯基]乙胺的报告，从而为手性胺的生物合成提供了宝贵的见解.