Honvári Máté Gergő, Kucsinka Bence Attila, Mócza Levente András, Csuka Pál, Bódai Viktória, Poppe László, Hornyánszky Gábor
Department of Organic Chemistry and Technology, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Műegyetem Rkp. 3, 1111, Budapest, Hungary.
Fermentia Microbiological Ltd, Berlini Út 47 - 49, 1049, Budapest, Hungary.
Appl Microbiol Biotechnol. 2025 Apr 30;109(1):108. doi: 10.1007/s00253-025-13486-2.
This study explored the bioreduction of N-(3-oxobutyl)heterocycles with (partially) saturated heterocyclic moieties using whole-cell forms of wild-type yeast strains and commercially available baker's yeast (Saccharomyces cerevisiae). Eleven wild-type yeast strains and baker's yeast were screened for ketoreductase activity on a series of five flexible N-heterocycles with prochiral carbonyl group in the N-(3-oxobutyl) substituent. Among the yeast strains tested, Candida parapsilosis (WY12) proved to be the most efficient biocatalyst in the bioreductions, resulting in the corresponding enantiopure alcohols-being promising chiral fragments with high level of drug-likeness-with good to excellent conversions (83-99%) and high enantiomeric excess (ee > 99%). Other strains, such as Pichia carsonii (WY1) and Lodderomyces elongisporus (WY2), also showed promising ketoreductase activities with certain substrates. After screening as lyophilized whole cells, C. parapsilosis cells were immobilized in the form of calcium, zinc, nickel, and copper alginate beads. The whole-cell immobilization enabled recycling, with considerable residual activity of the biocatalyst over multiple cycles. Additionally, the study explored the scalability of these bioreductions, with immobilized C. parapsilosis delivering promising results. The use of immobilized cells simplified the work-up process and resulted in chiral alcohols with similar or even higher conversions to those observed in the screening reactions. Molecular docking of the five flexible N-heterocycles with prochiral carbonyl group into the active site of the experimental structure of the carbonyl reductase of C. parapsilosis rationalized their biocatalytic behavior and confirmed the assigned (S)-configuration of forming enantiopure alcohols. KEY POINTS: • Ketoreductase activity of eleven wild-type yeast strains and baker's yeast were examined. • Candida parapsilosis was subjected to whole-cell immobilization and recycling. • Enantiopure alcohols with flexible N-heterocyclic units were produced at preparative scale.
本研究利用野生型酵母菌株的全细胞形式和市售面包酵母（酿酒酵母），探索了具有（部分）饱和杂环部分的N-（3-氧代丁基）杂环的生物还原反应。针对一系列五个在N-（3-氧代丁基）取代基中带有前手性羰基的柔性N-杂环，对11种野生型酵母菌株和面包酵母进行了酮还原酶活性筛选。在所测试的酵母菌株中，近平滑假丝酵母（WY12）在生物还原反应中被证明是最有效的生物催化剂，生成了相应的对映体纯醇——具有高水平类药性的有前景的手性片段——转化率良好至优异（83-99%），对映体过量值高（ee＞99%）。其他菌株，如卡氏毕赤酵母（WY1）和长孢洛德酵母（WY2），对某些底物也表现出有前景的酮还原酶活性。在筛选为冻干全细胞后，将近平滑假丝酵母细胞固定在钙、锌、镍和铜藻酸盐珠的形式中。全细胞固定化实现了循环利用，生物催化剂在多个循环中具有相当的残余活性。此外，该研究还探索了这些生物还原反应的可扩展性，固定化的近平滑假丝酵母取得了有前景的结果。使用固定化细胞简化了后处理过程，并生成了与筛选反应中观察到的转化率相似甚至更高的手性醇。将五个带有前手性羰基的柔性N-杂环与近平滑假丝酵母羰基还原酶实验结构的活性位点进行分子对接，合理化了它们的生物催化行为，并确认了生成对映体纯醇的指定（S）-构型。要点：• 检测了11种野生型酵母菌株和面包酵母的酮还原酶活性。• 对近平滑假丝酵母进行了全细胞固定化和循环利用。• 以制备规模生产了具有柔性N-杂环单元的对映体纯醇。
