Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, PR China.
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, PR China.
Bioorg Chem. 2020 Oct;103:104228. doi: 10.1016/j.bioorg.2020.104228. Epub 2020 Aug 26.
Aldo-keto reductase KmAKR-catalyzed asymmetric reduction offers a green approach to produce dichiral diol tert-butyl 6-substituted-(3R,5R/S)-dihydroxyhexanoates, which are important building blocks of statins. In our previous work, we cloned a novel gene of NADPH-specific aldo-keto reductase KmAKR (WT) from a thermotolerant yeast Kluyveromyces marxianus ZJB14056 and a mutant KmAKR-W297H/Y296W/K29H (Variant III) has been constructed and displayed strict diastereoselectivity towards tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1) but moderate activity and stability. Herein, to further co-evolve its activity and thermostability, we performed semi-rational engineering of Variant III by using a combinational screening strategy, consisting of tertiary structure analysis, loop engineering, and alanine scanning. As results, the "best" variant KmAKR-W297H/Y296W/K29H/Y28A/T63M (Variant VI) was acquired, whose K, k/K towards (5R)-1 was 0.66 mM and 210.77 s mM, respectively, with improved thermostability (half-life of 14.13 h at 40 °C). Combined with 1.5 g dry cell weight (DCW) LExiguobacterium sibiricum glucose dehydrogenase (EsGDH) for NADPH regeneration, 4.5 g DCW LVariant VI completely reduced (5R)-1 of up to 450 g L within 7.0 h at 40 °C, yielding the corresponding optically pure tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-3, >99.5% d.e.) with a space-time yield (STY) of 1.24 kg L day, and this was the highest level documented in literatures so far on substrate loading and STY of producing (3R,5R)-3. Besides (5R)-1, Variant VI displayed strong activity on tert-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-2). 4.5 g DCW LVariant VI completely reduced 400 g L (5S)-2, within 5.0 h at 40 °C, yielding optically pure tert-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-4, >99.5% d.e.) with a STY of 1.34 kg L day. In summary, Variant VI displayed industrial application potential in statins biomanufacturing.
醛酮还原酶 KmAKR 催化的不对称还原为生产手性二羟基叔丁酯 6-取代-(3R,5R/S)-二氢己酸酯提供了一种绿色方法，这些化合物是他汀类药物的重要构建块。在我们之前的工作中，我们从耐热酵母 Kluyveromyces marxianus ZJB14056 中克隆了一种新型 NADPH 特异性醛酮还原酶 KmAKR（WT）基因，并构建了 KmAKR-W297H/Y296W/K29H 突变体（变体 III），该突变体对叔丁基 6-氰基-(5R)-羟基-3-氧代己酸酯((5R)-1)表现出严格的立体选择性，但活性和稳定性适中。在此基础上，为了进一步协同进化其活性和热稳定性，我们采用组合筛选策略对变体 III 进行了半理性工程设计，该策略包括三级结构分析、环工程和丙氨酸扫描。结果获得了“最佳”变体 KmAKR-W297H/Y296W/K29H/Y28A/T63M（变体 VI），其对(5R)-1 的 K 和 k/K 值分别为 0.66mM 和 210.77s mM，热稳定性提高（在 40°C 下半衰期为 14.13 小时）。与 1.5g 干细胞重量（DCW）的西伯利亚极端小球藻葡萄糖脱氢酶（EsGDH）一起用于 NADPH 再生，在 40°C 下 4.5g DCW LVariant VI 可在 7.0 小时内完全还原高达 450g L 的(5R)-1，生成相应的光学纯叔丁基 6-氰基-(3R,5R)-二羟基己酸酯((3R,5R)-3，>99.5%d.e.），时空产率（STY）为 1.24kg L day，这是迄今为止文献中底物加载和（3R,5R）-3 的 STY 的最高水平。除了(5R)-1，变体 VI 对叔丁基 6-氯-(5S)-羟基-3-氧代己酸酯((5S)-2)也具有很强的活性。在 40°C 下，4.5g DCW LVariant VI 可在 5.0 小时内完全还原 400g L 的(5S)-2，生成光学纯叔丁基 6-氯-(3R,5S)-二羟基己酸酯((3R,5S)-4，>99.5%d.e.），STY 为 1.34kg L day。总之，变体 VI 在他汀类药物生物制造中具有工业应用潜力。
