tert-Butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) is a valuable chiral synthon, which is used for the synthesis of the cholesterol-lowering drugs atorvastatin and rosuvastatin. To date, only the alcohol dehydrogenases from Lactobacillus brevis (LbADH) and Lactobacillus kefir (LkADH) have demonstrated catalytic activity toward the asymmetric reduction of tert-butyl 6-chloro-3,5-dioxohexanoate (CDOH) to (S)-CHOH. Herein, a tetrad mutant of LkADH (LkTADH), A94T/F147L/L199H/A202L, was screened to be more efficient in this bioreduction process, exhibiting a 3.7- and 42-fold improvement in specific activity toward CDOH (1.27 U/mg) over LbADH (0.34 U/mg) and wild-type LkADH (0.03 U/mg), respectively. The molecular basis for the improved catalytic activity of LkTADH toward CDOH was investigated using homology modeling and docking analysis. Two major issues had a significant impact on the biocatalytic efficiency of this process, including (i) the poor aqueous stability of the substrate and (ii) partial substrate inhibition. A fed-batch strategy was successfully developed to address these issues and maintain a suitably low substrate concentration throughout the entire process. Several other parameters were also optimized, including the pH, temperature, NADP(+) concentration and cell loading. A final CDOH concentration of 427 mM (100 g/L) gave (S)-CHOH in 94 % yield and 99.5 % e.e. after a reaction time of 38 h with whole cells expressing LkTADH. The space-time yield and turnover number of NADP(+) in this process were 10.6 mmol/L/h and 16,060 mol/mol, respectively, which were the highest values ever reported. This new approach therefore represents a promising alternative for the efficient synthesis of (S)-CHOH.

中文翻译：

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（S）-6-氯-5-羟基-3-氧代己酸叔丁酯与开菲尔乳杆菌的突变醇脱氢酶的高效酶促合成。

（S）-6-氯-5-羟基-3-氧代己酸叔丁酯（（S）-CHOH）是一种有价值的手性合成子，可用于合成降胆固醇药物阿托伐他汀和罗苏伐他汀。迄今为止，仅来自短乳杆菌（LbADH）和开菲乳杆菌（LkADH）的醇脱氢酶显示出对6-氯-3，5-二氧己酸叔丁基酯（CDOH）不对称还原为（S）-CHOH的催化活性。在此，筛选出LkADH（LkTADH）的四元突变体A94T / F147L / L199H / A202L在该生物还原过程中更有效，对CDOH（1.27 U / mg）的比活性提高了3.7到42倍。分别超过LbADH（0.34 U / mg）和野生型LkADH（0.03 U / mg）。使用同源模型和对接分析研究了LkTADH对CDOH的催化活性提高的分子基础。两个主要问题对该方法的生物催化效率产生了重大影响，包括（i）底物的不良水稳定性和（ii）部分底物抑制作用。已成功开发了分批补料策略，以解决这些问题并在整个过程中保持适当的低底物浓度。还优化了其他几个参数，包括pH值，温度，NADP（+）浓度和细胞负载。在与表达LkTADH的全细胞反应38小时后，最终的CDOH浓度为427 mM（100 g / L），得到（S）-CHOH的产率为94％，ee为99.5％。在此过程中，NADP（+）的时空产率和周转数分别为10.6 mmol / L / h和16,060 mol / mol，这是有史以来的最高值。