Semi-rational design to enhance short-chain alcohol dehydrogenases in the synthesis of (S)-1-(4-fluorophenyl)ethanol

doi:10.16085/j.issn.1000-6613.2024-1851

Abstract

Abstract:

(S)-1-(4-fluorophenyl)ethanol is an important chiral drug intermediate, which is widely used in the synthesis of various drugs such as Alzheimer’s disease therapeutics. Currently, preparation inefficiencies are prevalent in the synthesis of this chiral alcohol using bioasymmetric reduction. In order to establish an enzymatic and efficient synthesis process of (S)-1-(4-fluorophenyl)ethanol, this study screened a laboratory enzyme library containing 323 alcohol dehydrogenases, obtained DpADH, a short-chain alcohol dehydrogenase possessing high stereoselectivity (e.e. value of 99.9%) and relative activity, and further improved the catalytic performance of the enzyme by semi-rational design. The excellent variant M₃ (N164C/S195W/F157A) with a 30.8-fold increase in catalytic efficiency was successfully obtained using multiple rounds of iterative mutagenesis. To improve the efficiency of coenzyme delivery, a co-expression strain of alcohol dehydrogenase variant M₃ with glucose dehydrogenase BsGDH was constructed, which showed 98.5% conversion of 4-fluoroacetophenone (100g/L) within 7h of reaction. Molecular dynamics simulations showed that the variant M₃ increased the favourable π-π interactions with the substrate and stabilised the substrate-bound conformation. This study provides an economical and efficient synthetic route for (S)-1-(4-fluorophenyl)ethanol, and provides guidance for the molecular modification and mechanistic elucidation of DpADH, which enhances the potential of the enzyme for industrial applications.

Key words: (S)-1-(4-fluorophenyl)ethanol, semi-rational design, alcohol dehydrogenase, asymmetric reduction, co-expression

摘要：

(S)-1-(4-氟苯基)乙醇是一种重要的手性药物中间体，被广泛用于阿尔兹海默症治疗等多种药物的合成当中。目前，利用生物不对称还原法合成该手性醇时普遍存在制备效率低下的问题。为建立(S)-1-(4-氟苯基)乙醇酶法高效合成工艺，本研究对实验室包含323种醇脱氢酶的酶库进行筛选，得到了具备高立体选择性（e.e.=99.9%）和相对活性的短链醇脱氢酶DpADH，随后通过半理性设计经过多轮迭代突变成功获得了催化效率提高30.8倍的优良变体M₃（N164C/S195W/F157A）。为进一步提高生物催化效率，构建了醇脱氢酶突变体M₃与葡萄糖脱氢酶BsGDH的共表达菌株，在100g/L底物投料下可于7h内实现99.6%的转化率。分子动力学模拟结果显示，M₃变体增加了与底物间有利的π-π相互作用并稳定了底物结合构象。本研究为(S)-1-(4-氟苯基)乙醇的制备提供了经济高效的合成途径，并为醇脱氢酶DpADH的分子改造和机制解析提供了指导，提高了该酶的工业应用潜力。

关键词: (S)-1-(4-氟苯基)乙醇, 半理性设计, 醇脱氢酶, 不对称还原, 共表达

CLC Number:

Q816

XIE Jingwen, MENG Yifang, YE Wenjie, WANG Hualei, WEI Dongzhi. Semi-rational design to enhance short-chain alcohol dehydrogenases in the synthesis of (S)-1-(4-fluorophenyl)ethanol[J]. Chemical Industry and Engineering Progress, 2025, 44(5): 2515-2523.

谢静雯, 孟仪方, 叶文杰, 王华磊, 魏东芝. 半理性设计提高短链醇脱氢酶在(S)-1-(4-氟苯基)乙醇合成中的应用[J]. 化工进展, 2025, 44(5): 2515-2523.

Figures/Tables 12

References 13

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序号	酶	转化率/%	e.e./%
28	ADH31	40.2	82.4（R）
39	ADH42	36.0	83.0（S）
70	ADH82	41.6	99.9（R）
71	ADH83	46.2	99.9（R）
72	ADH84	39.8	99.9（R）
73	ADH85	44.6	99.9（R）
105	ADH117	39.2	69.8（S）
113	ADH125	31.4	98.5（S）
114	ADH126	31.6	96.1（R）
163	ADH175	69.4	99.9（R）
225	ADH237	36.7	99.9（S）

序号	酶	转化率/%	e.e./%
28	ADH31	40.2	82.4（R）
39	ADH42	36.0	83.0（S）
70	ADH82	41.6	99.9（R）
71	ADH83	46.2	99.9（R）
72	ADH84	39.8	99.9（R）
73	ADH85	44.6	99.9（R）
105	ADH117	39.2	69.8（S）
113	ADH125	31.4	98.5（S）
114	ADH126	31.6	96.1（R）
163	ADH175	69.4	99.9（R）
225	ADH237	36.7	99.9（S）

氨基酸	频率/%
氨基酸	164	195	157	101	152	151	201
T	12.40	7.07	10.85	2.15	1.37	11.25	31.11
I	0	6.06	4.65	2.15	0	26.25	2.22
L	0	13.13	11.63	8.60	3.42	12.50	6.67
E	0	0	0	2.15	0	0	0
A	17.36	16.16	10.08	9.14	58.90	5.00	13.33
Q	0.83	0	0	2.15	5.48	3.75	0
Y	0	6.06	10.85	1.08	0	0.63	2.22
R	0.83	1.01	4.65	7.53	0.68	0	2.22
P	0	8.08	2.33	16.67	0	0.63	0
M	0.83	2.02	2.33	1.08	0.68	3.75	0
V	5.79	6.06	4.65	4.84	6.16	27.50	2.22
G	5.79	7.07	0	5.38	4.79	1.88	17.78
S	33.88	9.09	9.30	5.91	10.27	5.00	8.89
D	0	0	0.78	9.14	0.68	0.63	0
N	12.40	6.06	8.53	2.15	2.74	0.63	4.44
H	0	0	0	3.23	2.05	0	0
W	0	4.04	1.55	3.23	0	0.63	8.89
K	0	0	1.55	1.61	0	0	0
C	9.92	0	0.78	0.54	1.37	0	0
F	0	8.08	15.50	11.29	1.37	0	0

氨基酸	频率/%
氨基酸	164	195	157	101	152	151	201
T	12.40	7.07	10.85	2.15	1.37	11.25	31.11
I	0	6.06	4.65	2.15	0	26.25	2.22
L	0	13.13	11.63	8.60	3.42	12.50	6.67
E	0	0	0	2.15	0	0	0
A	17.36	16.16	10.08	9.14	58.90	5.00	13.33
Q	0.83	0	0	2.15	5.48	3.75	0
Y	0	6.06	10.85	1.08	0	0.63	2.22
R	0.83	1.01	4.65	7.53	0.68	0	2.22
P	0	8.08	2.33	16.67	0	0.63	0
M	0.83	2.02	2.33	1.08	0.68	3.75	0
V	5.79	6.06	4.65	4.84	6.16	27.50	2.22
G	5.79	7.07	0	5.38	4.79	1.88	17.78
S	33.88	9.09	9.30	5.91	10.27	5.00	8.89
D	0	0	0.78	9.14	0.68	0.63	0
N	12.40	6.06	8.53	2.15	2.74	0.63	4.44
H	0	0	0	3.23	2.05	0	0
W	0	4.04	1.55	3.23	0	0.63	8.89
K	0	0	1.55	1.61	0	0	0
C	9.92	0	0.78	0.54	1.37	0	0
F	0	8.08	15.50	11.29	1.37	0	0

突变体	转化率/%	e.e./%
DpADH	36.7	99.9
N164C（M₁）	55.9	99.9
N164C/S195W（M₂）	82.0	99.9
N164C/S195Y	80.6	99.9
N164C/S195F	79.8	99.9
N164C/S195A	55.4	99.9
N164C/S195L	48.5	99.9
N164C/S195N	46.3	99.9
N164C/S195T	35.1	99.9
N164C/S195I	29.8	99.9
N164C/S195V	32.0	99.9
N164C/S195P	29.9	98.7
N164C/S195G	15.6	95.6
N164C/S195W/F157A（M₃）	95.5	99.9
N164C/S195W/F157T	90.0	99.9
N164C/S195W/F157R	89.6	99.9
N164C/S195W/F157S	89.5	99.9
N164C/S195W/F157M	88.4	99.9
N164C/S195W/F157L	88.2	99.9
N164C/S195W/F157V	88.0	99.9
N164C/S195W/F157Y	88.0	99.9
N164C/S195W/F157N	87.7	99.9
N164C/S195W/F157I	87.0	99.9
N164C/S195W/F157P	1.5	96.5