利用工程化亚胺还原酶实现生物基N-取代糠胺的规模化生产

doi:10.1016/S1872-2067(25)64765-6

摘要/Abstract

摘要：

N-取代糠胺(FAs)作为药物中间体和功能高分子前体化合物, 其绿色合成受到广泛关注. 亚胺还原酶(IREDs)能够催化生物基呋喃与胺供体还原胺化合成N-取代FAs. 通常, 化学法合成N-取代FAs面临生产安全和环境污染等问题. 相比之下, 生物催化具有反应条件温和, 选择性高和环境友好等优势. 当前, 生物催化合成N-取代FAs仍处于起步阶段, 存在底物载量低(20 mM)和无法催化大体积胺基供体(如芳基胺等)反应等问题. 此外, 当前IRED工程化改造中使用的高通量筛选方法依赖于昂贵的仪器设备,以及存在假阳性干扰. 因此, 建立快速、简单且抗干扰的高通量筛选方法具有重要的意义.

本文报道了一种能够高效合成N-取代FAs的IRED, 并建立了一种快速、简单且抗干扰的高通量IREDs筛选方法; 通过三轮酶工程改造野生型酶, 获得了高活性突变体M3, 并成功将其用于全细胞催化合成高浓度N-取代FA. 首先, 筛选得到一种来源于Streptomyces albidoflavus的SaIRED, 该酶具有较高活性和较广底物谱. 随后, 基于羰基化合物与2,4-二硝基苯肼间的颜色反应, 建立了高通量筛选方法. 为探究该方法的普适性, 将本研究获得的突变体组合成新的突变体文库, 用于筛选催化苯甲醛与苯胺, 苄基丙酮与环丙胺, 糠醛与2-氨乙基甲砜还原胺化反应的高活性突变体. 研究结果表明, 该方法能够准确筛选出催化不同反应的最优突变体, 证明其具有普适性. 通过分子对接和结构比对, 选定SaIRED活性空腔及周围的33个氨基酸作为突变位点; 使用NNK简并密码子引物进行饱和突变, 构建约3300个突变体的文库; 基于上述筛选方法, 获得了32个阳性单突变体, 分布于12个氨基酸位点. 其中, A242是一个新发现的突变热点, 活性最高的单突变体为A242T, 比活达到12.8 U mg^-1, 是野生型酶的2.6倍. 根据氨基酸位点的空间分布, 进行了第二轮组合突变, 发现D241和A242组合突变能显著提高突变体酶活, 双突变体D241A/A242T的比活为16.9 U mg^-1. 在第三轮迭代组合突变中, 发现双突变体D241A/A242T与I127位点具有明显的协同效应, 其中三突变体I127V/D241A/A242T (M3)的比活达到20.2 U mg^-1, 是野生型酶的4.2倍; 并且, M3对其它不同底物的活性均优于野生型酶, 其催化合成一系列N-取代FAs的产率高达99%. 机制研究表明, A242突变为苏氨酸可能促进了底物质子化, D214突变为丙氨酸拓宽了底物隧道, 而I127突变为缬氨酸则增大了酶活性空腔, 从而导致M3活性显著提高. 最后, 为实现高浓度N-取代FAs的合成, 本研究在Escherichia coli中共表达突变体M3和葡萄脱氢酶(GDH), 构建了全细胞催化剂; 经工艺优化后, 全细胞催化N-烯丙基糠胺(N-allyl-FA)合成的选择性显著提高, 可在7 h内将250 mmol L^-1糠醛与烯丙胺完全转化为N-allyl-FA, 时空收率为4.7 g L^-1 h^-1, 分离收率达91%, 实现了N-allyl-FA的克级制备.

总之, 本研究建立的高通量筛选方法不仅克服了前人方法的缺陷, 还具有较好的普适性, 其应用将有效缩短IREDs的工程改造周期. 基于SaIRED的挖掘和改造, 本研究为N-取代FAs的绿色合成提供了一个强有力的工具.

关键词: N-取代糠胺, 亚胺还原酶, 还原胺化, 高通量筛选, 蛋白质工程

Abstract:

AD-substituted furfurylamines (FAs) are valuable precursors for producing pharmacologically active compounds and polymers. However, enzymatic synthesis of the type of chemicals is still in its infancy. Here we report an imine reductase from Streptomyces albidoflavus (SaIRED) for the reductive amination of biobased furans. A simple, fast and interference-resistant high-throughput screening (HTS) method was developed, based on the coloration reaction of carbonyl compounds with 2,4-dinitrophenylhydrazine. The reductive amination activity of IREDs can be directly indicated by a colorimetric assay. With the reductive amination of furfural with allylamine as the model reaction, SaIRED with the activity of 4.8 U mg^-1 was subjected to three rounds of protein engineering and screening by this HTS method, affording a high-activity tri-variant I127V/D241A/A242T (named M3, 20.2 U mg^-1). The variant M3 showed broad substrate scope, and enabled efficient reductive amination of biobased furans with a variety of amines including small aliphatic amines and sterically hindered amines, giving the target FAs in yields up to >99%. In addition, other variants were identified for preparative-scale synthesis of commercially interesting amines such as N-2-(methylsulfonyl)ethyl-FA by the screen method, with isolated yields up to 87% and turnover numbers up to 9700 for enzyme. Gram-scale synthesis of N-allyl-FA, a valuable building block and potential polymer monomer, was implemented at 0.25 mol L^-1 substrate loading by a whole-cell catalyst incorporating variant M3, with 4.7 g L^-1 h^-1 space-time yield and 91% isolated yield.

Key words: N-substituted furfurylamines, Imine reductases, Reductive amination, High-throughput screening, rotein engineering

王剑朋, 路光辉, 吴倩, 代建容, 李宁. 利用工程化亚胺还原酶实现生物基N-取代糠胺的规模化生产[J]. 催化学报, 2025, 76: 210-220.

Jian-Peng Wang, Guang-Hui Lu, Qian Wu, Jian-Rong Dai, Ning Li. Toward scalable production of biobased N-substituted furfurylamines by engineered imine reductases[J]. Chinese Journal of Catalysis, 2025, 76: 210-220.

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链接本文: https://www.cjcatal.com/CN/10.1016/S1872-2067(25)64765-6

https://www.cjcatal.com/CN/Y2025/V76/I9/210

图/表 7

Scheme 1. Representative pharmaceuticals containing a FA moiety and catalytic synthesis of N-substituted FAs.

Fig. 1. The activity of SaIRED in the synthesis of various N-substituted FAs. Assay conditions: 15 mmol L-1 furan, 60 mmol L-1 amine, 0.2 mmol L-1 NADPH, 5-50 μg mL-1 enzyme, 1 mL phosphate buffer (0.1 mol L-1, pH = 7), 30 °C. a The addition of 10% (v/v) DMSO.

Fig. 2. Screening of the target IREDs: the HTS method (A), SSM on A242 residue (B), the synthesis of N-benzylaniline (C), N-(4-phenylbutan-2-yl)cyclopropanamine (D), and N-2-(methylsulfonyl)ethyl-FA (E).

Table 1 Thermostability, specific activity, and kinetic parameters of WT and M3 a.

Enzyme	T_m (°C)	Specific activity (U mg^-1)	K_m (mmol L^-1)	k_cat (s^-1)	k_cat/K_m (L mmol^-1 s^-1)	K_m (mmol L^-1)	k_cat (s^-1)	k_cat/K_m (L mmol^-1 s^-1)
Enzyme	T_m (°C)	Specific activity (U mg^-1)	1	1	1	d	d	d
WT	45.1	4.8	5.4	8057.2	1520.2	50.0	8921.3	178.4
M3	45.5	20.2	5.3	33668.8	6349.7	35.7	29878.6	836.1

Fig. 3. Substrate binding mode analysis of furfural in the WT enzyme (A) and variant M3 (B). The active cavity profiles of the WT enzyme (C) and variant M3 (D). The selected distances are shown in Angstroms.

Fig. 4. Reductive amination of biobased furans with amines by the variant M3. Reaction conditions: 20 mmol L-1 furan, 10 equiv. amine, 1 g L-1 purified variant M3, 0.8 g L-1 purified GDH, 1 mmol L-1 NADP+, 30 mmol L-1 glucose, 2 mL phosphate buffer (0.1 mol L-1, pH = 7), 30 °C, 150 rpm, 12 h. a The addition of 10% (v/v) DMSO.

Fig. 5. The scheme of N-allyl-FA synthesis (A) and preparative-scale synthesis of N-allyl-FA by a fed-batch process (B). Reaction conditions: 150 mmol L-1 furfural, 4 equiv. allylamine, 1.5 equiv. glucose, 40 g L-1 E. coli RARE_M3_GDH cells (wet weight), 30 mL Tris-HCl buffer (0.2 mol L-1, pH = 9), 30 °C, 150 rpm. The arrow shows the addition of 0.3 mmol furfural, 0.3 mmol allylamine, and 0.3 mmol glucose.

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