液-液相分离介导的双酶凝聚体增强辅因子循环效率与反应速率

doi:10.1016/S1872-2067(24)60172-5

催化学报 ›› 2025, Vol. 69: 135-148.DOI: 10.1016/S1872-2067(24)60172-5

液-液相分离介导的双酶凝聚体增强辅因子循环效率与反应速率

刘佳旭^a, 陈佳鑫^a, 张晓彦^a, 范代娣^b, 白云鹏^a^,^b^,^*()

^a华东理工大学, 生物反应器工程国家重点实验室, 上海生物制造产业省部共建协同创新中心, 上海 200237
^b西北大学化工学院, 陕西省生物材料与发酵工程技术研究中心, 陕西西安 710069

收稿日期:2024-09-03 接受日期:2024-10-17 出版日期:2025-02-18 发布日期:2025-02-10
通讯作者: *电子信箱: ybai@ecust.edu.cn (白云鹏).
基金资助:
国家重点研发计划(2023YFA0913600);国家自然科学基金(22378120);国家自然科学基金(22078096)

Enhanced cofactor recycling and accelerated reaction rate via liquid-liquid phase separation in dual-enzyme condensates

Jiaxu Liu^a, Jiaxin Chen^a, Xiaoyan Zhang^a, Daidi Fan^b, Yunpeng Bai^a^,^b^,^*()

^aState Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China
^bShaanxi R&D Centre of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an 710069, Shaanxi, China

Received:2024-09-03 Accepted:2024-10-17 Online:2025-02-18 Published:2025-02-10
Contact: *E-mail: ybai@ecust.edu.cn (Y. Bai).
Supported by:
National Key R&D Program of China(2023YFA0913600);National Natural Science Foundation of China(22378120);National Natural Science Foundation of China(22078096)

摘要/Abstract

摘要：

酶催化作为一种绿色的生物制造方法,凭借其高选择性和在温和反应条件下工作的特性, 成为了合成手性化学品的重要且优选的方法.近年来, 酶催化技术取得了显著进展, 但是许多反应需要昂贵的辅因子, 较低的辅因子循环效率导致反应成本高、速率低, 在工业催化中的应用受到限制. 细胞中辅因子的再生和循环过程非常高效, 这是因为细胞内部具有高度有序的微纳结构和酶的空间组织. 然而, 在体外构建具有类似辅因子高效循环效率的催化系统仍然是一个巨大的挑战.

本文报道了一种基于蛋白质内在无序区域(IDRs)介导的液-液相分离(LLPS)制备双酶凝聚体的策略. IDRs分别与负责催化反应的羰基还原酶(SmCR_V4)和负责生成辅因子的葡萄糖脱氢酶(BmGDH)融合表达, 获得既具有催化功能又可以自发发生液-液相分离的融合蛋白. 对四种不同来源但均能在体外发生LLPS的IDRs进行了筛选, 得到酶活力最高的组蛋白H3K27去甲基化酶X-连锁基因突变蛋白融合酶. 共聚焦显微镜表征结果表明, 融合蛋白形成液液相分离的凝聚体, 且蛋白质浓度越高, 相分离程度越大. 凝聚体对pH的耐受性高, 在pH = 5.0‒9.0均可发生LLPS. 荧光漂白实验证明凝聚体内部流动性良好, 分子扩散不受限制. 共聚焦图像分析结果证明两种融合蛋白共定位在同一个凝聚体内. 辅因子使用荧光标签标记后, 共聚焦显微镜下观察到辅因子在双酶凝聚体中富集, 表明邻近效应促进了辅因子在凝聚体内部和周围再生, 从而提升辅因子循环的效率. 催化4-氧代癸酸甲酯不对称还原的实验结果表明, 双酶凝聚体体系中辅因子循环效率是游离酶体系的20倍, 时空产率是3.4倍. 在辅因子浓度低至1 µmol/L时, 双酶凝聚体可以在50 min内催化16 µmol底物完全转化. 双酶凝聚体展现出了良好的稳定性, 在连续反应12 h后, 凝聚体的形貌并未改变. 在放大合成实验中, 与工业生物催化标准相比, 辅因子的消耗减少了50倍, 同时双酶凝聚体仍然保持了高效的催化能力. 实验结果表明, 双酶凝聚体对反应的加速效果与相分离程度正相关, 邻近效应使得辅因子循环效率加快从而提高了反应速率. 粗颗粒模拟实验进一步证明了邻近效应对辅因子富集的促进作用. 该方法操作简便, 辅因子循环效率高, 为生物催化领域提供了新的反应技术.

综上所述, 本文不仅展示了基于IDRs的液-液相分离在体外生成双酶凝聚体的潜力, 还揭示了该凝聚体在提高辅因子循环效率和反应动力学方面的重要作用. 本文结果为设计新型的多酶催化系统提供了重要的理论基础, 并为绿色和可持续的生物制造过程开辟了新的道路.

关键词: 辅因子循环, 凝聚体, 邻近效应, 内在无序区域, 酶催化, 手性化学品

Abstract:

Enzyme catalysis is a promising way to produce chiral products in a green and sustainable way. However, the high cost of cofactors and their relatively low recycling efficiency hinder the widespread application of enzyme catalysis in industry. In contrast, cofactor regeneration and recycling in cells is highly efficient, mainly due to physical effects caused by the ordered spatial organization of enzymes in vivo. The construction of similar catalytic systems with high cofactor recycling in vitro remains challenging. Here, we present a facile method to generate dual enzyme condensates in vitro based on intrinsically disordered region-mediated liquid-liquid phase separation. Typically, a carbonyl reductase from Serratia marcescens (SmCR_V4) and a glucose dehydrogenase from Bacillus megaterium (BmGDH) were co-localized in the condensates. This resulted in an up to 20-fold increase in cofactor recycling efficiency (substrate converted per cofactor per unit time), and a 3.4-fold increase in space-time yield compared to the free enzyme system. The reaction enhancement was shown to be highly correlated with the degree of condensation of the dual enzymes. Fluorescence confocal microscopy showed that the cofactor, nicotinamide adenine dinucleotide phosphate (NADPH), was enriched between neighboring enzymes during the reaction due to the proximity effect, facilitating its regeneration and recycling within the condensate. In a scaled-up synthesis, the consumption of NADPH was reduced 50-fold compared to industrial biocatalytic standards, while the condensate still maintained efficient product synthesis. Concentrating multiple enzymes in a nano- and micro-condensate to increase the reaction rate may provide a general and inexpensive method for improving cofactor-involved enzymatic reactions.

Key words: Cofactor recycling, Condensate, Proximity effect, Intrinsically disordered region, Enzyme catalysis

刘佳旭, 陈佳鑫, 张晓彦, 范代娣, 白云鹏. 液-液相分离介导的双酶凝聚体增强辅因子循环效率与反应速率[J]. 催化学报, 2025, 69: 135-148.

Jiaxu Liu, Jiaxin Chen, Xiaoyan Zhang, Daidi Fan, Yunpeng Bai. Enhanced cofactor recycling and accelerated reaction rate via liquid-liquid phase separation in dual-enzyme condensates[J]. Chinese Journal of Catalysis, 2025, 69: 135-148.

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链接本文: https://www.cjcatal.com/CN/10.1016/S1872-2067(24)60172-5

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图/表 9

Scheme 1. Illustration of enzyme catalysis and cofactor recycling in single aqueous phase and in dual-enzyme condensates in LLPS.

Fig. 1. Construction and assay of IDR-enzyme fusion proteins. (a) IDRs linked to enzymes were expressed to form condensates. (b) Catalytic activities of condensates with UTXIDR linked to the C- and N-termini of SmCRV4 and BmGDH. Assay conditions for IDR-SmCRV4: 4 mmol/L 1c, 200 μmol/L NADPH, 100 mmol/L phosphate-buffered saline (PBS), pH = 7.0, 40 °C. Assay conditions for IDR-BmGDH: 15 mmol/L glucose, 100 μmol/L NADP+, 100 mmol/L PBS, pH = 7.0, 40 °C. (c) The catalytic efficiencies of four IDR-SmCRV4 proteins. Assay conditions: 0.5?8 mmol/L 1c, 200 μmol/L NADPH, 5% dimethylsulfoxide, 100 mmol/L PBS, pH = 7.0, 40 °C. (d) The catalytic efficiencies of four IDR-BmGDH proteins. Assay conditions: 10?40 mmol/L glucose, 100 μmol/L NADP+, 100 mmol/L PBS, pH = 7.0, 40 °C. (e) IDR-mediated phase separation upon expression of fusion proteins in E. coli BL21(DE3) containing plasmids pET28a+ (UTXIDR-S-G) and pBAD His B (UTXIDR-B-m) were observed under a confocal fluorescence microscope. Scale bar, 10 μm.

Fig. 2. Characterization of single-enzyme condensates. (a) Confocal fluorescence images of UTXIDR-S-G with concentrations from low to high. Scale bar, 50 μm. (b) The turbidity of purified UTXIDR-S at OD600 was measured at different concentrations (μmol/L). (c) Confocal fluorescence images of UTXIDR-S-G in 100 mmol/L PBS, pH 5.0?9.0. Scale bar, 10 μm. (d) Analysis of the particle size of UTXIDR-S-G at different concentrations. (e) Size distribution of condensates generated by 10 μmol/L UTXIDR-S and 10 μmol/L UTXIDR-S-G. (f) Confocal fluorescence images of 19 μmol/L UTXIDR-S-G after the addition of different concentrations of NaCl. Scale bar, 10 μm. (g) Statistical analysis of the number of condensates in Fig. 2(f). (h) FRAP experiments using UTXIDR-S-G. Scale bar, 5 μm. Each sample in the above assays contained 5% PEG.

Fig. 3. Characterization of condensates of dual enzymes. (a) Confocal images of equal mixing of UTXIDR-S-G and UTXIDR-B-m in different channels. Scale bar, 10 μm. Each sample contained 5% PEG. (b) Fluorescence intensity analysis of the protein-rich phase (red box) of Fig. 3(b). (c) Comparison of particle size analysis between single-enzyme condensates and dual-enzyme condensates. (d) Fluorescence changes of different ratios of UTXIDR-S-G to UTXIDR-B-m. (e) Variation in activity of UTXIDR-S and UTXIDR-B upon mixing at different ratios. Reaction condition: 4 mmol/L 1c (15 mmol/L glucose), 200 μmol/L NADPH (100 μmol/L NADP+), PBS 7.0, 40 °C.

Fig. 4. The initial reaction rates and substrate conversions in the free enzyme and dual-enzyme condensates. Reaction conditions: 8 mmol/L 1c, 100 μmol/L NADP+, 10 mmol/L glucose, carbonyl reductase = 1 U, glucose dehydrogenase = 1 U, 2 mL PBS (pH = 7.0, 100 mmol/L), 30 °C. (a) Free enzymes and dual-enzyme condensates. (b) Dual-enzyme condensates added with NaCl. (c) Dual-enzyme condensates are added with different cell lysates. (d) Confocal fluorescence images of different systems. Scale bar, 10 μm. (e) Distribution of NADPH in different reaction systems. Reaction conditions: SmCRV4/UTXIDR-S = 1 U, BmGDH/UTXIDR-B = 1 U, 8 mmol/L 1c, 10 mmol/L glucose, 100 μmol/L NADP+, and 10 μmol/L TCF-MQ. Scale bar, 10 μm. (f) Fluorescence analysis of the green box in Fig. 4(e).

Fig. 5. The enhanced NADPH recycling in the condensation system. (a) Confocal fluorescence images were acquired during the reduction of 1c (4 mmol/L) by UTXIDR-S + UTXIDR-B in 1 mL of PBS (100 mmol/L, pH 7.0) containing 100 μmol/L NADP+, 10 mmol/L glucose, and 10 μmol/L TCF-MQ. Scale bar, 10 μm. (b) Radial distribution functions of the amounts of NADPH [g(r)] in a simulated 40 × 40 × 40 (σ) box, where r is the distance between the position of the NADPH and the center of the box. (c) The conversion of 1c over time in various experimental conditions. Reaction conditions: 1c (8 mmol/L), NADP+ (100, 10, or 1 μmol/L), glucose (10 mmol/L), 100 mmol/L PBS, pH = 7.0, 30 °C, UTXIDR-S = UTXIDR-B = 1 U, 0.25 g of UTYIDR lysate; SmCRV4 = BmGDH = 1 U.

Fig. 6. Asymmetric synthesis of chiral alcohols. (a) Reaction condition: 100 mmol/L PBS, pH 6.0/7.0, 200 μmol/L NADPH, 4.0 mmol/L substrate at 40 °C. (b) Conversion reaction conditions: 0.2 g/L crude extracts of E. coli/UTXIDR-S, UTXIDR-B, 4.0 mmol/L substrate, 0.1 mmol/L NADP+, 10 mmol/L glucose, in PBS (pH = 6.0/7.0), at room temperature for 12 h. The product enantiomeric excesses (ees) generated by the condensate and free SmCRV4 + BmGDH (in parentheses) were determined by chiral gas chromatography.

Fig. 7. Time courses of reduction of 1c, 1d, 1h catalyzed by free enzymes and dual-enzyme condensates. Reaction conditions: 2 mL PBS (pH 7.0, 100 mmol/L), 30 °C. (a) 8 mmol/L 1c, 100 μmol/L NADP+, carbonyl reductase = 1 U, glucose dehydrogenase = 1 U, free enzymes, UTXIDR-S + UTXIDR-B and UTXIDR-S + UTXIDR-B + (UTYIDR lysate). (b) 50 mmol/L 1d, 10 μmol/L NADP+, 100 mmol/L glucose, carbonyl reductase = 8.5 U, glucose dehydrogenase = 8.5 U, free enzymes, UTXIDR-S + UTXIDR-B and UTXIDR-S + UTXIDR-B + (UTYIDR lysate). (c) 8 mmol/L 1 h, 100 μmol/L NADP+, 10 mmol/L glucose, carbonyl reductase = 1 U, glucose dehydrogenase = 1 U, free enzymes, UTXIDR-S + UTXIDR-B and UTXIDR-S + UTXIDR-B + (UTYIDR lysate).

Table 1 Scaling-up experiments by the free enzyme and dual-enzyme condensates.

Product	Catalyst (U/mL)	Time (h)	Conv. (%)	Yield (%)	ee (%)	Cofactor TTN
3c	1.68^a	3	73.7	60.5	>99	3685
3c	1.68^b	3	99.9	89.8	>99	5000
3h	2^c	4	68.6	55.4	>99	3430
3h	2^d	4	99.9	86.2	>99	5000
3d	0.17^e	8	70.7	59.6	>99	3535
3d	0.17^f	8	82.0	73.5	>99	4100
3p	0.8^g	8	54.7	48.5	44	2735
3p	0.8^h	8	60.9	56.2	47	3045

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液-液相分离介导的双酶凝聚体增强辅因子循环效率与反应速率

Enhanced cofactor recycling and accelerated reaction rate via liquid-liquid phase separation in dual-enzyme condensates

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