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

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

Abstract

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

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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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(24)60172-5

https://www.cjcatal.com/EN/Y2025/V69/I2/135

Figures/Tables 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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