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Self-assembled multimeric-enzyme nanoreactor for robust and efficient biocatalysis Liang Yin, Xiang Guo, Lu Liu, Yong Zhang, and Yan Feng ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00279 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 13, 2018

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ACS Biomaterials Science & Engineering

Self-assembled multimeric-enzyme nanoreactor for robust and efficient biocatalysis

Liang Yin, Xiang Guo, Lu Liu, Yong Zhang*, and Yan Feng* State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.

*Corresponding authors: [email protected] (Yong Zhang) or [email protected] (Yan Feng); Tel. /Fax: +86-021-34207248

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ABSTRACT: The construction of artificial multienzyme nanodevices with desired spatial arrangements have shown great promise for improving the overall performance of targeted enzyme cascades. However, it is a challenge to rationally design and construct multiple oligomeric enzyme assemblies that can be used as stable and reusable catalysts. Herein, we report a novel approach to rapidly achieve ultra-stable multimeric enzyme nanoclusters (MENCs) based on enzymes property of oligomerization and the SpyTag/SpyCatcher system of orthogonally reactive split peptides. The SpyCatcher peptide and its binding partner SpyTag were fused to a dimeric cytochrome P450 monooxygenase mutant (P450BM3m) and a tetrameric glucose dehydrogenase (GDH), respectively. The fusion proteins self-assembled into the MENCs, forming a covalently coupled supramolecular multienzyme nanodevices that facilitated NADPH regeneration and converted indole into a pigment indigo. We investigated the morphology of the MENCs and found these multimeric enzymes assembled into two dimensional layer-like nanoscale architecture, ranging from a few to several hundred square microns in size. Importantly, enzymatic analysis revealed that the MENCs not only increased the initial rate by more than three times for the indigo synthesis, but also achieved significant improvements on stability and reusability compared to unassembled enzyme mixtures. This work demonstrates a versatile and efficient strategy to construct stable and multifunctional biocatalysts with potential applications in metabolic engineering and synthetic biology.

KEYWORDS: multimeric enzyme, SpyCatcher/SpyTag, self-assembly, P450 monooxygenase， glucose dehydrogenase, indigo

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INTRODUCTION Natural enzyme cascades are sometimes assembled into sophisticated multienzyme complexes, which feature well-controlled geometries to facilitate transfer of reaction intermediates and thereby improve catalytic efficiency.1-4 Learning from nature, the idea of designing highly ordered multienzyme nanodevices represents a promising strategy in metabolic engineering and synthetic biology for producing valuable bio-commodities.5-9 To date, multiple artificial enzyme assembly strategies have been explored, including enzyme fusion,10,11 scaffold-mediated enzyme co-localization.12-14 However, as the fact that many metabolic enzymes are multimeric and there are the increased complexity of the inter-subunit interactions, make the multimeric enzyme assemblies frequently suffer from steric hindrance or uncontrolled tethering with the integrated scaffold materials, which might cause inactive fusion enzymes15,16 or produce unstable and disordered scaffold-enzyme complexes.17 The rational design and construction of highly stable and reusable assembly of multimeric enzymes remains quite challenging. Although a multienzyme nanodevice has been achieved by combination of oligomeric enzymes and PDZ domain-ligand interacting modules.18 However, such nanodevices were based on reversible interactions,19 highly sensitive to pH, temperature, protein concentration, and ionic strength, commonly encountered problems from dissociation of weak coupling among cascade enzymes or collapse of artificial substrate channels.20,21 It is worth to anticipate that designing multienzyme nanoreactors by incorporating molecular defined irreversible linkage may provide a unique opportunity to shield these devices from deactivation, thereby improving their recyclability, operational stability and overall utility. The newly developed SpyTag/SpyCatcher conjugation system is based on the chemical interactions of a split protein domain CnaB2 from Streptococcus pyogenes that features two peptide fragments named SpyTag (13 amino acids) and SpyCatcher (116 amino acids).22 The SpyCatcher/SpyTag is capable of spontaneously reconstituting covalent conjugation under a wide range of temperatures, pH values (5–8), buffers (no specific anions or cations are required), even in the presence of non-ionic detergents.23 This system works well when fused at either N-terminus or C-terminus of functional proteins, which provides a covalent linkage to assemble diverse proteins into a multi-component complex, such as engineered catalytic biofilms,24 and covalent enzymes display.25 C



Here, we designed and constructed multimeric-enzyme nanoclusters (MENCs) that couple properties of multimeric enzyme spontaneous oligmerization assembly with the defined covalent pairing interaction of the SpyTag/SpyCatcher system (Figure 1A,1B). As a proof-ofconcept for MENCs, dimeric cytochrome P450 mutant (P450BM3m:A74G/F87V/L188Q) 26 from Bacillus megaterium and tetrameric glucose dehydrogenase (GDH) from Bacillus subtilis were chosen as model enzymes for the construction. Especially, the designed MENCs enables us to achieve an efficient nicotinamide cofactor NADP(H) regeneration system for producing the pigment indigo from indole, which potentially reduce the high cost of cofactors in biocatalysis (Figure 1C).

Figure 1. A) Strategy for self-assembled MENCs from dimeric P450BM3m and tetrameric GDH. B) The mechanism of isopeptide bond formation. C) The conversion of indole into indigo catalyzed by the MENCs.

The initial assembly step is the oligomerization of subunits of two separate multimeric enzymes in E.coli cells to generate nanoscale building blocks for further self-assembly of MENCs. In the subsequent conjugation, the SpyTag/SpyCatcher modules of the fused multimeric enzymes undergo a pairing-dependent irreversible covalent conjugation when D


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mixed these purified enzymes, thus producing the P450BM3m-GDH MENCs. To design the SpyTag/SpyCatcher fusion constructs, we dissected quaternary structures of constituent enzymes of the GDH-P450BM3m MENCs. The P450BM3m monomer consists of a hemedependent monooxygenase domain at N-terminus and a FAD/FMN containing reductase domain as well as NADPH binding sites at its C-terminus. In the construction, we fused the SpyCatcher at the N-terminus of P450BM3m with a flexible (G4S)2 linker to avoid impairment of the P450BM3m reductase activity (Figure S1A). As a cofactor-recycling component, the quaternary of the GDH was analyzed by a homology-based structural modeling27. It was indicated that the C-terminus of GDH might embed in the interior of proteins, close to the NADP+ binding site of adjacent subunit, while the N-terminus of enzyme exposed at its molecular surface (Figure S1B). Hence, the SpyTag was designed to fuse at the N-terminus of GDH subunit. To ensure that the SpyTag freely access and target to the SpyCatcher partner, a flexible hydrophilic peptide of [(AG)3 PEG]5 28 was used to link the SpyTag and the GDH subunit (Figure S2). The fused SpyTag-GDH and SpyCatcher-P450BM3m, and the non-fused corresponding enzymes were recombinant expressed in E. coli, respectively (Figure S3). Gel filtration chromatography analysis of the purified proteins demonstrated that SpyTag-GDH or SpyCatcher-P450BM3m showed a similar retention profile as that of the non-fused corresponding enzyme (Figure 2A, 2B), which suggested that the quaternary structure of multimeric enzymes were not appreciably compromised by the SpyTag or SpyCatcher fusion. Nearly

identical

UV/Vis-spectrum

between

the

SpyCatcher-P450BM3m

and

the

corresponding non-fused monooxygenase verified the essential heme and flavin incorporation in the P450BM3 variant (Figure 2C). Moreover, analysis of the consumption rate of cofactor NADP+/NADPH indicated that the SpyTag-GDH or the SpyCatcher-P450BM3m has a similar oxidation-reduction activity as that of the non-fused counterparts (Figure S4).

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Figure 2. Gel filtration chromatography analysis of the fused enzymes and UV/Vis spectra of

the monooxygenases. A) Elution profiles of SpyCatcher-P450BM3m and non-fused counterpart P450BM3m. B) Elution profiles of SpyTag-GDH and non-fused counterpart GDH. C) UV/Vis-spectra analysis of SpyCatcher-P450BM3m and P450BM3m. The enzyme solutions were bubbled with carbon monoxide (∼ 90 s) after the addition of sodium dithionite (10 mg), and the difference spectra (400 to 500 nm) were recorded.

The self-assembly of the P450BM3m-GDH MENCs was initiated by mixing purified SpyCatcher-P450BM3m and SpyTag-GDH proteins. Each mixed enzyme was calibrated to ensure 1:1 subunit molar ratio. The SDS-PAGE analysis showed that the desired covalent self-assembly of SpyCatcher-P450BM3/SpyTag-GDH migrated to a gel region corresponding to a molecular weight above 150 kDa, which is consistent to the theoretically calculated molecular weight of coupled subunit of 166 kDa (Figure 3A). The observed migration profile in SDS-PAGE indicated that the irreversible covalent conjugation already happened in these two multimeric enzymes. We further analyzed the formation process of MENCs using dynamic light scattering (DLS) measurements and found that around 80-90% components of the mixture converted into macromolecules within 10 minutes (Figure 3B). When we held the subunit ratio constant but altered the absolute concentration of the enzyme components in mixture, it was found that the concentration of the multimeric protein had a direct effect on growing rates of MENCs. The mixed enzymes at concentrations of 5-10 µM produced the MENCs of small size (~100 nm), while the mixture at a concentration of 2 µM yielded the relatively large MENCs (~1000 nm) within 10 minutes. Over the time, with the further assembly, the very large MENCs gradually appeared and roughly estimated approximately 20% MENCs generated precipitates in the bulk solution.

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Figure 3. A) SDS-PAGE analysis of the assembled P450BM3m-GDH MENCs. The assembled MENCs were subjected to 8% SDS-PAGE and stained with coomassie bright blue. The concentration of SpyCatcher-P450BM3m and SpyTag-GDH was 2µM. The black arrow indicates the isopeptide bond coupled P450BM3m-SpyCatcher-SpyTag-GDH subunits. B) DLS analysis of the assembly of P450BM3m-GDH MENCs. DLS assays were performed for 10 min after mixing the multimeic enzymes ([SpyCatcher-P450BM3m subunit]:[ SpyTagGDH subunit]= 1:1 molar ratio).

To examine the morphology of the MENCs, we used field-emission scanning electron microscope (FE-SEM), atomic-force microscope (AFM) and transmission electron microscope (TEM) to characterize P450BM3m-GDH MENCs. It was shown that soluble MENCs were self-assembled into two-dimensional nano-layers ranging from approximately 10 to 500 µm2 (Figure 4A, Figure S5). Similar structures were also observed under AFM (Figure S6). The morphology of the P450BM3m-GDH MENCs are planar with clear boundaries, which is different from the stacked structure observed in disulfide-bond-locked LPd-FPl MESDs.21 Moreover, TEM analysis revealed that the MENCs have interconnected porous network structures (Figure 4B).

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Figure 4. The representative electron microscope images of the P450BM3m-GDH MENCs. A) The FE-SEM images of MENCs. After assembly, samples were lyophilized and sputtercoated with nano gold prior to analysis. Two-dimensional nano-layer like structures in the MENCs were observed under SEM. B) The TEM images of MENCs. The assembled samples were stained with phosphotungstic acid prior to analysis. The porous interconnected network structures in the MENCs were visualized under TEM. We further analyzed the enzymatic activity of the P450BM3m-GDH MENCs. The P450BM3m-GDH MENCs showed a higher initial reaction rate than controls, unassembled enzymes mixture. The reaction equilibrium for the MENCs was reached in only 300 s, whereas the control mixture did not reach reaction equilibrium until 1,200 s (Figure 5A). Notably, when using a 2 µM concentration of NADP+, the initial catalysis reaction rate of the MENCs was ~3.1 times faster (19.6 ± 0.43 µM/min) than the unassembled enzymes mixture (6.3±0.19 µM/min) (Figure S7 ). The increased catalytic efficiency suggested that NADP (H) channeling might occur between the monooxygenase and the dehydrogenase in the P450BM3m-GDH MENCs.

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Figure 5. Enzyme kinetic analysis, storage stability and reusability of the MENCs. A) Profiles of indigo production catalyzed by P450BM3m-GDH MENCs and the mixture of the two multimeric enzymes. Synthesis of indigo at 10 µM NADP+ concentrations catalyzed by 2 µM of P450BM3m-GDH MENCs (Assembled) or P450BM3m and GDH (Unassembled control). B) The thermostability of the P450BM3m-GDH MENCs or unassembled enzymes mixture at various temperatures. C) The storage stability at 4°C. All of the measurements were executed at 5 µM MENCs concentration; the equal molar concentration for unassembled P450BM3m and GDH as control. D) The reusability of the P450BM3m-GDH MENCs after five cycles of recovery. The concentration of the P450BM3m-GDH MENCs was 20 µM. Data represent the mean ±standard deviation of three measurements.

To evaluate the stability of the P450BM3m-GDH MENCs, we conducted catalytic assays after storage for 1, 3, 5, 7, or 10 days at 4°C. The MENCs retained 80% of their initial activity after 3 days, and retained 40% activity after 10 days; the control mixture had only I



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40% of its full activity after 3 days storage and was inactive after 7 days (Figure 5B). We also tested thermostability by incubating MENCs and control enzyme mixtures at 4 °C, 20 °C, 30 °C and 45 °C for 24 hours, and then determined the residual activity of the samples. Under various temperature storage, the residual activities of the P450BM3m-GDH MENCs were significantly higher than that of the control mixtures (Figure 5C).

The MENCs could be

recovered by an ultrafiltration step, we thus tested the reusability of the MENCs. It is found that the MENCs retained around 67% of the initial activity even after five rounds of biocatalysis reaction. This dropoff might have been caused by ever-decreasing recovery rates of the MENCs from round to round (Figure 5D). In conclusion, the self-assembled P450BM3m-GDH MENCs not only showed higher catalytic efficiency than the unassembled enzymes, but also obtained better enzymatic properties like improved stability and reusability across multiple batches of biocatalysis. The increased reaction rate of the MENCs over the free enzymes maybe ascribed to formed substrate channeling in the MENCs. The enhanced stability of the MENCs over unassembled enzymes could have been a result of the formation of orderly nanocluster structures which restricted conformational changes of component enzymes during storage or exposure to various temperatures. Our work establishes the SpyTag/SpyCatcher-based MENCs concept as a novel platform for engineering architecturally sophisticated supramolecular catalytical systems that can improve biosynthesis efficiency and stability of cascade enzymes. We anticipate that this novel approach will find a broad range of uses in enzyme-based biomaterials or potentially applications in metabolic engineering and synthetic biology.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Detailed experimental procedures. Figure S1-S7, quaternary structure analysis of multimeric enzymes P450BM3 and GDH; the plasmid maps of enzyme constructs; the SDS-PAGE analysis of purified enzymes; the activity characterization of fusion enzymes; characterization of P450BM3m-GDH MENCs using SEM and AFM; the initial reaction rates of the MENCs

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and control at various NADP+ concentrations. Table S1-S2, DNA primers and amino acid sequence used in the research.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Yong Zhang), [email protected](Yan Feng). Tel./Fax: +86-021-34207248

ORCID Yan Feng: 0000-0002-2522-2115 Yong Zhang: 0000-0003-1497-5979

Author Contributions Y.F. designed the experiments. L.Y., X.G. and L.L. performed the experiments. Y.F and Y.Z. analyzed the data. L.Y., Y.Z. and Y. F wrote the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work is supported by the grants from Natural Science Foundation of China (31770846, 31620103901)

and

the

international

S&T

cooperation

Chinese Ministry of Science and Technology (2017YFE0103300).

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Self-assembled multimeric-enzyme nanoreactor for robust and efficient biocatalysis Liang Yin, Xiang Guo, Lu Liu, Yong Zhang*, and Yan Feng*

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