The Design And Construction Of Self-Assembled Artificial Multienzyme Nanoreactors

Natural enzyme cascades are often assembled into sophisticated nanoscale multienzyme complexes,which feature well-controlled geometries and substrate channeling to facilitate transfer of reaction intermediates and thereby improve catalytic efficiency.The idea of designing highly ordered multienzyme nanodevices represents a promising strategy in synthetic biology and metabolic engineering.Learning from nature,multiple artificial enzymes assembly strategies have been explored and used to produce valuable biocommodities.However,as the fact that many metabolic enzymes are oligomer and there is the increased complexity of the intersubunit interactions,construction of efficient and stable multienzyme complexes remains quite challenging.Most multienzyme nanodevices commonly encountered problems from lower hierarchical structures,controllability and stability.Aiming to solve above problems,we attempt to develop 2D and 3D supramolecular multienzyme nanodevices by using oxidation-reduction enzymes as model system and orthogonally reactive SpyCatcher/SpyTag system as self-assembling modules.We explored 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 fragment and its binding partner SpyTag peptide were fused to a dimeric cytochrome P450 monooxygenases mutant(P450BM3m)and a tetrameric glucose dehydrogenase(GDH),respectively.The multimeric fusion proteins self-assembled into supramolecular devices,forming a covalently linked enzymes cascade that facilitated NADPH regeneration and converted indole into the pigment indigo.We investigated the morphology of MENCs and found these multimeric enzymes assembled into twodimensional 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 indigo synthesis,the MENCs also had significantly improved stability and reusability compared to enzyme mixtures.Designing protein scaffold with higher order topological structures is necessary to fabricate a 3D multienzyme nanodevices.We choose E.coli Dps protein as scaffold,which folds into spherical protein shells composed of 12 subunits in four-helix bundles.In this study,we firstly constructed fusion building blocks SpyTag-Dps-ALR by fusing a monomeric aldehyde reductase(ALR)and SpyTag peptide to the C-terminus and Nterminus of Dps respectively.In addition,SpyCatcher was fused to the N terminus of dimeric formate dehydrogenase(FDH)for yielding SpyCatcher-FDH,and then we conducted SEC,EMSA and TEM analysis experiments,all of the results showed that SpyTag-Dps-ALR self-assembled into monodispersed and homogeneous nanoparticle in E.coli.The diameter is approximately 12 nm.After simply mixing the SpyTag-DpsALR and SpyCatcher-FDH in vitro,the two fusion proteins spontaneously fabricated into 3D nanodevices.We analyzed the assembly profiles by SDS-PAGE and DLS.It showed that the maximum assembly ratio is 1:12.The structures were further observed by AFM,revealing that the nanodevices were self-assembled into 3D porous superlattice networks ranging from a few to several hundred square microns in size.The 3D nanodevices efficiently converted the COBE into R-CHBE,and which obtained approximately 5-fold conversion efficiency using NADP+ as cofactor than NADPH.By controlling the assembly ratio of SpyTag-Dps-ALR and SpyCatcher-FDH,the yields of R-CHBE increased from 30.26% to 71.9 % with the assembly ratio increase from 1:3 to 1:12.The nanodevice also demonstrated good reusability,3D nanodevice retained approximately 80% of the initial activity was retained even after seven cycles.Our work establishes the SpyTag/SpyCatcher-based MENCs concept as a new platform for engineering architecturally sophisticated supramolecular catalysts,and we also developed 3D multienzyme nanodevice by using Dps as self-assembling blocks.The design concept underlying the Dps protein we demonstrated here can in theory be used for the supramolecular assembly of many other biomaterials.