Construction Of Self-assembled Multienzyme Complex By SpyTag/SpyCatcher To Achieve Efficient Biosynthesis

Multienzyme complex system has attracted much attention in synthetic biology owing to its excellent overall catalytic ability.The construction of highly-efficient and stable artificial multienzyme reactors has a broad application in biocatalysis.Simulated and learned from the strategy of multienzyme assembly in natural catalytic systems,a variety of assembly strategies have been established,including protein fusion,scaffold-mediated assembly and immobilization technology.With these strategies,efficient and stable artificial multienzyme complexes were successfully constructed in vitro and in vivo,and the catalytic efficiency of cascade reactions were improved markedly.However,there are many problems in the assembly modules described above,such as the instability of the multienzyme complexes which are easy to be affected by environmental factors;only assembled at specific terminal;restricted to in vitro systems.Therefore,exploring new assembly modules and building more stable and widely applicable multienzyme catalytic assembly system are of great practical significance.SpyCatcher can form an irreversible covalent linkage to its partner SpyTag via a spontaneous isopeptide bond under various conditions,which has been widely applied in protein engineering.To explore the potential application of SpyTag/SpyCatcher system in the self-assembly of multiple enzyme complex,SpyCatcher and SpyTag were fused with P450BM3m monooxygenase and glucose dehydrogenase?GDH?,respectively.The fusion proteins were expected to self-assemble into a double-enzyme complex with the ability of coenzyme regeneration and efficient biosynthesis of indigo.This study mainly included in vitro and in vivo researches.For the in vitro part,the fusion protein SpyTag-GDH,SpyCatcher-P450BM3m and the original enzyme GDH and P450BM3m were expressed and purified.Enzyme activities confirmed that the fusion had no effect on the catalytic activities of the original enzymes.And then the double-enzyme complex was obtained by mixing fusion protein SpyTag-GDH and SpyCatcher-P450BM3m.The self-assembly of multienzyme complex was successfully conformed by SDS-PAGE and dynamic light scattering experiments.The catalytic activity and temperature stability of the self-assembled double-enzyme complex and free double enzyme system were analyzed.The results showed that the self-assembled multienzyme complex could effectively improve the initial rate of cascade reaction.When the NADP⁺concentration was 20?mol/L,the catalytic rate of self-assembled multienzyme complex was1.7 times higher than that of the free enzyme system.What's more,self-assembled multienzyme complex showed better temperature stability.The longer the temperature was and the longer the storage time was,the better the stability was.For the in vivo part,the recombinant plasmids pACYCDuet1-GDH and pACYCDuet1-SpyTag-GDH were constructed firstly.And then pACYCDuet1-SpyTag-GDH and pET28a-SpyCatcher-P450BM3m were transferred to E.coli BL21?DE3?to construct recombinant bacteria co-expressing SpyTag-GDH and SpyCatcher-P450BM3m.The self-assembled multienzyme complex was confirmed by SDS-PAGE,Western Blotting and Nano-Liquid Chromatography.After that,the ability to synthesize indigo by multienzyme complex was systematically analyzed under different culture conditions.When cultured to OD₆₀₀ for 0.6-0.8 and induced at 16?with 0.5 mmol/L IPTG for 18 h,the engineered bacteria showed robust ability to catalyze indole?2 mmol/L?and glucose?4 mmol/L?.The yield of indigo produced by self-assembled complex cells was up to 258 mg/L,which was 1.9and 2.4 times higher than that of cells with free enzymes and P450BM3m single enzyme system accordingly.The conversion ratio was 52%when reaction was balanced after 70 min.It is confirmed that engineered bacteria containing self-assembly coenzyme regeneration system shows higher biosynthesis efficiency.These findings suggest that the SpyTag/SpyCatcher system can successfully assemble multienzyme complex for efficient biosynthesis of chemicals,which provide a new idea for the design of multienzyme complex assembly in vitro and in vivo.