Supramolecular Self-assembly Of Multiple Enzymes In Vitro And In Vivo

Multienzymatic cascade reactions allow synthesizing complex products from simple elements, which makes them become increasingly important roles in large scale biotransformation. However, developing strategies to improve cooperativity and catalysis efficiency of cascaded enzymes remains a challenge. In cells, multienzymatic pathways are offen spatially organized into clusters or microcompartments, which would bring enzymes together and increase yield of metabolic reactions. Recently, inspired by nature, researchers have developed many methods for multienzymatic assembly which could improve the efficiency of multienzymatic cascade reactions. In this study, we attempt to develop a simple, economical, and efficient multienzymatic assembly stategy by using a two-enzyme pathway for synthesizing L-tert-leucine as a technically relevent model. The detailed works are introduced as follows:(1) Construction and fusion of the two-enzyme pathway for synthesizing L-tert-LeucineLeucine dehydrogenase and formate dehydrogenase were constructed by PCR, and were experessed in E.coil BL21 (DE3). The reaction system was developed by screening different conditions, including temperature, pH, and concentration of substrate. Under the optimum condition, the two-enzyme pathway could transform 92% of 600 mM substrate to L-tert-leucine with 95% ee value.Fusion of leucine dehydrogenase and formate dehydrogenase was perfomed by gene fusion, and various linkers were used to connect these two enzymes. The results showed that fused enzymes with rigid ER/K linkers demonstrated higher level of soluable expression and higher catalysis efficiency. However, compared to coexpressed enzymes, fused enzymes got lower level of soluable expression and catalysis efficiency. Additionally, fused enzymes were unstable and prone to be effected by environmental conditions change.(2) Co-immoblization of the two-enzyme pathwayFormate dehydrogenase and leucine dehydrogenase were co-immobilized onto PD-IONPs for synthesis of L-tert-leucine. The results indicated that leucine dehydrogenase retained desired activity. However, formate dehydrogenase had only a marginal activity after immobilization. To enhance the activity of co-immobilized formate dehydrogenase, an enzyme-orientated strategy was developed by site-directed mutagenesis. Seven mutants were constructed based on homology modeling of formate dehydrogenase and immobilized on PD-IONPs to investigate their influence on immobilization. After the immobilization, mutant C-c demonstrated the highest immobilization yield and retained 90 percent of its initial activity, which was about 3-fold higher than that of wild-type formate dehydrogenase. Moreover, co-immobilization of formate dehydrogenase and leucine dehydrogenase was performed for the synthesis of L-tert-leucine. The catalytic efficiency of the co-immobilized mutant C-c and leucine dehydrogenase was increased dramatically compared to that of the co-immobilized wild-type formate dehydrogenase and leucine dehydrogenase.(3) Supermolecular self-assembly of the two-enzymes pathwayTo realize the ordered self-assembly of formate dehydrogenase and leucine dehydrogenase in vitro and in vivo, a scaffold-free self-assembly strategy was developed by exploiting enzyme oligomerization and protein-protein interaction properties. A PDZ domain and its ligand were fused to leucine dehydrogenase and formate dehydrogenase, respectively. Assembly of the two-enzyme pathway was performed successfully by mixing fused protein in vitro or co-expressing them in vivo. The multienzyme supramolecular devices formed in vitro and in vivo were characterized by using DLS, SDS-PAGE, field-emission scanning electron and atomic force microscopy, and fluorescence complementation label. The best molar ratio of subunit used for assembly in vitro was 1:1. Assemblies formed in vitro assumed two-dimensional layer-like structures, and demonstrated protein concentration dependency. Assembly that performed in vivo mainly occurred between 9-18 h after induction. The resultant assemblies assumed micron-sized layer-like structures, and existed in the form of inclusible bodies which were confined to the poles of cells. Moreover, both in vitro and in vivo assemblies showed higher NAD(H) recycling efficiency and structural stability than did unassembled structures when applied to synthesize L-tert-leucine, which suggested that transfer channeling for NAD(H) occurred between these two enzymes after assembly.(4) Modification of multienzyme supramolecular devicesAs the multienzyme supramolecular devices were large in size, they could be used as solid biocatalysts. To enhance the reuseability of assemblies, the assemblies were modified by disulfide bonds engineering. By analyzing the three-dimensional structural details of the PDZ-ligand interface, Asn24 of the PDZ domain and Val2 of the PDZ ligand, which were in close proximity for potential disulfide bond formation, were selected to be substituted by cysteine. After mutation, the disulfide bonds were successfully introduced to the assembly interfaces.The resultant multi-enzyme supramolecular device demonstrated good reusability, and approximately 80% of its initial catalytic activity was retained even after eight cycles of reuse.A strategy was developed for the self-assembly of multiple oligomeric enzymes that was successfully applied to in vitro and in vivo systems, and the generated assemblies have highly ordered structure and efficient function of cascaded enzymes. This study paved a novle and simple way for assembling and immobilizing multiple enzymes, compartmentalizing metabolic enzyme cascades in living cells, and constructing bio-nano-materials.