Engineered repeat proteins as scaffolds to assemble multi-enzyme systems for efficient cell-free biosynthesis

Abstract

Multi-enzymatic cascades with enzymes arranged in close-proximity through a protein scaffold can trigger a substrate channeling effect, allowing for efficient cofactor reuse with industrial potential. However, precise nanometric organization of enzymes challenges the design of scaffolds. In this study, we create a nanometrically organized multi-enzymatic system exploiting engineered Tetrapeptide Repeat Affinity Proteins (TRAPs) as scaffolding for biocatalysis. We genetically fuse TRAP domains and program them to selectively and orthogonally recognize peptide-tags fused to enzymes, which upon binding form spatially organized metabolomes. In addition, the scaffold encodes binding sites to selectively and reversibly sequester reaction intermediates like cofactors via electrostatic interactions, increasing their local concentration and, consequently, the catalytic efficiency. This concept is demonstrated for the biosynthesis of amino acids and amines using up to three enzymes. Scaffolded multi-enzyme systems present up to 5-fold higher specific productivity than the non-scaffolded ones. In-depth analysis suggests that channeling of NADH cofactor between the assembled enzymes enhances the overall cascade throughput and the product yield. Moreover, we immobilize this biomolecular scaffold on solid supports, creating reusable heterogeneous multi-functional biocatalysts for consecutive operational batch cycles. Our results demonstrate the potential of TRAP-scaffolding systems as spatial-organizing tools to increase the efficiency of cell-free biosynthetic pathways.

Constructing Protein-Scaffolded Multienzyme Assembly Enhances the Coupling Efficiency of the P450 System for Efficient Daidzein Biosynthesis from (2S)-Naringenin

“…Anisotropy measurements were registered employing 6 nm slits with excitation at 340 nm and emission at 463 nm. The acquired data underwent analysis utilizing GraphPad Prism 8, where fitting was performed employing One Site-Specific binding model …”

“…The acquired data underwent analysis utilizing GraphPad Prism 8, where fitting was performed employing One Site-Specific binding model. 34 2.13. Real-Time PCR Measurements for Transcriptional Analysis.…”

“…In contrast, when performing the protein assay at a concentration of 2 μM, the G-CPR, S-CYP82D26, and Alrd-P were separated by approximately 94 nm, as calculated and reported previously. 32,34 The proximity effect between G-CPR, S-CYP82D26, and Alrd-P, facilitated by the SGP scaffold, may enhance NADPH accessibility to the SGP-scaffolded assembly of the P450 system.…”

“…Previous research has indicated that the catalytic efficiency of a scaffolded multienzyme system can be enhanced through electrostatic interaction between protein scaffolds and cofactors, resulting in a diffusion-limited effect of cofactor and higher local concentrations of cofactors, in addition to proximity effects. 34 Based on the electrostatic potential calculations illustrated in Figure 5B, the surface of the SGP scaffold is found to contain positively charged areas, which could be conducive to electrostatic interactions with the phosphate groups of NADP + /NADPH. Besides, the computational docking studies suggest that a positively charged region of the SGP scaffold could bind NADP + and NADPH (Figure 5B).…”

Programming interchangeable and reversible heterooligomeric protein self-assembly using a bifunctional ligand

“…Then, the chemical space for articially designed protein-assembled structures and functions remains limited. Alternatively, self-assembly of two protein components can be achieved through gene fusion, 18,19 disulphide bond, 20 gene fusion and covalent linkage, 21 host-guest interaction, 22 and computational design. [23][24][25][26] However, their components are not easily interchangeable unless their gene constructs or PPIs are redesigned from scratch.…”