Engineering substrate channeling in biosystems for improved efficiency

Abstract

The efficiency of a biochemical reaction is contextually regulated in the landscape of the metabolic network of a cell. The availability and activity of the corresponding enzyme are maintained at a level that favors the overall fitness of the cell, constituting a big issue for biotechnology application that a substantial accumulation of specific product is principally expected. Substrate channeling mechanism naturally existing in multiple active site enzymes, enhances the catalytic efficiency of the involved enzymes via the proximity effect enabled by the'evolutionally optimized'orientation of the enzymes. In metabolic engineering or in vitro cascade biocatalysis, improved efficiency can be obtained when the involved enzymes are rationally organized, to simulate substrate channeling mechanism in artificially constructed systems. In this paper, we reviewed the available approaches adopted in constructing in vitro cascade biocatalysis or in vivo engineered pathways for the production of valuable products. Among these approaches, assembling enzymes onto the designed scaffolds (protein, DNA or RNA) has been proved effective in many metabolic engineering studies. Encapsulating enzymes in virus capsid or encapsulin can be used to create specialized 'organelle-like' microreactors in cell by simulating the naturally existing bacterial microcompartment. Assembling enzymes on lipid droplet or thylakoid membrane of chloroplasts, will be of particular significance in practice in plant cell bioreactors. Inclusion of a feedback mechanism as an addition to these engineered systems may promise further increased efficiency.

Substrate Channeling by a Rationally Designed Fusion Protein in a Biocatalytic Cascade

“…Substrate channeling is a phenomenon by which an intermediate in a multistep catalytic cascade is prevented from reaching equilibrium with the bulk solution and is instead directed toward subsequent reaction centers. , Within biological systems, substrate channeling between enzymes in cascade reactions results in increased flux through preferred pathways and prevention of side reactions; this is a critical feature considering the plethora of catalysts and substrates all present in the same aqueous solution in typical organisms. , As research into and the utilization of multistep enzyme cascades continues to increase in recent years, methods by which substrate channeling can be applied to these cascade reactions are being actively pursued …”

“…There is some disagreement about whether fusion proteins with simple linker sequences intended to provide only proximity and/or orientation of the enzymes constitute substrate channeling in a formal sense . In systems such as those which employ fusion proteins, , protein/DNA scaffolds, − or surface coimmobilization of constituents, , significant catalytic rate enhancements have been observed which exceed expectations when proximity alone is considered; Sigman et al illustrated that, for a typical enzyme (e.g., turnover frequency, k , of 10 s –1 ) and a typical aqueous-phase small molecule (e.g., diffusion coefficient, D , of 10 –5 cm 2 s –1 ), there is no appreciable enhancement of local concentrations of the intermediate species at subsequent reaction centers compared to the bulk solution . That is, diffusion typically occurs at time scales that are orders of magnitude faster than enzyme turnover events, so proximity alone should not produce an observable overall rate increase for most cascade reactions.…”

Adjustable Bioorthogonal Conjugation Platform for Protein Studies in Live Cells Based on Artificial Compartments

“…In naturally occurring metabolic pathways, enzymes are not randomly distributed in the cytosol, but are highly organized to ensure rapid and efficient processing of intermediates. 1 Therefore, the investigation and modification of natural cellular processes through well characterized conjugation reactions or the construction of artificial pathways in synthetic biology can benefit from bioorthogonal protein conjugation to spatially confined synthetic scaffolds. For instance, by defined tethering of enzymes catalyzing consecutive reactions on a synthetic scaffold, natural substrate channeling can be mimicked, thereby accelerating or increasing product formation and also preventing toxic intermediates from diffusion into the cell lumen.…”

“…Spatial organization of proteins through conjugation and scaffolding strategies are fundamental features of life, crucial to orchestrate important cellular processes. In naturally occurring metabolic pathways, enzymes are not randomly distributed in the cytosol, but are highly organized to ensure rapid and efficient processing of intermediates . Therefore, the investigation and modification of natural cellular processes through well characterized conjugation reactions or the construction of artificial pathways in synthetic biology can benefit from bioorthogonal protein conjugation to spatially confined synthetic scaffolds.…”

“…(iii) The fusion of large conjugation tags to enzymes can compromise enzyme activity or expression level, which is difficult to predict . (vi) Bioorthogonal conjugation reactions in vivo are mainly characterized qualitatively rather than quantitatively …”