Synthetic Scaffold Systems for Increasing the Efficiency of Metabolic Pathways in Microorganisms

Abstract

Microbes have been the preferred hosts for producing high-value chemicals from cheap raw materials. However, metabolic flux imbalance, the presence of competing pathways, and toxic intermediates often lead to low production efficiency. The spatial organization of the substrates, intermediates, and enzymes is critical to ensuring efficient metabolic activity by microorganisms. One of the most common approaches for bringing the key components of biosynthetic pathways together is through molecular scaffolds, which involves the clustering of pathway enzymes on engineered molecules via different interacting mechanisms. In particular, synthetic scaffold systems have been applied to improve the efficiency of various heterologous and synthetic pathways in Escherichia coli and Saccharomyces cerevisiae, with varying degrees of success. Herein, we review the recent developments and applications of protein-based and nucleic acid-based scaffold systems and discuss current challenges and future directions in the use of such approaches.

Inducible plasmid copy number control for synthetic biology in commonly used E. coli strains

“…In addition to the application examples we considered in this paper, we envision dynamic PCN control to be deployed in a wide range of settings. Potential contexts could include dynamic spatial organization of metabolic pathways with the plasmid acting as a scaffold to localize fusion proteins consisting of DNA binding domains and metabolic enzymes 56 , 57 , and alternative mechanisms for gene expression regulation, for instance, using plasmids to carry decoy operator sequences as a method for altering gene circuit dynamics and mitigating toxicity 58 , 59 , or interacting with the host’s native TFs for "soft” regulation to alter metabolism 60 . In addition, dynamic PCN control not only provides us with a straightforward way to simultaneously modulate the expression of multiple gene targets, it can also offer an economical alternative complementing transcriptional, translational, and post-translational control 37 .…”

Theoretical and Practical Aspects of Multienzyme Organization and Encapsulation

“…Notable successes include the production of artemisinic acid in yeast and 1,3-propanediol (1, in Escherichia coli (71,82). As the suite of accessible compounds expands with novel enzyme discovery and enzyme engineering, efforts to generate new products can be hindered by bottlenecks that can arise from cofactor imbalances or mismatched enzyme kinetics (29,73,86). These bottlenecks can lead to intermediate accumulation, resulting in toxicity or undesired side products.…”

Exploring linker's sequence diversity to fuse carotene cyclase and hydroxylase for zeaxanthin biosynthesis

“…Microcompartments allow for the sequestration of enzymes and hydrogenases have previously been targeted in a repurposed carboxysome ( Li et al, 2020 ). Synthetic scaffolds made of proteins or nucleic acids are widely used to anchor enzymes ( Geraldi et al, 2021 ; Park et al, 2022 ). Finally, at a scale limited to two or three enzymes, protein fusions allow to bring enzymes together in a one-to-one ratio ( Elleuche, 2015 ).…”