Theoretical and Practical Aspects of Multienzyme Organization and Encapsulation

Abstract

The advent of biotechnology has enabled metabolic engineers to assemble heterologous pathways in cells to produce a variety of products of industrial relevance, often in a sustainable way. However, many pathways face challenges of low product yield. These pathways often suffer from issues that are difficult to optimize, such as low pathway flux and off-target pathway consumption of intermediates. These issues are exacerbated by the need to balance pathway flux with the health of the cell, particularly when a toxic intermediate builds up. Nature faces similar challenges and has evolved vspatial organization strategies to increase metabolic pathway flux and efficiency. Inspired by these strategies, bioengineers have developed clever strategies to mimic spatial organization in nature. This review explores the use of spatial organization strategies, including protein scaffolding and protein encapsulation inside of proteinaceous shells, toward overcoming bottlenecks in metabolic engineering efforts. Expected final online publication date for the Annual Review of Biophysics, Volume 52 is May 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Characterization and heterologous reconstitution of Taxus biosynthetic enzymes leading to baccatin III

“…The spatial organization of enzymes was assumed to benefit metabolite synthesis and regulation ( 45 , 46 ). To investigate the subcellular location of the six cytochrome P450 enzymes, we fused the green fluorescent protein (GFP) gene with the genes individually at their C termini and performed a thorough analysis.…”

Towards using bacterial microcompartments as a platform for spatial metabolic engineering in the industrially important and metabolically versatile Zymomonas mobilis

“…The potential for encapsulating synthetic biochemical pathways into BMC shells in industrially important bacterial hosts has many applications in metabolic engineering (Kerfeld and Sutter, 2020;Stewart et al, 2021;Liu, 2022;Abrahamson et al, 2023). It is especially relevant to pathways with poor enzyme kinetics, or prone to crosstalk with endogenous host pathways or those that generate volatile or toxic intermediates that might disrupt the metabolism of the cell.…”

Tuning Multistep Biocatalysis through Enzyme and Cofactor Colocalization in Charged Porous Protein Macromolecular Frameworks

“…The diverse enzymatic structures and processes inside cells have inspired us to develop artificial materials at multiple length scales to direct and enhance in vitro catalysis. , The naturally occurring complexes composed of enzyme cascades (known as metabolons) are of particular interest, as they are often able to colocalize coupled reactions for high efficiency and selectivity. , By mimicking these structures, a variety of materials capable of multistep biocatalysis have been developed, ,− with continuous regeneration of enzyme cofactors being one important aspect. ,, Enzyme cofactors usually shuttle between functionally coupled enzymes and act as reaction intermediates to transfer functional groups: for example, nicotinamide adenine dinucleotide (NAD) transfers hydride . Because of the high cost of the cofactors for acellular catalysis, recent work has focused on multienzyme systems that replenish the cofactor stock, where a cofactor-regenerating enzyme is coupled with a cofactor-consuming enzyme capable of catalyzing a reaction of interest. , However, these systems still require investment of a large quantity of cofactor to maintain a high overall rate .…”