Modular enzyme assembly for enhanced cascade biocatalysis and metabolic flux

Abstract

Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated. Enzyme engineers seek to construct multienzyme complexes to prevent intermediate diffusion, to improve product yield, and to control the flux of metabolites. Here we choose a pair of short peptide tags (RIAD and RIDD) to create scaffold-free enzyme assemblies to achieve these goals. In vitro, assembling enzymes in the menaquinone biosynthetic pathway through RIAD–RIDD interaction yields protein nanoparticles with varying stoichiometries, sizes, geometries, and catalytic efficiency. In Escherichia coli, assembling the last enzyme of the upstream mevalonate pathway with the first enzyme of the downstream carotenoid pathway leads to the formation of a pathway node, which increases carotenoid production by 5.7 folds. The same strategy results in a 58% increase in lycopene production in engineered Saccharomyces cerevisiae. This work presents a simple strategy to impose metabolic control in biosynthetic microbe factories.

Enzyme assembly guided by SPFH‐induced functional inclusion bodies for enhanced cascade biocatalysis

“…Organizing interrelated cellular components on a temporal and spatial scale is crucial for improving the efficiency of the cellular processes such as metabolism and signal transduction (Agapakis, Boyle, & Silver, 2012; Young et al, 2017). With the advent and development of synthetic biology, the design of efficient microbial cell factories using spatial organization within cellular environment to enhance the metabolic flux has attracted extensive attentions (Kang et al, 2019; Lee et al, 2018; Young et al, 2017). To develop a scaffold that can be better applied to the living cells, the researchers paid attention to the proteins that can self‐assemble into defined, nano to macromolecular architectures (Howorka, 2011).…”

Production of Terpenoids by Synthetic Biology Approaches

“…However, scaffolded enzyme assemblies have different limitations, as enzymes fused in large structures may encounter a decrease or complete loss of the activity (Jia et al, 2014). Recently, Kang W. et al (2019) developed a scaffold-free modular enzyme assembly by employing a pair of short peptide tags. The GGPP synthase and IDI were modularly assembled, which increased carotenoid production by 5.7-folds in E. coli and yielded a titer of 2.3 g/L lycopene in S. cerevisiae.…”

Enzyme Assembly for Compartmentalized Metabolic Flux Control

“…Kang et al created scaffold-free enzyme assemblies by using a pair of short peptide tags RIAD and RIDD [28], which originate from the cAMP-dependent protein kinase (PKA) and the A kinase-anchoring proteins (AKAPs), respectively [43][44][45]. RIDD refers to a docking and dimerization domain of the R subunits of PKAs, and the RIAD peptide is derived from an amphipathic helix of the anchor domain of an AKAP that specifically binds to the RIDD dimer [46,47].…”

“…Further analysis showed that with RIAD-RIDD interaction, the assembly of enzymes for the menaquinone biosynthetic pathway yielded protein nanoparticles with varying stoichiometries, geometries, sizes, and catalytic efficiencies in vitro. In addition, a similar strategy was used to assemble the last enzyme of the mevalonate pathway with the first enzyme of the carotenoid pathway ( Figure 1), resulting in an increase in carotenoid production by 5.7-fold in E. coli [28]. In general, Kang et al proposed a simple metabolic control strategy, which provides a new direction for scaffold-free enzyme assembly; however, when the target enzyme is a monomer rather than an oligomer, the RIAD-RIDD trimer in the cell can only aggregate up to three enzymes (two kinds of enzymes).…”

“…Therefore, a variety of rational enzyme assembly or colocalization strategies have been developed to synthesize high-value products [12,13,18,[20][21][22]. These engineered systems range from simple fusion proteins to scaffold-free self-assemblies, synthetic scaffolds, or physical compartments [23][24][25][26][27][28].…”