Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm

Abstract

We have developed a system for producing a supramolecular scaffold that permeates the entire Escherichia coli cytoplasm. This cytoscaffold is constructed from a three-component system comprising a bacterial microcompartment shell protein and two complementary de novo coiled-coil peptides. We show that other proteins can be targeted to this intracellular filamentous arrangement. Specifically, the enzymes pyruvate decarboxylase and alcohol dehydrogenase have been directed to the filaments, leading to enhanced ethanol production in these engineered bacterial cells compared to those that do not produce the scaffold. This is consistent with improved metabolic efficiency through enzyme colocation. Finally, the shell-protein scaffold can be directed to the inner membrane of the cell, demonstrating how synthetic cellular organization can be coupled with spatial optimization through in-cell protein design. The cytoscaffold has potential in the development of next-generation cell factories, wherein it could be used to organize enzyme pathways and metabolite transporters to enhance metabolic flux.

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).…”

“…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). Bacterial microcompartments (BMCs) could improve bioproduction by encapsulating related enzymes within a unique self‐assembled protein shell (Kerfeld & Erbilgin, 2015, Lee et al, 2018). However, for complex metabolic pathways containing multiple enzymes, the role of BMCs is limited because of the random cross‐linking between enzymes and scaffolds (Moon, Dueber, Shiue, & Prather, 2010).…”

“…In synthetic biology and metabolic engineering field, increasing attentions focus on the enzyme clustering, such as the self‐assembly of multiple enzymes or the formation of intracellular supramolecular scaffolds (Lee et al, 2018; Price, Chen, Whitaker, Papoutsakis, & Chen, 2016). Enzyme clustering can assemble multiple enzymes to balance intracellular metabolism and increase pathway efficiency by reducing substrate loss (Price et al, 2016; Tippmann et al, 2016).…”

“…For example, Price et al (2016) reported a self‐assembly strategy to organize methanol dehydrogenase, 3‐hexulose‐6‐phosphate synthase, and 6‐phospho‐3‐hexuloseisomerase into a supramolecular enzyme complex, to enhance methanol conversion to fructose‐6‐phosphate. In another example, Lee et al (2018) found that ethanol production can be improved by tethering pyruvate decarboxylase and alcohol dehydrogenase to an intracellular filamentous arrangement formed by bacterial microcompartment protein PduA and two complementary coiled‐coil peptides. Further analysis indicated that enzyme clustering could control local concentrations of reactants, intermediates, and enzymes in synthetic pathways to overcome the inherent challenges of intracellular metabolism, thereby improving reaction flux and minimizing cross‐reactions.…”

“…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). Bacterial microcompartments (BMCs) could improve bioproduction by encapsulating related enzymes within a unique selfassembled protein shell (Kerfeld & Erbilgin, 2015, Lee et al, 2018.…”