Encapsulin Nanocontainers as Versatile Scaffolds for the Development of Artificial Metabolons

Abstract

The construction of non-native biosynthetic pathways represents a powerful, modular strategy for the production of valuable synthons and fine chemicals. Accordingly, artificially affixing enzymes that catalyze sequential reactions onto DNAs, proteins, or synthetic scaffolds has proven to be an effective route for generating de novo metabolons with novel functionalities and superior efficiency. In recent years, nanoscale microbial compartments known as encapsulins have emerged as a class of robust and highly engineerable proteinaceous containers with myriad applications in biotechnology and synthetic biology. Herein we report the concurrent surface functionalization and internal packaging of encapsulins from Thermotoga maritima to generate a catalytically competent two-enzyme metabolon. Encapsulins were engineered to covalently sequester up to 60 copies of a dihydrofolate reductase (DHFR) enzyme variant on their exterior surfaces using the SpyCatcher bioconjugation system, while their lumens were packaged with a tetrahydrofolate-dependent demethylase enzyme using short peptide affinity tags abstracted from the encapsulin’s native protein cargo. Successful cross-talk between the two colocalized enzymes was confirmed as tetrahydrofolate produced by externally tethered DHFR was capable of driving the demethylation of a lignin-derived aryl substrate by packaged demethylases, albeit slowly. The subsequent introduction of a previously reported pore-enlarging deletion in the encapsulin shell was shown to enhance metabolite exchange such that the encapsulin-based metabolon functioned at speeds equivalent to those of the two enzymes freely dispersed in solution. Our work thus further emphasizes the engineerability of encapsulins and their potential use as flexile scaffolds for biocatalytic applications.

Nanotechnological Applications Based on Bacterial Encapsulins

“…Simply mixing the purified nanocompartment with proteins fused to a SpyCatcher domain covers the nanocompartment surface with the recombinant protein. A similar strategy has been reported using a SpyCatcher fusion on the C terminus of the EncTm encapsulins [83].…”

“…The mutant ∆9Gly2, which generates the largest pore size (~11 Å) [81], showed contradictory results, either with mass transport increased by sevenfold [81] or with minimal differences in the ion flux kinetics [82]. In an unrelated study, the ∆9Gly2 EncTm mutant showed a fivefold increase in the relative performance of a nanoreactor-catalyzed enzyme cascade when compared to wild type EncTm [83]. These results underscore the difficulties inherent to accurately quantifying flux into protein cages and warrant further investigation.…”

“…Pores in the nanocompartment regulate the molecular flux through the shell by size and by charge [6,53,81,82], and constitute one of the key parameters in controlling encapsulated protein activity [83]. Their role as a molecular sieve is thought to assist the flux of correct substrates [36,49], preventing undesired reactions.…”

“…The high catalytical activity of encapsulated enzymes requires adaptation between pore characteristics and metabolite size and charge [63,83]. Two studies analyzed the influence of pore size and charge on mass transport [81,82] by systematically mutating the loop region that delineates the pores at the fivefold axis of the EncTm nanocompartment.…”

“…Family 1 encapsulins are in essence natural nanoreactors with a simple, CLP-based mechanism for internalizing enzymes and other cargo proteins; this has fostered the engineering of nanocompartments as nanoreactors using a variety of enzymes [50,63,83,88,97,103,111]. Confinement of enzymatic reactions within encapsulin shells is particularly suited to overcoming problems associated with toxicity of intermediate products and competition by metabolic activities [97].…”