Dynamically regulating metabolic fluxes with synthetic metabolons

Abstract

Enzyme–enzyme assemblies commonly occur naturally, yet the factors that lead to their transient nature are not fully understood. Mitkas et al. have shown how clustered regularly interspaced short palindromic repeats (CRISPR) enzymes and RNA scaffolds allow synthetic enzyme complexes to be formed and disassembled as needed, providing powerful new tools for metabolic engineering. Enzyme–enzyme assemblies commonly occur naturally, yet the factors that lead to their transient nature are not fully understood. Mitkas et al. have shown how clustered regularly interspaced short palindromic repeats (CRISPR) enzymes and RNA scaffolds allow synthetic enzyme complexes to be formed and disassembled as needed, providing powerful new tools for metabolic engineering. Considerable evidence supports the contention that dynamic interaction between enzymes represents an important form of metabolic control and thereby has considerable influence in both controlling and coordinating metabolic fluxes [1.Sweetlove L.J. Fernie A.R. The role of dynamic enzyme assemblies and substrate channelling in metabolic regulation.Nat. Commun. 2018; 9: 2136Crossref PubMed Scopus (218) Google Scholar]. Given this, metabolic engineers have looked to exploit this natural innovation to improve natural product yields [2.Delebecque C.J. et al.Organization of intracellular reactions with rationally designed RNA assemblies.Science. 2011; 333: 470-474Crossref PubMed Scopus (510) Google Scholar, 3.Agapakis C.M. et al.Natural strategies for the spatial optimization of metabolism in synthetic biology.Nat. Chem. Biol. 2012; 8: 527-535Crossref PubMed Scopus (305) Google Scholar, 4.Dueber J.E. et al.Synthetic protein scaffolds provide modular control over metabolic flux.Nat. Biotechnol. 2009; 27: 753-759Crossref PubMed Scopus (985) Google Scholar, 5.Han D.R. et al.Single-stranded DNA and RNA origami.Science. 2017; 358eaao2648Crossref Scopus (160) Google Scholar]. It has previously been argued that post-translational modification would be an ideal strategy given that it circumvents the need to alter enzyme levels [6.Venayak N. et al.Engineering metabolism through dynamic control.Curr. Opin. Biotechnol. 2015; 34: 142-152Crossref PubMed Scopus (152) Google Scholar]. The dynamic regulation of enzyme–enzyme assemblies (or metabolons) would represent one such route [3.Agapakis C.M. et al.Natural strategies for the spatial optimization of metabolism in synthetic biology.Nat. Chem. Biol. 2012; 8: 527-535Crossref PubMed Scopus (305) Google Scholar], but its application is currently hindered by our lack of understanding of the mechanisms by which (dis)assembly of such complexes is controlled. Metabolons are enzyme–enzyme assemblies that practice substrate channeling the transfer of intermediates from active site to active site of two or more enzymes. Such complexes increase pathway flux as well as minimizing the effects of competing pathways and facilitate rapid turnover of labile or toxic intermediates [1.Sweetlove L.J. Fernie A.R. The role of dynamic enzyme assemblies and substrate channelling in metabolic regulation.Nat. Commun. 2018; 9: 2136Crossref PubMed Scopus (218) Google Scholar]. Synthetic metabolons have been generated using artificial protein or DNA scaffolds as a means to colocalize enzymes in a controlled manner, facilitating superior product synthesis [4.Dueber J.E. et al.Synthetic protein scaffolds provide modular control over metabolic flux.Nat. Biotechnol. 2009; 27: 753-759Crossref PubMed Scopus (985) Google Scholar,7.Conrado R.J. et al.Engineering the spatial organization of metabolic enzymes: mimicking nature's synergy.Curr. Opin. Biotechnol. 2008; 19: 492-499Crossref PubMed Scopus (287) Google Scholar]. However, to date, such synthetic metabolons, whilst successful in elevating yields, are static and lack the ability to respond to cellular circumstance. As such, the terminology ‘synthetic metabolon’ is actually inaccurate, as they are rather synthetic multienzyme complexes, much more akin to the stable enzymes that have evolved to constitutively operate in complex with one another. The work of Mitkas and colleagues, however, describes a true synthetic metabolon, the activity of which is controlled dynamically by RNA [8.Mitkas A.A. et al.Dynamic modulation of enzyme activity by synthetic CRISPR-Cas6 endonucleases.Nat. Chem. Biol. 2022; 18: 492-500Crossref PubMed Scopus (7) Google Scholar]. In this study, the authors demonstrate that synthetic metabolons can be turned on or off in response to RNA binding and illustrate this application by the production of the phytohormone auxin as well as the production of malate. Driven by the hope of improved production, the idea of synthetic metabolons has long captured the imagination of metabolic engineers, with protein and RNA enzyme scaffolds being demonstrated to result in enhanced natural product yields [2.Delebecque C.J. et al.Organization of intracellular reactions with rationally designed RNA assemblies.Science. 2011; 333: 470-474Crossref PubMed Scopus (510) Google Scholar,4.Dueber J.E. et al.Synthetic protein scaffolds provide modular control over metabolic flux.Nat. Biotechnol. 2009; 27: 753-759Crossref PubMed Scopus (985) Google Scholar]. More recently, a light-based control system has been developed to fine-tune the assembly and disassembly of enzyme clusters [9.Zhao E.M. et al.Light-based control of metabolic flux through assembly of synthetic organelles.Nat. Chem. Biol. 2019; 15: 589-597Crossref PubMed Scopus (127) Google Scholar]. However, as we commented earlier, these approaches do not truly mirror metabolons since they do not dynamically regulate their abundance in response to molecular cues. Mitkas and colleagues have managed, via the repurposing of CRISPR/CRISPR-associated protein 6 (Cas6) and toehold-mediated RNA interactions, to more faithfully synthetically replicate a metabolon [8.Mitkas A.A. et al.Dynamic modulation of enzyme activity by synthetic CRISPR-Cas6 endonucleases.Nat. Chem. Biol. 2022; 18: 492-500Crossref PubMed Scopus (7) Google Scholar]. To do so, they created enzyme fusions to two Cas6 proteins: Cse3 from Escherichia coli and Csy4 from Pseudomonas aeruginosa. Both these proteins recognize RNA scaffold strands with distinct sequences and bind to form distinct RNA–enzyme complexes. The assembly and disassembly of the resultant synthetic metabolons is regulated via single-strand RNA sticky ends, termed toeholds [10.Green A.A. Synthetic bionanotechnology: synthetic biology finds a toehold in nanotechnology.Emerg. Top. Life Sci. 2019; 3: 507-516Crossref PubMed Scopus (9) Google Scholar]. Such toeholds provide a nucleation point for RNA interaction and once base-pairing is established here, RNA strands can be released, which promotes exposure of previously inaccessible regions of RNA. The ingenious strategy of Mitkas and coworkers was to exploit the programmable nature of RNA to allow different trigger molecules to regulate the metabolon on demand (Figure 1). As such, they could turn on the metabolon by triggering the assembly of both RNA–enzyme complexes or turn it off by using a trigger RNA of one of the scaffold RNAs in order to kick it out of the metabolon. In doing so, they were able to demonstrate the possibility of increasing auxin and malate yield by three- and ninefold, respectively. This work collectively thus represents a considerable achievement. Whilst the authors themselves acknowledge that it is currently limited to the assembly of a two-enzyme metabolon [8.Mitkas A.A. et al.Dynamic modulation of enzyme activity by synthetic CRISPR-Cas6 endonucleases.Nat. Chem. Biol. 2022; 18: 492-500Crossref PubMed Scopus (7) Google Scholar], it is important to note that whilst enzyme–enzyme assemblies are often much larger in nature, proof of substrate channeling is often limited to enzyme pairs [1.Sweetlove L.J. Fernie A.R. The role of dynamic enzyme assemblies and substrate channelling in metabolic regulation.Nat. Commun. 2018; 9: 2136Crossref PubMed Scopus (218) Google Scholar]. This fact notwithstanding, it is clear that the search for additional natural or synthetic Cas6 proteins alongside the development of more sophisticated RNA scaffolds means that the extension of this approach, and potentially also of its effect on biosynthetic efficiencies, will represent an important frontier of such approaches in the coming years. In addition, whether other regulatory and/or structural elements, such as post-translational modifications and protein–lipid interactions, can be modified in tandem are aspects worthy of future attention. That said, as they themselves demonstrate, the prospects for biotechnology of even the relatively simply synthetic metabolon are highly promising in their own right. This work was supported by funding from the Max-Planck Society (Y.Z. and A.R.F.) and A.R.F and Y.J.Z would like to thank the European Union’s Horizon 2020 research and innovation programme, project PlantaSYST (SGA-CSA No. 664621 and No. 739582 under FPA No. 664620) for supporting their research.

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