Spatial organization of enzymes for metabolic engineering
Introduction
Metabolic engineering holds promise as an alternative to synthetic chemistry for cheaply and renewably producing molecules of value. Although construction of novel pathways out of enzymes from different organisms proves to be a powerful strategy for synthesizing a variety of compounds (reviewed in Keasling, 2010), achieving commercially viable productivity remains challenging. Some of these challenges are attributable to low enzymatic activities and flux imbalances, while others often arise from unintended and difficult-to-characterize interactions between synthetic pathways and the cellular environment of host organisms. For example, pathway metabolites may be toxic to the host organism, get diverted by endogenous reactions, or be lost via secretion (Fig. 1A). An emerging strategy to combat these issues is to organize enzymes of a synthetic pathway into multi-enzyme complexes. Co-localizing pathway enzymes into complexes with optimal enzyme stoichiometries increases the local concentrations of pathway metabolites and enzymes, potentially limiting the accumulation of pathway intermediates and decreasing the probability of unintended interactions with other cellular components (Fig. 1B).
Multi-enzyme complexes are frequently seen in nature. Enzymes catalyzing the last two steps of tryptophan synthesis form a complex where the intermediate indole is channeled from one active site to the other (Miles, 2001). Larger enzyme complexes are prevalent in eukaryotes (An et al., 2008, Narayanaswamy et al., 2009, Noree et al., 2010). An especially interesting case is the purinosome, which assembles dynamically depending on the cellular purine level (An et al., 2008). Bacteria encapsulate vital pathways such as carbon fixation in polyhedral nanostructures called microcompartments (Shively and English, 1991, Yeates et al., 2010), and eukaryotes have multiple membrane-bound organelles, which insulate various biochemical processes. While enzyme co-localization occurs in all of these examples, naturally occurring enzyme complexes appear to employ a variety of mechanisms by which they confer a selective advantage. For instance, bacterial microcompartments in Salmonella enterica trap a toxic aldehyde intermediate and limit its buildup in the cell (Sampson and Bobik, 2008). Evolutionarily related microcompartments in cyanobacteria concentrate CO2 around carbon fixation enzymes to increase reaction rates (Dou et al., 2008, Marcus et al., 1992). Mechanisms of other natural enzyme complexes were reviewed recently (Conrado et al., 2008). Better understanding of the mechanisms behind enzyme complexes will help inform and inspire more effective engineering of multi-enzyme complexes.
As a first attempt to organize enzymes into complexes, fusions of metabolic enzymes catalyzing successive reactions were shown to enhance pathway flux in certain instances (reviewed in Conrado et al., 2008). However, two notable disadvantages are inherent to the enzyme fusion strategy: it is not readily amenable to pathways containing more than two enzymes, and it cannot be easily used to balance enzyme stoichiometry. We therefore focus our review on two emerging alternatives. The first is synthetic scaffolding, a flexible method to co-localize two or more pathway enzymes and balance their stoichiometry at the complex. Although still under active characterization, we propose here one possible mechanism through which scaffolded enzymes could reduce intermediate buildup within the cell. In the second section, we discuss the potential for the use of protein shells to enable more advanced spatial organization of metabolic enzymes. Shell proteins create a physical barrier to prevent intermediates from escaping, while pores allow substrate(s) to enter and product(s) to exit. We conclude with a discussion of the advantages and associated challenges inherent to both of these technologies.
Section snippets
Modular scaffold strategy for improving metabolic flux
Recently the utilization of synthetic scaffold proteins to organize enzymes into complexes was shown to considerably increase titers of the mevalonate and glucaric acid biosynthetic pathways (Dueber et al., 2009, Moon et al., 2010). In both cases, a scaffold protein carrying multiple protein–protein interaction domains was used to co-localize sequential pathway enzymes that had been tagged with peptide ligands specific for the domains on the scaffold (Fig. 2A). By varying the number of domains
Physical compartments for pathway sequestration
Regardless of the mechanisms at work in scaffolded systems, a more direct approach for limiting cross-talk between engineered pathways and the cellular milieu is to physically encapsulate pathway enzymes into distinct compartments. As discussed previously, compartmentalization is observed at the molecular level in substrate-channeling enzymes all the way up to organelles in eukaryotic cells. Compartmentalized pathways benefit from physical barriers that prevent metabolite exchange and protect
Conclusions
In this review, we've discussed two emerging strategies for spatially organizing metabolic pathways into multi-enzyme complexes: enzyme scaffolding and protein shells. Both have their potential advantages and associated challenges (Table 1). The modular nature of protein interaction domains allows relatively easy construction of synthetic scaffolds. Scaffolding has been used to improve product titer in multiple engineered pathways, and in each case, scaffold architecture proved to be a critical
Update
While this article was under review, a new report (Delebecque et al., 2011) from Pamela Silver's laboratory demonstrated that RNA-based scaffold can be utilized in vivo to co-localize hydrogenases and ferrodoxins, which increases H2 production by ∼48-fold compared to non-scaffolded proteins. In this design, sequence-symmetric RNA building blocks were constructed to allow polymerization into one- or two-dimensional scaffolds isothermally. A PP7 and a MS2 aptamer domain were included in each RNA
Acknowledgments
The authors would like to thank Stanley Lei Qi, Weston Whitaker, Michael Lee, Katelyn Connell and the rest of the Dueber laboratory for discussions in preparing this review. This work is supported by Energy Biosciences Institute to Hanson Lee, by Graduate Research Fellowship from National Science Foundation to William DeLoache, and by National Science Foundation to John Dueber (Grant # CBET-0756801).
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