engineering of metabolic pathways using synthetic enzyme complexes1… · engineering of metabolic...

Update on Engineering Metabolic Pathways

Engineering of Metabolic Pathways Using SyntheticEnzyme Complexes1[OPEN]

Nicholas Smirnoff2,3

Biosciences, College of Life and Environmental Sciences, University of Exeter, Geoffrey Pope Building, StockerRoad, Exeter EX4 4QD, United Kingdom

ORCID ID: 0000-0001-5630-5602 (N.S.).

Plants provide a source of enzymes for metabolicengineering to produce valuable or useful products inmicro-organisms; furthermore, plants can be engi-neered (Andre et al., 2016; Vickery et al., 2016; Moseset al., 2017). Production of high-value compounds (e.g.pharmaceuticals) and nutraceuticals (e.g. omega-3 fattyacids, carotenoids, tocochromanols, ascorbate, and an-thocyanins) involves either the introduction of inno-vative pathways into a convenient host species oroptimization of endogenous pathways. Other manip-ulations include engineering protective, secondary-compound production for pest and pathogenresistance and osmolytes for stress resistance. Manip-ulation of central metabolic pathways such as photo-synthesis (e.g. Calvin-Benson cycle, alternative carbonsinks, introduction of CO2 concentrating mechanisms,photorespiratory bypasses, and xanthophyll cycle) orstarch and lipid synthesis has much potential to con-tribute to yield improvement. The use of plants asmetabolic engineering vehicles to produce valuablecompounds, as opposed to transferring plant pathwaysto microbes, will depend on feasibility and economicfactors. Specialized cells and tissues (glandular tri-chomes, resin ducts and lactifers, or oilseeds) adaptedto synthesize and store toxic and hydrophobic com-pounds involved in defense may make production ofcertain classes of compounds (e.g. isoprenoids and al-kaloids) more advantageous in plants (Huchelmannet al., 2017). On the other hand, plants present bottle-necks in terms of the number of genes that can beconveniently manipulated and a long time frame foroptimizing pathway engineering (Sweetlove et al.,2017). As an alternative to stable transformation, themethod of transient expression (for example in Nicoti-ana) provides a rapid route to optimizing engineering

and could act as a production platform (Reed andOsbourn, 2018). Also, it has become apparent that cam-bial (stem) cells are easily cultured and produce highyields of secondary compounds, such as taxol from yew(Taxus cuspidata; Lee et al., 2010). This finding could leadto a resurgence in the use of plant cell cultures. Recentdevelopments in metabolic engineering and the applica-tion of a synthetic biology approach have been summa-rized (Stewart et al., 2018). Key tools and requirements formetabolic engineering in plants are a set of promoters forvarious functions. For example, they drive purposes suchas expression in specific cell types; the ability to introducemultiple enzymes that are expressed at the appropriate

1This work was supported by the Biotechnology and BiologicalSciences Research Council (BB/M011429/1).

2Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is: .Nicholas Smirnoff ([email protected]).

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level; and ensuring that the supply of reductant and co-factors is not limiting. Furthermore, they drive the tar-geting of the pathway to specific subcellular locations/organelles; an example of the importance of location isillustrated by the production of dhurrin in transgenic to-bacco (Nicotiana benthamiana). Dhurrin is a cyanogenicglycoside produced by sorghum, and the enzymes arenormally anchored to the endoplasmic reticulum (ER).Targeting the enzymes to the thylakoid membrane in acomplex enables ferredoxin to be used as an alternativereductant and improves performance of the pathway(Gnanasekaran et al., 2016; Henriques de Jesus et al.,2017). This example also serves as an introduction to thepotential of synthetic enzyme complexes to assist meta-bolic engineering.

ENZYME COMPLEXES: OCCURRENCEAND SIGNIFICANCE

Metabolons and Substrate Channeling

The possibility that enzymes are not randomly dis-tributed but are associated into potentially dynamiccomplexes consisting of enzymes in a metabolic path-way (metabolons) has a long history. The term“metabolon” was introduced by Srere (1985) to denotea “supramolecular complex of sequential metabolicenzymes and cellular structural elements” (Srere, 1987).He proposed that metabolons would enable channelingof pathway intermediates between enzymes. (The nextparagraph defines “channeling.”) The original defini-tion included ribosomes and the DNA replicationcomplex. However, more recent usage excludes thesehighly organized structures, and there is a tendency forenzyme complexes to be termed metabolons in the ab-sence of evidence for channeling or other functionalattributes. There are immense technical challenges indetecting potentially loose and dynamic enzyme in-teractions (for example, by pulldowns, yeast two-hybrid, and in vivo using fluorescent proteins) andassessing their in vivo functionality. In plants, there areexamples of enzyme associations detected by these vari-ous methods, and these examples have been reviewed(Laursen et al., 2015; Sweetlove and Fernie, 2018).Examples include flavonols/isoflavonols (Achnineet al., 2004; Crosby et al., 2011; Lee et al., 2012b;Dastmalchi et al., 2016; Diharce et al., 2016), polyamines(Panicot et al., 2002), sporopollenin (Lallemand et al.,2013; Qin et al., 2016), alkanes (Bernard et al., 2012),indole acetic acid (Müller andWeiler, 2000; Kriechbaumeret al., 2016), carotenoids (Nisar et al., 2015), and dhurrin(Møller and Conn, 1980; Laursen et al., 2016). In centralmetabolism, the best studied examples are glycolysisand the tricarboxylic acid (TCA) cycle (Giegé et al.,2003; Graham et al., 2007; Zhang et al., 2017). Theglycolytic enzymes are associated with the mito-chondrial membrane and show dynamic behavior;complex formation increases with high respiratorydemand (Graham et al., 2007). Similarly, in mammalian

cells, the purinosome, an assembly of enzymes in-volved in purine biosynthesis, assembles when thereis high demand for product (Pedley and Benkovic,2017; Baresova et al., 2018). In only a few cases hasthe functional significance of these enzyme com-plexes been established. In this review, “metabolon”will be used in cases where channeling is demonstrated.The phrase “enzyme complex” will be used where twoormore enzymes in ametabolic pathway are physicallyassociated.The functional significance of metabolons has been

debated, but the principles are becoming clearer, in partbecause of additional insights derived from syntheticenzyme complexes. To be effective, an enzyme complexmust enable channeling (Castellana et al., 2014;Sweetlove and Fernie, 2018). Channeling is the move-ment of an intermediate between active sites of suc-cessive enzymes with much decreased escape into thebulk cytoplasmic solution (Fig. 1A). Channeling couldinvolve direct tunneling of intermediates between ac-tive sites and/or electrostatic guidance (Elcock et al.,1997). Channeling occurs in highly organized com-plexes such as Trp synthase (Dunn et al., 2008), malatedehydrogenase/citrate synthase (Bulutoglu et al., 2016),and bacterial Pro oxidation. In the latter example, Pro isconverted to Glu through Pro dehydrogenase (PRODH),which produces 1-pyrroline-5-carboxylate (P5C). P5Cspontaneously hydrates to form L-Glu-semialdehyde(GSA), which is then oxidized by P5C dehydrogenase(P5CDH) to form Glu. In many bacteria, PRODH andP5CDH comprise a bifunctional enzyme, and kineticstudies indicate direct channeling of P5C/GSA be-tween the active sites. However, in other cases, suchas Thermus thermophilus, the enzymes PRODH andP5CDH are on distinct proteins. Kinetic studies, sub-strate trapping, and surface plasmon resonance analysisof protein-protein interaction showed an orientation-dependent association between the enzymes and sub-strate channeling (Sanyal et al., 2015). Therefore, weakbut specific interactions between these enzymes haveevolved to enable channeling. This concept is an ex-ample of the Rosetta Stone hypothesis (Marcotte et al.,1999). The hypothesis suggests that if two separateproteins have homologs in another genome that arelocated on a single polypeptide, then the separate pro-teins are likely to interact with each other. A largeproportion of the identified fusion proteins are en-zymes (Enright et al., 1999; Marcotte et al., 1999). Froman engineering point of view, synthetic fusion enzymesmay or may not be effective. This is most likely becausethe enzymes have not coevolved complementarystructures that enable effective channeling. Or, fusioncould interfere with correct folding.Considering less-organized metabolons, the essential

and comprehensive analysis by Sweetlove and Fernie(2018) identifies the key point that the close associationof sequential enzymes (in the absence of specific inter-actions) cannot be effective at channeling because sub-strate diffusion rate is much faster than enzymecatalysis, so the intermediate can escape (Fig. 1B). Only

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a few enzymes operate at diffusion limited rates (kcat/KM ; 109 M21 s21), including triose phosphate isom-erase, carbonic anhydrase, superoxide dismutase, cat-alase, and acetylcholine esterase. Therefore, simplypairing noncoevolved enzymes will not in itself be ef-fective; and, even if it could be effective, it would in-crease initial rate but not steady-state rate (Sweetloveand Fernie, 2018). As noted previously, direct chan-neling requires coevolved enzymes. This is unlikely tobe the case when heterologous enzymes are used forengineering. Channeling requires that intermediatesare not in equilibrium with the bulk solvent, and thissituation could be achieved by a large cluster of en-zymes, not necessarily arranged in a specificmanner, sothat “probabilistic” channeling occurs (Castellana et al.,2014; Sweetlove and Fernie, 2018). Because of localizedhigh enzyme concentration, the probability that a sub-strate binds to an active site before it leaves the cluster is

increased, and an increase in flux is also predicted(Fig. 1D). It is suggested that high enzyme concentra-tion can influence the thermodynamic feasibility ofa pathway and its direction (Angeles-Martinez andTheodoropoulos, 2015). Evidence for effective chan-neling in enzyme complexes in vivo is scarce, althoughthe wide range of central and secondary metabolismpathways with interacting enzymes suggests that it islikely. Demonstration of channeling is challenging, andthe various approaches have been reviewed (Zhanget al., 2017; Sweetlove and Fernie, 2018). Isotopic dilu-tion is a useful technique: If channeling is occurring, anadded unlabeled pathway intermediate will not equil-ibrate with the labeled intermediate derived from a la-beled precursor. Channeling has been demonstratedin vitro for the isolated ER-bound dhurrin biosynthesismetabolon (Møller and Conn, 1980) and validatedin vivo when a biosynthetic complex is introduced into

Figure 1. Enzyme Assemblies and Their Influence on Substrate Channeling. A, Two closely associated (“co-evolved”) enzymesenabling direct channeling of the intermediate between active sites. The active sites could be located on separate proteins or on asingle bifunctional protein. B, Tagged enzymes attached to a synthetic scaffold protein, nucleic acid scaffold, or lipid scaffold(Table 1). There is little channeling because the diffusion rate of the intermediate is much faster than enzyme activity. C, The sameassembly as (B) but showing howmultimeric scaffolded enzymes can form larger aggregates of high enzyme concentration. D, Alarge assembly of enzymes providing high local enzyme concentration enables probabilistic channeling. Here, the high enzymeconcentration increases the chance that the intermediate binds to an enzyme active site before diffusing away. E, An encapsulatedenzyme assembly is identical to (D), but a self-assembling protein coat provides an additional diffusion barrier with pores at thevertices to allow (selective) exchange of substrates and products. The enzymes could be tethered to the coat proteins. Examplesare bacterial microcompartments (BMCs) specialized for use of carbon sources in pathways involving reactive intermediates(metabolosomes) and for CO2 fixation with encapsulated carbonic anhydrase and Rubisco (carboxysomes). Eukaryotes lackBMCs but have pyrenoids (an aggregation of Rubisco surrounded by a loose starch sheath found in algae and hornworts) andperoxisomes. Peroxisomes house enzymes that produce toxic products and could be considered analogous to metabolosomes.They are bounded by a membrane that is relatively permeable to small molecules, and they exhibit channeling.

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Enzyme Complexes for Metabolic Engineering



chloroplasts (Henriques de Jesus et al., 2017). In plants,isotope dilution experiments have shown channeling inthe glycolytic pathway bound to the surface of mito-chondria (Giegé et al., 2003; Graham et al., 2007). Acomprehensive study of the plant TCA cycle showed158 binary protein-protein interactions that were con-firmed by the channeling of citrate and pyruvate usingisotope dilution experiments (Zhang et al., 2017). Thiskey study provides strong evidence for physical asso-ciation between enzymes and the occurrence of chan-neling. It is also of significance because TCA cycleenzymes were the first enzymes involved in the initialcharacterization of metabolons (Srere, 1987; Vélot et al.,1997; Bulutoglu et al., 2016). The other two conse-quences of channeling include decreasing the loss ofpotentially reactive and toxic intermediates into thebulk solution and influencing flux at branchpoints(Zhang et al., 2017; Sweetlove and Fernie, 2018). It isnotable that a large proportion of metabolons aremembrane-associated. As well as the prior examples,glycolytic enzymes associate with the cytoskeleton inyeast and Arabidopsis (Araiza-Olivera et al., 2013;Garagounis et al., 2017). It is possible that channeling isaided by the physical and chemical properties in thecytoplasm in proximity to surfaces such as membranesor cytoskeletal elements (Theillet et al., 2014). It is pro-posed that bacterial cytoplasm is divided into a super-crowded “cytogel” extending 20 to 70 nm from theplasmamembrane andmore dilute cytosol (Spitzer andPoolman, 2013). While not likely to influence the dif-fusion rate of small molecules significantly, the forma-tion of protein complexes may be favored near surfaces,suggesting that anchoring synthetic complexes to amembrane could be an advantageous strategy. Protein-protein interactions are driven by several mechanismsnot covered here (Williamson, 2012). A fresh suggestionis that enzymes show chemotactic movement alongtheir substrate gradient, an effort that could drive theircolocalization (Wu et al., 2015; Illien et al., 2017; Agudo-Canalejo et al., 2018; Zhao et al., 2018). These experi-ments use fluorophore-tagged enzymes to followmovement in microfluidic devices. However, the in-terpretation of the fluorescence correlation spectros-copy, on which the conclusions are based, has beencriticized (Günther et al., 2018).

SYNTHETIC ENZYME COMPLEXES

Construction and Functioning

The existence of enzyme complexes and the possi-bility that they are important in influencing metabolicpathways have provided the drive to explore the use ofsynthetic enzyme complexes in metabolic engineering.There are essentially two approaches: anchoring enzymeson scaffold molecules of various kinds or encapsulatingenzymes in protein-coated microcompartments based onbacterial microcompartments and viral capsids. Manyreviews have discussed and advocated synthetic enzyme

complexes and possibly outnumber actual examples of itsapplication. The reader is referred to these reviews formoreinformation: (Conrado et al., 2008; Boyle and Silver, 2012;Lee et al., 2012a; Singleton et al., 2014; Chessher et al., 2015;Pröschel et al., 2015; Siu et al., 2015; Polka et al., 2016;Plegaria and Kerfeld, 2018; Qiu et al., 2018). A selection ofexamples of synthetic enzyme complexes is reviewed herein relation to the methods used and outcome.

Protein and Protein-Lipid Scaffolds

The initial report of the construction of a synthetic en-zyme complex in metabolic engineering was the assemblyof three enzymes required for synthesizingmevalonic acid(acetoacetyl-CoA thiolase, hydroxy-methylglutaryl-CoAsynthase, and hydroxymethylglutaryl-CoA reductase)on a synthetic protein scaffold (Dueber et al., 2009;Table 1). The enzymes were linked to scaffolds usinghigh-affinity mammalian protein-protein interactiondomains (SH3, GBD, and PDZ) assembled in variouscombinations in a synthetic scaffold protein with cog-nate binding domains. Each enzyme was fused to SH3,GBD, and PDZ ligands. Expression in E. coli resulted inassembly of the scaffolded proteins and an increase inmevalonate accumulation when scaffolded. Followingthis success, further scaffolding experiments have beenreported (Table 1). These display an increasing diver-sity and ingenuity of methods used to scaffold en-zymes. Other high-affinity protein-protein interactiondomains have been harnessed (e.g. dockerin-cohesin,Leu zippers, synthetic coiled-coil proteins). Generally,two to three enzymes have been assembled on the scaf-folds. In cases where enzymes aremultimeric, attachmentof one enzyme to several scaffolds could allow cross-linking to form larger structures (Fig. 1C). Larger con-glomerations have been achieved by scaffolding to verylarge proteins (Price et al., 2016) or to proteins liable toform inclusion bodies (Han et al., 2017). A very promisingapproach is the production of a network of cytoskeleton-like synthetic protein filaments to which enzymes arescaffolded (Lee et al., 2018a, 2018b). Some naturally oc-curring metabolons are membrane bound as noted pre-viously. In this context, enzyme complexes anchored inlipid droplets have been produced by using scaffoldproteins that associate with lipid-binding proteins, suchas oleosin (which is the coat protein for lipid droplets inoilseeds) and certain virus coat proteins (Myhrvold et al.,2016; Lin et al., 2017).

Nucleic Acid Scaffolds

DNA and RNA have been explored as enzyme scaf-folds (Delebecque et al., 2011; Conrado et al., 2012;Rahmana et al., 2014; Zhu et al., 2016). Enzymesare tagged with DNA-binding proteins (e.g. zinc fin-ger proteins and transcription activator-like effectors[TALEs]) and DNA scaffolds are synthesized withspecific binding site arrangements and spacing. These


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Table 1. Examples of Metabolic Pathway Engineering Using Scaffolded Enzyme Complexes

Product Host Organism Approach Outcome Assay Conditions Reference

Ethyl acetate S. cerevisiae Dockerin tags + cohesion/oleosin scaffold.

Enzymes colocalize tolipid droplet membranes(FRET). 1.8-fold increasein product in cell lysateassay. Channeling nottested.

Initial activityin vitro?

Lin et al.,2017

Ethanol (pyruvatedecarboxylase andalcoholdehydrogenase)

E. coli Filamentous scaffoldproteins formed frombacterialmicrocompartment coatprotein (PduA) fused tosynthetic self-assembling coiled-coilproteins. Enzymestagged with coiled-coilproteins. Also attachedto inner membrane.

Network of cytoplasmicfilaments visualized byTEM. Proteincolocalizationconfirmed by taggedfluorescent proteins andmicroscopy.

Ethanol yieldincreased 2-foldby 20 h, butinitial rate ofincrease samewith or withoutscaffold.

Lee et al.,2018b

Dhurrin N. benthamiana Fusion of three enzymes toTatB and TatC (thylakoidmembrane proteins).Transient expression,chloroplast targeted.

Dhurrin increases 5-fold.Channeling suggested bydecreased side products.Confirmed thylakoidlocation, but notenzyme proximity.

Productsmeasured 5 dpost-Agrobacteriuminfiltration

Henriques deJesus et al.,2017

Butan-1-ol E. coli Enzymes attached toClostridiumexoglucanase cellulose-binding domain-induced inclusionbodies via Leu zippertags.

2-fold increase in butanolformation. Enzymesshown to be present ininclusion bodies.

Stable transgeniclines.

Han et al.,2017

Indole-3-acetic acid E. coli Enzymes (or split GFP) fusedto DNA-binding TALEproteins assembled on aplasmid with variousdistances between DNA-binding sites.

GFP fluorescence indicatesassembly onDNA scaffold.IAA production increasedup to 8-fold in scaffold andbinding site spacing-dependent manner.

Overnight IPTGinduction.

Zhu et al.,2016

Methanol to Fru-6-phosphate viaformaldehyde

E. coli A multisubunit malatedehydrogenase fused toSH3 plus a two-enzymefusion protein with aSH3 ligand.

Assembly into a complexconfirmed by TEM anddynamic light scattering.A 97-fold increase in F6Pin vitro and a 2.4-foldincrease in methanolconsumption in vivo.

Faster initialMeOHconsumptionrate in vivo upto 5 h postaddition.

Price et al.,2016

Indigo E. coli Bacteriophage Ø P9 andP12 proteins assembledinto protein-lipidvesicles. Enzymes orfluorescent proteinsfused to N terminus ofP9.

Colocalization offluorescent proteins andfractionation of cellextracts show assembly oflipid-protein droplets(; 20 nm diameter).Indigo productionincreased 2.5-fold in thecomplex (P12-dependent).

Enzymeexpressioninduced“overnight.”

Myhrvoldet al., 2016

2,3-butanediol fromphosphoenolpyruvate(PK) anda-acetolactatesynthase

S. cerevisiae Enzymes tagged withcohesin and dockerin toassemble via cohesin-dockerin interaction.

Complex formationconfirmed byimmunoprecipitation. A1.3-fold increase inbutanediol. Evidence forchanneling: pyruvateproduced by PK is lessavailable for ethanolformation.

Faster product yield(g/L culture) upto 24 h afterinitiating aculture bydilution.

Kim et al.,2016

(Table continues on following page.)





have been expressed in E. coli with the scaffolds onplasmids. The plasmid copy number determines theamount of scaffold. RNA scaffolds have also beentested on the basis that RNA can fold into potentiallyuseful geometries to provide binding sites (aptamers)for tagged proteins (Delebecque et al., 2011). Theseapproaches are appropriate for bacteria but would bemore problematic in plants due to the need for acces-sible DNA and potential RNA instability.

Why Is Scaffolding Successful?

The examples of scaffolding in Table 1 show an in-crease in product because of enzyme scaffolding,

although the benefit is sometimes modest. E. coli andSaccharomyces cerevisiae are the predominant hosts fortesting scaffolds, with the only plant example being thetargeting of dhurrin biosynthesis enzymes to the thy-lakoid membrane by transient expression in N. ben-thamiana (Henriques de Jesus et al., 2017). Why doesscaffolding work? As discussed earlier, dispersed scaf-folded enzyme units would be unlikely to exhibitchanneling and, if they did, faster initial reactionrates—but not increased steady-state rates—would beexpected (Sweetlove and Fernie, 2018). Considering theexamples shown in Table 1, it is generally not possibleto determine if the system is at steady state becausemany of the measurements are made hours or daysafter inducing scaffolding. Therefore, it is tempting to

Table 1. (Continued from previous page.)

Product Host Organism Approach Outcome Assay Conditions Reference

Branchpoint betweencarbamoyl phosphate(carbamoyl phosphatesynthetase) use for Arg(Orncarbamoyltransferase)and carbamoyl-Aspsynthesis (Aspcarbamoyltransferase)

E. coli Carbamoyl phosphatesynthetase and Aspcarbamoyltransferasefusion protein expressedat high level.

Increase in phase-brightcytoplasmic structurestypical of protein-denseclusters. Evidence fordiversion of carbamoylphosphate away fromthe competing Argsynthesis pathwaydependent on clustering.

Castellanaet al., 2014

Alkanes (acyl-ACP-reductase and fattyaldehydedecarbonylase)

E. coli Fusion protein or enzymestagged with zinc-fingerDNA binding proteinsassembled on a plasmidDNA scaffold.

Enzyme scaffold assemblynot assessed. Fusionprotein increasesalkanes 4.8-fold andDNA scaffold up to 8.8-fold (dependent onenzyme stoichiometry).

24 h post-IPTGinduction.

Rahmanaet al., 2014

Resveratrol S. cerevisiae GBD, SH3, and PDZcombined in proteinscaffolds and enzymestagged with theirligands.

Resveratrol increased byup to 5-fold.

Measured 36 h(5-fold) and96 h (2-fold)after induction.

Wang andYu, 2012

Resveratrol, 1, 2-propanediol, andmevalonate

E. coli Enzymes tagged with zinc-finger DNA bindingproteins assembled on aplasmid DNA scaffold.Random scaffoldcontrol.

Assembly shown in vitro(split YFP) and in vivo.Up to 5-fold increase inproduct depending onenzyme proximity/pathway.

24 h postinduction(resveratrol andpropane diol).50 h postinduction(mevalonate).

Conradoet al., 2012

Hydrogen production(ferredoxin andhydrogenase)

E. coli RNA scaffolds withaptamers plus proteinstagged with aptamer-binding proteins.

Assembly indicated by splitGFP. Mutant aptamersite controls. Up to 48-fold increase in product(dependent on scaffoldgeometry).

16 h afterinduction.

Delebecqueet al., 2011

Glucaric acid E. coli GBD, SH3, and PDZcombined in proteinscaffolds and enzymestagged with theirligands.

Up to 5-fold increase (g/L).Enzyme stoichiometryeffects observed.

48 h postinduction.

Moon et al.,2010

Mevalonate E. coli GBD, SH3, and PDZcombined in proteinscaffolds and enzymestagged with theirligands.

Up to 77-fold increase (g/L). Enzymestoichiometry effectsobserved..

Up to 3 dpostinduction.

Dueber et al.,2009


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propose that most of the manipulations inadvertentlyinduce the formation of sufficiently large complexesthat increase local enzyme concentration, enablingprobabilistic channeling and increased rate at steadystate (Fig. 1C). This proposal is essentially how pyre-noids work (as will be shown later in this study). Theexample of Castellana et al. (2014); Table 1) is also im-portant because it shows that high expression of a bi-functional enzyme to form a complex big enough tovisualize enables channeling and diverts intermediatesat a branchpoint. Controlling flux at a branchpoint isalso seen in the example of butanediol formation (Kimet al., 2016). Channeling was demonstrated whendhurrin biosynthesis enzymes were anchored to thethylakoid membrane. The pathway intermediates arereactive; and when the enzymes are not anchored, LC-MS analysis detects many compounds derived fromthem, and anchoring greatly reduces their accumu-lation (Henriques de Jesus et al., 2017). This point isimportant for engineering pathways that involve re-active intermediates, where the benefit could beprotection against toxicity, a value could be equal tothe benefit of greater yield. It is evident that normalmetabolism causes “metabolite damage” (the pro-duction of unintended compounds), and it is sug-gested that metabolite repair enzymes could be partof the metabolic engineering tool kit (Sun et al., 2017).Channeling between critical enzymes would alsocontribute to damage-limitation. In the scaffold ex-amples, it is likely that channeling is enabled by ag-gregation of the individual scaffolds into largerclusters. In some of the cases, this prospect has beendemonstrated (Table 1). IAA and alkane biosynthesisenzymes have been detected in complexes in plants(Müller and Weiler, 2000; Bernard et al., 2012;Kriechbaumer et al., 2016) but without specific evi-dence for channeling, so it is noteworthy that scaf-folding increases production of these compounds inmicro-organisms (Table 1).

Most of the examples shown in Table 1 demon-strate that assembly has occurred by using tech-niques such as coimmunoprecipitation (co-IP), fluorescentproteins (bimolecular fluorescence complementation[BiFC] and Förster resonance energy transfer[FRET]) and transmission electron microscopy (forlarger assemblies and encapsulated enzymes). Pos-sibly, super-resolution microscopy and transmissionelectron cryomicroscopy (cryoEM) will be useful inproviding more detailed information on the size andstructure of complexes. Another factor, not explicitlytackled in any of the studies, is the possibility thatthe scaffolding has a favorable influence on the totalamount of enzyme (perhaps by decreased rate ofproteolysis) or influences specific activity and ki-netic properties. Again, the characterization is rarelysufficiently detailed to assess these possibilities. Fi-nally, it is reasonable to suppose that unsuccessfulattempts at scaffolding have not been published,making it impossible to assess the probability ofsuccess.

SYNTHETIC MICROCOMPARTMENTS

Nanoreactors

Many bacteria produce microcompartments (BMCs),protein-coated nanostructures that encapsulate en-zymes (Fig. 1E). Their structure and functions havebeen well-reviewed (Kerfeld et al., 2018). They can bedivided into two categories by function. Metab-olosomes contain catabolic enzymes for the use of car-bon sources through pathways that produce reactiveintermediates. Carboxysomes function as part of theCO2-concentrating mechanism (CCM) of photosyn-thetic bacteria, and they contain ribulose bisphosphatecarboxylase-oxygenase (Rubisco) and carbonic anhydrase.They occur in two distinct forms: a-carboxysomes in pro-teobacteria, and some cyanobacteria and b-carboxysomesin cyanobacteria. BMCs function by concentrating en-zymes (Rubisco and carbonic anhydrase) in a restrictedspace that enables channeling (as described previouslyfor other enzyme complexes). The shell presumablyevolved because there is an additional benefit to a dif-fusional barrier. Our understanding of how BMCs as-semble, encapsulate the correct enzymes, and allowsubstrate and product exchange via pores has advancedto the point where synthetic BMCs that self-assemblehave been expressed in bacterial cells. Assembly ofenzymes for encapsulation is assisted by incorporationof encapsulation peptides (EPs; Gonzalez-Esquer et al.,2016; Plegaria and Kerfeld, 2018). In plant research,there has been a focus on the possibility of introducingcarboxysomes into chloroplasts to mimic the cyano-bacterial CCM. As in cyanobacteria, this process wouldalso need transporters to concentrate bicarbonate intothe chloroplast stroma. Bicarbonate would enter thecarboxysomes, where CO2 production is catalyzed byencapsulated carbonic anhydrase. The high local con-centration of Rubisco drives rapid CO2 fixation andoutcompetes the oxygenase reaction (Rae et al., 2013).The beginnings of this goal have been achieved bysuccessful assembly of b-carboxysome shells in chlo-roplasts by transient expression of five shell proteins inNicotiana (Lin et al., 2014). YFP, tagged with a smalltargeting peptide from the carboxysome-organizingprotein, CcmN, was incorporated into the shells. In arecent breakthrough, a minimal functional carbox-ysome was expressed in tobacco chloroplasts (Longet al., 2018). This result was achieved by introducingtwo a-carboxysome coat proteins and the large andsmall subunits from the cyanobacterium Cyanobium.Chloroplasts were transformed to enable knockout ofthe endogenous Rubisco large subunit. The resultingplants were able to grow, carrying out CO2 assimilationwith the encapsulated Cyanobium Rubisco. The resultsshow that this minimal carboxysome allows encapsu-lated Rubisco to function; therefore, the pores inthe protein coat enable exchange of substrates andproducts.

BMCs are related to viral capsid proteins. Capsidproteins can self-assemble in heterologous hosts and





have the potential to be used to encapsulate enzymes.An interesting recent example is encapsulation of anindigo biosynthesis pathway from Trp in a virus capsidprotein. The enzymes were anchored to the capsidproteins using SpyTag/SpyCatcher protein fusions(Giessen and Silver, 2016). This system is based on theCnaB2 domain from the fibronectin-binding proteinFbaB from Streptococcus pyogenes, and the processworks by a spontaneous reaction between a Lys residueon SpyCatcher and an Asp on SpyTag to form an iso-peptide bond (Reddington and Howarth, 2015). Tag-ging the enzymes with SpyCatcher and bacteriophageMS2 capsid protein with SpyTag resulted in assemblyof particles in E. coli that increased indigo production by60% compared to controls (Giessen and Silver, 2016).Pores of BMCs and capsids can be engineered to controlsubstrate uptake specificity (Glasgow et al., 2015). Iso-lated capsids showed that the enzymes were markedlymore stable in vitro because of the covalent linkages.

HARNESSING AND MODIFYING OTHERNATURALLY OCCURRING STRUCTURES

Synthetic Organelles

Pyrenoids and peroxisomes could be consideredlarge enzyme complexes that enable probabilisticchanneling. Peroxisomes contain oxidases that producehydrogen peroxide along with catalase, which decom-poses the peroxide to water. In leaves, photorespirationgenerates a large flux of glycolate, which is oxidized inperoxisomes to produce glyoxylate (a reactive alde-hyde) and hydrogen peroxide. By cooperation betweenperoxisomes and mitochondria, photorespiration pro-duces glycerate for recycling into the Calvin-Bensoncycle (Hagemann and Bauwe, 2016). Isolated spinachleaf peroxisomes produce glycerate at the same ratewith and without an intact membrane; and, in bothcases, the intermediates glyoxylate and hydroxypyr-uvate are not detected in the suspension medium(Heupel and Heldt, 1994). The results indicate that theleaf peroxisome is a protein complex that maintains itsintegrity without the membrane boundary and thatexhibits channeling. More recently, interaction betweenglycolate oxidase and catalase was shown by BiFC andco-IP (Zhang et al., 2016). The relative simplicity ofperoxisomes (lack of a genome and a single membranepermeable to small molecules) makes them a temptingbasis for production of a synthetic organelle housingengineered metabolic pathways. As noted previously,the evidence for channeling in leaf peroxisomes (even inthe absence of a membrane) provides a useful startingpoint. Various pathways have been engineered into per-oxisomes, for example, to produce polyhydroxyalkanoatesin Arabidopsis (Mittendorf et al., 1999; Kessel-Vigeliuset al., 2013). Yeast peroxisomes have been engineeredto efficiently produce alkanes and fatty alcohols fromacyl-CoAs, with evidence that the high enzyme con-centrations enabled channeling (Zhou et al., 2016).

These pathways use the acyl-CoA metabolizing ca-pacity of peroxisomes. Modification of the existingperoxisomal protein import system can increase its ef-ficiency for importing enzymes (DeLoache et al., 2016).However, the recent creation of a different protein im-port system that runs in parallel with the endogenoussystem provides a step toward synthetic peroxisomes(Cross et al., 2017). Deeper understanding of prolifera-tion mechanisms and the protein-protein interactionsthat hold the peroxisomal matrix together will also as-sist in reaching this goal.The pyrenoid could provide another starting point

for producing a protein aggregate with high enzymeconcentration that enables channeling. This structureconsists of an aggregate of Rubisco in the chloroplastsof algae and some liverworts, and it is required for theirCO2 concentrating mechanism (CCM). It traps CO2produced by carbonic anhydrase, allowing improvedRubisco activity (Meyer et al., 2017; Küken et al., 2018).The protein components of this structure have beenidentified. The protein EPYC1, present in high con-centration, interacts with Rubisco to form a scaffold(Mackinder et al., 2016, 2017). Additionally, the result-ing structure is liquid, rather than crystalline, and itundergoes a phase transition and fission during celldivision (Freeman Rosenzweig et al., 2017). Thesestructures suggest the possibility of making syntheticorganelle-like structures without walls or membranesfor metabolic engineering. The introduction of pyre-noids into plant chloroplasts is a potential route forimproving photosynthesis (Mackinder, 2018).


Smirnoff



CONCLUSION

It is evident that the amount of final product inengineered metabolic pathways can be increased byvarious ingenious scaffolding approaches. The mostlikely explanation is that the resulting enzyme aggre-gates are (often inadvertently) large enough to enableprobabilistic channeling due to increased local enzymeconcentration. Pyrenoids work in the same manner,while encapsulation (in BMC and capsid coat proteins)increases enzyme concentration and provides an addi-tional (potentially selective) diffusion barrier. Leafperoxisomes are robust protein complexes that can holdtogether without their membrane and exhibit channel-ing. Peroxisomes and pyrenoids could form the basisfor engineering multienzyme metabolic pathways thatbenefit from channeling. Ultimately, the widespreaduse of channeling in plant metabolic engineeringwill bedetermined by a balance between the extra time re-quired to introduce and optimize enzyme assembliescompared to the potentially modest benefit in productyield. Channeling could provide a critical advantage ifit enables production of compounds with highly reac-tive and toxic pathway intermediates or improves di-version of central metabolism intermediates into theengineered pathway.

ACKNOWLEDGMENTS

Thank you to the Biotechnology and Biological Sciences Research Councilfor funding.

Received October 12, 2018; accepted November 1, 2018; published November19, 2018.

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