Delft University of Technology
Dynamics in redox metabolism, from stoichiometry towards kinetics
Verhagen, Koen JA; van Gulik, Walter M.; Wahl, Sebastian Aljoscha DOI
10.1016/j.copbio.2020.01.002 Publication date
2020
Document Version Final published version Published in
Current Opinion in Biotechnology
Citation (APA)
Verhagen, K. JA., van Gulik, W. M., & Wahl, S. A. (2020). Dynamics in redox metabolism, from stoichiometry towards kinetics. Current Opinion in Biotechnology, 64, 116-123.
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Dynamics
in
redox
metabolism,
from
stoichiometry
towards
kinetics
Koen
JA
Verhagen,
Walter
M
van
Gulik
and
Sebastian
Aljoscha
Wahl
Redoxmetabolismplaysanessentialroleinthecentral
metabolicnetworkofalllivingcells,connecting,butatthesame
timeseparating,catabolicandanabolicpathways.Redox
metabolismisinherentlylinkedtotheexcretionofoverflow
metabolites.Overflowmetabolismallowsforhighersubstrate
uptakerates,potentiallyoutcompetingothermicroorganisms
forthesamesubstrate.Withindynamicallychanging
environments,overflowmetabolismcanactasstorage
mechanism,asisshowninmanyrecentlydescribed
processes.However,forcompleteunderstandingofthese
mechanisms,theintracellularstateofthemetabolismmustbe
elucidated.Inrecentyears,progresshasbeenmadeinthefield
ofmetabolomicstoimprovetheaccuracyandprecisionof
measurementsofintracellularandintercompartmental
metabolites.Thisarticlehighlightsseveraloftheserecent
advances,withfocusonredoxcofactormeasurements,both
fluorescenceandmassspectrometrybased.
Address
DepartmentofBiotechnology,DelftUniversityofTechnology,Vander Maasweg9,2629HZDelft,theNetherlands
Correspondingauthor:Wahl,SebastianAljoscha(s.a.wahl@tudelft.nl)
CurrentOpinioninBiotechnology2020,64:116–123
ThisreviewcomesfromathemedissueonAnalyticalbiotechnology EditedbyYinjieTangandLudmillaAristilde
https://doi.org/10.1016/j.copbio.2020.01.002
0958-1669/ã2020TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons. org/licenses/by/4.0/).
Introduction
The redox metabolism is commonly studied and interpreted under steady state conditions, where the transfer of electrons and metabolites inside the cell is balanced.Naturalenvironments,aswellasincompletely mixedbioreactorsaremostlynotatsteady-state,requiring flexibility of metabolism, including redox metabolism. Thetransfer ofelectrons betweendifferentreactionsof themetabolicnetwork isfacilitated byelectroncarriers suchas NADH,NADPH, FADH2,quinones and ferre-doxins. Each electron carrier fulfils different functions within the cell, with NADH being used primarily for catabolism, such as respiration and fermentation.
NADPH is the cofactor used for anabolism like amino acidand lipidsynthesis [1]. Thisseparationof electron carriers allows for additional functionality within the network, as ratios of the reduced and oxidized forms of the different cofactors can vary independently. For example,itiswellestablishedthatNADPH/NADPratios arehighercomparedtoNADH/NADtogeneratehigher thermodynamicdrivingforcesinthereductivedirection inanabolism.
Engineered as well as natural environments are frequently dynamic – with perturbation timescales in theorder of seconds to regime changes in theorder of hours. Dynamics can arise from fluctuations in the availability of carbon source(s), electron donor(s) resp. acceptor(s).Toobtainastablemetabolismtheredoxstate withinthecellhastobebalancedusingavailableelectron sinksandsources.
Themechanismsbehindoverflowmetabolism,suchasthe Crabtreeeffectinyeast,andtheirlinktoredoximbalances have been extensively studied under steady state conditions [2–4], where electrons are essentially channelled towards fermentation products and excreted from the cells. Next to the competitive advantage of a highergrowthrate,alsohighersubstrateuptakeratesare achieved,potentiallyoutcompetingothermicroorganisms forthesamesubstrate.Suchadvantagescanbecomeeven more relevant in dynamically changing environments wheresubstrateisonlyavailableforshortperiodsoftime. Sofaronlyafewdynamicsystemshavebeenstudiedwith respect to sudden changes of the redox state. Under dynamic conditions, the temporary storage of electrons can be an essential mechanism to obtain a competitive advantage by increasing metabolic flux [5]. Electron overflow has been observed for various prokaryotes andeukaryotesinresponseto substratepulses,resulting in excretion of compounds which cannot easily be furthermetabolizedorareeventoxic,suchasethanolor lactate[6].
While these phenomena can be explained from a ‘strategic’ point of view, it is not clear how such behaviours are regulated on a metabolic level. Here, accurate intracellular measurements are required that cancapture thefast dynamicsofredoxmetabolism.
Withthelow concentrations andhigh turnover rates of intracellular redox cofactors, the measurements are
challenging,especiallyfordynamicsystems[7].Further complexity arisesfromsubcellularcompartmentation of thesemetaboliteswithineukaryotes.Compartmentation allows the establishment of different environments withinacell,therebyprovidingawiderrangeof thermo-dynamicpossibilities[8].DifferentratiosofNADH/NAD in different compartments can allow for simultaneous oxidizing and reducing environments within the same organism. Unfortunately, limited quantitative data are available for compartment specific concentrations. In addition,metabolitechannellingmayovercome thermo-dynamicconstraintsbycreatinglocalhighconcentration environments near enzyme reaction centres, enabling thermodynamic pathway feasibility which may not be observed from themeasured overall cellularmetabolite concentrations [9,10].
Here, we will review recent studies providing insights intothedynamicsoftheredoxmetabolismuponchanges in the environment, discuss the relevance of the understanding of the redox metabolism for the design and optimization of microbial production strains and processes, and describe how technical challenges in measuringredoxstatesindifferentcellularcompartments canbeapproached.
Stoichiometry
and
dynamics
of
redox
coupling
–
a
link
between
overflow
and
storage
mechanisms
Recent works haveanalysed theimpact of dynamically fluctuating environments in thecontext of (very) large-scale microbial cultivations. Within large-scale reactors gradients insubstrate,oxygenandpHwilloccurdueto mass-transfer limitations (poor mixing) [11]. A microor-ganismtravelingthroughthesegradientswillexperience fluctuations in substrate and oxygen supply. Such fluctuations can be mimicked using scale-down approaches, such as two reactor STR/PFR systems [12]or microfluidiccultivations[13].
During anaerobic cultivation products such as acetate and ethanol are excreted. Thesecontain electrons that could not be transferred to an external acceptor and consequently ‘store’ a significant amount of electrons intheextracellularspace.Suchanexcretionofelectrons may also occur under aerobic conditions, known as overflow metabolism. Electron (as well as carbon and energy) overflow allows for higher growth rates under protein allocationlimitingconditions [3,14,15].Yeast, grown underglucose limitingconditions and subjected toaerobicandanaerobiccycles,showedcyclicexcretion and (re)consumptionof electrons.Ethanol isproduced under anaerobic conditions, and reconsumed when oxygen becomes available [16]. Under such dynamic conditions, ethanolcould beregardedasastorage pool that is generated under electron excess conditions and consumed when other donors (like glucose) are
depleted. A similar behaviour was observed for Corynebacteriumglutamicum.Whencultivatedinaglucose fedtwo-compartmentreactorsystem,lactateproduction is observed under O2 limiting conditions, which was reconsumedonceoxygenwaspresent[17].Inthissense, fermentation products can be seen as storage metabolites.
Accepting theviewthat fermentationproductscanalso beinterpretedas(external)storagemetabolites,overflow metabolismbetweendifferentorganismscanbeseenina morecommonframework.Forexample,Plasticicumulans acidivoransgrowninbatchculturewithacetateascarbon sourceaccumulatesintracellularPHAinsteadofbiomass. This mechanism circumvents a limitation in the respiratory capacity — if acetate is directly converted to biomass, the oxygen requirement per mol acetate consumed is muchhigher compared to PHA synthesis. Using PHA as electron sink, the substrate can be consumedatamuchhigherratebecauseelectrontransfer toanexternaldonorviarespirationisnotrequired.Thus, PHA is storage acts as an overflow valve, generating a competitive advantage atthe expense of biomass yield [18,19]. This is mechanistically similar to overflow metabolism in yeast, with the difference that the producedoverflowmetabolitein yeastisexcreted.This hastheadvantageofamuchlargerspacetostorebutwith the disadvantage of being shared with putative other organisms.Therefore,P.acidivorans,withitsintracellular storage of PHA, is especially able to prevail over its competitors within environments with short-term fluctuatingavailability ofsubstrate.
Anotherexampleofintracellularoverflowmetabolismcan befoundincyanobacteria.Cyanobacteriautilizeglycogen as carbon storage metabolite during light/dark cycles. Knock-out ofglycogensynthesishoweverhas shownthat glycogenalsohasanimportantfunctionasstorageofATPto maintainenergyhomeostasiswhenexcessATPisproduced at the photosynthetic electron transport chain [20]. Cyanobacteria incapable of storing glycogen will instead produceextracellularorganicacidsasoverflowproductsto balance their energy homeostasis, thereby preventing unnecessary reduction of redox cofactors and ensuing obstructionofthephotosyntheticelectrontransportchain.
The ‘art’ of dynamicredoxbalancingis exemplifiedby so-called phosphate accumulating organisms (PAOs), such as Candidatus Accumulibacter phosphatis. These organisms play a crucial role in biological phosphate removalwithinwastewatertreatmentsystems[21].PAOs aregrowninacyclicanaerobic/aerobicenvironmentwith volatilefattyacids,suchasacetate,assubstrateswhichare fedduringtheanaerobicphase.Acetatecatabolismunder anaerobicconditionscanonlygeneratealimitedamount of Gibbs free energy, and is thus not favourable. In contrast, under aerobic conditions, full oxidation of
acetate generates a much higher amount of ATP [22]. PAO’s are able to internalize acetate rapidly and carry carbonandelectronstotheaerobicphaseusingastorage polymer, polyhydroxybutyrate (PHB). However, two challengesneed to betackled: (1) Redox: PHBcarries moreelectronsperamountofcarboncomparedtoacetate, (2)Energy:conversionofacetatetoPHBrequiresATP. PAO metabolism is adapted to these challenges by utilizing intracellular storage pools such as glycogen and polyphosphate that serve as sources for electrons intheformof NADHandenergyasATP,respectively. Such a variety of storage pools allow for a flexible, dynamicbalancingofredoxandenergymetabolismunder anaerobicconditions. Howcells have implementedthe regulationandoptimizationofthesedifferentsourcesand sinks is still unknown. The achieved accumulation of PHBfromextracellularacetatethenservesascarbonand electronsourceduringtheaerobicphase,bothforbiomass synthesisaswellasrefuellingoftheintracellularglycogen andpolyphosphatepools[5,23].
Theseexamples (see also Table 1) show that dynamic balancing of electron sinks (and sources), including intracellularstorageaswellasoverflowproducts,facilitate the maximization of substrate uptake rates, potentially generating a competitive advantage. Quantitative descriptions of the metabolic processes, including the metabolicand geneticregulation, are limited to model organismslikeSaccharomycescerevisiae[3],Escherichiacoli orLactococcuslactis[24].Additionally,onlyafewdynamic conditions have been studied, like diurnal cycles [25]. First modelling studies for microbial communities are available[26],delivering comprehensive insightsto the putativecompetitiveadvantagesofdynamicandflexible redoxmetabolism.
Redox
coupling
as
metabolic
engineering
strategy
Commonly,productsofinterestareeithermorereduced ormoreoxidisedthanthesubstrate.Consequently,redox couplings and interactions are basically inevitable and have to be taken into account [31] in metabolic engineering.Such aredoxcouplingcanbeusedto link biomass synthesis with the product pathway. This
strategy can especially be exploited for products that generateasurplusofATP(oftenreferredtoascatabolic products) and several approaches can be used [32]: (1) changing the cofactor specificity of either the product pathway or the catabolic pathway [33–35], (2) utilizing transhydrogenasestointerconvertdifferentelectron car-riers [36], (3) enforcing (electron and carbon) flux to the product by eliminating other electron sinks (like by-product pathways to glycerol or ethanol, but also respiration) [37], (4) providing external secondary sourcesofelectrons,suchasformate,toincrease intracel-lular reducing power [38].Additionally, in the case of eukaryotes, compartmentation has to be taken into account.
Manycommoditychemicals,relevantfortheproduction ofbiofuelsorpolymers,aremorereducedthanthecarbon sources, such as sugars, which are used in these bioprocesses.Thismeansthatadditionalreducingpower has to be generated to drive the respective product pathways. An example of one of these commodity chemicalsis 1,4-Butanediol (BDO). Implementation of theBDOpathwayinE.coli,however,withoutadditional metabolic engineering, shows thatinsufficient reducing powerisavailabletoproduceBDO[39].Toprovidethis additional reducing power, Yim et al. [39] eliminated fermentationpathwaystowardsethanol,lactate,formate, succinateandinadditionenabledgrowthatmicro-aerobic conditions allowing for limited respiration and thus limitedtransfer ofelectronsto oxygen.
Anexampleforredoxpathwayengineeringineukaryotes istheimplementationof anefficient isobutanol produc-tionpathway in yeast.The isobutanolproduction path-waybranchesofffromtheintermediate2-ketoisovalerate ofthevalinesynthesispathwaywhichislocalizedinthe mitochondria. The mitochondrial localization requires additionaltransportsteps fortheproductbutalsoredox cofactors. The synthesis of isobutanol from pyruvate requires additional input of 4 electrons, in theform of 1NADPHand1NADH[40].Withtheimplementation ofthefirststepsofthevalinepathwayinthecytosolthe theoretical yield for isobutanolon glucose is 1mol/mol comparedto0.63for themitochondriallocalization[41].
Table1
Examplesofdualoverflowandstoragemetabolisminarangeofprocessesunderdynamiccultureconditions,sortedbytimescaleofthe dynamics
Timescale Organism IC/ECelectronacceptor Cultureconditions Reference
30s–20min S.cerevisiae Extracellular Aerobic/Anaerobiccycles [16,27]
100s C.glutamicum Extracellular AeratedSTR,unaeratedplug-flowloop [17]
360s P.chrysogenum Intracellular Aerobic,Repetitivesubstratepulse [28]
400s S.cerevisiae Intracellular Aerobic,Repetitivesubstratepulse [29,30]
6hour CandidatusAccumulibacter phosphatis
Intracellular Aerobic/Anaerobiccycles,Repetitive substratepulse
[22,23]
The observed low yields are caused by competitionof multiple cellularpathways for therequiredelectrons in theformofNADHandNADPH.Byknockingoutgenes transcribing for pyruvate decarboxylase and glycerol dehydrogenase, ethanol and glycerol production were blocked, and NADH produced in the glycolysis is forcedintotheisobutanolpathway,essentiallyachieving redoxcouplingbetweenthecatabolicglycolysisandthe isobutanol production pathway. In addition, competing pathways,such as valine and 2,3-butanediol biosynthesis, were eliminated to further improve the production of isobutanol [40]. In addition, the cofactor specificity of the NADPH dependent enzymatic step was changed to NADH, allowing for further redox coupling[41].
Measurement
challenges
Accurately monitoring dynamic changes in the redox cofactor ratios is essential for understanding their overall role within the metabolism [42]. Current techniquesusedforquantificationofNAD(P)H,FADH2 and quinones rely on liquid chromatography–mass spectrometry(LC–MSincludingapproachesusing isoto-pically labelledNAD(P)H/FADH2/quinones as internal
standardimprovemeasurementaccuracy[43]),enzymatic assays orfluorescence[44,45–48].However,becauseof theunstablenatureofNAD(P)H,FADH2andquinones, especially duringsamplepreparation,accurate measure-ment of the in-vivo redox ratios is challenging. In-vivo measurements using fluorescence can be applied to circumventsamplepreparation,butthis techniquedoes not differentiate between NADH and NADPH and additionally suffers from high signal to noise ratios. The redoxchargeofferredoxinscanbemeasured using near infrared spectroscopy, although this does present additional challengesin deconvolutionof themeasured signal[49].
Inaddition,eukaryoticcellsdifferfromprokaryoticcells in that they have separated different parts of their metabolic reaction network in compartments, with different redox ratios. Canelas et al. [50] performed a thermodynamicanalysisoftheglycolyticreactionsusing wholecellaswellascompartmentspecificNAD/NADH ratio measurements. With whole cell measurements, glycolysis appeared thermodynamically not feasible, which clearly showed the impact of redox factor compartmentation generating different potentials. To
DynamicsinredoxmetabolismVerhagen,vanGulikandWahl 119
Figure1
Current Opinion in Biotechnology
Left:Cytosolicfluorescence-basedsensor(adaptedfromRef.[61]):ThissensorworksbasedontheprincipleofFo¨rsterresonanceenergytransfer (FRET),thatis,energyistransferredfromadonortoanacceptorfluorophore.Theenergytransferonlyoccurswhenbothfluorophoresarein proximityofeachother,andthedistanceisinfluencedbybindingoftheligand.ThefluorescencesignalsofYFPversusCFParesubsequently usedtoevaluatetheligandconcentration[61].Right:Cytosolicequilibrium-basedsensor:Byoverexpressinganenzymecatalysingacloseto equilibriumreaction,equilibriumbetweenmetabolitesofinterestandmeasurablepoolscanbeestablished.Fromtheequilibriumconstant(Keq)
andinvolvedmeasuredmetaboliteconcentrations,themetaboliteofinterestcanbedetermined.Withtheequilibriumreactionexpressedonlyin thecytosol,andthemeasuredmetabolites(blue)exclusivelypresentinthecytosol,thecompartmentspecificconcentrationofthemetaboliteof interest(red)canbedetermined[50].
assess the actual redox ratio driving the various intracellular redox reactions, the compartment-specific NADH/NAD+ and NADPH/NADP+ ratios have to be determined. This presents an additional challenge for measuring the actual intracellular redox ratios in eukaryoticcells.
To address these issues, fluorescent biosensors can be used.Thesebiosensors,composedoffluorescentproteins and allosteric binding domains, change in fluorescent signal based on the binding of NADH to the binding domain (Figure 1). The fluorescence output signal is changed upon binding due to conformational changes in the sensor complex [51,52]. These biosensors are utilizedas screeningmethodsusingFACS,allowing for high-throughput screening oflarge mutant libraries for increasedpathwayactivity[53,54].Inaddition,NADPH biosensors havealso been employed to studythe influ-ence of cellular processes on NADPH availability [55] and dynamic changes in NADPH concentrations [56]. However, a disadvantage of these biosensors is that a fluorescence microscope is required to provide a live readoutofthesinglecellbiosensorfluorescence,andthus these biosensors cannot be used to monitor the redox ratiosincells inlargercultivations,suchas reactors.
Another optionis to utilizeso-called equilibrium-based sensor reactions [50] (Figure 1). By overexpressing an enzymecatalysinganequilibrium-basedreaction,anear equilibrium can be established between different metabolite pools involved in the reaction. From the equilibriumconstantandinvolvedmeasured metabolite concentrations, an undetermined metabolite concentra-tioncanbedetermined.Tobesurethatsuchareaction operates close to equilibrium it should have a high capacitycomparedtothein-vivoreactionrate.Sometimes nativereactionscanbeusedforthispurpose[57].Ifnot,a heterologous enzymecan beexpressed to act as sensor reaction;however,itshouldbeverifiedthatthisdoesnot interfere with the metabolism. An example is the expressionofmannitol-1-phosphatedehydrogenasefrom E. coli in S. cerevisiae, converting fructose-6-phosphate and NADH into mannitol-1-phosphate and NAD+ and viceversa,whichappeared tobeessentiallyadeadend reactioninS.cerevisiae[50].Ifthisreactionisexpressed exclusivelyinaspecificcompartment,andthemeasured metabolitesareexclusivelypresentinthisspecific com-partment,thenthecompartmentspecificconcentrationof theundeterminedmetabolitecouldbedetermined. Sev-eralofthesesensorreactionshavebeendeveloped,both formeasuringthecytosolicNADH/NAD+andNADPH/ NADP+ratios[50,58].Usingmannitol-1-phosphate dehy-drogenase expressed in the cytosol as sensor reaction highlydynamic changes in thecytosolic NADH/NAD+ ratio,asresultofglucoseandcombined glucose/acetalde-hydepulsesto asteadystateglucoselimitedchemostat, could be measured [50]. Disadvantage of this sensor
reactionbasedtechniqueisthatonlypopulationaverage signalscanbeobtained.Anysinglecellvariationinredox ratioscannotbeobserved.
Theultimateaimistobeabletomeasurethecomplete spatialandtemporalstateoftheentiremetabolism.The described sensors, both equilibrium-based and fluores-cence-based,areabletoprovidethesemeasurements,but arelimitedto onlyaselectamountof metabolites.The current GC–MS and LC–MS techniques are able to measure the full scope of the metabolism within a discrete temporal space, but are unable to provide the spatial property of metabolites of the cell, especially important within eukaryotic cells. However, even with future improvements in resolution and accuracy of MS technology, the spatialproperty of the metabolism will not be resolved using regular extraction techniques. Promising developmentsto resolve this issue are made inthefieldof single-cellmetabolomics[59]. MALDI-MSI (matrix assisted laser desorption/ionization mass spectrometry imaging) is utilized to sample the spatial distributionofmetaboliteswithinsamples.Withalateral resolution down to 1.4mm [60], this technique was utilizedtoanalysesingle-cellorganismswithsubcellular resolution. Because of the need of sample preparation, thistechnique,as of yet,cannot beutilized to measure single-cell and subcellular metabolome changes under dynamicconditions.However,withfuturedevelopments in metabolome quenching techniques and organelle purification, MS imaging may well help provide full insideintotheactualmetabolicspatialandtemporalstate ofsingle cells.
Concluding
remarks
Redoxcofactors,availableredoxsinksandsourcesgenerate relevant couplings within the metabolic networks and influencethe rangeofreactionsthatarethermodynamically feasiblewithinthecell.Ashighlighted,manyintracellular storageprocessescanbeconsideredasoverflowprocesses, similar to fermentative pathways, which are essentially extracellular storage processes. Mechanisms redirecting electron flows,like overflow metabolism or intracellular storagepools,canacceleratemetabolicfluxesandincrease competitivenessundermanydynamicconditions.Atthe sametimeelectronstorage incellularorextracellularcan increasemetabolicflexibility[23].
Future research will have to consider the limitations imposed by the redox metabolism on the metabolism as a whole. Especially in compartmented systems, the quantificationoftheinvivosubcellularredoxratiosisof paramountimportanceforrealisticmodellingand under-standingof cellularmetabolism andfor design of novel strainsandprocesses.Furtherdevelopmentsinthefield ofsinglecellmetabolomicsmayhelptoprovideinsightin themetabolic spatial and temporal state of single cells and their subcellular components. In addition, many
enzyme cofactor specificities, especially for non-model organisms,areunknownandthusmoreenzymaticassays are required for elucidation of novel enzymes for metabolicengineering.Furthermore,additional measure-mentsandcomputationalpredictionsofelectrontransfer between compartments through protein-protein com-plexes [62] or redox shuttles, as well as membrane processes,suchaselectronbifurcation[63],areessential to gain insight into dynamic switches withinthe redox metabolism.
Conflict
of
interest
statement
Nothing declared.
CRediT
authorship
contribution
statement
Koen JA Verhagen: Conceptualization, Investigation, Writing - original draft, Writing - review & editing, Visualization. Walter Mvan Gulik:Writing -review & editing. Sebastian Aljoscha Wahl: Conceptualization, Writing -review& editing,Supervision.
Acknowledgements
ThisworkwassupportedbytheNederlandseOrganisatievoor WetenschappelijkOnderzoek(NWO)[projectnumber737.016.001].We thankPascaleDaran-LapujadeandLeonorGuedesdaSilvafortheirinput anddiscussionsduringthewritingofthisreview.
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