Model of central and trimethylammonium metabolism for optimizing l-carnitine production by E. coli

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doi:10.1016/j.ym

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Metabolic Engineering 7 (2005) 401–425

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Model of central and trimethylammonium metabolism foroptimizing L-carnitine production by E. coli

Angel Sevillaa, Joachim W. Schmidb, Klaus Mauchb, Jose L. Iborraa,Mathias Reussb, M. Canovasa,�

aDepartamento de Bioquımica y Biologıa Molecular B e Inmunologıa, Facultad de Quımica, Universidad de Murcia,

Apdo. Correos 4021, 30100 Murcia, SpainbInstitute of Biochemical Engineering, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany

Received 14 February 2005; received in revised form 27 May 2005; accepted 13 June 2005

Available online 10 August 2005

Abstract

The application of metabolic engineering principles to the rational design of microbial production processes crucially depends on

the ability to make quantitative descriptions of the systemic ability of the central carbon metabolism to redirect fluxes to the

product-forming pathways. The aim of this work was to further our understanding of the steps controlling the biotransformation of

trimethylammonium compounds into L-carnitine by Escherichia coli. Despite the importance of L-carnitine production processes,

development of a model of the central carbon metabolism linked to the secondary carnitine metabolism of E. coli has been severely

hampered by the lack of stoichiometric information on the metabolic reactions taking place in the carnitine metabolism. Here we

present the design and experimental validation of a model which, for the first time, links the carnitine metabolism with the reactions

of glycolysis, the tricarboxylic acid cycle and the pentose-phosphate pathway. The results demonstrate a need for a high production

rate of ATP to be devoted to the biotransformation process. The results demonstrate that ATP is used up in a futile cycle, since both

trimethylammonium compound carriers CaiT and ProU operate simultaneously. To improve the biotransformation process, resting

processes as well as CaiT or ProU knock out mutants would yield a more efficient system for producing L-carnitine from

crotonobetaine or D-carnitine.

r 2005 Elsevier Inc. All rights reserved.

Keywords: Metabolism link; Central carbon and secondary carnitine metabolism; Biotransformation; Escherichia coli; L-carnitine

1. Introduction

In human cells, L-carnitine (R(-)-3-hydroxy-4-tri-methylaminobutyrate) transports long-chain fatty acidsthrough the inner mitochondrial membrane, which iswhy several clinical applications for L-carnitine havebeen identified. Consequently, the demand forL-carnitine has increased worldwide (Seim et al., 2001)and chemical and biological processes have beendeveloped for its production (Cavazza, 1981; Hoekset al., 1996; Kleber, 1997; Kulla, 1991). Strains

e front matter r 2005 Elsevier Inc. All rights reserved.

ben.2005.06.005

ing author.

esses: [email protected] (J.L. Iborra),

stuttgart.de (M. Reuss).

belonging to the genera Escherichia, Proteus andSalmonella racemice D-carnitine, a waste product andan environmental problem resulting from the chemicalsynthesis of L-carnitine, and/or the biotransformation ofcrotonobetaine (dehydrated D-carnitine) to produce L-carnitine (Canovas et al., 2002; Castellar et al., 1998;Kleber, 1997; Obon et al., 1999). However, thebioprocesses involved need to be further studied if theyare to be optimized. Although the protective capacity ofcarnitine or other trimethylammonium compounds hasbeen studied in Escherichia coli, since it allows cells towithstand osmotic up-shocks, the entire process is stillnot fully understood (Eichler et al., 1994a, b; Elssneret al., 2001; Kleber, 1997). This secondary metabolismhas been used for L-carnitine production using E. coli

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Nomenclature

Metabolites

2pg 2-phosphoglycerate3pg 3-phosphoglycerate6pg 6-phosphogluconateaccoa acetyl-coenzyme Aadp adenosindiphosphateaicar 5-amino-4-imidazolecarboxamideribo-

tideakg. a-ketoglutarateaki a-ketoisovalerateamp adenosinmonophosphatearg arginineasn asparagineasp aspartateatp adenosintriphosphatecarp carbamoylphosphatecr crotonobetainecrcoa crotonobetainylCoAcdp cytideindiphosphatecho chorismatecmp cytidinemonophosphateco2 carbondioxidecoa coenzyme Acr crotonobetainecrcoa crotonobetainylCoActp cytidinetriphosphatecys cysteinedala D-alaninedamp deoxyadenosinmonophosphatedcmp deoxycytideinmonophosphatedglu D-glutamatedgmp deoxyguanosinmonophosphatedhap dihydroxyacetonephosphatedna deoxyribonucleic aciddtmp deoxythymidinmonophosphatedipim diaminopimelatee4p erythrose-4-phosphateea electron aceptor oxidizedeah2 electron aceptor reducedetamp phosphatidyl-ethanolaminef6p fructose-6-phosphatefad flavin-adenine-dinucleotide, oxidizedfadh2 flavin-adenine-dinucleotide, reducedfattyn,i fatty acid containing n carbon atoms and

i double bondsfdp fructose-1,6-bisphosphatefthf formyltetrahydrofolatefum fumarateg1p glucose-1-phosphateg6p glucose-6-phosphategap glyceraldehyde-3-phosphateglc glucosegln glutamine

gly glycineglycp glycerol-3-phosphategmp guanosinmonophosphateh protonh2o waterh protonhis histidinehom homoserineile isoleucineimp inosinemonophosphateisocit isocitratekival a-ketoisovaleratelala L-alaninelc L-(-)carnitinelccoa L-carnitinylCoAleu leucinelglu L-glutamatelys lysinemal malatemet methioninemethf methylentetrahydrofolatemythf methyltetrahydrofolatemurunit subunit of mureinenad diphosphopyridindinucleotide, oxidizednadh diphosphopyridindinucleotide, reducednadp diphosphopyridindinucleotide-phos-

phate, oxidizednadph diphosphopyridindinucleotide-phos-

phate, reducednh4 ammoniumoac oxaloacetateorn ornithinep inorganic phosphatepep phosphoenolpyruvatephe phenylalaninepgp 1,3-diphosphoglyceratepro prolineprpp phosphoribosylpyrophosphatepyr pyruvaterib5p ribose-5-phosphateribu5p ribulose-5-phosphaterna ribonucleic acidsed7p sedoheptulose-7-phosphateser serineso4 sulfatesuc succinatesuccoA succinyl-coenzyme Athf tetrahydrofolatethr threoninetrp tryptophantyr tyrosineudp uridindiphosphateump uridinmonophosphateutp uridintriphosphateval valinexyl5p xylulose-5-phosphate

A. Sevilla et al. / Metabolic Engineering 7 (2005) 401–425402

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Enzymes

ALD aldolaseATPase ATPaseCaiB enoyl-CoA transferaseCaiD enoyl-CoA hydrataseCaiT betaine antiporterCIS citrate synthaseDCT dicarboxylate transporterDHAPS dhap synthasesDiff diffusionENO enolaseFUMR fumarate reductaseG1PAT g1p adenyltransferaseG3PDH glycp dehydrogenaseG6PDH g6p dehydrogenaseGAPDH gap dehydrogenaseGLYK gly kinaseLDH lactate dehydrogenaselipidSynth i lipid synthesis reaction iMurSynth i mureine synthesis reaction i

PEPCX pep carboxylasePDH pyr dehydrogenasePFK phosphofructokinasePGDH 6pg dehydrogenasePGI glucose-6-phosphoisomerasePGK phosphoglycerate kinasePGlucoM phosphoglucomutasePGM phosphoglycerate mutasePK pyr kinaseProtSynth i protein synthesis reaction iProU betaines transporter ATP dependentPTA PhosphotransacetylasePYRFL Pyruvate formate-lyaseR5PI ribose-phosphate isomeraseRPPK ribose-phosphate pyrophosphokinaseRu5P ribulose-phosphate epimeraseTA transaldolaseTIM triosephosphate isomeraseTKa transketolase reaction aTKb transketolase reaction b

A. Sevilla et al. / Metabolic Engineering 7 (2005) 401–425 403

cells in growing and resting state (Canovas et al., 2002).In brief, crotonobetaine is transformed into L-carnitinethrough the involvement of CoA esters and two newenzymes, an enoyl-CoA hydratase and a CoA transferase,while it is reduced to g-butyrobetaine by a crotonobetainereductase and a CoA transferase (Elssner et al., 2001;Preusser et al., 1999), which are induced anaerobically inthe presence of the D,L-carnitine mixture and/or croto-nobetaine. Genetic studies have been conducted toelucidate carnitine metabolism in E. coli O44 K74(Kleber, 1997), where it has been found that twodivergent structural operons, caiTABCDE and fixABCX,are coexpressed, the former responsible for the enzymesof carnitine metabolism and the latter being required toprovide electrons for carnitine reduction (Eichler et al.,1996; Walt and Kahn, 2002). Although a genome-scalepathway database of E. coli is available (Karp et al.,2004), it will be necessary to understand the stoichio-metric links between the production pathways and thecentral metabolism if we are to understand the cellbiochemistry and physiology to optimize this bioprocess.Resting cell processes are an important issue sincebiotransformation occurs in a simple medium containingonly the substrate dissolved in buffer, thus loweringproduction costs and simplifying product purificationcompared with growing cell processes. In a previouspaper, Canovas et al. (2003b) presented for the first timethe link between the central and the carnitine metabolismmodel in E. coli. Also in a previous paper Chassagnole etal. (2002) presented a large-scale stationary model,although this is not the only possibility (Pramanik andKeasling, 1997; Reed et al., 2003).

The objective of this study was to investigate theinterconnection of the central carbon and the carnitinemetabolism of E. coli. The need for a quantitativeunderstanding of the pathways responsible for produ-cing the precursors of biosynthetic pathways has beendemonstrated in the case of the production of aromaticcompounds by several research groups (Berry, 1996;Flores et al., 1996; Gosset et al., 1996; Liao et al., 1996;Lu and Liao, 1997; Patnaik et al., 1995; Patnaik andLiao, 1994; Stephanopoulos et al., 1998; Stephanopou-los and Simpson, 1997). Due to the importance of thecentral carbon route, several contributions have at-tempted to developing a kinetic model using differentcell types (Chassagnole et al., 2002; Gosset et al., 1996;Liao et al., 1996; Stephanopoulos and Simpson, 1997;Torres et al., 1997). Recently, it has been pointed outthat, besides the supply of precursor molecules, thesupply of cofactors is an important issue for metabolicengineering (Flores et al., 2004; Koebmann et al., 2002;Marx et al., 2003; Oliver, 2002; Wittmann and Heinzle,2002). Since the central carbon metabolism and carni-tine production are mainly linked because of the supplyand demand of energy equivalents, and regeneration ofreduction equivalents plays an important role in energysupply during anaerobic fermentation, a large-scalemodel describing cofactor balances for the centralmetabolism of E. coli is required. The model presentedherein is based on the E. coli large-scale stationarymodel of Chassagnole et al. (2002), which incorporatesthe model of carnitine metabolism described by Canovaset al. (2003b). The present work represents a firstapproach to understanding the link between the central

ARTICLE IN PRESSA. Sevilla et al. / Metabolic Engineering 7 (2005) 401–425404

carbon (or primary metabolism) and the metabolism ofthe secondary trimethylammonium compounds involvedin the production of L-carnitine by E. coli in theirgrowing and resting states for optimizing the bioprocess.The choice of appropriate biocatalyst state and reactorconfiguration is of prime importance for L-carnitineformation and process optimization (Canovas et al.,2003a, c). The bioprocesses carried out in continuousand high cell density membrane reactors were thereforechosen and implemented in the complete model of thebiotransformation. Furthermore, the activities of theenzymes, crotonobetaine hydrating reaction activity(CDH) and pyruvate dehydrogenase (PDH) and trans-porters, CaiT and Pro U, as well as the levels ofcarnitine, crotonobetaine and ATP and the NADH/NAD+ ratio were measured, in order to resolve theinner degrees of freedom in the metabolic flux distribu-tion. To validate the complete model, experimentalfluxes are compared with outputs. ATP balances andL-carnitine yields during anaerobic conditions withdifferent fermentation products were calculated afterdetermining elementary flux modes.

2. Materials and methods

2.1. Bacterial strain and culture media

The bacterial strain used, E. coli O74 K74 (DSM8828), contained the complete cai and fix operons andwas stored in a minimal medium (MM) containingglycerol (20%) at �20 1C. The MM was that describedby Obon et al. (1999), while the complex medium (CM)contained (g/L): bacteriological peptone, 20; NaCl, 5;glycerol (carbon source), 12.6; and crotonobetaine, 4.The pHs of both media were adjusted to 7.5 with 1MKOH prior to autoclaving.

2.2. Reactor operation

2.2.1. Growth of the bacteria

Batch and continuous experiments were performed inreactors equipped with temperature, pH, oxygen andpump controllers (Biostat B, Braun Biotech Interna-tional GMBH, Melsungen, Germany). A 1L culturevessel of 0.5–0.8 L working volume was used. E. coli 044K74 was grown under different conditions, in order tooptimize the induction of the carnitine metabolismenzymes. The culture was inoculated with 3% (v/v) ofthe liquid culture stored at �20 1C in 20% (v/v) glycerol,while the medium employed was the CM mentionedabove. The cells were grown in batch or continuousfeeding reactors under anaerobic conditions at 37 1C.Anaerobic conditions were maintained to induce theenzymes involved in the carnitine metabolism (Kleber,1997), while D,L-carnitine mixture, D-carnitine or croto-

nobetaine were supplied as inducers. Nitrogen was usedto ensure anaerobiosis during the experiments. Datawere worked at steady state so as to be used whenconstructing the model. Resting cells experiments inbatch and continuous membrane high cell recyclereactors were used to determine reactor macroscopicand cell metabolic fluxes, enzyme activities, cell energeticcontent (ATP pool and NADH/NAD+ ratio) andextracellular metabolites (crotonobetaine, acetate, etc.).

2.2.2. Resting cell experiments

For the resting cell experiments, microaerobic oranaerobic conditions were used. Then cultures wereharvested at the end of the exponential growth phase,centrifuged at 16,000g for 10min and washed twice with75mM phosphate buffer, pH 7.5. The final pellet was re-suspended in the same initial volume but containing50–500mM crotonobetaine in 50mM phosphate buffer,pH 7.5 at 37 1C and left in the reactor system for15–24 h. All experiments were performed at least intriplicate and under sterile conditions. The valuesreported are the means of the assays performed.

2.2.3. Membrane reactor operation

The reactor vessel was also coupled to a cross-flowfiltration module (Minitan, Millipore, USA) equippedwith four 0.1 mm hydrophilic polyvinylidene difluorideDurapore plates of 60 cm2 area (Millipore, USA)(Canovas et al., 2002). The cell broth was recycled intothe reactor with a peristaltic pump adjusted to a highflow rate (70ml/min) to minimize membrane fouling.E. coli cells for the inoculum were grown as explainedpreviously and transferred to the fermenter. Continuousoperation was set at 37 1C and started by feeding withthe CM medium (anaerobically by bubbling nitrogenpreviously passed through a water trap). The system wasrun at different dilution rates ranging from 0.1 to 1 h�1.Experiments were run to work out the cell decayconstant, which was seen to be 0.01 h�1. Beside this,resting cell experiments were also run in this system byswitching the CM medium over to a biotransformationmedium containing 50–500mM crotonobetaine in50mM phosphate buffer at pH 7.5.

2.3. Enzyme assays

The crotonobetaine hydration reaction assay wascarried out according to Jung et al. (1989), and thecrotonobetaine reduction reaction (CRR) assay accord-ing to Preusser et al. (1999), both started by usingcrotonobetaine as substrate. Enzyme activity wasdefined either as the total mmols of substrate consumedper minute (U) or as specific activity, which is the mmolof substrate consumed per minute and mg of protein(U/mg). In certain experiments, fumarate was used to

ARTICLE IN PRESSA. Sevilla et al. / Metabolic Engineering 7 (2005) 401–425 405

inhibit the CRR activity and so increase L-carnitineproduction.

The enzyme activity assays were optimized for theconditions and media. In each case, reactor bulk liquidsamples were withdrawn and centrifuged at 16,000g at4 1C. The supernatant was removed and cells were re-suspended within the corresponding extraction buffer,depending on the enzyme. Cells were sonicated for 6cycles (10 s each), at 10 mm amplitude, with a probe of1 cm diameter and below 20 1C. The extract wascentrifuged for 15min at 16,000g and 4 1C to removecell debris. Protein content was determined by themethod of Lowry et al. (1951).

PDH complex: The method was that of de Graef et al.(1999). The extraction buffer was 50mM potassiumphosphate, pH 7.5, 0.1mM EDTA, 5mM b-mercap-toethanol and 3mM MgCl2. The enzyme activity wasfollowed as the decrease in potassium ferricyanideabsorbance at 430 nm (eferricianide ¼ 1.030M�1 cm�1)and 37 1C. One unit of enzyme activity was the amountof enzyme required for the generation of 2 mmol offerricyanide per min.

2.4. Substrate consumption for growth and

biotransformation processes

L-carnitine concentration was determined enzymati-cally with the carnitine acetyl transferase method (Junget al., 1989). Glycerol, crotonobetaine and g-butyrobe-taine were analysed by HPLC with a Tracer Spherisorb-NH2 column, 3 mm, 25� 0.46 cm, supplied by Teknok-roma (Barcelona, Spain) as reported (Obon et al., 1999).The isocratic mobile phase was 0.05mol/L acetonitrile/H3PO4 (65/35) pH 5.5 at a flow rate of 1ml/min.Bacterial growth was followed spectrophotometricallyat 600 nm, using a Novaspec II from Pharmacia-LKB(Uppsala, Sweden), and converted to dry weightaccordingly.

2.5. Determination of the central metabolite

concentrations required

ATP content and NADH/NAD+ ratio: The energycontent per unit of cell was determined as the ATP leveland NADH/NAD+ ratio throughout the experiments.For ATP measurement, the HS II bioluminescence assaykit from Boehringer (Mannhein, Germany), based onluciferase enzyme using a FluoStar fluorimeter (BGP,Germany) in the conditions stated and after cell DMSOlysis, was used.

Reducing power in the form of NADH/NAD+ ratiowas calculated as in Snoep et al. (1990). The measurementswere made using an enzymatic method based on thealcohol dehydrogenase enzyme. The extraction of NADHand NAD+ was carried out by two different methods,involving alkali or acid extraction depending on whether

the reduced or the oxidized form was obtained. The cellcontent was determined after biomass optical densitytransformation as dry weight and assuming either anintracellular volume of 1.63ml/mg (Emmerling et al., 2000)or 1.72ml� 10�13/cell (worked out by flow cytometry inthis work). For Acetyl-CoA and CoA, 1ml was takenfrom the culture, collecting cells by centrifugation at11.800g for 10min at 4 1C. The pellets were re-suspendedin 100ml 1N formic acid, shaking for 15 s every 5min for30min. Cells were kept on ice after collection and duringextraction. Samples were neutralized with 100ml of 1MKH2PO4. Finally, the samples were centrifuged at 11.800g

for 5min, using the supernatant to analyse the acetyl-CoAand HS-CoA cell content. The cell level of bothmetabolites was determined by HPLC using the methoddescribed by Debuysere and Olson (1983). The systemwas that used in Section 4, equipped with a m-Bonda-pakTM C18 (Millipore), 4.5mm� 25 cm column. Themobile phase was as follows: phase A (85%), 120mMphosphate buffer, pH 4 and 0.05% b-mercaptoethanol andphase B (15%), 98% methanol with 2% clorophormeluted at 0.8ml/min. Acetyl-CoA and CoA were detectedat 254nm.

E. coli anaerobic metabolite production: The acetate,fumarate, lactate, formate and pyruvate contents of thebulk liquid reactor were determined by HPLC using thesame system as in Section 4. A Tracer Spherisorb-NH2

(3 mm, 25� 0.46 cm) column supplied by Teknokroma(Barcelona, Spain) was used. The isocratic mobile phasewas 0.05mol/L acetonitrile/H3PO4 (65/35) pH 4.7 at aflow rate of 0.5ml/min. The effluent was monitored at207 nm. Samples were withdrawn from the reactor andcentrifuged at 12,000g for 10min at 4 1C. The super-natant was used for analyses.

2.6. Software used for model building

The software used was INSILICO discovery, version1.03, from Insilico biotechnology GmbH, which is acomputational tool for the network-oriented in silico

analysis and design of cellular properties, which allowedus to perform the Metabolic Flux Analysis for thedifferent conditions and the Elementary Flux Modes.

2.7. Model building of the central carbon and carnitine

metabolism in E. coli

The present model is based on a previous large-scalestationary model proposed by Chassagnole et al. (2002).A short description follows:

1.
The carbon and energy source is glucose, whereasammonium and sulfate are used as the nitrogen and Ssources, respectively. Phosphate is used as the sourceof phosphorus. The energy requirements for theirtransport into the cell are considered in the model.


2.
Glucose dissimilation involves glycolysis, the pen-tose-phosphate pathway, and tricarboxylic acid cycle.
3.
The model includes the synthesis of biomass, which iscomposed of lipids, proteins, DNA, RNA, poly-sacharides and murein. Polymer synthesis starts fromprecursors provided by the central catabolic path-ways.
4.
Electron transport chain for the regeneration of thecofactors as well as exchange reactions for O2, CO2,and H2O are included.
5.
Finally, synthesis and excretion of acetate areconsidered.
2.8. Link between the central metabolism and carnitine

metabolism in E. coli

Since carnitine metabolism in E. coli is repressed byglucose, we will consider glycerol as the only centralcarbon source. Primary metabolism has been adaptedaccordingly. Thus, the large-scale stationary model ofChassagnole et al. (2002) was modified by taking intoaccount (i) the central metabolism under anaerobicconditions and (ii) the link with L-carnitine metabolism.Certain considerations have been taken into account tomodify the E. coli central stationary metabolism model:

1.
In order to use glycerol as the carbon and energysource, a glycerol transporter (Burns and Lewis,1997; Sweet et al., 1990), the glycerol kinase(Thorner, 1975), required for its entrance in theglycolysis pathway, as well as the glycerol-phosphatedehydrogenase required for the production of G3Pwas included.The following fermentation products were consideredas well: Lactate (Zhou et al., 2003) and formiate(Dawes and Foster, 1956; Gupta and Clark, 1989).Ethanol was not included as a fermentation product(Clark, 1989; Kessler et al., 1991, 1992; Knappe andSawers, 1990; Rudolph et al., 1968; Shafqat et al.,1999; Shone and Fromm, 1981), since experimentalresults did not point to its production during thebiotransformation processes.
2.
The gluconeogenesis pathway was included by sub-stituting the phosphofructokinase reaction by thebisphosphatase catalysed reaction (Babul and Guixe,1983; Coll et al., 1988; Hamilton et al., 1988), toenable the synthesis of glucose derivatives.
3.
In the UMP de novo synthesis (Karibian, 1978;Larsen and Jensen, 1985; Owen and Kaback, 1979), itwas necessary to substitute oxygen by a differentelectron acceptor, menaquinone (Karibian, 1978),and its later oxidation by means of fumaratereduction to succinate (Brandsch and Bichler, 1989;Cecchini et al., 2002; Cole et al., 1985; Darlison and
Guest, 1984; Iverson et al., 2002, 1999; Luna-Chavezet al., 2000; Maklashina and Cecchini, 1999; Matteviet al., 1999; Westenberg et al., 1993; Wood et al.,1984).

4.
It was also necessary to include an electron acceptor,since the fermentative pathways were unable toassimilate the reducing power arising from glyceroldissimilation. In the fermentation processes describedhere, the reduction of fumarate to succinate was usedfor this purpose, since it acts as an electron acceptorby inhibiting the crotonobetaine reductase enzyme(Canovas et al., 2002; Kleber, 1997). Fumarate iscarried into the cell by means of an antiport withsuccinate. The DcuC transporter is one of threetransporters known to be responsible for the uptakeof C4-dicarboxylates, such as fumarate, underanaerobic conditions. A knockout mutant of dcuCin a dcuA/dcuB double mutant lost the ability togrow by fumarate respiration and was unable tomediate fumarate/succinate exchange (Zientz et al.,1996). Whole cell experiments using dcuA-C indi-cated that DcuC can mediate both fumarate/succi-nate exchange and fumarate/proton symport (Zientzet al., 1996). Fumarate is not assimilated by the celland it is not a carbon source. Our experimentalresults indicated that succinate was produced andexcreted to the medium during the L-carnitineproduction process.
5.
The carnitine pathway is detectable not only in cellspreviously grown anaerobically but also in somespecies, such as E. coli ATCC 25922 and DSM 8828,P. vulgaris and P. mirabilis, grown under aerobiosisin the presence of inducers such as D,L-carnitinemixture or crotonobetaine (Canovas et al., 2002;Elssner et al., 2000; Kleber, 1997; Obon et al., 1999).It was first postulated that L-carnitine dehydratasereversibly catalysed the conversion of L-carnitine intocrotonobetaine and that crotonobetaine reductasenon-reversibly transformed crotonobetaine into g-butyrobetaine as an electron sink (Jung et al., 1989;Kleber, 1997; Roth et al., 1994), even though thislatter in E. coli can be inhibited by fumarate additionas alternative electron sink (Obon et al., 1999).Functions have been assigned to each putativeprotein of the caiTABCDE operon, and it is knownthat CaiT is an exchanger (antiporter) for L-carnitineand g-butyrobetaine in E. coli (Jung et al., 2002),which also transports crotonobetaine (Canovas et al.,2002, 2003c). The enoyl-CoA hydratase (CaiD) iscomposed of two identical subunits, requiring CoAtransferase activity (CaiB) (Fig. 1A). It has been verifiedthat the hydration reaction of crotonobetaine to L-carnitine (CDH) proceeds at the CoA-level in two steps:the protein CaiD-catalysed hydration of crotonobetai-nyl-CoA to L-carnitinyl-CoA, followed by CoA-transferfrom L-carnitinyl-CoA to crotonobetaine, catalysed by

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Fig. 1. (A) Carnitine metabolism in E. coli based on the model of

Canovas et al. (2003b). Anaerobic metabolism of L-carnitine in E. coli.

CaiT, L(-) carnitine /crotonobetaine protein transporter; CaiB,

crotonobetainyl-CoA/CoA transferase; CaiC, crotonobetaine or L-

carnitine CoA ligase; CaiD, enoyl-CoA hydratase or D-carnitine

racemase activity, ProU (energy-dependent betaine transporter),

intracellular crotonobetaine, CRint, extracellular crotonobetaine,

CRext, crotonobetainylCoA, CRCoA, intracellular L(-)-carnitine,

LCint, extracellular L(-)-carnitine, LCext and LcarnitinylCoA, LCCoA.

(B) The representation was obtained from the software INSILICO

discovery version 1.03 using experimental results. Numbers in white

boxes denote fluxes in mM/h of intracellular volume.

Fig. 2. Anaerobic central metabolism in E. coli based on the model of

Chassagnole et al. (2002) including glycerol as the substrate and

carnitine metabolism. The representation was obtained from the

software INSILICO discovery version 1.03.


CaiB (Elssner et al., 2001). Thus, CaiD and CaiB fromE. coli have been found to catalyse the reversiblebiotransformation of crotonobetaine to L-carnitine inthe presence of a co-substrate, either g-butyrobetainyl-CoA or crotonobetainyl-CoA (Elssner et al., 2001).CaiD was also postulated to be involved in theracemization of D-carnitine (Eichler et al., 1996).Furthermore, CaiC has been suggested as a CoA-trimethylammonium ligase (Eichler et al., 1996),activating crotonobetaine/g-butyrobetaine/L-carnitinewhen it reaches the cell. The function of protein CaiE

is not totally understood and further studies must beundertaken. With all this information, we haveproposed a model to describe the entire activity of E.

coli able to produce L-carnitine from crotonobetaineunder anaerobic conditions in bioreactors (Fig. 1A).Thus the L-carnitine metabolism was implementedwithin the INSILICO software (Fig. 1B). Moreover, itwas linked to the central metabolism representation byintegrating the following processes (Fig. 2):

1. Crotonobetaine as the substrate entering thesystem,

2. Crotonobetaine as the substrate leaving thesystem,

3. L-carnitine as the product leaving the system,4. Cai T antiport of crotonobetaine/L-carnitine,5. Pro U active transport of crotonobetaine,6. Diffusion of L-carnitine,7. CaiB enoyl-CoA transferase,8. CaiC CoA ligase (function being explained in the

next section),9. CaiD enoylCoA hydratase.

The complete adapted model of central metabo-lism and carnitine production is shown in Fig. 2and Appendix A.

2.9. Flux analysis of the biotransformation of

crotonobetaine into L-carnitine

First it was decided to study the carnitine metabolismseparately. The coupling to the central metabolism wasinvestigated in a second step.


1. Carnitine metabolism

The subsystem presented 5 degrees of freedom, 3internal and 2 external, meaning that at least 5 fluxes ofthe system were required for it to be completelydetermined. The available information is listed inTable 1. The experimental results allowed us todetermine the influx of crotonobetaine, the efflux ofcrotonobetaine and the transport of this compound.ProU is a crotonobetaine transporter depending on ATPand during the biotransformation process it presentsonly a basal level (Canovas et al., 2003c). However,during reactor operation, CaiT is the main group oftransporter proteins carrying the betaine derivatives,since it is expressed when the carnitine operon,caiTABCDE, is induced in the presence of crotonobe-taine or L-carnitine (Kleber, 1997). Thus from Canovaset al. (2003c):

MProU ¼Ratebasal transport

Rateinduced transport¼

2:2mmol=mgprotein min

26mmol=mgprotein min

¼ 0:0846. ð1Þ

Table 1

Experimental and theoretical fluxes obtained in the biotransformation

of crotonobetaine into L-carnitine by E. coli in a continuous high cell

density reactor (see Materials and Methods)

Transformation Experimental rate

(mM/h)

Output rate from

the model (mM/h)

Effluent CR

(extracellular)

29.49 29.49

Influent CR

(extracellular)

50.00 50.00

CaiT(intracellular) CB

Effluenta,b,c3048 3048

Pro U (intracellular) CB

Influentb4378 4378

CDH (intracellular)a,b 1030 1030

PDH (intracellular)b 7.21 7.21

Cellular growth

(extracellular)

4.88 2.06

Influent glycerol

(extracellular)

100.00 100.00

Effluent glycerol

(extracellular)

43.28 43.30

Effluent acetate

(extracellular)

27.08 27.13

Effluent lactate

(extracellular)

26.20 26.25

aThese fluxes were redundant though experimentally obtained.

Therefore the degrees of freedom for carnitine metabolism were

established.bTo calculate theses fluxes a relationship between the intracellular

volume and the reactor volume of 0.0154 Lcell/Lreactor was used.cFrom Canovas et al. (2002, 2003a) is known the portion of the CB

excretion flux (729.47mmol/Lreactor.h ) and the portion of CB influent

flux (776.41mmol/Lreactor.h) carried out by CaiT. That means a net

flux of 46.94mmol/Lreactor h or 3048mmol/Lcell h.

MProU should be showing the portion of flux throughProU taking into account that CaiT was induced andrepresented a rate of 26mmol/mg proteinmin whileunder basal conditions and without CaiT inductiononly a rate of 2.2mmol/mg proteinmin was worked outusing radio-labelled carnitine (Canovas et al., 2003c).Taking into account that the total crotonobetaineuptake flux was 796.89mmol/Lreactor h, the flux throughProU can be calculated. The relation of cell volume toreactor volume was 0.0154 Lcell/Lreactor, resulting in aProU-catalysed crotonobetaine uptake flux of4378mmol/Lcell h with respect to intracellular volume.

As the second step, the CaiC activity uses eithercrotonobetaine or L-carnitine and CoA as substrate(Fig. 1A). In fact CaiC catalyses the following twoirreversible reactions (Elssner et al., 2001):

LCþ CoAþATPþH2O�!Cai C

LCCoAþADPþ Pi;

CRþ CoAþATPþH2O�!Cai C

CRCoAþADPþ Pi;

ð2Þ

where LC is L-carnitine, LCCoA is L-carnitinyl-CoA,CR is crotonobetaine and CRCoA is crotonobetainyl-CoA, respectively. Both reactions involve the irrever-sible covalent binding of one CoA molecule. Since noreaction is included in the subsystem that allows for therelease of free CoA, it follows from a CoA balance thatthe fluxes through both CaiC catalysed reactions have tobe zero in the subsystem. What is more, CRCoA andLCCoA subsequently form a conserved moiety, which isstill present in the model if it is coupled to the centralmetabolism model. Therefore, the finding that the fluxthrough both CaiC catalysed reactions is zero still holdsfor the complete model.

2. Link between the E. coli central and the carnitine

metabolism under anaerobiosis.The complete system presented 11 degrees of freedom,

3 internal and 8 external, so that at least 11 system fluxeswere required to determine it completely. The fluxesderived from the carnitine metabolism are those alreadydetermined in the previous section. Below, we show therest of the fluxes required to solve the system:

a.
Enzyme PDHUsing the experimental setup presented in Materialsand Methods, it was determined that the activity ofpyruvate dehdrogenase in the high cell densityreactor at the steady state was 10.0mU/mg prot.Therefore, a flux through this reaction can becalculated using a relationship between the proteinweight and the cellular volume. In E. coli therelationship between the cellular volume and thedry weight is 1.63 ml/mg cdw (Emmerling et al., 2000)and the relationship between the protein weight andthe dry weight is 0.0199mg prot/mg cdw (Pramanik


and Keasling, 1997). Thus, a value of protein-to-cellvolume of 83.24 mL/mg prot was obtained and theflux through the PDH enzyme was 7.21mM/h.

b.

Fig. 3. Flux analysis of the anaerobic central metabolism in E. coli

based on the link of the model of Chassagnole et al. (2002) and the

carnitine metabolism model of Canovas et al. (2003b). Numbers

denote fluxes in mM/h of intracellular volume.

Cell growthTaking into account that the process is carried out ina continuous high cell density cell recycle reactor andthat in the steady state the cellular concentration isconstant, the only possible growth is due to cellgeneration. It was also known that the cellular decayconstant is 0.01 h�1 (Canovas et al., 2002) and thecellular density in the steady state was 12.183 g/L.Therefore, a flux for cell growth of 4.88mM/h can becalculated (see Appendix B).The rest of the fluxes (influent glycerol, effluentglycerol, efluent acetate, effluent lactate) were experi-mentally determined. In Table 1, the fluxes used inthe model are shown. With these data the system istotally defined and identified.

3. Results and discussion

The work carried out represented a modification ofthe large-scale stationary model developed by Chassag-nole et al. (2002) for E. coli metabolism under anaerobicconditions and with glycerol as the carbon source (Figs.1 and 2). For this reason, the transport of glycerol intothe cell and an electron acceptor to consume thereducing power generated by the reduction of glycerolwere taken into consideration. Thus the steps takenwere: first, linking the carnitine metabolism, second,carrying out an energy balance for the process and thirdfollowing the L-carnitine yield in different scenarios.

3.1. Link of the carnitine to the central metabolism of E.

coli

The carnitine model developed by Canovas et al.(2003a, b) was linked to the central E. coli metabolism(Fig. 1). For the transport of the substrate and theproduct, two systems were considered: ProU, which isdependent on ATP and able to introduce eithercrotonobetaine or L-carnitine into the cell in a symportmanner, and CaiT, which is not dependent on ATP, andwhich operates as an antiport of crotonobetaine/L-carnitine allowing the production of L-carnitine fromcrotonobetaine with no need for energy. The firsttransporter is fully operational under osmotic stress(Canovas et al., 2003c), together with ProP, anothertrimethylammonium/H+ antiporter, and also energy-dependent. The CaiT transporter operates fully when thecarnitine metabolism has been induced in the presence ofeither L-carnitine or crotonobetaine. The reactions thatwere taken into account are presented in Figs. 1 and 2and listed in Appendix A. The model was able to describethe process associated with the entry of the substrate and

the exit of the product. Moreover, both metabolismmodels were integrated so that growth and maintenanceof the cells together with the biotransformation processcould be shown, in agreement with experimental results.The distribution of intracellular fluxes is shown in Fig. 3and the most important results are summarized below:

(a)
The flux from fumarate to succinate compared withthat of other reactions in the TCA cycle wasextremely high since, under anaerobiosis, the TCAflux is inhibited to decrease the extend of NADHreduction. Nevertheless, a high flux of fumaratereduction is necessary to assimilate the high reduc-tion power of the C source, glycerol.
(b)
The enormous flux through the lower part ofglycolysis and then towards acetate and lactatecompared with the TCA cycle and anabolic fluxeswas a consequence of an anaerobic process. Acetateis necessary to generate energy without NAD+
reduction, whereas lactate to regenerate the NADHformed during the glycerol dissimilation. Moreover,the flux through TCA must be very low to reduce thegeneration of NADH and the anabolic fluxes arealso low as a consequence of the depletion of ATP.


3.2. ATP balance for E. coli growth and L-carnitine

production during the biotransformation

Based on stoichiometric calculations and applying theMFA, a balance on ATP was performed by taking intoconsideration the MFA around the complete model anddetermining the ATP consumed by any of the metabolicparts considered. Glycerol dissimilation generated en-ergy equivalents in terms of ATP from ADP, one of theproducts of the metabolism. The fractions of ATP usedfor various processes in the metabolism are summarizedin Table 2. More than half of the energy was possiblywasted in a futile cycle associated with the entrance ofcrotonobetaine by means of ProU, while the transporterCaiT (without using up energy) is able to carry L-carnitine into the cell with the simultaneous excretion ofcrotonobetaine, generating no L-carnitine. Finally, toclose the cycle, L-carnitine is excreted by diffusion, andthe final result is the consumption of ATP. Figs. 4A–Cdepict the biotransformation with and without con-sumption of ATP and the use of ATP in the futile cyclewith no L-carnitine production.

3.3. Topological analysis

According to the topological analysis, the modelpresented 11 total degrees of freedom, with nounbalanced reactions and unused transformation steps,and 11 elementary flux modes, the most representativeof which are shown in Figs. 4, 5 and 7. Elementaryfluxes were obtained principally for the consumption ofglycerol to produce energy in the form of ATP; to closethe balance, the ATP was consumed by the ATPasesystem. It may be remarked that these solutions are idealresponses of the system but they are extreme solutions.The real solution would be a linear combination of allthe above possibilities, which illustrate the possiblesolution space of the system. The maximal yield of ATPon glycerol differed, depending on the final product ofglycerol dissimilation and on the energy yields, and it isevident that the yields of carnitine production are alsoaffected by the final product of glycerol dissimilation(Table 3), as long as crotonobetaine is taken up usingthe energy-driven reaction of ProU. Possible productsare CO2, acetate or lactate. When one mole of glycerol

Table 2

Distribution of ATP consumption in the central and carnitine

metabolism

Process % ATP

Futile cycle 58

L-carnitine production 25

Biomass production 14

Other metabolic processes 3

was consumed to produce CO2, two moles of ATP wereobtained. The reducing power generated needed sevenfumarate moles as electron acceptor (Fig. 5A). Second,when one mole of glycerol was consumed, producingone mole of acetate, three fumarate moles wereconsumed to cancel the reducing power used (Fig. 5B).This means that the excretion of acetate allows for anunchanged yield of energy on glycerol, while therequirements for fumarate reduction are substantiallylower. In the third case, where lactate is produced, onlyone mole of ATP is produced with the consumption ofone mole of fumarate (Fig. 5C). Excretion of lactatethus involves lower requirements for fumaratereduction and a very low yield of energy on glycerol.The maximal yields of carnitine production are sum-marized in Table 3. The experimental values resulted ina yield of L-carnitine on glycerol of 0.36mole/mole,which meant that the L-carnitine yield could beincreased 3–6 times.

3.4. Starting point of the biotransformation. Process with

resting cells

When the concentration of the substrate was muchhigher than the product (e.g., at the beginning of thebiotransformation process), no energy was devoted tothe transport of the substrate. In this case, ProUprobably does not work and CaiT takes a mole ofcrotonobetaine in antiport with a mole of L-carnitine.Thus, the biotransformation proceeds and the relation-ship would be 1 crotonobetaine:1 L-carnitine in anideal case reaching an equilibrium with 50% maximumconversion. The experimental results using restingcells coincided with model predictions, since biotrans-formations resulted in a 50% conversion of crotonobe-taine into L-carnitine. This yield is based onthe equilibrium constant of the hydration reaction,Keq ¼ 1 (Elssner et al., 2001). From this point thebiotransformation could only transform crotonobetaineinto L-carnitine until a 50% yield. In contrast, higheryields were found because ProU transports crotonobe-tine in an irreversible and ATP-dependent way. Sohigher concentrations inside the cell could be reachedand higher yields found. Therefore, higher conversionrates can only be achieved with the aid of ProU activity(Fig. 6).

Another elementary flux mode that has to beconsidered is that which enables cell growth andmaintenance (Fig. 7). For this to happen, underanaerobiosis, 4.14 moles of glycerol was used to produce2.86 moles of CO2 and the reducing power required19.43 moles of fumarate to accept the electronsgenerated in the process in order to reach a flux of 1moles of biomass. These fluxes were calculatedfrom those presented in Fig. 7 and then referredto the external volume. See the Flux analysis of the

ARTICLE IN PRESS

Fig. 4. The most representative elementary flux modes obtained from the model related to the energy devoted to the carnitine metabolism. Numbers

denote fluxes in mM/h of intracellular volume. (A) Representation of the dissimilation of glycerol and the final product CO2. Two moles of ATP and

six moles of NADH per mole of glycerol were generated in the process and consumed by the carnitine metabolism and the fumarate reduction,

respectively, in order to close the balance. (B) Representation of the carnitine synthesis without energy consumption; thus, crotonobetaine is introduce

by CaiT inside the cell, biotransformed into L-carnitine and excreted also by CaiT. (C) Representation of the dissimilation of glycerol and the final

product CO2. Two moles of ATP and six moles of NADH per mole of glycerol were generated in the process and consumed by the carnitine futile

cycle and the fumarate reduction, respectively, in order to close the balance.


ARTICLE IN PRESS

Table 3

Theoretical maximal yields of carnitine synthesis when different final

products from glycerol and fumarate dissimilation (CO2, acetate and

lactate) are obtained and the experimental result obtained using a high

cell density reactor

Fermentation product YLc/glyc YLc/Fum

CO2 2 1/3.5

Acetate 2 1/1.5

Lactate 1 1

Experimental 0.3 1/1.7


biotransformation of crotonobetaine into L-carnitinesection in Materials and Methods for more details in thecalculations.

In Fig. 8, the link of both metabolisms in E. coli at thelevel of the CoA pool and the ATP level is shown. Theresults obtained with the model matched experimentalresults and indicated points where metabolic improve-ment could be achieved. The most representativeelementary flux modes within the developed model werethose related with (a) glycerol dissimilation to generateATP with three final products (CO2, lactate andacetate), (b) the production of L-carnitine (with orwithout the use of energy, showing the importance ofATP, since the lower the energy used the less L-carnitineproduced) and (c) the fact that there exists a futile cyclerelated to the existence of two transporters for thesubstrate and product (CaiT and ProU).

When a balance of ATP was performed, 58% of ATPwas consumed in the mentioned futile cycle (Table 2).The possible waste of energy was associated to theentrance of crotonobetaine and/or L-carnitine, affectingthe yield of L-carnitine production. In this process,under anaerobiosis, energy is used by transporter ProUwhile transporter CaiT is simultaneously acting. Thus aspreviously mentioned, the entrance of either crotonobe-taine as substrate (always at higher concentrations thanL-carnitine), or of L-carnitine (the product of thebiotransformation), generates a waste of energy, sincetransporters do not recognize or show any preferencefor either of them. This suggests a way of applyingmetabolic engineering strategies to inhibit the activity ofCaiT or ProU in order to favour the entrance ofsubstrate and the production of the product. Genedeletion has been shown to be suitable in certainconditions to improve the system response (Zhao etal., 2004). These results show that two transportersworking for some time in the opposite direction andwasting energy might not be suitable for cell metabolicfunctioning. When the carnitine metabolism is inducedunder anaerobiosis so as to perform the biotransforma-tion, besides the required enzyme activities, CaiD andCaiB (Fig. 1A), CaiT is also expressed for the transportof trimethylammonium compounds. In this case the

carnitine metabolism accepts electrons from the anae-robic metabolism (Kleber, 1997). However, ProU isexpressed when the cell is under osmotic stress toaccumulate trimethylammonium compounds inside thecell (L-carnitine and crotonobetaine) as a way ofprotecting itself from the extreme osmoticsituation (Verheul et al., 1998). When the cellcarnitine metabolism has been induced, ProU is alsoexpressed at a basal level, and so a combined transportof L-carnitine/crotonobetaine is affected by CaiT andProU during the biotransformation process (Canovaset al., 2003c).

In addition, experimental and model results con-firmed that, as a result of ATP cell content shortages,the transport of both the substrate (crotonobetaine) andthe product (L-carnitine) decreased. In fact, we havecalculated a production of one mole of L-(-)carnitinesynthesized from one mole of crotonobetaine, consum-ing 1 ATP mole, but only 1.13 ATP moles are availablefor L-(-)carnitine synthesis (see Appendix B). Therefore,the cell is allowed to carry out this biotransformationbut the process almost leads to ATP depletion.However, CaiT has been shown to work in an antiportmanner without the need for ATP (Jung et al.,2002), and so, in transport processes, only ProU isdependent on ATP. Furthermore, during resting cellprocesses the importance of the cell-stored material forenergy and intermediary production was also obvious,since the level of acetyl-CoA was maintained, withparallel high activities of the enzymes ICL and PDH(Canovas et al., 2003b), suggesting that cell-mainte-nance compounds and energy are synthesized fromstored material. Besides this, in the present work we sawthat cell metabolism was hardly devoted to the produc-tion of new cells, since a small amount of glycerol wasused for cell growth during the biotransformation(Fig. 7). Moreover, in a previous work using restingcell processes, cell ATP levels fell quickly from thestart (Canovas et al., 2003b). Therefore, production ofL-carnitine is bound to the use of energy, which acts as alimiting factor.

From the work carried out, it was demonstrated thatthe model of the link between the carnitine metabolismand the central metabolism generated outputs consistentwith the experimental results. Experiments with theoptimal values of the macroscopic magnitudes for thebiotransformation of crotonobetaine into L-carnitine,such as glycerol, crotonobetaine and the dilution rate ofthe high cell density reactor, were carried out. The datasummarized in Table 1 show that experimental resultscoincided with those simulated in different conditionsusing this model, meaning that the model matched thecell behaviour adequately. Moreover, simulation al-lowed certain key metabolic points to be identified,where the cell metabolism and thus the L-carnitineproduction process could be improved by metabolic

ARTICLE IN PRESS

Fig. 5. The most representative elementary flux modes related to energy production obtained from the model representing the consumption of

glycerol to produce CO2, acetate and lactate. Numbers denote fluxes in mM/h of intracellular volume. (A) Representation of the dissimilation of

glycerol with the final product, CO2. Thus, two moles of ATP and six moles of NADH per mole of glycerol were generated in the process and

consumed by the ATPase complex and fumarate reduction, respectively, in order to close the balance. (B) Representation of the dissimilation of

glycerol with the final product, acetate. In this case two moles of ATP and three moles of NADH per mole of glycerol were generated in the process

and consumed by the ATPase complex and the fumarate reduction, respectively, in order to close the balance. (C) Representation of the dissimilation

of glycerol with the final product, lactate. One mol of ATP and one mol of NADH per mol of glycerol were generated in the process and consumed

by the ATPase complex and fumarate reduction, respectively, in order to close the balance.


engineering. The next step is to obtain E. coli mutantswith the optimal modifications in the key enzymes and/or the carriers of the L-carnitine metabolism. This will

prove the theoretical results obtained, since the modelshows that in the steady state the production of L-carnitine could be nearly six times as high as the

ARTICLE IN PRESS

Fig. 6. L-carnitine yields when glycerol is assimilated to generate as the

final product. Numbers denote fluxes in mM/h of intracellular volume.

(A) CO2 or acetate: then two moles of L-carnitine were obtained per

mollecule of glycerol. (B) Lactate: then one mole of L-carnitine was

obtained per mole of glycerol.


production experimentally obtained. Beside this, thebalance of ATP obtained demonstrated that in order toproduce L-carnitine from glycerol metabolic engineeringstrategies should be taken, since the experimental value

for YLc/glyc was 0.36mol/mol in growing cell systems wasfar from the model output (nearly six times this value).According to the results shown, 58% ATP was used in afutile cycle related with the L-carnitine production sincesome was used in the symport carrier (ProU), while theCaiT carrier worked in an antiport manner forcrotonobetaine/L-carnitine transport. The result was awaste of ATP that could be avoided if either CaiT orProU was knocked out from the genome of the cell(see Fig. 1A). On the other hand, 25% ATP was used forL-carnitine synthesis while 14% was used for biomassand 3% for other processes (Table 2). This is animportant fact that needs to be considered forcell optimization, since a high ATP content is used forcell growth that could be used for the biotransformationprocess. Thus, one solution to improve the processwould be a CaiT or ProU knock out mutantand possibly the use of resting cells to avoid the useof energy for cell growth during the biotransformation.The results obtained from the model showed thatthe use of resting cells always meant 1.52 times moreL-carnitine than in growing cells systems. In fact, theexperiments performed showed that the growing cellsystems always allowed 25% L-carnitine conversion,whereas resting cell systems generated 50% conversion.Thus, the cause for the low L-carnitine productionmight be the high biomass production, which consumes14% of the ATP in growing cell systems, apart from theATP used in the above-mentioned futile cycle (Figs. 4and 7), which suggests the need for strain metabolicimprovement.

4. Conclusions

We have presented the design and experimentalvalidation of a model, which, for the first time, linkscarnitine metabolism at the level of ATP and inter-mediaries with glycolysis reactions, the pentose-phos-phate pathway and the rest of the metabolic pathwaysintroduced. The results provided the first evidence todemonstrate the need for high levels of ATP to maintainmetabolite transport and biotransformation. The mod-eling results also demonstrated that there is a futile cyclefor ATP due to the simultaneous operation of thetrimethylammonium compound carriers, CaiT andProU. However, in order to better demonstrate theexistence of this futile cycle, deletion of the caiT geneand observation of the desired effect are necessary. Thisis why we are currently engaged in obtaining both CaiToverexpressing and knock out mutants so as to fullyprove what we are claiming. Furthermore, in order toimprove the process of biotransformation, restingprocesses as well as CaiT knock out mutants wouldyield a more efficient system which could produce either6 or 1.52 times as much L-carnitine production from

ARTICLE IN PRESS

Fig. 7. Glycerol dissimilation to biomass synthesis without carnitine metabolism expression. Numbers denote fluxes in mM/h of intracellular volume

and they were normalized to reach 1mM/h of biomass.


ARTICLE IN PRESS

Fig. 8. Link between the central and carnitine metabolisms of E. coli. ACK, acetate kinase; ACS, acetyl-CoA synthetase; CS, citrate synthetase; CTe-,

electron transport chain; ICDH, isocitratedehydrogenase; ICL, isocitrate lyase; LDH, lactate dehydrogenase; PEP, phosphoenolpyruvate; PEPCK,

PEP carboxykinase; PEPCX, PEP carboxylase; PFL, pyruvate formate-lyase; PK, pyruvate kinase; PTA, phosphotransacetylase. CaiT, L-carnitine/

crotonobetaine protein transporter; CaiB, acetyl-CoA/CoA transferase; CaiC, crotonobetaine or L-carnitine, CoA ligase; CaiD, enoyl-CoA

hydratase D-carnitine racemase activity and ProU betaines energy-dependent transporter (Canovas et al., 2003b; Karp et al., 2004).


crotonobetaine or D-carnitine as in the present condi-tions. Thus, with a CaiT knock out mutant, energywould be dedicated to getting the substrate into the cellusing ProU. However, the product must leave the cell byany means, such as cell permeabilization or diffusion.The aim of further work, therefore, would be to studythe possibility of increasing crotonobetaine biotransfor-mation into L-carnitine by using permeabilized cells ofE. coli strains. By permeabilizing the cell, the substrateuptake and product efflux controlled by both the outermembrane in E. coli and cell envelope diffusion(Canovas et al., 2003c), along with the transport systemitself (ProU), could be improved and so lead to a higherdegree of substrate conversion using a CaiT deletionmutant. Moreover, a ProU deletion mutant would meanless ATP used in the transport process and moreredirected to activating the substrate which generatescrotonobetainyl-CoA to be transformed into L-Carniti-nylCoA (Fig. 1A). However, a lot of work remains,since certain enzymes of the L-carnitine metabolismneed to be studied in depth (e.g., CaiC, and CaiB,with trimethylammonium/CoA ligase and/CoA trans-ferase activities, respectively) before we can fullyunderstand the L-carnitine function and role in E. coli.

Furthermore, the connection of both metabolismssketched in Figs. 1, 2 and 8 suggests the existence ofcontrol points, not only at the central but also at thecarnitine metabolism, where it would be possible to actto redirect the metabolic fluxes (i.e., energy metabolism).Therefore, future studies to optimize the biotransforma-tion should also be addressed at redirecting themetabolic fluxes towards an increase in energy levelsand the levels of metabolites required for biotransfor-mation. In summary, the step that has been taken in thiswork will contribute to the optimization of themetabolism of L-carnitine in E. coli to increase itsproduction.

Acknowledgments

This work has been supported by CICYT projectsBIO2002-04157-C02-01 and SENECA-CARM PB/10/FS02. We would like to thank Prof. H-P. Kleber(University of Leipzig, Germany) for valuable discus-sions. Biosint S.p.A. (Italy) is also acknowledged for thekind gift of the substrate.


Appendix A

List of reaction stoichiometries for the steady-state flux model according to ECOCYC (Karp et al., 2004) andNeidhardt et al. (1996). The cometabolites, amp, adp and atp, are treated as external.

Substances crossing the boundaries of the system were: glycerol, acetate, formiate, lactate, biomass, o2, co2, protons,h2o,L-carnitine, crotonobetaine, fumarate and succinate (the last two by symport):

so4 þ hþ adpþ p so4 þ atpþ h2o

2pþ hþ adp pþ atpþ h2o

nh4 þ 3 hþ 3adpþ 3p nh4 þ 3atpþ 3h2o

crþ adpþ p crþ atpþ h2o

Emden– Meyerhof– Parnas pathway:

g6p2g1p

g6p2f6p

f6pþ p fdpþ h2o

fdp2dhapþ gap

dhap2gap

gapþ pþ nad2pgpþ nadhþ h

pgpþ adpþ h23pgþ atp

3pg22pg

2pg2pepþ h2oþ h

pepþ adpþ h! pyrþ atp

pyrþ coaþ nad! accoaþ co2 þ nadh

Pentose-phosphate pathway:

g6pþ nadpþ h2o! 6pgþ nadphþ 2h

6pgþ nadp! ru5pþ nadphþ co2

ribu5p2rib5p

ribu5p2xyl5p

xyl5pþ rib5p2gapþ sed7p

gapþ sed7p2e4pþ f6p

xyl5pþ e4p2f6pþ gap

Tricarboxylic acid cycle:

oacþ accoaþ h2o! isocitþ coaþ h

isocitþ nadp2akgþ nadphþ co2


akgþ coaþ nad! succoaþ nadhþ co2

succoaþ adpþ p2sucþ atpþ coa

malþ nad2oacþ nadhþ h

Phosphoenolpyruvate carboxylase:

pepþ co2 þ h2o! oacþ pþ h

Amino acid synthesis:

pyrþ lglut2lalaþ akg

lala2dala

2h2oþ co2 þ glnþ 2atp2carpþ lglutþ pþ 2adpþ 3h

2lglutþ accoaþ atpþ nadphþ h2o! ornþ akgþ coaþ adpþ pþ nadpþ acetateþ h

ornþ carpþ aspþ atpþ h2o2argþ fumþ ampþ 3pþ 2h

aspþ nh4 þ atpþ h2o2asnþ 2hþ ampþ 2p

oacþ lglut2aspþ akg

so4 þ 2atpþ 4nadphþ 2h2sulfideþ 4nadpþ 2adpþ 2pþ 2h2o

serþ accoaþ sulfideþ h2cysþ coaþ acetate

nh4 þ akgþ nadphþ h2lglutþ nadpþ h2o

lglut2dglut

lglutþ nh4 þ atp2glnþ adpþ pþ h

serþ thf2glyþmethf þ h2o

prppþ glnþ atpþ 2nadþ 5h2o! hisþ aicarþ akgþ 2nadhþ 7hþ 5p

aspþ atpþ 2nadphþ 2h2homþ adpþ pþ 2nadp

thrþ pyrþ nadphþ lglutþ 2h! ileuþ nh4 þ nadpþ h2oþ co2 þ akg

homþ succoaþ cysþmythf þ h2o2metþ coaþ sucþ pyrþ nh4 þ hþ thf

aspþ pyrþ lglutþ succoaþ atpþ 2nadphþ h! dipimþ akgþ sucþ coaþ adpþ 2nadpþ p

2pyrþ nadphþ 2h2akiþ co2 þ nadpþ h2o

akiþ lglutþ accoaþ h2oþ nad2leuþ akgþ coaþ nadhþ hþ co2

dipimþ h2lysþ co2

pepþ e4pþ h2oþ h! dahpþ p

dahpþ pepþ nadphþ atp! choþ adpþ 3pþ nadpþ h2oþ h

choþ lglutþ h! phenþ akgþ co2 þ h2o


lglutþ atpþ 2nadphþ 2h! proþ adpþ pþ h2oþ 2nadp

3pgþ lglutþ nadþ h2o2serþ akgþ pþ nadhþ 2h

homþ atpþ h2o2thrþ adpþ pþ h

choþ glnþ prppþ ser! trypþ 2pþ co2 þ gapþ lglutþ hþ pyrþ h2o

choþ lglutþ nad! tyrþ akgþ co2 þ nadh

akiþ lglut2valþ akg

Nucleotide metabolism:

rib5pþ atp! prppþ ampþ h

prppþ 2glnþ glyþ 5atpþ aspþ fthf þ 4h2oþ co2! aicarþ 5adpþ 7pþ 2lglutþ thf þ fumþ 9h

aicarþ fthf2thf þ impþ h2o

impþ aspþ atp2ampþ adpþ pþ fumþ 2h

ampþ atp22adp

h2oþ atp! adpþ pþ h

impþ nadþ atpþ glnþ 3h2o2gmpþ ampþ nadhþ 2pþ 4hþ lglut

carpþ aspþmenaquinoneþ prpp2umpþmenaquinoneh2 þ co2 þ 3p

umpþ atp2udpþ adp

udpþ atp2utpþ adp

utpþ glnþ atpþ h2o! ctpþ adpþ pþ 2hþ lglut

ctpþ adp2cdpþ atp

cdpþ adp2cmpþ atp

adpþ atpþ nadphþ h2datpþ adpþ h2oþ nadp

gmpþ 2atpþ nadphþ h2dgtpþ 2adpþ nadpþ h2o

cdpþ atpþ nadphþ h2dctpþ adpþ nadpþ h2o

udpþ 3atpþ 2nadphþ h2oþmethf2dttpþ 3adpþ 2nadpþ 2pþ thf

Synthesis of lipid precursors:

dhapþ nadhþ h2glycpþ nad

glycpþ serþ ctpþ h2o2ethohaminpþ cmpþ co2 þ 2pþ h

5accoaþ 4atpþ 8nadphþ 3hþ h2o! fatty10;0 þ 5coaþ 4adpþ 8nadpþ 4p


5accoaþ 5atpþ 10nadphþ 4hþ h2oþ succoaþ pyr! fatty13;0 þ 6coaþ 5adpþ 10nadpþ 5pþ oac













8accoaþ 8atpþ 16nadphþ 7hþ h2oþ succoaþ pyr! fatty190 þ 9coaþ 8adpþ 16nadpþ 8pþ oac

Polymerization reactions:

b1lalaþ b2argþ b3asnþ b4aspþ b5cysþ b6glnþ b7lglnþ b8glyþ b9hisþ b10ileþ b11leuþ b12lysþ b13metþ

b14phenþ b15proþ b16serþ b17thrþ b18trpþ b19tyrþ b20val þ b21atpþ b22h2o! proteinþ b21adpþ b21pþ b21h

w1ampþ w2gmpþ w3umpþ w4cmpþ w5atpþ w1h2o! rnaþ w5adpþ w5pþ w5h

d1datpþ d2dgtpþ d3dttpþ d4dctpþ d5atp! dnaþ d5adpþ d5pþ d5h

�1fatty10;0 þ �2fatty12;0þ�3fatty13;0þ�4fatty13;1þ�5fatty14;0þ�6fatty14;1þ�7fatty15;0þ�8fatty15;1þ�9fatty16;0þ�10fatty16;1þ

�11fatty17;0þ�12fatty17;1þ�13fatty18;0þ�14fatty18;1 þ �15fatty19;0 þ �16glycpþ �17etamp! lipid þ �18h2o

ðf1þ1Þg6pþ f1atpþ f1h2o! polysaccharidesþ f1adpþ f1hþ 2f1p

2f6pþ 2glnþ 2accoaþ 7atpþ pepþ nadphþ lalaþ dglutþ dipimþ dalaþ 5h2o! murunitþ 2coaþ 2lglutþ

6adpþ ampþ nadpþ 1pþ 9h

g1murunit! mureinþ g2h2oValues of bi, wi, di, ei, fi and gi according to the biomass composition.

Virtual biomass formation reaction:

a1proteinþ a2rnaþ a3dnaþ a4lipidsþ a5polysaccharidesþ a6mureine! biomass

Values of ai according to the biomass composition.

Regeneration of C1-transfer cometabolites:

thf þ atpþ nadhþ co22fthf þ adpþ pþ nad

thf þ co2 þ 3nadhþ 3h2mythf þ 3nadþ 2h2o

nadhþ nh4 þ co2 þmethf2nadþ glyþ thf


Anaerobic respiration:

pyrþ coa! formateþ accoa

pyrþ nadhþ h2lactateþ nad

formateþ h! co2 þ h2

pþ accoa2acpþ coa

acpþ adp2acetateþ atp

Glycerol dissimilation:

glycþ atp2glycpþ adpþ h

Fumarate reduction:

fumþ eah22sucþ ea

fumþmenaquinoneh22sucþmenaquinone

fumþ h2o2mal

nadhþ hþ ea2nadþ eah2

Carnitine metabolism:

lcþ crcoa2lccoaþ cr

lccoa2crcoaþ h2o

Appendix B

B.1. Maintenance coefficients

An ATP balance of the E. coli central and carnitine metabolism was performed to check that the results obtainedcoincided with the model’s output. In the analysis the following processes are considered for the ATP balance:

1.
Catabolism of the substrate, assuming maximal yield for glycerol degradation:
CH8=3OþH2O�!rS

CO2 þ23ATPþ 7

3‘‘H2’’.

2.
Reactions leading to the production of biomass. A membrane cell-recycle reactor in which biomass is produced wasused to compensate the endogenous metabolism or cell decay and to maintain the stationary state.
3.
Specific maintenance requirements of ATP. 4. Reactions leading to product formation. In these reactions, the number of ATP moles available for product
formation is unknown.

CRþ a3;1ATPþH2O�!rP

LC.

The ATP balance results in

2

3rS �

rX

YmaxATP

�mATPCX � a3;1rP ¼ 0,

where:Degradation rate of glycerol:

rS ¼ 56:72 ðmole=m3 hÞ.

Rate of cell formation:

rX ¼ 4:88 ðmole� CÞðDW=m3 hÞ.


Coefficient of ATP consumed for cell growth:

YmaxATP ¼ 0:33 ðmole� CÞDW=mole.

Coefficient of ATP consume for cell maintenance:

mATP ¼ 0:00055 mole=ðmole� CÞDW h.

The cellular concentration:

CX ¼ 487:91 ðmole� CÞDW=m3.

The rate of L-carnitine synthesis:

rP ¼ 20:51 mole=m3 h.

Then

a3;1 ¼rS � ðrX=Ymax

ATPÞ �mATPCX

rP¼ 1:13ATP.

Thus, 1.13 ATP moles are available for L-(-)carnitine synthesis. It is known that 1 mole of ATP per 1 mole of L-carnitineis necessary and so the cell is allowed to carry out this biotransformation even though the process comes very close to ATPdepletion (Canovas et al., 2003b). Thus, a probable limiting factor for this bioprocess is the ATP concentration.

In the following previous values are calculated:

1.
rs the degradation rate of glycerol is known experimentally:
rS ¼ 56:72mM

h¼ 56:72

mole

m3 h.

2.
rX when the high cell density reactor is in the steady state the rate of cell formation is the same as for cell decay, so:
rX ¼ CXme ¼ 487:91� 0:01ðmole� CÞDW

m3 h

¼ 4:88ðmole� CÞDW

m3 h.

YATPmax was taken from Roels (1983)

YmaxATP ¼ 0:33

ðmole� CÞDW

mole.

3.
In the case of mATP, the value from Roels (1983) was not considered, since it was not compatible with high cell densitycultures. Therefore, this coefficient was calculated using data from Canovas et al. (2003b) with cells performing thebiotransformation in resting state. From 5 to 24h no carnitine transformation was detected and the cellularconcentration was, approximately constant. It was assumed that all the ATP was used for cellular maintenance:
mATP ¼ 0:00684mole ATP

m3 h

1

12:54

m3

ðmole� CÞDW

¼ 0:00055mole

ðmole� CÞDW h.

4.
CX, from experimental data it was known that
CX ¼ 12:183g DW

L

1

24:97

ðmole� CÞDW

g DW� 103

dm3

m3

¼ 487:91ðmole� CÞDW

m3 h.

5.
Finally, rP, the rate of L-carnitine synthesis was known from experiments:
rS ¼ 20:51mM

h¼ 20:51

mole

m3h.


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Pediococcus acidilactici. Appl. Environ. Microbiol. 69, 2237–2244.

Zientz, E., Six, S., Unden, G., 1996. Identification of a third secondary

carrier (DcuC) for anaerobic C-4-dicarboxylate transport in

Escherichia coli: roles of the three Dcu carriers in uptake and

exchange. J. Bacteriol. 178, 7241–7247.

Further Reading

Jung, H., Jung, K., Kleber, H.P., 1990. L(-)carnitine uptake by

Escherichia coli. J. Basic Microbiol. 30, 507–514.

Documents

Model of central and trimethylammonium metabolism for optimizing l-carnitine production by E. coli