The pyruvate dehydrogenase complex of Corynebacterium glutamicum: An attractive target for metabolic...

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Journal of Biotechnology xxx (2014) xxx– xxx

Contents lists available at ScienceDirect

Journal of Biotechnology

jo u r n al homep age: www.elsev ier .com/ locate / jb io tec

he pyruvate dehydrogenase complex of Corynebacteriumlutamicum: An attractive target for metabolic engineering

ernhard J. Eikmannsa, Bastian Blombachb,∗

Institute of Microbiology and Biotechnology, University of Ulm, 89069 Ulm, GermanyInstitute of Biochemical Engineering, University of Stuttgart, 70569 Stuttgart, Germany

r t i c l e i n f o

rticle history:eceived 4 November 2013eceived in revised form2 December 2013ccepted 16 December 2013vailable online xxx

eywords:orynebacterium glutamicumyruvate dehydrogenase complex

a b s t r a c t

The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative thiamine pyrophosphate-dependent decarboxylation of pyruvate to acetyl-CoA and CO2. Since pyruvate is a key metabolite ofthe central metabolism and also the precursor for several relevant biotechnological products, metabolicengineering of this multienzyme complex is a promising strategy to improve microbial production pro-cesses. This review summarizes the current knowledge and achievements on metabolic engineeringapproaches to tailor the PDHC of Corynebacterium glutamicum for the bio-based production of l-valine,2-ketosiovalerate, pyruvate, succinate and isobutanol and to improve l-lysine production.

© 2014 Elsevier B.V. All rights reserved.

etabolic engineeringyruvateuccinate-Ketoisovalerate-Valine-Lysine

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sobutanol

. Introduction

Industrial biotechnology is regarded as one of the current keyechnologies with an expected market volume of more than $300illion by 2030, only considering microbial, non-biopharmaceuticalroducts (Festel, 2010; Neubauer, 2011). Limited fossil resourcesepresent the main driver for the development of cost effec-ive processes for the production of chemicals from renewableeedstocks. Microbial fermentation is an established industrialpplicable technology and advances more and more to an economiceasible alternative to crude-oil based production processes (Bozellnd Petersen, 2010). Accordingly, the portfolio of biotechnologicalroducts increases rapidly (reviewed in Straathof, 2013). Thesechievements are occasionally possible due to the steadily increas-ng knowledge on metabolism and pathway regulation of industrial

Please cite this article in press as: Eikmanns, B.J., Blombach, B., The pyruvate dtarget for metabolic engineering. J. Biotechnol. (2014), http://dx.doi.org/10

elevant organisms. However, metabolic engineering approachesery often represent defined applications for one target productnd are not generally suited for other applications or further target

∗ Corresponding author at: Institute of Biochemical Engineering, University oftuttgart, Allmandring 31, 70569 Stuttgart, Germany. Tel.: +49 711 685 64549;ax: +49 711 685 65164.

E-mail address: [email protected] (B. Blombach).

168-1656/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jbiotec.2013.12.019

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products. Most relevant microbial products (native or not) derivefrom central metabolites, i.e., share a common precursor such asacetyl-CoA, oxaloacetate or pyruvate. Therefore, engineering thecentral metabolism is attractive not only for a certain product butmoreover for the complete product class derived from the givencentral metabolite.

Pyruvate is a central intermediate of all organisms and repre-sents the turntable distributing carbon into amino acid synthesis,the citric acid cycle (oxidative or reductive), fatty acid synthesis,anaplerosis or is used for regeneration of NAD+ by the formation oflactate under anaerobic conditions. Under ordinary aerobic growthconditions, a major pyruvate converting enzyme in most of theknown microorganisms is the pyruvate dehydrogenase complex(PDHC), which represents an attractive target for metabolic engi-neering.

Corynebacterium glutamicum is a Gram-positive facultativeanaerobic organism that grows on a variety of sugars, organicacids, and alcohols as single or combined carbon and energysources (Eggeling and Bott, 2005; Liebl, 2006; Nishimura et al.,2007; Takeno et al., 2007). The organism is generally regarded as

ehydrogenase complex of Corynebacterium glutamicum: An attractive.1016/j.jbiotec.2013.12.019

safe (GRAS status) and is the workhorse for large scale produc-tion of amino acids, such as l-glutamate (world-wide productionby fermentation > 2.5 million t/a) and l-lysine (>1.9 million t/a)(Eggeling and Bott, 2005; Takors et al., 2007; Ajinomoto, 2013).

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http://www.sciencedirect.com/science/journal/01681656

http://www.elsevier.com/locate/jbiotec

mailto:[email protected]


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n order to improve the production performance by metabolicngineering approaches, the central carbon metabolism, the phys-ology and the regulation of relevant pathways of C. glutamicum

ere analyzed in detail and genetic tools as well as systems biol-gy approaches on the ‘omics’ level have been developed andmployed (overviews in Kirchner and Tauch, 2003; Eggeling andott, 2005; Sauer and Eikmanns, 2005; Wendisch et al., 2006;ott, 2007; Takors et al., 2007; Burkowski, 2008; Brinkrolf et al.,010; Becker and Wittmann, 2011; Teramoto et al., 2011; Vertèst al., 2012). Recent studies explored the usefulness of C. glu-amicum for the production of other commodity chemicals, suchs the biofuels isobutanol and ethanol (Inui et al., 2004; Smith


t al., 2010; Blombach and Eikmanns, 2011; Blombach et al., 2011),he diamines cadaverine and putrescine (Mimitsuka et al., 2007;chneider and Wendisch, 2010, 2011; Kind et al., 2010a,b; Kindnd Wittmann, 2011), the sugar alcohol xylitol (Sasaki et al., 2010),

ig. 1. Schematic presentation of the central carbon metabolism of C. glutamicum includi-valine and isobutanol (not native). Ellipses represent proven enzymes present in C. glutamiven in brackets. ADH (adhA), alcohol dehydrogenase A; AHAIR (ilvC), acetohydroxyacid isolaT (alaT), alanine aminotransferase; ALD (ald), acetaldehyde dehydrogenase; AvtA (avtA

fum), fumarase; GAPDH (gapA), glyceraldehyde-3P dehydrogenase; GPDH (zwf, opcA), glyase; KIVD, 2-ketoacid decarboxylase from L. lactis; LDH (ldhA), l-lactate dehydrogena

alate:quinone oxidoreductase; MS (aceB), malate synthase; ODHC (odhA, aceF, lpd), 2Cx (pyc), pyruvate carboxylase; PDHC (aceE, aceF, lpd), pyruvate dehydrogenase comparboxylase; PGDH (gnd), 6P-gluconate dehydrogenase; PGI (pgi), phosphoglucose isomerrom E. coli; PQO (pqo), pyruvate:quinone oxidoreductase; PTA (pta), phosphotransacetyl

PRESSiotechnology xxx (2014) xxx– xxx

gamma-amino butyric acid (Takahashi et al., 2012), polyhydroxy-butyrate (Song et al., 2012), the chemical chaperone ectoine (Beckeret al., 2013), carotenoids (Heider et al., 2012, 2013) and also severalorganic acids such as succinate, d-lactate and 2-ketoisocaproate(reviewed in Wieschalka et al., 2012a; Bückle-Vallant et al., 2013).

Since the common precursor of several products mentionedabove is pyruvate, the optimization of its availability has a highpotential to improve microbial production processes. Despite themajor importance of pyruvate as precursor for relevant biotechno-logical products and the central role of the PDHC in the metabolismof industrially important organisms, only the PDHC of C. glutamicumwas intensively engineered to establish a microbial platform with


completely abolished or with reduced PDHC activity useful for thebio-based production of not only pyruvate but also for its derivedproducts l-lysine, succinate, 2-ketoisovalerate, l-valine and isobu-tanol (Fig. 1). This review summarizes the current knowledge and

ng pathways for the production of pyruvate, 2-ketoisovalerate, succinate, l-lysine,icum. Rectangles represent heterologous enzymes. Abbreviations: coding genes aremeroreductase; AHAS (ilvBN), acetohydroxyacid synthase; AK (ack), acetate kinase;

), valine-pyruvate aminotransferase; DHAD (ilvD), dihydroxyacid dehydratase; FUMucose-6P dehydrogenase; ICD (icd), isocitrate dehydrogenase; ICL (aceA), isocitratese; MalE (malE), malic enzyme; Mdh (mdh), malate dehydrogenase; MQO (mqo),-ketoglutarate dehydrogenase complex; ODx (odx), oxaloacetate decarboxylase;

lex; PEP phosphoenolpyruvate; PEPCk (pck), PEP carboxykinase; PEPCx (ppc), PEPase; PK (pyk), pyruvate kinase; PntAB (pntAB), membrane bound transhydrogenasease; SDH (sdhABC), succinate dehydrogenase; TA (ilvE), transaminase B.

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Fig. 2. Illustration of the genomic organization of the lpd (cg0441), aceF (cg2421)aPg

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nd aceE (cg2466) genes encoding the E3, E2 and E1 subunits of the C. glutamicumDHC, respectively. The promoter of each gene is indicated by P. The upper numbersive the genomic localization (Kalinowski et al., 2003).

ecent achievements on engineering the PDHC of C. glutamicumor establishment and/or improvement of aerobic and anaerobicroduction processes.

. The PDHC of C. glutamicum: biochemical properties,egulation and genomic organization of the genes

The PDHC belongs to a multienzyme complex family that alsoomprises the 2-ketoglutarate dehydrogenase complex (ODHC)nd the branched-chain 2-ketoacid dehydrogenase complex. TheDHC catalyzes the oxidative decarboxylation of pyruvate to acetyl-oA and CO2 and generally is composed of multiple copies ofhree subunits: pyruvate decarboxylase (E1) catalyzes the thiamineyrophosphate- (TPP-) dependent, irreversible, oxidative decar-oxylation of pyruvate, followed by the acylation of the lipoylrosthetic group attached to the dihydrolipoamide acyltransferaseE2). The lipoic acid-containing E2 subunit catalyses the transferf the acyl group from the lipoyl group to coenzyme A (CoA). Theesulting dihydrolipoyl group is reoxidized by the flavin-containingipoamide dehydrogenase (E3), generating NADH + H+ from NAD+

reviewed in de Kok et al., 1998; Nevelin et al., 1998).In C. glutamicum, the E1, E2 and E3 subunits of the PDHC are

ncoded by aceE (cg2466), aceF (cg2421) and lpd (cg0441), respec-ively (Fig. 2). Recently, it was shown that the E1 subunit of theDHC, encoded by odhA, forms together with the E2 and E3 subunitsf the PDHC an unusual supercomplex, which possesses both PDHCnd ODHC activity (Niebisch et al., 2006; Hoffelder et al., 2010). Inontrast to the chromosomal (operon) organization in Escherichiaoli and Bacillus subtilis, the genes encoding the PDHC in C. glutam-cum are not clustered, but distributed over the genome and theyre transcribed as monocistronic genes from their own promoterFig. 2). The transcriptional start sites of the aceF and aceE genes areocated 61 bp and 121 bp upstream of the translational start (ownnpublished results; Schreiner et al., 2005). The lpd mRNA repre-ents a leaderless transcript, i.e., transcription of the lpd gene startst the translational start (Schwinde et al., 2001).

The PDHC of C. glutamicum shows a Km of about 1.7 mM foryruvate, requires Mg2+ and TPP for maximal activity, seems not toe subject for significant allosteric control, and shows in the mid-xponential growth phase a specific activity of about 30–36 mU/mgrotein when grown in minimal or complex medium containinglucose or acetate (Schreiner et al., 2005). However, in complexedium without any additional carbon source PDHC activity isore than two times higher. This activation is mediated by the

ranscriptional regulator RamB (Gerstmeir et al., 2004) whichnhances transcription of the aceE gene resulting in increased over-ll PDHC activity during growth in complex medium (Blombacht al., 2009a). Whereas the PDHC of C. glutamicum seems to be onlyeakly regulated by the growth medium, a severe influence of the

rowth phase on the specific activity was found (Schreiner et al.,005). The specific PDHC activity was highest (87 ± 3 mU/mg pro-ein) when the cells were harvested in the mid- to late exponential


rowth phase. About 8-fold lower activities were found in cells ofhe stationary phase. In view of the central position of the PDHC inhe metabolism, this regulation certainly is highly relevant for thearbon flux in C. glutamicum.

Fig. 3. Batch fermentation of C. glutamicum �aceE on CGXII medium containingglucose and acetate (Blombach et al., 2007a). �, growth; �, glucose; �, acetate; �,pyruvate; ©, l-alanine; �, l-valine.

3. Characteristics of PDHC-deficient C. glutamicum strains

Schreiner et al. (2005) identified and functionally character-ized the E1 subunit of the PDHC in C. glutamicum and showed thatthe activity of this complex is essential for growth of this organ-ism on glucose, pyruvate and lactate as carbon and energy source.This result also demonstrated that the pyruvate:quinone oxidore-ductase (PQO), which in principle can form a bypass togetherwith acetate kinase and phosphotransacetylase (see Fig. 1), is notable to provide sufficient acetyl-CoA required for growth. For thegrowth of C. glutamicum in medium containing acetate as the soleor as additional carbon and energy source, PDHC activity is dis-pensable (Schreiner et al., 2005). However, in medium containingglucose and acetate, C. glutamicum �aceE showed a characteristicbehaviour: as long as acetate was present in the medium the PDHC-deficient strains grew exponentially but did not secrete organic oramino acids into the medium. After depletion of the acetate, thestrain stopped growing but continued to metabolize glucose and itshowed a relatively high intracellular pyruvate concentration (up to26 mM; Blombach et al., 2007a,b). Furthermore, the PDHC-deficient

mutant produced 25–35 mM of pyruvate, l-valine and l-alaninefrom 222 mM of glucose (Fig. 3; Blombach et al., 2007a,b). In a set offurther investigations, this promising PDHC-deficient platform wasused to engineer C. glutamicum for the production of pyruvate andof pyruvate-derived products, such as l-valine, 2-ketoisovalerate,succinate and isobutanol and for improvement of l-lysine produc-tion (see below).

4. Growth-decoupled l-valine, 2-ketoisovalerate andpyruvate production and improvement of l-lysineproduction under aerobic conditions

l-Valine is an essential amino acid for vertebrates and it is usedfor infusion solutions, for cosmetics and as precursor for the chem-ical synthesis of herbicides (Eggeling, 2001; Leuchtenberger, 1996;Park et al., 2007). Efficient l-valine production was achieved byoverexpression of the ilvBNCE genes encoding the l-valine biosyn-thetic enzymes acetohydroxyacid synthase, isomeroreductase andtransaminase B (Fig. 1) in C. glutamicum �aceE. The ilvBNCE over-expression led to an about ten-fold lower intracellular pyruvateconcentration (26 vs. 2 mM) and a clear shift of the product spec-trum towards l-valine (Blombach et al., 2007a). The resultingstrain C. glutamicum �aceE (pJC4ilvBNCE) produced in an inter-


mittent fed-batch process up to 195 mM l-valine with a substratespecific product yield (YP/S) of 0.39 mol l-valine/mol of glucosein the production phase (Blombach et al., 2008). C. glutamicum�aceE (pJC4ilvBNCE) was further improved by deletion of the PQO

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Table 1Characteristics of relevant C. glutamicum production strains with engineered PDHC.

Strain Medium Titre (mM; g/l) YP/Sa

(mol/mol)QP

b

(mM/h)Reference

l-ValineC. glutamicum �aceE (pJC4ilvBNCE)c Minimal medium, glucose, acetate, 0.5% BHId 195; 22.8 0.39 7.9 Blombach et al. (2008)C. glutamicum �aceE �pqo(pJC4ilvBNCE)c

Minimal medium, glucose, acetate, 0.5% BHId 225; 26.4 0.52 9.5 Blombach et al. (2008)

C. glutamicum �aceE �pqo �pgi(pJC4ilvBNCE)c


C. glutamicum �aceE �pqo �pgi �pyc(pJC4ilvBNCE)c


C. glutamicum aceE A16 �pqo �ppc(pJC4ilvBNCE)

Minimal medium, glucose 714; 83.6 0.36 12.8 Buchholz et al. (2013)

2-KetoisovalerateC. glutamicum �aceE �pqo �ilvE(pJC4ilvBNCD)c

Minimal medium, glucose, acetate,l-isoleucine, l-leucine, l-valine, 0.1% yeastextract

188; 21.8 0.47 4.6 Krause et al. (2010)

C. glutamicum aceE A16 �pqo �ppc�ilvE (pJC4ilvBNCD)

Minimal medium, glucose, l-isoleucine,l-leucine, l-valine, 1% yeast extract

290; 33.7 0.33 6.0 Buchholz et al. (2013)

PyruvateC. glutamicum �aceE �pqo �ldhA �C-TilvN �alaT �avtAc

Minimal medium, glucose, acetate, l-alanine 512; 45 0.97 4.9 Wieschalka et al. (2012a)

SuccinateC. glutamicum �aceE �pqo �ldhA �C-TilvN �alaT �avtAc

Minimal medium, glucose, acetate, l-alanine 330; 38.9 1.02 5.6 Wieschalka et al. (2012b)

IsobutanolC. glutamicum �aceE �pqo �ilvE �ldhA�mdh (pJC4ilvBNCD-pntAB)(pBB1kivD-adhA)c

Minimal medium, glucose, acetate,l-isoleucine, l-leucine, l-valine, 0.5% yeastextract

175; 13.0 0.48 4.4 Blombach et al. (2011)

a YP/S is given as mol product/mol of glucose consumed.

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b QP is given as mmol product produced/h l.c YP/S and QP are given for the production phase, when acetate was completely cod BHI, brain heart infusion.

ene (pqo) and, to improve NADPH availability, by deletion ofhe phosphoglucose isomerase gene pgi (Fig. 1). These modifica-ions stepwise increased l-valine production. C. glutamicum �aceE

pqo �pgi (pJC4ilvBNCE) produced about 412 mM l-valine with YP/S of about 0.75 mol/mol glucose with a volumetric produc-ivity (QP) of about 5.6 mM/h (Table 1; Blombach et al., 2008).urther analysis of C. glutamicum �aceE �pqo �pgi (pJC4ilvBNCE)howed that inactivation of PGI is accompanied by high intracel-ular NADPH concentrations and reduced by-product formationBartek et al., 2010). The maximal theoretical YP/S of 0.86 mol/mollucose (Bartek et al., 2010) was finally obtained by additional dele-ion of the pyc gene encoding pyruvate carboxylase in C. glutamicum

aceE �pqo �pgi (pJC4ilvBNCE) (Table 1; Blombach et al., 2008).lternatively, NADPH availability was improved by introduction of

he E. coli membrane-bound transhydrogenase PntAB, catalyzinghe reversible conversion of NADH to NADPH, into a l-valine-roducing strain. Metabolic flux analyses of C. glutamicum �aceEpqo (pJC4ilvBNCE) and the derivative with plasmid-bound pntAB

xpression showed that introduction of PntAB activity resultedn a significantly reduced flux into the pentose phosphate path-

ay (PPP) and in accordance, in a significantly increased YP/S ofp to 0.92 mol l-valine/mol of glucose (Bartek et al., 2011).2-etoisovalerate is used in pharmaceutical applications and servess a substitute for l-valine and l-leucine in chronic kidney dis-ase patients (Aparicio et al., 2009, 2012). The PDHC-deficient C.lutamicum �aceE was engineered for 2-ketoisovalerate produc-ion by deletion of the transaminase B gene ilvE and additionalverexpression of the ilvBNCD genes (ilvD encodes dihydroxyacidehydratase; Fig. 1; Krause et al., 2010). 2-Ketoisovalerate produc-


ion was further improved by deletion of the pqo gene. The finaltrain C. glutamicum �aceE �pqo �ilvE (pJC4ilvBNCD) producedn intermittent fed-batch fermentations at high cell densities upo 188 mM 2-ketoisovalerate with a YP/S of about 0.47 mol/mol

ed for growth purposes.

glucose and showed a QP of about 4.6 mM/h in the overall produc-tion phase (Krause et al., 2010; Table 1).

Pyruvate has manifold application ranges, e.g., for the synthesisof chemicals and polymers or as ingredient or additive in food, cos-metics and pharmaceuticals (Li et al., 2001; Zhu et al., 2008). Basedon the �aceE �pqo double mutant, C. glutamicum was engineeredfor efficient production of pyruvate from glucose by additionaldeletion of the ldhA gene, encoding l-lactate dehydrogenase (LDH;Fig. 1), and introduction of a leaky variant of the acetohydroxyacidsynthase (�C-T IlvN), which possesses an about twofold-lower Km

for the substrate pyruvate (4.7 mM vs. 7.8 mM) and an about four-fold lower Vmax (Blombach et al., 2009b). The latter modificationabolished overflow metabolism towards l-valine, prevented thestrain for branched-chain amino acid auxotrophy and shifted theproduct spectrum towards pyruvate production. In shake-flasks,the resulting strain C. glutamicum �aceE �pqo �ldhA �C-T ilvNproduced about 190 mM pyruvate with a YP/S of 1.36 mol/mol ofglucose, however, still secreted significant amounts of l-alanine.Additional deletion of the genes encoding the transaminases AlaTand AvtA (Fig. 1) reduced l-alanine formation by about 50%. In fed-batch fermentations at high cell densities with adjusted oxygensupply during growth and production (0–5% dissolved oxygen), thenewly constructed strain C. glutamicum �aceE �pqo �ldhA �C-TilvN �alaT �avtA produced more than 500 mM pyruvate with amaximum yield of 0.97 mol/mol of glucose and a QP of 4.9 mM/h inthe production phase (Table 1; Wieschalka et al., 2012b).l-Lysine is an essential amino acid for vertebrates and widely

used as feed additive in swine and poultry production with anannual production of about 1.9 million t (Kelle, 2005; Ajinomoto,


2013). A lot of efforts have been made to improve l-lysine pro-duction by engineering the l-lysine biosynthetic pathway andoptimizing precursor and cofactor (NADPH, oxaloacetate) avail-ability (reviewed in Blombach and Seibold, 2010; Becker and

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ittmann, 2011; van Ooyen et al., 2012). Inactivation of the PDHCn the defined l-lysine producer C. glutamicum DM1729 led to anbout fourfold higher biomass-specific l-lysine yield and a morehan 40% higher YP/S. However, this strain also secreted significantmounts of l-alanine, l-valine and pyruvate into the medium, sug-esting a surplus of precursor availability and a further potential tomprove l-lysine production (Blombach et al., 2007b).

. Growth-decoupled succinate and isobutanol productionnder anaerobic conditions

Succinate has a very broad scope of application and can be useds precursor for known petrochemical bulk products, such as 1,4-utanediol, tetrahydrofuran, g-butyrolactone, adipic acid, maleicnhydride, various n-pyrrolidinones, and linear aliphatic estersreviewed in Wieschalka et al., 2012a). Employing the pyruvate-roducing strain C. glutamicum �aceE �pqo �ldhA �C-T ilvN �alaTavtA (see above and Fig. 1), Wieschalka et al. (2012b) set up

n efficient succinate production process. In this one-stage fed-atch fermentation process with C. glutamicum �aceE �pqo �ldhAC-T ilvN �alaT �avtA, biomass formation and succinate produc-

ion was combined in a single bioreactor. The process includedhree phases: (i) an aerobic growth phase on glucose plus acetate,ii) a self-induced microaerobic phase at the end of the exponen-ial growth by minimal aeration, and (iii) an anaerobic productionhase, realized by gassing the fermenter with CO2. This optimizedrocess led to growth-decoupled succinate production of morehan 330 mM with a YP/S of 1.02 mol succinate/mol of glucoseTable 1; Wieschalka et al., 2012a). Due to the inactivation of theDHC, PQO and lactate dehydrogenase (LDH), C. glutamicum �aceEpqo �ldhA �C-T ilvN �alaT �avtA does not form significant

mounts of acetate or lactate as by-products under any aerobic andnaerobic condition tested (Wieschalka et al., 2012a).

Isobutanol is an attractive biofuel which possess severaldvantages, such as a lower hygroscopy and corrosivity, a fullompatibility with existing engines and pipelines and a highernergy density, allowing safer handling and a more efficientse compared to ethanol (Dürre, 2007). Furthermore, isobutanolan serve as precursor for the production of isobutene (Machot al., 2001; van Leeuwen et al., 2012) which is used as gaso-ine additive and for the production of butyl rubber and specialtyhemicals (Gogerty and Bobik, 2010). Based on the PDHC-deficient-ketoisovalerate production strain (see above), C. glutamicum wasngineered for the production of isobutanol from glucose underxygen deprivation conditions, by inactivation of LDH and malateehydrogenase (Mdh), implementation of ketoacid decarboxyl-se (KDC) from Lactococcus lactis, native alcohol dehydrogenaseADHA), and expression of the transhydrogenase genes pntAB from. coli (Fig. 1). In closed bottles the resulting strain producedsobutanol with a YP/S of 0.77 mol/mol of glucose. In fed-batch fer-

entations with an aerobic growth phase and an oxygen-depletedroduction phase, the most promising strain C. glutamicum �aceEpqo �ilvE �ldhA �mdh (pJC4ilvBNCD-pntAB) (pBB1kivD-adhA)

ormed about 175 mM isobutanol with a volumetric productivityf 4.4 mM/h, and showed an overall YP/S of about 0.48 mol/mol oflucose in the production phase (Table 1; Blombach et al., 2011).

. Overcoming the growth-decoupled productionhenotype of PDHC-deficient C. glutamicum strains

All PDHC-deficient production strains described above show the


ame characteristic production behaviour, i.e., as long as acetatebesides glucose) is present in the medium they grow exponen-ially and do not secrete any product(s) into the medium, andnly after complete depletion of the acetate, growth-decoupled

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in CGXII minimal medium containing glucose (Buchholz et al., 2013). �, OD600; �,glucose; ♦, l-alanine; �, l-valine; ©, phosphate.

production is initiated. Engels and coworkers identified thetranscriptional regulator SugR to repress ptsG [encoding theglucose-specific phosphotransferase system (PTS) enzyme II]expression in the presence of gluconeogenic carbon sources, suchas pyruvate, citrate or acetate (Engels and Wendisch, 2007). Dele-tion of the sugR gene in C. glutamicum �aceE �pqo (pJC4ilvBNCE)in fact resulted in growth-coupled l-valine production in the pres-ence of acetate (Blombach et al., 2009c). As glucose consumption ofC. glutamicum is not negatively affected in the presence of ethanol(Arndt and Eikmanns, 2007; Arndt et al., 2008), the replacement ofacetate by ethanol also led to l-valine production during growth(Blombach et al., 2009c). Interestingly, the replacement of acetateby ethanol as well as the inactivation of sugR led to about five timeshigher glucose consumption rates and in the latter case, to about50% lower acetate consumption rates, indicating some kind of crossregulation of carbon consumption (Blombach et al., 2009c). A fur-ther alternative to overcome the non-producing growth phenotypeof PDHC-deficient C. glutamicum strains was the use of the non-PTS-sugar maltose (Krause et al., 2009). It was found that maltosein the medium led to an increase of ptsG expression, and conse-quently improved glucose consumption in the presence of acetateand resulted in growth-coupled l-valine production in mediumcontaining glucose, maltose and acetate. However, the positiveeffect on glucose consumption was only potent as long as maltosewas present in the medium (Krause et al., 2009).

7. Reduction of PDHC activity in C. glutamicum

For growth in minimal glucose medium, the PDHC-deficientproduction strains require either acetate or ethanol as essentialadditional carbon source. To overcome this requirement, Buchholzet al. (2013) very recently made use of a previously generated pro-moter library with differing promoter activities (Vasicová et al.,1999) for aceE promoter substitution. Replacement of the nativepromoter of the chromosomal PDHC E1p gene aceE by a setof mutated promoter variants led to a series of C. glutamicumstrains with gradually reduced PDHC activities and growth rates onmedium containing glucose as sole carbon source (Buchholz et al.,2013). Additional overexpression of the ilvBNCE genes resulted inl-valine overproduction by all strains. Among these strains, C. glu-tamicum aceE A16 (pJC4ilvBNCE) showed the highest l-valine yieldand was further improved by additional deletion of the pqo andthe phosphoenolpyruvate carboxylase gene (ppc). In fed-batch fer-mentations in which growth at high cell densities was stoppedby a phosphate limitation, C. glutamicum aceE A16 �pqo �ppc


(pJC4ilvBNCE) produced up to 83.6 g l-valine/l (i.e., 714 mM) withan overall YP/S of 0.36 mol/mol of glucose and a volumetric produc-tivity of 1.5 g/l h (Table 1; Fig. 4; Buchholz et al., 2013).

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To test the applicability of the novel C. glutamicum platformith attenuated PDHC activity for other products, C. glutamicum

ceE A16 �pqo �ppc was engineered for the production of 2-etoisovalerate (Buchholz et al., 2013) by additional deletion of the

lvE gene and overexpression of the ilvBNCD genes. The final strain. glutamicum aceE A16 �pqo �ppc �ilvE (pJC4ilvBNCD) produced

n a growth-coupled manner about 290 mM 2-ketoisovalerate with YP/S of a 0.33 mol/mol of glucose and showed a QP of about.0 mM/h (Buchholz et al., 2013). Replacement of the native aceEromoter in the defined l-lysine producers C. glutamicum DM1800nd DM1933 by the dapA-A16 promoter significantly improved l-ysine production. Both strains with reduced PDHC-activity showedn batch cultivations 100% and 50% increased YP/S and about 50%nd 100% improved biomass-specific production rates compared toheir parental strains DM1800 and DM1933 (Buchholz et al., 2013).

. Conclusion

C. glutamicum traditionally employed as an industrial aminocid producer has been successfully engineered to enlarge its prod-ct portfolio for novel product classes, including polymers, organiccids or biofuels. Since most relevant products derive from theentral metabolism, a lot of attention has been paid on the opti-ization of precursor availability. However, metabolic engineering

pproaches covering several products are rare. The above describedxamples demonstrate that PDHC-deficient C. glutamicum strainsnd C. glutamicum strains with reduced PDHC activity represent

promising and powerful platform for the production not onlyor pyruvate itself, but also for other pyruvate-derived amino andrganic acids as well as isobutanol. Furthermore, the engineeredDHC-deficient platform can be applied under aerobic and anaer-bic conditions and the growth-decoupled production behaviouright be an expedient attribute for the production of cytotoxic or

rowth inhibiting products.

cknowledgements

The support of the Fachagentur Nachwachsende Rohstoffe (FNR)f the BMELV (FNR Grant 220-095-08A; BioProChemBB project inhe frame of the ERA-IB programme) is gratefully acknowledged.he authors further gratefully acknowledge the funding by theeutsche Forschungsgemeinschaft (DFG; Grant TA 241/5-1).

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dx.doi.org/10.1021/cr400309c

The pyruvate dehydrogenase complex of Corynebacterium glutamicum: An attractive target for metabolic...

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