Expression of thepfl Gene and Resulting Metabolite Flux Distribution in nuo andackA-pta E. coliMutant Strains

Randeep Singh,† Yea-Tyng Yang,‡ Biqing Lu, § George N. Bennett,§ and Ka-Yiu San*,†,‡

Department of Bioengineering, Department of Chemical and Biomolecular Engineering, and Department of Biochemistry andCell Biology, Rice University, P.O. Box 1892, MS 142, Houston, Texas 77251-1892

Our laboratory previously studied the interaction betweennuoand the acetate-producing pathwayencoded byackA-ptain Escherichia coli.We examined metabolic patterns, particularly the ethanoland acetate production rates, of several mutant strains grown under anaerobic growth conditions.Since the pyruvate formate-lyase (PFL) pathway is the major route for acetyl-CoA and formateproduction under anaerobic conditions, we examined the effects ofnuoandackA/ptamutationson the expression of pyruvate formate-lyase (pfl) under anaerobic conditions. TheackA-ptamutanthas apfl::lacZ expression level much higher than that of the wild-type strain, and cultures alsoexhibit the highest ethanol production. Real-time PCR demonstrated that theadhEgene expressionin theack-ptamutant strain was approximately 100 fold that of the same gene in theackA-ptanuo mutant strain. This result correlates with the observed ethanol production rates in culturesof the strain. However, the lack of exact correlation between the ethanol production rates andthe RT-PCR data suggests additional regulation actions at the posttranslation level. In addition,the activity of thepfl gene as indicated by mRNA levels was also considerably greater in theack-pta mutant. We can conclude that deletions ofnuo and ack/pta can partially affect theexpression of the genes encodingadhEandpfl under anaerobic conditions.

Introduction

The conversion of pyruvate to formate and acetyl-CoA iscatalyzed by the enzyme pyruvate formate-lyase (PFL) underanaerobic conditions. The acetyl-CoA is converted to acetatethrough the major acetate production pathway encoded by thegenes for acetate kinase (ackA) and phosphotransacetylase (pta)and to ethanol through the alcohol dehydrogenase (ADH)pathway (Figure 1). Previously, our laboratory examined theeffects of removing the ACK-PTA pathway with and withoutan additional mutation in thenuo system on the redistributionof metabolic fluxes inE. coli (Yang et al., 1999c). The nuosystem encodes the NADH:ubiquinone dehydrogenase (NDH-I), which functions to couple redox chemistry to the generationof the proton motive force (PMF) necessary for providing energyfor ATP synthesis and solute transport (Calhoun and Gennis,1993; Matsushita and Kaback, 1986; Weidner et al., 1993).

It was observed that the three strains studied (parent strain,an ackA-ptamutant, and anackA-pta nuomutant) exhibiteddrastic differences in their specific formate, ethanol, acetate,and lactate synthesis rates in chemostat cultures. This isdemonstrated in the respective profiles of metabolites producedafter 24 h for chemostat cultures of each strain. Cultures of theparent GJT001 strain and the singlenuomutant both showed anegligible lactate production rate and equimolar ethanol andacetate synthesis rates in an anaerobic chemostat (Yang et al.,1999c). Under the same experimental conditions, the specificacetate production rate of theackA-pta mutant decreasedsignificantly, with a corresponding large increase in the lactate

rate and a slight drop in the ethanol rate (Yang et al., 1999c).However, for theackA-pta nuomutant, the formate, acetate,and ethanol excretion rate declined significantly. As a result,most of the carbon flux from pyruvate was channeled to thelactate pathway. This observation suggests inactivation of thePFL pathway under these conditions. However, this decreasethrough the formate (PFL) and ethanol (ADH) pathways wasnot the result of genetic mutation disrupting the formation offunctional PFL or ADH. The introduction of aBacillus subtilisacetolactate synthase (als) gene returned the ethanol and lactatelevels to those of its parent strain GJT0001 (Yang et al., 1999a).

To further understand the effect of thenuo and ackA/ptamutations onpfl expression, strains carrying apfl::lacZ fusionwere constructed to monitorpfl gene expression. In addition,the NADH/NAD+ ratio was measured to provide further insight

* To whom correspondence should be addressed. Fax: (713) 737-5877.Tel: (713) 285-5361. Email: ksan@rice.edu.

† Department of Bioengineering.‡ Department of Chemical and Biomolecular Engineering.§ Department of Biochemistry and Cell Biology.

Figure 1. Pyruvate dissimulation pathway ofE. coli. LDH is lactatedehydrogenase, PFL is pyruvate formate-lyase, PTA is phosphotrans-acetylase, ACK is acetate kinase, and ADH is alcohol dehydrogenase.

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into the regulation process. Thenuo gene encodes a NADHdehydrogenase; therefore thenuostrain carrying a mutation mayhave an altered NADH/NAD+ ratio. For growth on minimalmedia, the phenotype exhibited by anuo strain was attributedto a large NADH/NAD+ ratio (Pruss et al., 1994). Furthermore,a correlation exists between the NADH/NAD+ ratio and ADHenzyme synthesis (Leonardo et al., 1996). The deactivase activityof the ADH enzyme has been shown to be inhibited by highNADH and pyruvate levels (Kessler and Knappe, 1992).

The enzyme PFL requires both a complex transcriptionalscheme for expression and posttranslational regulation foractivity. Posttranslational regulation includes the interconversionof PFL between active and inactive forms. Conversion to theactive form is catalyzed by a PFL activase, which generatesthe oxygen-sensitive free radical element essential for itscatalytic activity (Knappe et al., 1969, 1974; Wagner et al.,1992). This activase requires reduced flavodoxin and adenosylmethionine as substrates. In the opposing role, alcohol dehy-drogenase (ADH) not only reduces acetyl-CoA to ethanol viaacetaldehyde but also functions as a PFL deactivase (Kessleret al., 1991, 1992; Knappe and Sawers, 1990). This communica-tion, however, focuses on transcriptional level events. Multiplepromoters coordinate anaerobic induction ofpfl expression withinfluence from transcriptional factors such as ArcA and Fnr. Inaddition, several metabolites and glycolytic intermediates havebeen implicated in modulation of the transphosphorylation ofArcA and the induction ofpfl transcription (Iuchi, 1993; Sawersand Bock, 1988).

Our experimental result indicated thatpfl expression and theNADH/NAD+ ratio do not correlate with the observed ethanolproduction rates in the three strains. If PFL is inactivated, acetyl-CoA is not produced for reduction to ethanol. Pyruvate is knownto inducepfl expression; however, in this case the buildup inglycolytic intermediates resulting fromackA-ptamutation maylead to an induction ofpfl expression, but the enzyme remainsinactive.

Materials and Methods

E. coli strains used in this work are listed in Table 1. Theconstruction of the strains GJT001, YBS125, and YBS121 hasbeen described previously (Tolentino et al., 1992; Yang et al.,1999a, 1999b). In this study, three additional strains withpfl::lacZ fusions were constructed (the host strain GJT001(pfl::lacZ),theackA-ptamutant YBS121(pfl::lacZ), and theackA-pta nuomutant YBS125(pfl::lacZ)) to examine the expression of thepyruvate formate-lyase (pfl) gene. Plasmid pRM23 (Sawers andBock, 1988) contains the 5′ untranslated leader region of thepfl gene with the sequence encoding 131 amino acids of thestuctural gene in apfl::lacZ fusion. With this construct, Sawersand Bock (1988) observed a 12-fold anaerobic induction andan additional induction with the addition of pyruvate or formate.The plasmid was transformed intoE. coli P90C. The selectedtransformants were infected withλ RS45, a vector for transfer-ring fusions to the chromosome as described by Simons and

Kleckner (1987). The resulting phage lysate was used totransduceE. coli P90C cells, and recombinant clones wereselected as lac+, Km resistant colonies. A homogeneous lysatefrom a single clone was prepared by UV stimulation of lysogeninduction (Silhavy et al., 1984). Lysates were used to infectE.coli strains GJT001, YBS121, and YBS125 by using a multi-plicity of infection of 0.001 to ensure integration of singleprophage at theλ attachment site. Colonies exhibiting Kmresistance and a lac+ phenotype were selected. Single copyintegration was confirmed by PCR analysis.

Medium. For all cultivations, complex Luria-Bertani broth(LB) medium or minimal media (M9) were supplemented with20 g/L of glucose and 1 g/L NaHCO3. Tetracycline, chloram-phenicol, and kanamycin (Sigma Chemical, St. Louis, Missouri)were added at concentrations of 25, 34, and 50 mg/L, respec-tively, to provide positive selective pressure as required.

Cultivation Conditions. Two different cultivation methods,anaerobic tubes and an anaerobic bioreactor in batch mode, wereused in this study. Cultivation of anaerobic tubes was conductedin 15 mL centrifuge tubes filled to capacity with complex orminimal media. Samples were taken during the exponentialphase (OD600 less than 1.0) forâ-galactosidase expression assaysand after 12 h for quantification of fermentation products. Forbioreactor runs, the cells were cultivated in a 2.5 L bioreactor(New Brunswick Scientific, Bioflow III) in batch mode with1.3 L working volume and a small constant flow of nitrogen inthe headspace to maintain anaerobic conditions (Yang et al.,1999a). The pH, temperature, and agitation were maintained at7.0, 37 °C, and 250 rpm, respectively. During exponentialgrowth phase, samples were taken periodically for analysis.

Analytical Techniques. Fermentation broth samples wereprocessed as previously reported (Yang et al., 1999a). Acetateand ethanol were quantified using a Varian 3000 gas chromato-graph (Varian Inc., Palo Alto, CA) operated as reportedpreviously (Yang et al., 1999a). Cell density was monitored at600 nm using a spectrophotometer. The optical density (OD600)can be related to cell dry weight (Yang et al., 1999a). Cellextracts were prepared by sonication. All assays were done intriplicate.â-Galactosidase was assayed, the activity was calcu-lated by the method of Miller (1972), and results were calculatedand reported in Miller units (MU). The NAD+ and NADHcycling assays were based on the protocol described byLeonardo et al. (1996) and Snoep et al. (1990) and modifiedby Berrios et al. (1999). Real-time quantitative PCR (RT-PCR)was conducted using a reverse transcription kit (Promega,Madison, WI) and the ABI 7000 Sequence Detection System(Applied Biosystems, Foster City, CA). The genes that werestudied in this work and their primers (Shalel-Levanon et al.,2005) are listed in Table 2. The RT-PCR method for determiningmRNA activities and calculating relative expression levels weredescribed in an earlier study (Shalel-Levanon et al., 2005).

Results and DiscussionThe integration of thepfl::lacZ fusion does not affect the

fermentation patterns of ethanol and acetate for the strains listed

Table 1. Strains and Plasmids Used

strains significant genotype reference

pRM23 pfl::lacZ vector Sawers and Bo¨ck, 1988GJT001 spontaneouscadRmutant of MC4100 (ATC35695)

∆lac(arg-lac)U169rpsL150 relA1 ptsFTolentino et al., 1992

YBS121 GJT001pta, ack, CmR Yang et al., 1999cYBS125 GJT001pta, ack, nuo,KmR Yang et al., 1999aGJT001(λRS45) GJT001 (pfl::lacZ), KmR this studyYBS121(λRS45) YBS121 (pfl::lacZ), KmR this studyYBS125(λRS45) YBS125 (pfl::lacZ), KmR this study

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in Table 1, since the strains retain the normalpfl gene. Ethanolproduction was the primary focus since formate production wasshown to parallel ethanol production and acetate production isnegligible in strains containing anackA-ptamutation (Yang etal., 1999c). The specific ethanol and acetate production of thesestrains (Figure 2) shows that parent,ack-pta, andackA-pta nuostrains were essentially unaffected by the genetic manipulations.This verification is necessary since samples forâ-galactosidaseexpression were taken during the exponential phase whereethanol and acetate levels are too low for detection. Thesepatterns are very similar to our earlier chemostat observations(Yang et al., 1999a, 1999b). Compared to the parent strain, theackA-ptamutant YBS121 has a slightly lower ethanol level anda nondetectable quantity of acetate due to the lack of the acetateproduction pathway. TheackA-pta nuomutant YBS125 alsoshowed negligible acetate formation and very little ethanolproduction as previously observed.

The ackA-ptaandnuo mutations affect theâ-galactosidaseexpression in aerobic shake flask cultures. The parent strainshowed a relatively low expression of about 70 MU, while theackA-pta nuomutant has the highest level, five times that ofGJT001. TheackA-pta mutant strain showed an intermediateexpression level of 180 MU.

The â-galactosidase expression levels for all three strainsincrease significantly under anaerobic conditions (Figure 4). Theparent strain, GJT001, is induced 8-fold. This observation isconsistent with earlier reports that the expression ofpfl isactivated under anaerobic conditions (Kessler and Knappe,1996). A second level of regulation also exists in substrateinduction of the gene (Sawers and Bo¨ck, 1988).

Theâ-galactosidase expression levels for both mutant strainsare much higher than their parent strain. This is unexpectedsince the ackA-pta nuo mutant shows very little ethanolproduction. The experiments were repeated with controlledanaerobic bioreactor batch cultures, and the results are sum-marized in Figure 4 (data shown are the averages of 5 samplepoints during the exponential growth phase). Theâ-galactosidaseexpression levels for the three strains in the bioreactor show apattern similar to those found in the anaerobic tube experiment.

The above observation suggests that the ethanol synthesispathway from pyruvate is not limited by regulation at thetranscription level under these conditions. The lack of ethanolproduction despite highâ-galactosidase expression controlledby the pfl::lacZ fusion in the ackA-pta nuo mutant strainindicates that PFL activity might be regulated in this instanceat the translational or at the enzymatic level. Possible PFLinactivation can be due to a redox potential shift to above100mV, which would deactivate PFL (Kessler et al., 1992). Thelack of dihydroflavodoxin reductant is also believed to keepPFL in its nonradical inactive form (Kessler and Knappe, 1996).A suppression of the activase would have a similar effect.

Real-time PCR was conducted to observe the relative geneexpression levels of thepfl, ldh, andadh in the ackA-pta andackA-pta nuo mutants (Figure 3). Relative to the wild type(GJT0001), theackA-ptamutant exhibits higher levels of activityfor pfl, ldh, andadh.As noted earlier, there is evidence of PFL

inactivation in theackA-pta nuomutant, shown by the low levelof metabolic pathway activity. The difference in gene expressionof adhEactivity is also notable between the two strains studied(P e 0.05) (Figure 3). Coinciding with the results of the analysisof expression of thepfl::lacZ fusion in anaerobic tube experi-ments, theackA-pta nuo mutant showed virtually noadhexpression, as analyzed by evaluating mRNA levels throughRT-PCR. This provides further explanation for the absence ofethanol produced by theackA-pta nuomutant in the anaerobicexperiments. There is a negligible difference inldh geneexpression between the two strains, relative to the wild type(P e 0.05). However, the slight increase in lactate productionin the anaerobic tubes (P e 0.05) may be explained by the slightincrease inldh gene expression in theackA-pta nuo mutantrelative to that in theackA-pta mutant (Figure 4).

The intracellular NADH/NAD+ ratios for the three strainswere also measured. As the NADH/NAD+ ratio increases thelevel of ADH enzyme also increases. (Leonardo et al., 1993).However, the deactivase activity of the ADH enzyme is inhibitedby high NADH and pyruvate levels (Kessler and Knappe, 1992).

Table 2. List of Genes, Primer Pairs, and Amplified PCR Products

gene enzyme or function primer pairs PCR products (bp)

ldhA D-lactate dehydrogenase 5′-AGTCCGTGTTCCAGCCTATG-3′5′-CGGTCAGACCTTCCAGAGAG-3′

134

pflA pyruvate formate-lyase activating enzyme 5′-TACGATCCGGTGATTGATGA-3′5′-TCACATTTTTGTTCGCCAGA-3′

151

adhE alcohol dehydrogenase/Pfl-deactivase 5′-CTGGCAGGCTTCTCTGTACC-3′5′-TACCGCGTCTTCGAAATCTT-3′

141

Figure 2. Fermentation patterns in anaerobic tubes after 12 h ofanaerobic growth in tubes containing LB media (pH) 7.5) supple-mented with 20 g/L of glucose and 1 g/L NaHCO3.

Figure 3. Analyzed gene expression (RT-PCR) in cells of YBS121and YBS125 relative to GJT001 after 12 h of anaerobic growth. Geneexpression level is calculated by (2∧(-∆∆Ct)‚100) method (see Shalel-Levanon et al., 2005). Value of 1 corresponds to equal gene expressionrelative to wild-type strain GJT001, and values above 1 correspond tofold-increase.

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The results for the anaerobic tube and bioreactor experimentsare summarized in Figure 4. Both experiments show similarqualitative trends. The parent strain GJT001 has the highestNADH/NAD+ ratio and the lowest level ofpfl::lacZ-controlledâ-galactosidase activity. The NADH/NAD+ ratios, however,cannot explain the lack of ethanol production in the cultures ofackA-pta nuomutant strain since both this mutant strain andthe ackA-pta mutant strain exhibited similar NADH/NAD+

levels.â-Galactosidase expression levels were also tested in anaero-

bic tube cultures using minimal media with glucose as thecarbon source (data not shown). The results also show increasedpfl::lacZ-generatedâ-galactosidase levels in theackA-ptaandackA-pta nuomutant strains as compared to the parent.

Metabolites have been shown to affectpfl expression andactivity at the transcriptional and enzymatic levels. Metabolitessuch as lactate, acetate, and NADH were shown to stimulatetransphosporylation of ArcA, a transcription factor involved inthe anaerobic response (Iuchi, 1993; Iuchi and Linn, 1992; Linand Iuchi, 1994). Pyruvate and other glycolytic metabolites havebeen implicated as inducers ofpfl transcription (Sawers andBock, 1988). The integration host factor is responsible for theinduction observed by pyruvate addition (Sirko et al., 1993).Addition of pyruvate increasesâ-galactosidase levels for theparent strain GJT001 (pfl::lacZ) but not theackA-pta nuostrains(data not shown). Strains containing theackA-pta nuomutationalready excrete pyruvate due to intracellular buildup and arenot affected by addition of pyruvate (Yang et al., 1999c).Pyruvate is also a positive allosteric activator for the PFLenzyme (Kavanagh and Cole, 1976). The presence of high levelsof pyruvate and adequate NADH to suppress the deactivationfunction of AdhE protein (Leonardo et al., 1993) suggest the

cause of the low PFL activity may reside in an inability toactivate the enzyme under the relatively lower NADH/NAD+

ratios existing in theackA-pta nuomutant strain under theseconditions.

Summary

The presence of theackA-ptamutation has a large effect onpfl::lacZ expression and the NADH/NAD+ ratio. However, nocorrelation can be found between the observed ethanol produc-tion rates and theâ-galactosidase expression levels in thepfl::lacZ strains. The parent strain GJT001, which exhibited thehighest ethanol production, has the lowestpfl::lacZ-generatedâ-galactosidase expression level. On the other hand, theackA-pta nuomutant strain has a higherâ-galactosidase expressionlevel and yet it showed very little ethanol formation. Instead,higher intracellular pyruvate levels are probably responsible forthe increase theâ-galactosidase level in strains containing theackA-ptamutation.

The lack of positive correlation between the observed ethanolproduction rates and theâ-galactosidase expression levels inthe pfl::lacZ strains indicates the PFL enzyme activity is notcontrolled by limitations imposed at the transcription level underthese circumstances. There is, however, a strong correlationbetween the observed ethanol production rates and the geneexpression levels ofpfl andadhEin the YBS121 and YBS125strains. These observations illustrate that analysis solely at thetranscription level, such as using quantitative real-time PCR,might not be sufficient to give an accurate picture of thefunctioning pathway. The results also demonstrate the impor-tance of in vivo enzyme activity in the overall understandingof cellular responses relevant to metabolic engineering.

Figure 4. Expression patterns and NADH/NAD+ in cells of cultures grown in anaerobic tubes and in anaerobic bioreactors. Samples were takenduring the exponential phase (OD600 less than 1.0) forâ-galactosidase expression assays. For Specificâ-galactosidase activity MU units are[(mmol ONPG consumption)/(mg-protein‚h)].

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Accepted for publication March 23, 2006.

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