ARTICLE IN PRESS
Metabolic Engineering 10 (2008) 234– 245
Contents lists available at ScienceDirect
Metabolic Engineering
1096-71
doi:10.1
� Corr
E-m1 Th
journal homepage: www.elsevier.com/locate/ymben
A new model for the anaerobic fermentation of glycerol in enteric bacteria:Trunk and auxiliary pathways in Escherichia coli
Ramon Gonzalez �, Abhishek Murarka 1, Yandi Dharmadi 1, Syed Shams Yazdani
Department of Chemical and Biomolecular Engineering, Rice University, P.O. Box 1892, Houston, TX 77251-1892, USA
a r t i c l e i n f o
Article history:
Received 31 July 2007
Received in revised form
3 May 2008
Accepted 13 May 2008Available online 27 May 2008
Keywords:
Glycerol fermentation
Escherichia coli
Enteric bacteria
76/$ - see front matter & 2008 Elsevier Inc. A
016/j.ymben.2008.05.001
esponding author. Fax: +1713 348 5478.
ail address: [email protected] (R. Gon
ese authors contributed equally to this work
a b s t r a c t
Anaerobic fermentation of glycerol in the Enterobacteriaceae family has long been considered a unique
property of species that synthesize 1,3-propanediol (1,3-PDO). However, we have discovered that
Escherichia coli can ferment glycerol in a 1,3-PDO-independent manner. We identified 1,2-propanediol
(1,2-PDO) as a fermentation product and established the pathway that mediates its synthesis as well as
its role in the metabolism of glycerol. We also showed that the trunk pathway responsible for the
conversion of glycerol into glycolytic intermediates is composed of two enzymes: a type II glycerol
dehydrogenase (glyDH-II) and a dihydroxyacetone kinase (DHAK), the former of previously unknown
physiological role. Based on our findings, we propose a new model for glycerol fermentation in enteric
bacteria in which: (i) the production of 1,2-PDO provides a means to consume reducing equivalents
generated in the synthesis of cell mass, thus facilitating redox balance, and (ii) the conversion of glycerol
to ethanol, through a redox-balanced pathway, fulfills energy requirements by generating ATP via
substrate-level phosphorylation. The activity of the formate hydrogen-lyase and F0F1-ATPase systems
were also found to facilitate the fermentative metabolism of glycerol, and along with the ethanol and
1,2-PDO pathways, were considered auxiliary or enabling. We demonstrated that glycerol fermentation
in E. coli was not previously observed due to the use of medium formulations and culture conditions
that impair the aforementioned pathways. These include high concentrations of potassium and
phosphate, low concentrations of glycerol, alkaline pH, and closed cultivation systems that promote the
accumulation of hydrogen gas.
& 2008 Elsevier Inc. All rights reserved.
2 ADH, alcohol dehydrogenase; AKR, aldo-keto reductase; COSY, COrrelation
SpectroscopY; DHA, dihydroxyacetone; DHAK, DHA kinase; DHAP, DHA phosphate;
FHL, formate-hydrogen lyase complex; F0F1-ATPase, F0F1-H+-translocating ATP
(hydrol-/synthet-)ase; GLYC, glycerol; glyDH, glycerol dehydrogenase; G3P,
1. Introduction
Glycerol has become an inexpensive and abundant carbonsource due to its generation as inevitable by-product of biodieselfuel production. Worldwide surplus of glycerol has prompted theshutdown of facilities dedicated to its production or refining andthe economic viability of the biodiesel industry has been greatlyaffected (Yazdani and Gonzalez, 2007 and references therein).Given the highly reduced state of carbon in glycerol, its conversionto fuels or reduced products could result in yields higher thanthose obtained with the use of common sugars. Realizing thispotential, however, would require the anaerobic fermentation ofglycerol in the absence of external electron acceptors.
The ability to conduct fermentative metabolism of glycerol inthe Enterobacteriaceae family is shared by only a few memberssuch as Citrobacter freundii and Klebsiella pneumoniae (Booth,2005; Bouvet et al., 1995). This metabolic process is mediated by a
ll rights reserved.
zalez).
.
two-branch pathway (Fig. 1A), which results in the synthesis ofglycolytic intermediate dihydroxyacetone-phosphate (DHAP: seeFootnote2 for all acronyms) and fermentation product 1,3-propanediol (1,3-PDO) (Booth, 2005). In the oxidative branch,glycerol is dehydrogenated to dihydroxyacetone (DHA) by atype I, NAD-linked glycerol dehydrogenase (glyDH-I). DHA is thenphosphorylated by ATP- or PEP-dependent DHA kinases (DHAK) togenerate DHAP. Through the parallel reductive branch, glycerol isdehydrated by the coenzyme B12-dependent glycerol dehydrataseto form 3-hydroxypropionaldehyde (3-HPA). 3-HPA is thenreduced to the major fermentation product 1,3-PDO by the
sn-glycerol-3-phosphate; G3PDH, G3P dehydrogenase; a-G3PDH, aerobic G3PDH;
an-G3PDH, anaerobic G3PDH; HA, hydroxyacetone; LAL, lactaldehyde; MG,
methylglyoxal; MGR, MG reductase; MGS, MG synthase; MM, minimal medium;
NMR, nuclear magnetic resonance; PEP, phosphoenolpyruvate; PYR, pyruvate;
1,2-PDO, 1,2-propanediol; 1,2-PDOR, 1,2-PDO reductase; 1,3-PDO, 1,3-propanediol;
1,3-PDODH, 1,3-PDO dehydrogenase; 3HPA, 3-hydroxypropionaldehyde.
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GLYC
DHAP
3HPA
1,3-PDO DHA
H
G3P
H
GlycolysisPYR
GK
ATP ADPglyD
H2O
DHAK
ATP/PEP ADP/PYR
glyDH-I
1,3-PDODH
GLYC
G3PDH
DHAP
MG
LAL
HA
1,2-PDO
NA(P)D +
NAD(P)H
NAD+NADH
NA(P)D +NAD(P)H
NAD+
NADH
MGS(mgsA)
MGR
(fucO)
glyDH (gldA )
AKR ( yeaE, yghZ, yafB )
1,2-PDOR
Fig. 1. Glycerol dissimilation and 1,3- and 1,2-propanediol synthesis. (A) Glycerol-
fermenting species in the Enterobacteriaceae dissimilate glycerol via a two-branch
pathway (shaded): the reductive, 1,3-PDO-producing branch acts as a sink for the
reducing equivalents generated in the oxidative branch. This metabolic process
represents the established model for glycerol fermentation in enteric bacteria.
Metabolism of glycerol in species unable to synthesize 1,3-PDO, such as E. coli,
takes place through a respiratory pathway that requires an external electron
acceptor (enclosed in an oval). (B) Metabolic pathways leading to the synthesis of
1,2-propanediol (1,2-PDO) from dihydroxyacetone-phosphate in E. coli. Reactions
catalyzed by MGS, MGR, AKR, glyDH, and 1,2-PDOR are as previously reported
(Altaras and Cameron, 1999; Boronat and Aguilar, 1979; Cooper, 1984; Ko et al.,
2005; Misra et al., 1996; Saikusa et al., 1987; Truniger and Boos, 1994). Thick lines
indicate the route used by E. coli MG1655 for 1,2-PDO synthesis during glycerol
fermentation, as inferred from the experimental evidence summarized in Figs. 3
and 4. Abbreviations: AKR, aldo-keto reductases; DHA, dihydroxyacetone; DHAK,
DHA kinase; DHAP, DHA phosphate; GK, glycerol kinase; GLYC, glycerol; glyD,
glycerol dehydratase; glyDH-I, glycerol dehydrogenase, type I; G3P, glycerol-3-
phosphate; G3PDH, G3P dehydrogenase; H, reducing equivalents (H ¼ NADH/
NADPH/FADH2); HA, hydroxyacetone; LAL, lactaldehyde; MG, methylglyoxal; MGR,
methylglyoxal reductase; MGS, methylglyoxal synthase; PEP, phosphoenolpyru-
vate; PYR, pyruvate; 1,2-PDOR, 1,2-propanediol reductase; 1,3-PDO, 1,3-propane-
diol; 1,3-PDODH, 1,3-PDO dehydrogenase; 3HPA, 3-hydroxypropionaldehyde.
Metabolites shown in bold are extracellular.
R. Gonzalez et al. / Metabolic Engineering 10 (2008) 234–245 235
NADH-linked 1,3-PDO dehydrogenase (1,3-PDODH), thereby re-generating NAD+ (Fig. 1A). Only eight taxa of the Enterobacter-
iaceae grow fermentatively on glycerol and in all cases theyproduce 1,3-PDO and possess the enzymes glyDH-I and 1,3-PDODH (Bouvet et al., 1995). Although several types of glyDHshave been found in species unable to ferment glycerol (includingtype II, glyDH-II, in Escherichia coli), their role remains unknown(Bouvet et al., 1995).
As no 1,3-PDO-producing capacity has been identified in wild-type E. coli strains, it is believed that the metabolism of glycerol inthis organism requires the presence of electron acceptors (Booth,2005; Bouvet et al., 1994, 1995; Lin, 1976; Quastel et al., 1925;Quastel and Stephenson, 1925). The respiratory pathway mediat-ing glycerol utilization involves a glycerol transporter, a glycerolkinase, and two respiratory glycerol-3-P dehydrogenases(G3PDHs) (Booth, 2005; Borgnia and Agre, 2001; Lin, 1976;Pettigrew et al., 1990; Schryvers and Weiner, 1982; Walz et al.,
2002) (Fig. 1A). Under aerobic conditions a homodimeric enzymeassociated with the cytoplasmic membrane (aerobic G3P dehy-drogenase: a-G3PDH, encoded by the glpD gene) is known to beessential for the metabolism of glycerol. In the absence of oxygenand presence of other electron acceptors such as fumarate, athree-subunit enzyme (anaerobic G3P dehydrogenase: an-G3PDH,encoded by the glpABC operon) converts glycerol-3-phosphate(G3P) into DHAP. The lack of a G3PDH activity in the absence ofelectron acceptors is thought to result in the accumulation of G3Pat levels that become inhibitory for cell growth, a condition that isrelieved by the presence of fumarate (Booth, 2005). Therefore, theinability of E. coli to metabolize G3P under fermentative condi-tions has been suggested as the reason for this organisms’inability to ferment glycerol (Booth, 2005).
Despite the belief that the metabolism of glycerol in E. coli isrestricted to respiratory conditions, recent studies in our laboratoryshowed that this organism can ferment glycerol in the absence ofexternal electron acceptors (Dharmadi et al., 2006). Since glycerolfermentation was dependent on the supplementation of themedium with tryptone or other nutrients, we conducted furtherstudies to demonstrate the fermentative nature of this metabolicprocess and the use of glycerol in the synthesis of cell mass(Murarka et al., 2008). Our results also indicate that wild-typeE. coli strains might have the ability to produce 1,2-propanediol(1,2-PDO) during glycerol fermentation. Fig. 1B shows the pathwaysthat have been reported to mediate the synthesis of 1,2-PDO fromglycolytic intermediate DHAP in E. coli (Altaras and Cameron, 1999;Boronat and Aguilar, 1979; Cooper, 1984; Ko et al., 2005; Misraet al., 1996; Saikusa et al., 1987; Truniger and Boos, 1994).
In the studies described here we report the conditions,pathways, and mechanisms mediating the fermentative metabo-lism of glycerol in E. coli. Based on our findings, a new model forthe 1,3-PDO-independent fermentation of glycerol in entericbacteria is proposed. Genetic and environmental determinantsof the fermentative metabolism of glycerol are discussed in thecontext of the proposed model.
2. Materials and methods
2.1. Strains, plasmids, and genetic methods
Wild-type E. coli strains MC4100 (ATCC 35695), W3110 (ATCC27325), and E. coli B (ATCC 11303) and enteric bacteriaEnterobacter cloacae subsp. cloacae NCDC 279-56 (ATCC 13047),Buttiauxella agrestis (ATCC 33994), Serratia plymuthica (ATCC15928), and Leminorella richardii (ATCC 33998) were obtainedfrom the American Type Culture Collection (ATCC, Mannassas,VA). K12 strain MG1655 (F-l-ilvG-rfb-50 rph-1) along with thefollowing otherwise isogenic derivatives (mutation details inparentheses) were obtained from the University of WisconsinE. coli Genome Project (www.genome.wisc.edu): FB21196(glpDHTn5KAN-I-SceI), FB20724 (glpAHTn5KAN-I-SceI), FB22899(gldAHTn5KAN-I-SceI), FB21569 (dhaKHTn5KAN-I-SceI), FB21424(atpDHTn5KAN-I-SceI), FB21425 (atpFHTn5KAN-I-SceI), FB21975(mgsAHTn5KAN-I-SceI), FB20935 (fucOHTn5KAN-I-SceI), FB20044(yafBHTn5KAN-I-SceI), FB22576 (yeaEHTn5KAN-I-SceI), andFB23263 (yghZHTn5KAN-I-SceI). These strains carry a transposon(Tn5) insertion mutation in the specified gene (Kang et al., 2004).Disruption of genes/operons fdhF, gldA, dhaKLM, and glpABC werecreated in both MG1655 and W3110 backgrounds using themethod described by Datsenko and Wanner (2000). Informationabout plasmids (pKD4, pKD46, and pCP20) and primers (d-fdhF,v-fdhF, d-gldA, v-gldA, d-dhaKLM, v-dhaKLM, d-glpABC, andv-glpABC) used in their construction is provided in Table 1. Allmutants were verified through genomic PCR after construction to
ARTICLE IN PRESS
Table 1Plasmids and primers used in this study
Plasmid/primer Structure or description Source
Plasmids
pKD4 repR6KgApR FRT KmR FRT Datsenko and Wanner (2000)
pKD46 reppSC101ts ApR ParaBAD g b exo+ Datsenko and Wanner (2000)
pCP20 reppSC101ts ApR CmR cI857l PR flp+ Datsenko and Wanner (2000)
pZSKLM E. coli dhaKLM under control of PLtetO-1 (tetR, oriR SC101*, cat) Gutknecht et al. (2001)
pZSKLcf C. freundii dhaKL under control of PLtetO-1 (tetR, oriR SC101*, cat) Gutknecht et al. (2001)
pZSgldA E. coli gldA under control of PLtetO-1 (tetR, oriR SC101*, cat) This study
pZSKLMgldA E. coli dhaKLM and gldA under control of PLtetO-1 (tetR, oriR SC101*, cat) This study
pZSmgsAgldA E. coli mgsA and gldA under control of PLtetO-1 (tetR, oriR SC101*, cat) This study
pCA24NgldA E. coli gldA under control of IPTG-inducible promoter pT5/lac (oriR SC101*, cat) Kitagawa et al. (2005)
pCA24NfdhF E. coli fdhF under control of IPTG-inducible promoter pT5/lac (oriR SC101*, cat) Kitagawa et al. (2005)
pCA24NatpF E. coli atpF under control of IPTG-inducible promoter pT5/lac (oriR SC101*, cat) Kitagawa et al. (2005)
Primersa
d-fdhF gaagggttattatggctgggacttcattaacgatacgtgtaggctggagctgcttc This study
gcagtatttgtactccggcgttttcgtaatccatatgaatatcctccttag
v-fdhF cgtaatatcagggaatgaccc This study
gggcaaagaatgtcaaaaacaa
d-gldA gcagtgtggcgcaattctcggtatcggtggcggaaaaacgtgtaggctggagctgcttc This study
gacatcttctttaatatccagttgagcgagagttattggcaaacctacatatgaatatcctccttag
v-gldA atggaccgcattattcaatcac This study
gcctacaaaagcacgcaaattc
d-dhaKLM gctggagcaaaataatgaaaaaattgatcaatgatgtgcaagacggtgtaggctggagctgcttc This study
actgcgggagttcttctttcgtttgggtcaggtggcatatgaatatcctccttag
v-dhaKLM tatcccgcatcccttatgac This study
aaaccattagtgctgagtaaatt
c1-gldA agtcacggtaccatggaccgcattattcaatcac This study
gatcgtctgcagttattcccactcttgcaggaaac
c2-gldA gacactgcagaggagcaattatggaccgca This study
caagctacgcgtttattcccactcttgcagga
c3-gldA atactgggtaccatggaactgacgactcgc This study
gatcgtctgcagttacttcagacggtccgc
a Priming sequences for plasmid pKD4 are underlined. ‘‘d’’ and ‘‘v’’ indicate that the primer sequences (50 to 30) were used for deletion (‘‘d’’) and verification (‘‘v’’)
purposes during the creation of disruption mutants as previously described (Datsenko and Wanner, 2000). A ‘‘c’’ indicates that the primer was used for cloning purposes,
‘‘c1’’ to clone gldA alone (pZSgldA), ‘‘c2’’ to clone gldA along with dhaKLM (pZSKLMgldA), and ‘‘c3’’ to clone gldA along with mgsA (pZSmgsAgldA). The forward sequence
follows the reverse sequence in each case. Genes or operons deleted or cloned are apparent from primer names.
R. Gonzalez et al. / Metabolic Engineering 10 (2008) 234–245236
ensure that the gene of interest had been disrupted. By usingplasmid pKD4 as template, we ensured the creation of in-framegene deletions thus minimizing polarity effects on the expressionof downstream genes (Datsenko and Wanner, 2000). Throughoutthe paper, gene disruption mutants are referred to using thefollowing nomenclature: Dgene_name(s), where ‘‘gene_name(s)’’indicates the disrupted gene(s).
Table 1 describes other plasmids used in this study, whichinclude: (i) pZSKLM, expressing E. coli DHA kinase subunitsDhaKLM; (ii) pZSKLMgldA, expressing both E. coli DHA kinasesubunits DhaKLM and E. coli glycerol dehydrogenase (GldA); (iii)pZSgldA, expressing E. coli glycerol dehydrogenase (GldA); (iv)pZSmgsAgldA, expressing E. coli methylglyoxal synthase (MgsA)and glycerol dehydrogenase (GldA); and (v) derivatives of plasmidpCA24N carrying the genes fdhF, atpF, and gldA. Plasmid pZSKLMwas kindly provided by Dr. B. Erni, Universitat Bern, Switzerland(Gutknecht et al., 2001). The expression vector pZSgldA wasconstructed as follows. The coding region of the gldA gene wasPCR amplified using genomic DNA of E. coli MG1655 as templateand primers described in Table 1 (‘‘c1-gldA’’ primers). Therestriction enzyme sites KpnI and PstI were introduced throughthe forward and reverse primers, respectively, to facilitate cloningof PCR product in the expression vector pZSKLM (Bachler et al.,2005). The PCR was performed using Pfu turbo DNA polymerase(Stratagene, CA, USA) under standard conditions as described bythe supplier. The amplified product (1.1 kB) was digested withKpnI and PstI and used for ligation at the corresponding sites ofthe pZSKLM plasmid. The ligated product was used to transformE. coli DH5aT1 (Invitrogen, CA, USA). Positive clones werescreened by plasmid isolation and restriction digestion. The
expression vectors pZSKLMgldA and pZSmgsAgldA were con-structed in a similar fashion. For plasmid pZSKLMgldA, the gldA
gene was amplified along with its ribosome-binding site from thegenomic DNA of E. coli MG1655 using the primers described inTable 1 (‘‘c2-gldA’’ primers). The amplified product was digestedwith PstI and MluI and cloned at the corresponding sites ofpZSKLM, downstream of the dhaKLM gene (Bachler et al., 2005).For plasmid pZSmgsAgldA, the coding region of mgsA was PCRamplified using genomic DNA of E. coli MG1655 as template andthe primers described in Table 1 (‘‘c3-gldA’’ primers). Theamplified product was digested with KpnI and PstI and clonedat the corresponding sites of expression vector pZSKLMgldA(replacing the dhaKLM genes). pCA24N derivatives were obtainedfrom the Genome Analysis Project Japan (http://ecoli.aist-nara.ac.jp/), which provides clones of each ORF predicted from thegenome sequence of E. coli W3110. Every ORF has been cloned intoplasmid pCA24N, which contains the IPTG-inducible promoterpT5/lac (Kitagawa et al., 2005). Derivatives of pCA24N expressingformate dehydrogenase, the b subunit of the F0 complex ofF0F1-ATPase, and glycerol dehydrogenase are referred to here aspCA24NfdhF, pCA24NatpF, and pCA24NgldA, respectively. Plas-mids were transformed into E. coli strains and selected on LuriaBertani (LB) plates containing appropriate antibiotics. Geneexpression in the aforementioned plasmids was induced with100 ng/mL anhydrotetracycline (pZS series plasmids) or 10 mMIPTG (isopropyl-b-D-thiogalactopyranoside) (pCA24N derivatives).Standard recombinant DNA procedures were used for plasmidisolation, electroporation, and polymerase chain reaction (Sam-brook et al., 1989). The strains were kept in 32.5% glycerol stocksat �80 1C. Plates were prepared using LB medium containing 1.5%
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R. Gonzalez et al. / Metabolic Engineering 10 (2008) 234–245 237
agar. Antibiotics were included as needed at the followingconcentrations: 100 mg/mL ampicillin, 34mg/mL chloramphenicol,50mg/mL kanamycin, and 12.5 mg/mL tetracycline.
2.2. Culture medium and cultivation conditions
The minimal medium (MM) designed by Neidhardt et al.(1974) supplemented with 2 g/L tryptone (Difco, USA), 110 mMglycerol, 5 mM sodium selenite, and 1.32 mM Na2HPO4 in place ofK2HPO4 was used, unless otherwise specified. MOPS (morpholino-propanesulfonic acid) was used only in the inoculum preparationphase, which was conducted in tubes with no external control ofpH. When indicated, the medium was supplemented withspecified concentrations of the following compounds: monobasicand dibasic sodium and potassium phosphates, monobasic anddibasic ammonium phosphates, sodium chloride, sodium sulfate,potassium chloride, and potassium sulfate. Chemicals wereobtained from Fisher Scientific (Pittsburgh, PA) and Sigma-AldrichCo. (St. Louis, MO).
Fermentations were conducted in a SixFors multi-fermentationsystem (Infors HT, Bottmingen, Switzerland) with six 500 mLworking volume vessels and independent control of temperature(37 1C), pH (externally controlled with NaOH and H2SO4), andstirrer speed (200 rpm) (Dharmadi et al., 2006). Anaerobicconditions were maintained by initially sparging the mediumwith ultrahigh purity argon (Matheson, Tri-Gas, Houston, TX) andthereafter flushing the headspace with the same gas at 0.01 LPM.An oxygen trap (Alltech Associates, Inc., Deerfield, IL) was used toeliminate traces of oxygen from the gas stream. In someexperiments (specified in each case), carbon dioxide or hydrogenwere also included in the gas phase.
Prior to use, the cultures (stored as glycerol stocks at �80 1C)were streaked onto LB plates (with appropriate antibiotics ifrequired) and incubated overnight at 37 1C in an Oxoid anaerobicjar with the CO2 gas generating kit (Oxoid Ltd., Basingstoke,Hampshire, UK). A single colony was used to inoculate 17.5-mLHungate tubes (Bellco Glass, Inc., Vineland, NJ) completely filledwith MM supplemented with 10 g/L tryptone, 5 g/L yeast extract,and 110 mM glycerol. The tubes were incubated at 37 1C until anOD550 of �0.4 was reached. An appropriate volume of this activelygrowing pre-culture was centrifuged and the pellet washed andused to inoculate 350 mL of medium in each fermenter, with thetarget starting optical density of 0.05 at 550 nm.
2.3. Analytical methods
Optical density was measured at 550 nm and used as anestimate of cell mass (1 O.D. ¼ 0.34 g dry weight/L). Aftercentrifugation, the supernatant was stored at �20 1C for HPLC(High-Performance Liquid Chromatography) and NMR (nuclearmagnetic resonance) analysis (see next section for NMR experi-ments). To quantify concentration of glycerol, organic acids, andethanol, samples were analyzed with ion-exclusion HPLC usinga Shimadzu Prominence SIL 20 system (Shimadzu ScientificInstruments Inc., Columbia, MD) equipped with an HPX-87Horganic acid column (Bio-Rad, Hercules, CA). Operating condi-tions to optimize peak separation (30 mM H2SO4 in mobile phaseat 0.3 mL/min, column temperature 42 1C) were determinedusing a previously described approach (Dharmadi and Gonzalez,2005).
2.4. NMR experiments
The identity of the fermentation products was determinedthrough NMR experiments. Sample preparation and initial
characterization through 1D 1H NMR spectroscopy was conductedin a Varian 500 MHz Inova spectrometer equipped with a Pentaprobe (Varian, Inc., Palo Alto, CA), as previously described(Dharmadi et al., 2006) (Murarka et al., 2008). Further character-ization of the samples was achieved through a 2D 1H-1H COSY(COrrelation SpectroscopY) NMR experiment. A double-quantumfiltered COSY spectrum with watergate solvent suppression wasobtained using the wgdqfcosy pulse sequence that is part of theBioPack suite of pulse sequences (Varian, Inc., Palo Alto, CA). Thefollowing parameters were used: 6000 Hz sweep width; 0.5 sacquisition time; 600 complex points in t1 dimension; 32transients; 5.5 ms pulse width; and 1 s relaxation delay. Theresulting spectra were analyzed using FELIX 2001 software(Accelrys Software Inc., Burlington, MA).
2.5. Enzyme activities
Cells from anaerobic cultures (OD550 of �0.7) were harvestedby centrifugation (2 min, 10,000� g), washed twice with 9 g/LNaCl, and stored as cell pellets at �20 1C. For enzyme assays, cellswere resuspended in 0.2 mL of the buffer used in the specific assayand permeabilized by vortex mixing with chloroform (Osmanet al., 1987; Tao et al., 2001). Oxidative glycerol dehydrogenaseactivity (i.e., toward glycerol) was assayed by measuring thechange in absorbance at 340 nm and 25 1C in a 1 mL reactionmixture containing 2 mM MgCl2, 500 mM NAD+, 100 mM glycerol,30mL crude cell extract, and 100 mM of the appropriate bufferaccording to the pH of the assay (see below) (Truniger andBoos, 1994). Reductive glycerol dehydrogenase activity (i.e.,toward hydroxyacetone, HA) was measured in a similar mixture,but with HA and NADH replacing glycerol and NAD+, respectively.Since strain DgldA did not grow in a medium supplementedwith 2 g/L tryptone, a 10 g/L tryptone and 5 g/L yeast extractsupplementation was used for the purpose of measuringenzyme activities in this strain. Methylglyoxal (MG)-reducingactivities (e.g., MG reductase and aldo-keto reductases) weremeasured in a 1 mL reaction mixture containing 10 mM MG,0.1 mM NAD(P)H, and appropriate buffer (see below) (Ko et al.,2005). 1D proton NMR spectroscopy was used to identify theproducts of MG- and HA-reducing reactions. NMR measurementswere carried out after cell debris was removed from a reactionmixture containing cell extract (30 mL), MG/HA (10 mM), coen-zymes (1 mM NADH or 1 mM NADPH), buffer (100 mM potassiumphosphate, pH 7.0), and D2O. The NMR data were collected 1.5 hafter incubation at 25 1C. To study the effect of pH on enzymeactivities, the following buffers were used (pH in parenthesis):sodium phosphate (pH 6–8), potassium carbonate (pH 9.5), andsodium citrate (pH 5–6). Linearity of reactions (protein concen-tration and time) was established for all preparations. Thenonenzymatic rates were subtracted from the observed initialreaction rates. Results are expressed as mmoles of substrate/minute/mg of cell protein and represent averages for at least threecell preparations.
2.6. Calculation of fermentation parameters
Maximum specific growth rates (mM, h�1) were estimated byplotting total cell concentration versus the integral of cellconcentration, and fitting these plots to polynomial functions.The slope of the curves thus obtained (a straight line duringexponential growth) was used as the average specific rate. Growthyield (YX/S, mg cell/g glycerol) was calculated as the increase incell mass per glycerol consumed once the cultures reachedstationary phase.
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R. Gonzalez et al. / Metabolic Engineering 10 (2008) 234–245238
3. Results
3.1. 1,2-Propanediol as a product of glycerol fermentation: pathways
mediating its synthesis and significance for the fermentative
utilization of glycerol
During the analysis of fermentation samples in a previous studyof glycerol fermentation by E. coli MG1655, we noted two NMRpeaks at the same chemical shifts as that of methyl protons of 1,2-PDO (doublet at 1.15 ppm) (Murarka et al., 2008). These peaks wereobserved in the spectra of late fermentation samples (Fig. 2A) butwere absent in the initial samples (Fig. 2B). Since the peaks forother 1,2-PDO protons were masked by tryptone and glycerolsignals, and only part of those multiplet patterns were discernable,we conducted a 2D 1H-1H COSY (COrrelation SpectroscopY) NMRexperiment to further investigate the identity of the moleculegenerating the doublet at 1.15 ppm. The resulting spectra had thecross peaks corresponding to the C2 and C3 protons of 1,2-PDO,thereby confirming its synthesis (mutiplets at 1.15 and 3.88 ppm;Fig. 2C). Our findings represent the first report of 1,2-PDOproduction during the metabolism of glycerol in E. coli. 1,2-PDOsynthesis by wild-type E. coli was thought to be restricted to thefermentation of 6-deoxyhexose sugars fucose and rhamnose(Boronat and Aguilar, 1979; Hacking and Lin, 1976). We found thatthe concentration of 1,2-PDO in stationary phase cultures wasabout 0.570.15 mM. As we previously reported, ethanol was the
1.153 1.149 1.146 1.143 1.140
3.94
3.92
3.90
3.86
3.88
ppm
ppm
1.22 1.20 1.18 1.16 1.141.24ppm
1.22 1.20 1.18 1.16 1.141.24ppm
1,2-PDO
Ethanol
Fig. 2. Identification of 1,2-propanediol (1,2-PDO) as a product of glycerol
fermentation. (A) 1D-1H NMR spectrum of a late fermentation sample showing
two peaks at the same chemical shifts as those of methyl protons of 1,2-PDO
(doublet at 1.15 ppm). (B) 1D-1H NMR spectrum of a time zero sample, which
shows no evidence of these peaks. (C) 2D 1H-1H COSY (COrrelation SpectroscopY)
NMR spectrum of a late fermentation sample confirming the presence of 1,2-PDO:
COSY cross peaks of the C2 and C3 protons of 1,2-PDO at 1.15 and 3.88 ppm are
shown.
main product of glycerol fermentation along with minor amountsof succinic, acetic, and formic acids (Murarka et al., 2008). No otherproduct was found in the NMR spectra of fermentation samples.
The pathways that could mediate the synthesis of 1,2-PDOfrom DHAP, along with relevant enzymes and correspondinggenes, are summarized in Fig. 1B (Altaras and Cameron, 1999;Boronat and Aguilar, 1979; Cooper, 1984; Ko et al., 2005; Misraet al., 1996; Saikusa et al., 1987; Truniger and Boos, 1994). It isimportant to note that experimental evidence for many of thereactions is limited and a significant portion of it originates fromthe study of strains that were engineered to overproduce 1,2-PDOfrom sugars. Moreover, in many cases, genes encoding theproposed activities have not been identified and in none of thestudies was glycerol used as carbon source. Finally, note thatDHAP is a glycolytic intermediate generated during the utilizationof glycerol (see Fig. 1A and next section). In order to identify thespecific route mediating the synthesis of 1,2-PDO during thefermentative metabolism of glycerol, we used several genetic andbiochemical approaches and the results are described in thefollowing.
Since the synthesis of methylglyoxal (MG) from DHAP is acommon step in the 1,2-PDO pathway, regardless of the branchused for the conversion of MG to 1,2-PDO (Fig. 1B), we evaluated amutant devoid of the gene encoding MG synthase (MGS) anddemonstrated that 1,2-PDO synthesis was almost eliminated(Fig. 3A). Residual levels of 1,2-PDO in the MGS mutant could bedue to the spontaneous conversion of DHA to MG, as previouslyreported (Riddle and Lorenz, 1968). Since the main branch point inthis pathway is the reduction of MG (Fig. 1B), we also evaluatedmutants devoid of genes involved in each branch (Fig. 3A).A decrease in 1,2-PDO levels was observed in disruption mutantsof genes encoding aldo-keto reductase activities (AKR: DyghZ,DyeaE, and DyafB), which catalyze the conversion of MG to HA(Fig. 3A). However, wild-type levels of 1,2-PDO were produced bya DfucO mutant (Fig. 3A): fucO encodes a 1,2-PDO reductaseactivity, which converts lactaldehyde (LAL) to 1,2-PDO (Fig. 1B).These results suggest that the MG-HA-1,2-PDO branch is respon-sible for 1,2-PDO synthesis. To corroborate this hypothesis weassayed the cells for MG-reducing enzymes (MGR) and observedsignificant activity in the presence of either NADH or NADPH(Fig. 3B, panel I). Since these activities could be involved in theconversion of MG to either HA or LAL, we used 1D 1H NMRspectroscopy to characterize the reaction(s). Fig. 3B, panel II,shows that the product of MG-reducing activities is indeed HA(no LAL was detected). Similar results were obtained with mutantstrain DfucO (data not shown), which demonstrates that theabsence of LAL in the reaction mixture is not due to its fastconversion to 1,2-PDO. These results agree with the analysis ofgenetic mutants, which showed no changes in 1,2-PDO synthesisupon elimination of the activity that converts LAL to 1,2-PDO(Fig. 3A). We also demonstrated that cells fermenting glycerolexhibit significant HA-reducing activity (0.09570.002mmol/min/mg protein). In a study similar to the one discussed above, weidentified the HA-reducing activity as being NADH-dependent andresponsible for the conversion of HA to 1,2-PDO (data not shown).We also demonstrated that the gldA gene encodes this activity, asno HA reduction was observed with cell extracts of a DgldA
mutant. Given the central role of MG- and HA-reducing activitieson 1,2-PDO synthesis, and the significant effect of pH on glycerolfermentation (Dharmadi et al., 2006; Murarka et al., 2008), weassessed the effect of pH on these activities (Fig. 3C). In all cases,neutral to slightly alkaline conditions were found to be optimum.
The above results clearly show that the synthesis of 1,2-PDOduring glycerol fermentation occurs through the conversionof DHAP to MG to HA to 1,2-PDO (represented by thick lines inFig. 1B). We further investigated whether 1,2-PDO synthesis could
ARTICLE IN PRESS
MG1655 “1,2-PDO Mutants”
No
MG1655(pZSblank)
NoYesNoHydroxyacetone
MG1655(pZSmgsAgldA)
MG1655MG1655Strain
0.0
0.1
0.2
0.3
0.4
Cel
l gro
wth
(O
D55
0)
0
1
2
3
4
5
Gly
cero
l fer
men
ted
(g/L
)
0.3
0.4
0.5
0.6
Cel
l gro
wth
(O
D55
0)
0
40
80
120
1,2-
PDO
(%
of
wild
type
)
Fig. 4. Changes in cell growth and glycerol fermentation in response to
perturbations in 1,2-propanediol (1,2-PDO) synthesis. (A) Cell growth (white bars)
and 1,2-PDO synthesis (gray bars) in MG1655 and mutants of aldo-keto reductases
(AKR: DyeaE, DyghZ, and DyafB) and methylglyoxal synthase (MGS: DmgsA)
enzymes. Average performance for all mutants (referred to as ‘‘1,2-PDO mutants’’)
is shown. See Fig. 1B for role of these genes. (B) Effect of amplification of the 1,2-
PDO pathway on cell growth (white bars) and fermented glycerol (gray bars). All
cultures were conducted in MOPS minimal medium with no tryptone supple-
mentation and hydroxyacetone (20 mM) was included when indicated. Plasmid
pZSmgsAgldA expresses methylglyoxal synthase (mgsA) and glycerol dehydrogen-
ase (gldA), two key enzymes mediating the synthesis of 1,2-PDO (see Figs. 1B
and 3). Plasmid pZSblank was used as the control plasmid and contains the
backbone of plasmid pZSmgsAgldA without genes mgsA and gldA.
Fig. 3. Synthesis of 1,2-propanediol (1,2-PDO) during glycerol fermentation takes
place through the conversion of dihydroxyacetone-P to methylglyoxal (MG), and
further reduction of MG to hydroxyacetone (HA) and HA to 1,2-PDO. (A) Changes in
1,2-PDO levels in response to disruption of genes potentially involved in its
synthesis: see Fig. 1B for role of these genes. (B) Quantification of NAD(P)H-
dependent MG-reducing activities in cell extracts (panel I) and identification of HA
as the product of MG reduction (panel II). Spectra for initial (upper) and final
(lower) samples in the enzyme assays are shown. Symbols indicate MG (*), HA (.),
and acetate (#, impurity in MG). No lactaldehyde was detected. (C) Effect of pH on
MG- and HA-reducing activities. The use of NADPH or NADH as cofactor by MG
reductases (MGR) and HA as substrate by glycerol dehydrogenase (glyDH) is
indicated.
R. Gonzalez et al. / Metabolic Engineering 10 (2008) 234–245 239
play a role on glycerol fermentation. The analysis of mutants inwhich the production of 1,2-PDO had been reduced (Fig. 3A)revealed that lower 1,2-PDO levels correlated with decreased cellgrowth (Fig. 4A). Two additional experiments, in which amplifica-tion of the 1,2-PDO pathway allowed glycerol fermentation in theabsence of tryptone supplementation, provided conclusive evi-dence of the critical role of 1,2-PDO synthesis. In the first of theseexperiments, we supplemented the growth medium of strainMG1655 with HA and observed that glycerol fermentation tookplace in the complete absence of tryptone (an OD550 of 0.35 and3.8 g/L of glycerol fermented; Fig. 4B). Externally provided HA wascompletely converted to 1,2-PDO. In another experiment, we
increased 1,2-PDO synthesis by genetic means: that is, byoverexpressing MGS (mgsA) and glycerol dehydrogenase (gldA),two of the enzymes we had identified as key members of the 1,2-PDO pathway. Strain MG1655 (pZSmgsAgldA) grew to an OD550 of0.14 and fermented 1.6 g/L of glycerol in the absence of anysupplement (Fig. 4B). These results clearly demonstrate theenabling role the 1,2-PDO pathway plays on the ability of E. coli
to ferment glycerol.
3.2. Glycerol dehydrogenase and dihydroxyacetone kinase are
required for the anaerobic fermentation of glycerol in E. coli
Although the current model for glycerol metabolism in E. coli
assumes an absolute requirement of respiratory G3PDHs (Fig. 1),we have found that G3PDH-deficient strains DglpD and DglpA wereable to ferment glycerol (Murarka et al., 2008). These results notonly supported the fermentative nature of glycerol dissimilationbut, more importantly, indicated the existence of alternativepathways for the metabolism of this compound in the absence ofelectron acceptors.
Glycerol could be directly oxidized by the enzyme glyceroldehydrogenase (GldA, a type II glycerol dehydrogenase encodedby gldA) generating DHA. However, GldA is thought to be crypticin wild-type strains, not essential to any function, and itsphysiological role is uncertain (Liyanage et al., 2001; Trunigerand Boos, 1994). GldA activation was reported to require thedisruption of the respiratory route, followed by mutagenesis and
ARTICLE IN PRESS
R. Gonzalez et al. / Metabolic Engineering 10 (2008) 234–245240
selection of mutants that recovered the ability to aerobicallymetabolize glycerol (Jin et al., 1983; Tang et al., 1982a, b). Even insuch mutants, fermentative metabolism of glycerol was notfeasible. Interestingly, we found that GldA deficiency renderedthe cells unable to anaerobically grow on or ferment glycerol(Fig. 5, DgldA). Glycerol dehydrogenase activity was 0.22370.023 mmol/min/mg protein in MG1655 but undetectable in strainDgldA. Moreover, strain DgldA recovered its ability to fermentglycerol upon complementation with a plasmid encoded GldAactivity (Fig. 5: strain (DgldA(gldA+)). Since GldA is also involved inthe conversion of HA to 1,2-PDO (Fig. 1B), and this activity wasdetected in MG1655 and abolished in mutant strain DgldA (seeprevious section), the effect of the gldA mutation could be relatedto blocking either the conversion of glycerol to DHA or thesynthesis of 1,2-PDO. In the first case, the DHA produced by GldAwould need to be phosphorylated before entering the glycolyticpathway. A PEP-dependent, nontransporting enzyme II complexthat phosphorylates DHA (DHA kinase, DHAK) has been char-
0.0
0.2
0.4
0.6
0.8
1.0
Cel
l gro
wth
(O
D55
0)
0
2
4
6
8
10
Gly
cero
l fer
men
ted
(g/L
)
MG1655 ΔgldA ΔdhaKLM ΔdhaKLM (dhaKLM+)
ΔgldA(gldA+)
ΔgldA(gldA+,dhaKLM+)
0.00
0.13
0.26
5 6 7 8 9 10pH
GldA activity (μmoles/mg protein/min.)
Fig. 5. Identification of enzymes responsible for the fermentative dissimilation of
glycerol to glycolytic intermediate dihydroxyacetone-P (DHAP). Average perfor-
mance is shown for DgldA and DdhaKLM mutants created in MG1655 and W3110
backgrounds. Strains DgldA(gldA+), DdhaKLM(dhaKLM+), and DgldA(gldA+,dhaKLM+)
carry plasmids pCA24NgldA (or pZSgldA), pZSKLM, and pZSKLMgldA, respectively.
These plasmids express E. coli’s glycerol dehydrogenase (GldA) and dihydroxya-
cetone kinase (DhaKLM) enzymes. The data for DgldA(gldA+) represent the average
of independent experiments in which strain DgldA was transformed with either
pCA24NgldA or pZSgldA. Inset shows the effect of pH on the oxidative activity of
glycerol dehydrogenase (glyDH, encoded by gldA), which converts glycerol to
dihydroxyacetone. Glycerol fermentation (white bars) and cell growth (gray bars)
are shown: values represent the means and bars standard deviations for three
samples taken once the cultures reached stationary phase.
Table 2Relationship between the ability to ferment glycerol in the absence of external elect
inducibility of a type II glycerol dehydrogenase (glyDH-II) activity in E. coli strains and
Organism Parameter
mM7SD YX/S7SD
E. coli strains
MG1655 0.04070.003 32.972.9
E. coli B 0.03670.002 34.172.7
W3110 0.03170.002 32.273.1
MC4100 0.02970.004 54.978.8
Enteric bacteria
E. cloacae 0.02270.002 30.972.8
L. richardii NG NG
B. agrestis NG NG
S. plymuthica NG NG
Experiments were conducted in minimal medium supplemented with 2 g/L tryptone
exponential growth. YX/S: growth yield (mg cell/g glycerol) calculated as the increase
Synthesis of 1,2-PDO was identified through NMR as described in Section 2. glyDH type
hydroxyacetone; NG, no growth observed; SD, standard deviation.
acterized in E. coli (Gutknecht et al., 2001; Paulsen et al., 2000).We found that this enzyme is also required for glycerolfermentation, as a DHAK-deficient strain (DdhaKLM) did not growfermentatively on glycerol (Fig. 5). When strain DdhaKLM wastransformed with plasmid pZSKLM (Bachler et al., 2005), expres-sing E. coli DHA kinase subunits DhaKLM, it recovered the abilityto grow and ferment glycerol (Fig. 5). Since E. coli DHAK is a non-transporting enzyme and DHA was not found in the fermentationbroth, we inferred that the DHA produced by the action of GldAis not secreted into the medium but phosphorylated to DHAPby DHAK.
Although complementation of the gldA mutation with a plasmid-encoded GldA activity was weak (plasmid pZSgldA), the combinedexpression of GldA and DhaKLM from plasmid pZSKLMgldArestored glycerol fermentation in strain DgldA to those levelsobserved in MG1655 (Fig. 5). Given the central role of GldA onboth dissimilation of glycerol and 1,2-PDO synthesis, the significanteffect of pH on glycerol utilization (Dharmadi et al., 2006; Murarkaet al., 2008), and considering the unusual pH dependence reportedfor GldA activity (Truniger and Boos, 1994), we assessed the effect ofpH on the GldA-dependent oxidation of glycerol (Fig. 5, inset). Aspreviously reported (Truniger and Boos, 1994), the highest activitywas observed at the most alkaline pH tested.
The ability to ferment glycerol among enteric bacteria isthought to be limited to those species possessing type I glyceroldehydrogenase (glyDH-I) and 1,3-PDODH, the latter being anenzyme that facilitates the synthesis of 1,3-PDO from glycerol(Fig. 1A). However, as shown in this section, a type II glyceroldehydrogenase, glyDH-II (i.e., GldA) mediates the anaerobicfermentation of glycerol in E. coli, a metabolic process that doesnot involve a 1,3-PDO pathway. Since two other glyDHs ofunknown role have been identified in members of the Enter-
obacteriaceae (glyDH-III and glyDH-IV) (Bouvet et al., 1995), weinvestigated their potential involvement in the anaerobic fermen-tation of glycerol. To this end, we tested several enteric bacteriareported to possess glyDH types II (E. cloacae NCDC 279-56), III(L. richardii), and IV (B. agrestis), along with an organism reportednot to possess any glyDH activity (S. plymuthica) (Bouvet et al.,1995). Only strain E. cloacae NCDC 279-56, which like E. coli
possesses a glyDH-II, was able to ferment glycerol (Table 2). Wehave also demonstrated that the fermentative metabolism ofglycerol is a general characteristic of E. coli species as other testedstrains (W3110, MC4100, and E. coli B) were also able to conductthis metabolic process (Table 2). In all tested strains that were
ron acceptors, the synthesis of 1,2-propanediol (1,2-PDO), and the presence and
other members of the Enterobacteriaceae family
Property
1,2-PDO synthesis glyDH type glyDH inducer
Yes II GLYC/HA
Yes II GLYC/HA
Yes II GLYC/HA
Yes II GLYC/HA
Yes II GLYC/HA
No III GLYC
No IV HA
No None None
and 110 mM glycerol. mM: Maximum specific growth rate (h�1) calculated during
in cell mass per glycerol consumed once the cultures reached stationary phase.
and inducibility is as described elsewhere (Bouvet et al., 1995). GLYC, glycerol; HA,
ARTICLE IN PRESS
0.0
0.4
0.8
1.2
Cel
l gro
wth
(O
D55
0)
0
3
6
9
Gly
cero
l fer
men
ted
(g/L
)20% CO280% Argon
pH 7.5
MG1655
ΔfdhFΔatpF
MG1655
ΔfdhF (fdhF
+ )
ΔatpF (atpF
+ )ΔfdhF
ΔatpFΔfdhF
ΔatpF
Fig. 6. Effect of perturbations in the formate-hydrogen lyase (FHL) and F0F1-H+-
translocating ATP (hydrol-/synthet-)ase (F0F1-ATPase) systems on glycerol
fermentation. Strains DfdhF(fdhF+) and DatpF(atpF+) carry plasmids pCA24NfdhF
and pCA24NatpF, respectively. These plasmids express E. coli’s formate dehydro-
genase (pCA24NfdhF) and b subunit of the F0 complex of F0F1-ATPase (pCA24-
NatpF). Cell growth (white bars) and glycerol fermentation (gray bars) are shown.
Unless otherwise indicated, experiments were conducted at pH 6.3 and under an
argon atmosphere. Values represent the means and bars standard deviations for
three samples taken once the cultures reached stationary phase.
R. Gonzalez et al. / Metabolic Engineering 10 (2008) 234–245 241
able to ferment glycerol, the dissimilation of this carbon sourcewas accompanied by the synthesis of 1,2-PDO.
3.3. Formate-hydrogen lyase and F0F1-H+-translocating ATP
(hydrol-/synthet-)ase systems facilitate the fermentative metabolism
of glycerol
We previously reported that the anaerobic metabolism ofglycerol in a rich medium required an active formate hydrogen-lyase (FHL) system (Dharmadi et al., 2006). In a low-supplemen-ted medium (this study), cell growth and glycerol fermentation atacidic conditions were also impaired in FHL-deficient strain DfdhF
(Fig. 6) (FdhF is required for FHL activity; Bagramyan andTrchounian, 2003; Bagramyan et al., 2002; Self et al., 2004).Strain DfdhF recovered the ability to ferment glycerol uponcomplementation with a plasmid-borne fdhF gene (Fig. 6). As inthe case of rich medium (Dharmadi et al., 2006), the FHL-deficientstrain partially recovered its ability to ferment glycerol when CO2,a product of the FHL-mediated oxidation of formate, was includedin the gas atmosphere (Fig. 6). Hydrogen, the other product offormate oxidation, has a negative effect on glycerol fermentation,which we have demonstrated is due to its recycling and creationof a redox imbalance (Murarka et al., 2008).
Since coupling between FHL, the F0F1-H+-translocating ATP(hydrol-/synthet-)ase (F0F1-ATPase), and the low affinity K+
transport system TrkA has been suggested (Hakobyan et al.,2005; Bagramyan and Trchounian, 2003; Bagramyan et al., 2002),we investigated the potential involvement of F0F1-ATPase andTrkA on glycerol fermentation. While mutations on both subunitsof F0F1-ATPase (DatpF and DatpD, F0 and F1 subunits, respectively)impaired cell growth and glycerol fermentation (Fig. 6: only DatpF
mutant shown), disruption of the TrkA system had no effect (datanot shown). Strain DatpF recovered the ability to ferment glycerolupon complementation with a plasmid-borne atpF gene (Fig. 6).Unlike the FHL-deficient mutant, a CO2-enriched atmosphere didnot allow the F0F1-ATPase-deficient strain to recover its ability toferment glycerol (Fig. 6). It is noteworthy to mention that FHL-and F0F1-ATPase-deficient mutants are able to conduct fermenta-tive metabolism of other carbon sources (Hakobyan et al., 2005;Self et al., 2004; Bagramyan and Trchounian, 2003; Bagramyanet al., 2002).
In contrast to glycerol fermentation in rich medium (Dharmadiet al., 2006), the use of low supplementation allowed this process toproceed at alkaline pHs, albeit less efficiently (Fig. 6) (Murarka et al.,2008). FHL-mediated cleavage of formate to CO2 and H2 has alsobeen shown to proceed at alkaline conditions and formatedehydrogenase H (fdhF) is known to be required for the activity ofthe FHL complex (Bagramyan and Trchounian, 2003; Bagramyan etal., 2002). Our results also indicate that FHL is active at pH 7.5 as theamount of formate accumulated in the culture medium (56 mM) isonly 80% of that of ethanol (70 mM): considering that pyruvatedissimilation under fermentative conditions primarily takes placethrough the action of the enzyme pyruvate-formate lyase (whichconverts pyruvate to acetyl-CoA and formate), the molar concentra-tion of formic acid should equal that of ethanol (plus acetate) in theabsence of FHL activity. We then investigated the potential role ofFHL on glycerol fermentation at alkaline conditions. Unlike acidic pH,the fdhF mutation had no effect on cell growth or glycerolfermentation at pH 7.5 (Fig. 6). The lack of FHL activity in the fdhF
mutant is evident in the increase in formate accumulation observed:about 78 mM formate accumulated in the culture medium of strainDfdhF compared to 56 mM in wild-type MG1655. Also unexpectedwas the finding that disruption of the F0F1-ATPase systemcompletely impaired glycerol fermentation at pH 7.5 (Fig. 6). Theseresults indicate that at alkaline conditions the roles of FHL and F0F1-ATPase systems on glycerol fermentation appear to be unrelated.
3.4. Why was glycerol fermentation not previously observed in
E. coli?
In an attempt to identify environmental determinants ofglycerol fermentation, we investigated culture conditions pre-viously used in the study of glycerol metabolism in E. coli. Unlikeour medium, the media used in previous studies contained highlevels of potassium and sodium phosphates, presumably used tomaintain neutral to slightly alkaline conditions (pH in the 7–7.5range). This medium was first reported by Tanaka et al. (1967) andsubsequently used by other investigators (Bouvet et al., 1995;Freedberg et al., 1971; Richey and Lin, 1972; Sprenger et al., 1989;St. Martin et al., 1977; Tang et al., 1982a, b; Zwaig et al., 1970). Wefound that supplementing our medium with 34 mM NaH2PO4 and64 mM K2HPO4 (as in the above media) severely impaired glycerolfermentation (Fig. 7). To distinguish whether sodium, potassium,or phosphate contributed to the observed behavior, we addedthem individually to the culture medium at the concentrationsmentioned above. Addition of sodium, as either chloride or sulfatesalt, did not have any effect (Fig. 7). Addition of either potassiumor phosphate, however, greatly limited glycerol fermentation(Fig. 7), regardless of the counterion used. Previous mediumformulations also used concentrations of glycerol ranging from 20to 30 mM (Bouvet et al., 1995; Freedberg et al., 1971; Richey andLin, 1972; Sprenger et al., 1989; St. Martin et al., 1977; Tanakaet al., 1967; Tang et al., 1982a, b; Zwaig et al., 1970). The use of amedium containing 20 mM glycerol, along with high levels ofphosphate and potassium, completely impaired cell growth andglycerol fermentation at pH 7.5 (Fig. 7).
Another difference in the conditions used in our studiesrelates to the cultivation system. Our experiments were con-ducted in fully controlled fermenters in which anaerobic condi-tions are maintained by circulating oxygen-free argon through theheadspace. Most studies reported in the literature, however, wereconducted in closed tubes/flasks, often completely filled withmedium. Such cultivation systems promote the accumulation offermentation gas hydrogen, which we have shown to be detri-mental for glycerol fermentation (Murarka et al., 2008). In anexperiment in which a Hungate tube was completely filled with
ARTICLE IN PRESS
Table 3Analysis of redox balance for the conversion of glycerol into cell mass and selected
fermentation products
Pathway Stoichiometrya (kb) DKc (Hd)
Glycerol-cell mass C3H8O3(14/3)-3CH1.9O0.5N0.2(4.3)e 1.1 (0.55H)
Glycerol-ethanol+formatef C3H8O3(14/3)-C2H6O(6)+CH2O2(2) 0 (0H)
Glycerol-succinate C3H8O3(14/3)+CO2(0)-C4H6O4(14/4) 0 (0H)
Glycerol-1,2/1,3-PDO C3H8O3(14/3)-C3H8O2(16/3) �2 (�1H)
a Pathway stoichiometry accounts only for carbon balance between reactants
and products.b The degree of reduction per carbon, k, was estimated as described elsewhere
(Nielsen et al., 2003).c Degree of reduction balance (DK) is estimated as
Pover i reactantsniciki�P
over j productsnjcikj , where n and c are the stoichiometric coefficient and the
number of carbon atoms for each compound, respectively.d
I II IVIII0.00
0.09
0.18
0.27
0.36
0.45
Yie
ld (
g su
ccin
ic a
cid
/ g g
lyce
rol)
Fig. 8. Anaerobic fermentation of glycerol as a platform for the production of fuels
and reduced chemicals. Production of succinic acid from glycerol—(I) MG1655: pH
6.3, argon; (II) MG1655: pH 6.3, 10% CO2, (III) MG1655: pH 7.5, 20% CO2; and (IV)
DdhaKLM(pZSKLcf): pH 7.5, 20% CO2. Plasmid pZSKLcf expresses C. freundii DHA
kinase subunits DhaKL.
0.0
0.2
0.4
0.6
0.8
1.0
Cel
l gro
wth
(O
D55
0)
0
2
4
6
8
10
Gly
cero
l fer
men
ted
(g/L
)
All Na+None K+ PO43-
20 m
M g
lyce
rol
llAenoN All
Clo
sed
tube
, 100
mM
gly
cero
l
pH 6.3
Sodium, potassium or phosphate added
Fig. 7. Environmental determinants of glycerol fermentation in E. coli. Effect of
concentration of sodium (Na+), potassium (K+), phosphate (PO43�), and glycerol. Na+
(34 mM NaCl or 17 mM Na2SO4), K+ (128 mM KCl), and PO43� (98 mM NH4H2PO4)
were added to the medium to assess the individual impact of these ions on glycerol
fermentation. The combined effect of sodium, potassium, and phosphate was
evaluated by adding 34 mM NaH2PO4 and 64 mM K2HPO4 indicated. Unless
otherwise noted, experiments were conducted at pH 7.5, 37 1C, 110 mM glycerol,
2 g/L tryptone, and flushing the headspace with argon. Cell growth (white bars)
and glycerol fermentation are shown. Values represent the means and bars
represent the standard deviations for three samples taken once the cultures
reached stationary phase. The effect of Na+, K+, and PO43� was independent of the
counterion: data represent average behavior for cases in which different counter-
ions were used.
R. Gonzalez et al. / Metabolic Engineering 10 (2008) 234–245242
minimal medium supplemented with the aforementioned levelsof sodium, potassium, and phosphate, and in the presence of110 mM glycerol and 2 g/L of tryptone, no significant cell growthor glycerol fermentation was observed (Fig. 7). Taken together,these results clearly demonstrate that previous attempts toanaerobically ferment glycerol using E. coli were not successfulbecause the experiments were conducted at conditions thatnegatively affect glycerol fermentation.
Net redox units, H, are expressed per mole of glycerol (H�NAD(P)H ¼
FADH2 ¼ ‘‘H2’’).e Cell mass formula is the average reported for E. coli (Nielsen et al., 2003).
Conversion of glycerol into cell mass neglects carbon losses as 1-C metabolites. In
consequence, the degree of reduction balance in this case represents the minimum
amount of redox units generated.f Similar results are obtained by considering the conversion of glycerol to
ethanol and H2-CO2.
3.5. Implications of our findings for metabolic engineering
Since the feasibility of engineering E. coli for the production ofchemicals and fuels has been extensively documented, our findingscould enable the use of metabolic engineering strategies to developE. coli-based platforms for the anaerobic production of reducedchemicals from glycerol at yields higher than those obtained fromcommon sugars. An example is succinic acid, whose productionfrom sugars is limited by the availability of reducing equivalents.Fortunately, its synthesis from glycerol is feasible through a redox-balanced pathway (Table 3). However, very low production ofsuccinate from glycerol was observed in our experiments withMG1655 (Fig. 8), a consequence of glycerol dissimilation throughthe PEP-dependent GldA-DHAK pathway (i.e., low PEP availability).Using a combination of appropriate pH and concentration of CO2,along with the replacement of E. coli PEP-dependent DHAK (DdhaK
mutation) with C. freundii ATP-dependent DHAK (expression fromplasmid pZSKLcf), we achieved an almost 10-fold increase insuccinate yield (Fig. 8). These results clearly demonstrate thefeasibility of developing a metabolic engineering platform for theproduction of fuels and reduced chemicals from glycerol.
4. Discussion
4.1. A new model for the fermentative metabolism of glycerol in the
Enterobacteriaceae: trunk and auxiliary pathways in E. coli
The anaerobic fermentation of glycerol in enteric bacteria haslong been considered a privilege of species that have an active 1,3-
PDO pathway (Fig. 1). The synthesis of 1,3-PDO allows the cells toattain redox balance by consuming reducing equivalents gener-ated during the incorporation of glycerol into cell mass (Table 3:‘‘glycerol-1,3-PDO’’ and ‘‘glycerol-cell mass’’ pathways). Wehave demonstrated, however, that E. coli can fermentativelymetabolize glycerol in a 1,3-PDO-independent manner. The abilityto ferment glycerol was correlated to the cells’ capacity tosynthesize 1,2-PDO, a compound we identified as a product ofglycerol fermentation. Since the conversion of glycerol to 1,2-PDOconsumes 1 mole of reducing equivalents per mole of 1,2-PDOsynthesized (Table 3), it follows that the amount of 1,2-PDO foundin the fermentation broth (�0.5 mM) would be sufficient toprovide a sink for the reducing equivalents generated in thesynthesis of cell mass (0.6 mM reducing equivalents). The lattercalculation assumes that about 20% of the cell mass originatesfrom glycerol (Murarka et al., 2008), and makes use of the degreeof reduction analysis shown in Table 3 for the conversion ofglycerol to cell mass. Clearly, the incorporation of glycerol into cellmass generates excess reducing equivalents that E. coli can only‘‘dispose off’’ by the synthesis of 1,2-PDO. Given the low activity ofthe 1,2-PDO pathway, the utilization of building blocks containedin the tryptone reduces the use of glycerol in the synthesis of cellmass and the redox imbalance associated with it. In agreementwith this hypothesis, we found that stimulation of the 1,2-PDO
ARTICLE IN PRESS
O
O
OH
Cell MassGlycerol
Dihydroxyacetone
Dihydroxyacetone-P Phosphoenolpyruvate
Pyruvate
GldA(gldA)
NADH
DHAK
OH
OHHOEthanol
O
HO OH
OHO O
P OH
O
OH -OPO
HO OO
OH
OH
1,2-propanediol
NADH
2NADH
NAD(P)H
ATP
OH
OH
yNADH
xATP
(dhaKLM )
O
HO
Hydroxyacetone
Acetyl-CoA
AdhE(adhE )
PFL( pflB)
Formate
GldA( gldA) NADH
CoA
O
OOH
Glycolysis
CO2 + H2
FHL ( fdhF,hycB-I)
Fig. 9. A new paradigm for glycerol fermentation in E. coli and other enteric bacteria possessing a type II glycerol dehydrogenase (glyDH-II). The proposed trunk pathway for
the conversion of glycerol to glycolytic intermediate dihydroxyacetone-P in E. coli is composed of enzymes glycerol dehydrogenase (GldA, a glyDH-II) and dihydroxyacetone
kinase (DHAK). The pathways involved in the synthesis of 1,2-propanediol and ethanol are considered auxiliary as they enable glycerol fermentation by ensuring redox
balance and ATP generation, respectively. GldA is a key enzyme in this model and, as other type II glyDHs, it is induced by both glycerol and hydroxyacetone. The latter is an
intermediate in the synthesis of 1,2-propanediol. Coefficients x and y were used to represent the moles of ATP and NADH consumed and generated, respectively, in the
synthesis of cell mass from glycerol.
R. Gonzalez et al. / Metabolic Engineering 10 (2008) 234–245 243
pathway led to cell growth and glycerol fermentation in theabsence of tryptone supplementation (Fig. 4B).
Based on our findings, we propose a new model for thefermentative metabolism of glycerol in enteric bacteria in which:(i) the synthesis of 1,2-PDO provides a means to consumereducing equivalents generated during synthesis of cell mass,thus enabling redox-balanced conditions (Fig. 9; Table 3) and(ii) the conversion of glycerol to ethanol, through a redox-balanced pathway, fulfills energy requirements by generatingATP via substrate-level phosphorylation (Fig. 9; Table 3). Theproposed model includes an oxidative, GldA-DHAK-mediatedpathway that works in partnership with a reductive 1,2-PDO-producing pathway (Fig. 9). We propose that inducibility of GldA(a glyDH-II) by both glycerol and HA (Truniger and Boos, 1994)represents a ‘‘metabolic footprint’’ of the involvement of thisenzyme in both glycerol dissimilation and 1,2-PDO synthesis(Fig. 9).
Trunk and auxiliary pathways can be readily identified in theproposed model. Trunk pathways are those directly involved inthe generation of glycolytic intermediates. Auxiliary or enablingpathways, on the other hand, are those enabling glycerolfermentation by facilitating functions such as redox balance,ATP synthesis, CO2 generation, pH homeostasis, generation ofproton motive force, etc. In this work we identified GldA-DHAK asthe trunk pathway responsible for the conversion of glycerol intoglycolytic intermediate DHAP. Ethanol and 1,2-PDO pathwayswere considered auxiliary as they ensure ATP generation andredox-balanced conditions, respectively. Additional auxiliarypathways include the FHL-mediated conversion of formic acid toCO2 and H2 and the F0F1-ATPase system (Fig. 10). FHL might beimportant to maintain CO2 supply for cell growth (Dharmadi et al.,2006) (Fig. 6) or in establishing a proton-motive force (PMF)(Bagramyan and Trchounian, 2003; Hakobyan et al., 2005), thelatter required for cell growth and viability. We hypothesize thatF0F1-ATPase could take advantage of the PMF generated by FHL atacidic conditions (Fig. 10) to perform function(s) essential toglycerol fermentation. At alkaline conditions, on the other hand,the lower intracellular pH (pHi), compared to extracellular pH
(pHe), permits the generation of PMF independent of FHL (Fig. 10).For example, the diffusion of undissociated acids, such as formicacid, across the membrane and their dissociation at the higherpHe would result in the generation of PMF (Konings et al., 1995)(Fig. 10). This hypothesis would explain our findings that glycerolfermentation at alkaline conditions is independent of FHL but stillrequires F0F1-ATPase (Fig. 6). Given the fact that pHi is higher thanpHe at acidic conditions, generation of PMF would require anactive FHL, thus explaining why both systems are required (Fig. 6).The use of the aforementioned PMF by the F0F1-ATPase system inthe generation of energy, metabolite transport, or other functionsappears to be a key factor in the fermentative metabolism ofglycerol.
4.2. Relationship between proposed pathways and environmental
determinants of glycerol fermentation
The above pathways provide a framework to explain theobserved changes in cell growth and glycerol fermentation as afunction of pH, concentrations of potassium, phosphate, andglycerol, as well as the effect of cultivation systems that promoteor prevent the accumulation of fermentation gases hydrogen andcarbon dioxide (Fig. 7) (Dharmadi et al., 2006; Murarka et al.,2008).
High levels of phosphate promote the decomposition of bothDHA and HA (two key intermediates in the aforementionedpathways) and negatively affect GldA activity and its inducibilityby HA (Truniger and Boos, 1994). It is noteworthy that GldAis the most important enzyme in the proposed model forthe fermentative metabolism of glycerol, being required inboth trunk and auxiliary pathways (Fig. 9). In addition, MGsynthase, a key enzyme responsible for 1,2-PDO synthesis(Fig. 1B), is inhibited by high phosphate levels (Cooper, 1984;Hopper and Cooper, 1971; Zhu et al., 2001). High concentrationsof potassium, on the other hand, increase the toxicity of MG(Booth, 2005), a key intermediate in the synthesis of 1,2-PDO(Fig. 1B). The low-affinity of GldA for glycerol (Km is 3–40 mM;
ARTICLE IN PRESS
F0
F1
Periplasm
Cytoplasm
H+
H+ ATPADP+Pi
FocA/B
H2+CO2
H++HCOO-
H2+CO2
H2OH2CO3H++HCO3
-
H2O
H2CO3
CA
Hyd
H2 2H+
Formate
HCOOH
Q+2HQ+2H++
QHQH22
H++HCO3- Glycerol
Pyruvate/PEP
AcCoA
Fumarate +2H+ Succinate
H+
FHL
Q - Quinone pool
H+
FRD
Fig. 10. Relationship between formate-hydrogen lyase (FHL) and F0F1-H+-translocating ATP (hydrol-/synthet-)ase (F0F1-ATPase) systems and the metabolism of formic acid,
carbon dioxide, and hydrogen. FHL and F0F1-ATPase are auxiliary systems required for glycerol fermentation in E. coli. Equilibrium reactions for formic acid (pKa ¼ 3.74) and
CO2 in water (CO2/HCO3� pKa ¼ 6.3) are shown. Enzyme pyruvate formate lyase was assumed to mediate the conversion of pyruvate into AcCoA and formic acid (HCOOH).
Abbreviations/nomenclature: AcCoA, acetyl coenzyme A; CA, carbonic anhydrase; FHL, formate hydrogen-lyase; FocA/B, formate transporters; FRD, fumarate reductase;
Hyd, hydrogenases 1 and 2; and F0 and F1, subunits of the F0F1-ATPase.
R. Gonzalez et al. / Metabolic Engineering 10 (2008) 234–245244
Truniger and Boos, 1994) would explain both the requirementof high concentrations of glycerol for its fermentative meta-bolism (Fig. 7) and our observation that 10–30 mM glycerolremained unmetabolized in the medium of cultures that havereached stationary phase (Dharmadi et al., 2006; Murarka et al.,2008).
The effect of pH on glycerol fermentation (Dharmadi et al.,2006; Murarka et al., 2008) can also be related to its impact on theaforementioned pathways. GldA exhibits strong pH dependencewith higher oxidative activity at very alkaline pHs (Fig. 5) andreductive activity at neutral to alkaline conditions (Fig. 3C). Acidicconditions reduce not only the oxidative activity of glyDH, butalso MG-reducing activities (Fig. 3C), which are required for thesynthesis of 1,2-PDO (Fig. 1B). Alkaline conditions, on the otherhand, could increase both the oxidizing activity of GldA (Fig. 5)and the toxicity of MG (Booth, 2005), the latter a key intermediatein the synthesis of 1,2-PDO. Clearly, the intracellular pH needs tobe carefully controlled to avoid low activities of key enzymes andMG toxicity. For example, while an extracellular pH of 6.3apparently could prevent MG toxicity, the cells would still requirea system to prevent the pH from falling below the levelspermissible for glycerol-oxidizing and MG- and HA-reducingactivities (Figs. 4C and 6). By converting formic acid to CO2 andH2, the FHL system is known to prevent cytoplasmic acidification(Sawers and Clark, 2004), which could be a reason why FHL isrequired for glycerol fermentation at acidic conditions (Fig. 6).Interestingly, despite the requirement of FHL for glycerolfermentation under acidic conditions, its activity can generateexcess hydrogen that, if accumulated, would greatly impair thismetabolic process (see below).
The nature of the gas atmosphere determines, to a large extent,the feasibility of glycerol fermentation (Dharmadi et al., 2006;Murarka et al., 2008). This effect is also linked to the proposedpathways. Fig. 10 illustrates the interrelation between formic acid,carbon dioxide, and hydrogen metabolism and the FHL and F0F1-ATPase systems during glycerol fermentation. In a recent study wehave demonstrated that the negative effect of hydrogen, a productof formate oxidation via FHL, is due to its metabolic recycling,which in turn generates an unfavorable internal redox state(Murarka et al., 2008). The low activity of the 1,2-PDO pathway,which represents the only active, redox-consuming route would
explain the detrimental effect of the redox imbalance created byhydrogen recycling. The effect of CO2 could be related to theproposed roles of FHL and F0F1-ATPase, namely the generation(FHL) and utilization (F0F1-ATPase) of PMF (Fig. 10). If externallyprovided, diffusion of CO2 into the cells and its capture asbicarbonate through the action of carbonic anhydrase (Merlinet al., 2003) would result in a decrease of pHi (Fig. 10). Underconditions in which the pHe is controlled, like in our experiments,CO2 supplementation could then result in an increase in thePMF. Therefore, CO2 supplementation could at least partiallysubstitute for FHL in generating PMF. This would explain why theFHL mutant partially recovered its ability to ferment glycerol in aCO2-enriched atmosphere while no effect was seen on the F0F1-ATPase mutant (Fig. 6). CO2-enrichment also had a positive impacton glycerol fermentation by MG1655 at acidic conditions(Murarka et al., 2008), an effect very unlikely related to a CO2-limitation since oxidation of formate provides large amounts ofintracellular CO2.
Acknowledgments
This work was supported by grants from the National ResearchInitiative of the U.S. Department of Agriculture Cooperative StateResearch, Education and Extension Service (2005-35504-16698)and the U.S. National Science Foundation (CBET-0645188).We thank B.L. Wanner, F.R. Blattner, B. Erni, and H. Mori forproviding research materials, S. Moran for assistance with NMRexperiments, and K. Smith and Y. Moon for assistance with somegenetic constructs.
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