Journal of Industrial Microbiology & Biotechnology (2001) 27, 322–328D 2001 Nature Publishing Group 1367-5435/01 $17.00
Fermentation characterization and flux analysis of recombinantstrains of Clostridium acetobutylicum with an inactivated solRgene
LM Harris1, L Blank1, RP Desai1, NE Welker2 and ET Papoutsakis1
1Department of Chemical Engineering, Northwestern University, Evanston, IL 60208, USA; 2Department of Biochemistry,Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA
The effect of solR inactivation on the metabolism of Clostridium acetobutylicum was examined using fermentationcharacterization and metabolic flux analysis. The solR - inactivated strain ( SolRH ) of this study had a higher rate ofglucose utilization and produced higher solvent concentrations ( by 25%, 14%, and 81%, respectively, for butanol,acetone, and ethanol ) compared to the wild type. Strain SolRH( pTAAD ), carrying a plasmid - encoded copy of thebifunctional alcohol / aldehyde dehydrogenase gene ( aad ) used in butanol production, produced even higherconcentrations of solvents ( by 21%, 45%, and 62%, respectively, for butanol, acetone, and ethanol ) than strain SolRH.
Clarithromycin used for strain SolRH maintenance during SolRH( pTAAD ) fermentations did not alter productformation; however, tetracycline used for pTAAD maintenance resulted in 90% lower solvent production. Journal ofIndustrial Microbiology & Biotechnology ( 2001 ) 27, 322 – 328.
Keywords: Clostridium acetobutylicum; metabolic engineering; flux analysis; solvents; solR; tetracycline; clarithromycin
strain carries an additional plasmid pTAAD - encoded copy of theaad gene, which encodes a bifunctional butanol formation
Clostridium acetobutylicum, a Gram - positive, spore - forming,
enzyme [ 15 ]. SolRH was selected as the host strain in this study
obligate anaerobe, has long been of industrial interest for the
since it has a defined genetic modification; one non - replicative
production of the solvents acetone and butanol from renewable
plasmid is integrated into the chromosome of SolRH in contrast
sources [ 8 ]. Due to its ability to catabolize a wide range of
to strain SolRB, which has multiple non - replicative plasmids
substrates into solvents, C. acetobutylicum was in widespread use
integrated into its chromosome [ 16 ]. Additionally, the tetracy-
internationally for fermentative production of acetone and butanol
cline minimum inhibitory concentrations ( MICs ) in SolRH was
between 1910 and 1960. However, unprecedented growth of the
less than 5 g / ml compared to 10 g / ml in SolRB ( data not
petroleum industry during the middle of the 20th century led to a
shown ). Thus, use of the tetracycline resistance plasmid pTAAD
drastic decline in industrial C. acetobutylicum fermentations since it
( as reported here ) was more practical in strain SolRH. Strain
was more economical to produce solvents from petroleum - based
SolRH( pTAAD ) was constructed in order to determine if an
sources [ 8 ]. Metabolic engineering of this organism to construct
increased aad gene dosage would further enhance the solvent -
superior solvent - producing strains may be the key to a resurgence
producing capabilities of strain SolRH. The use of the antibiotics
of industrial - scale clostridial fermentations.
clarithromycin and tetracycline for selection was also examined
While the majority of genes directly responsible for formation
to examine their effects on product formation.
of acids and solvents in this strain have been cloned andstudied, less is known about mechanisms for controlling geneexpression in solventogenic clostridia. Recently, a putativerepressor of solvent formation genes, SolR, was identified and
a molecular characterization of solR - modified strains was
reported [ 16 ]. Inactivation of the solR gene ( located on the
Bacterial strains and plasmids are listed in Table 1.
pSOL1 megaplasmid ) [ 6 ], using genomic integration of a non -replicative plasmid, resulted in the generation of strains whichproduce higher levels of solvents. Preliminary characterization of
one of these strains ( renamed SolRB, formerly Mutant B )
Escherichia coli was grown aerobically at 378C in Luria – Bertani
included a single batch fermentation [ 16 ]. The present study
( LB ) medium, and C. acetobutylicum was grown anaerobically at
focuses on detailed fermentation characterization and metabolic
378C in Clostridium Growth Medium ( CGM [ 18,22 ] ). Colonies of
flux analysis [ 2 ] of a second strain ( SolRH; formerly Mutant
E. coli and C. acetobutylicum were obtained on agar - solidified LB
H ) and a derivative of that strain [ SolRH( pTAAD ) ]. The latter
or Reinforced Clostridial Medium ( RCM; Difco, Sparks, MD ),respectively. For recombinant strains, liquid media were appropri-ately supplemented with erythromycin ( Em; 100 g / ml ), tetra-cycline ( Tc; 10 g / ml ), and chloramphenicol ( Cm; 35 g / ml );
Correspondence: ET Papoutsakis, Department of Chemical Engineering, North-
40 g / ml of erythromycin and 10 g / ml of tetracycline were
western University, Evanston, IL 60208, USAReceived 12 September 2000; accepted 21 July 2001
used in solid media as needed. For bioreactor fermentations,
Analysis of C. acetobutylicum solR inactivation strainsLM Harris et al
( single copy of plasmid integrated into genome ),MIC 5 g / ml
( multiple copies of plasmid integrated into genome ),MIC 10 g / ml
aAbbreviations: solR putative repressor of sol operon genes; MLSr, macrolide lincosamide streptogramin B - resistant; recA, homologous recombinationabolished; lacZ, - galactosidase; mcrBC, methlycytosine - specific restriction system; Cmr, chloramphenicol - resistant; È3TI, È3T methylase; Tcr,tetracycline - resistant; Apr, ampicillin - resistant; AAD, aad, alcohol / aldehyde dehydrogenase.
bAmerican Type Culture Collection, Manassas, VA.
cNew England Biolabs, Beverly, MA.
erythromycin was substituted by its pH - stable derivative clari-
which point it was controlled through addition of 6 M NH4OH.
thromycin ( 100 g / ml ). E. coli strains were stored long - term at
À 858C in LB medium with 10% glycerol. For long - term storage of
acetone, ethanol, butanol, and acetoin, 3%; acetate and butyrate,
C. acetobutylicum, strains were maintained as spores on RCM agar
at a pH of 6.8, or frozen at À 858C in CGM with 15% glycerol.
Metabolic flux analysis involves the calculation of specific ( per
The isolation of plasmids from E. coli via the alkaline lysis
unit cell mass ) in vivo intracellular reaction rates for various
method and further manipulations of E. coli plasmid DNA were
enzymatic reactions ( which will be referred to as ‘‘fluxes’’ ). These
performed using standard protocols [ 9 ]. A modified alkaline
fluxes [ in units of mmol ( g cells ) À 1 h À 1 ] were calculated from
lysis method was used for isolation of plasmid DNA from
substrate utilization and product formation data using a system of
C. acetobutylicum [ 7 ]. A previously published method [ 14 ] was
linear equations developed from the metabolic reaction stoichiom-
etry, a widely used and validated technique [ 1 – 3,7,17 ]. A mass
methylated plasmid DNA [ 13 ] using a Bio - Rad Gene Pulser
extinction coefficient of 51 ( g cells ) À 1 cm À 1 was used to convert
optical density measurements ( A 600 ) into cell dry weight concen-trations ( g cells l À 1 ). The flux analysis was performed using
software developed specifically for analysis of C. acetobutylicumfermentation data [ 2,17 ], and the pathways fluxes considered are
Plasmid pTAAD was generated by cloning a 3.06 - kb DNA
depicted in Figure 1. For these analyses, the fermentation times
fragment containing aad gene with its two promoters into the
were re - scaled [ 1,3,7 ]: the normalized scale, T
tetracycline - resistant E. coli – C. acetobutylicum shuttle vector
pTLH1 [ 7 ]. These tetracycline - resistant vectors were suitable for
h at A 600 = 1. On this time scale, the transition from
exponential phase to stationary phase typically occurs at T
use in the MLSr ( macrolide lincosamide streptogramin B - resistant )
Therefore, the stages of the fermentations are classified as ‘‘Early’’
for T N < 10 h, and ‘‘Late’’ for T N > 10 h. The calculation of bothkinetic and integral fluxes and the errors of such calculations have
been discussed previously [ 1,3,7 ].
concentration analysis were performed as previously reported[ 3,7 ]. Batch fermentations were performed in either a BiostatM
( Braun Biotech, Allentown, PA ) or a BioFloII bioreactor ( NewBrunswick Scientific, Edison, NJ ) with working volumes of 1.5
Strain SolRH [ 16 ] and newly developed strain SolRH( pTAAD )
and 4.01, respectively. After inoculating the growth medium
were characterized using fermentation experiments and metabolic
( CGM pH 6.2 ), the culture pH was allowed to fall to 5.0, at
flux analysis to determine the impact of two genetic alterations, the
Analysis of C. acetobutylicum solR inactivation strains
Figure 1 C. acetobutylicum primary metabolic pathways and corresponding in vivo fluxes. The conversion between major carbon containing speciesis depicted without cofactors. Selected enzymes are shown in bold and abbreviated: PTA, phosphotransacetylase;AK, acetate kinase;CoAT, CoAtransferase;AADC, acetoacetate decarboxylase;PTB, phosphotransbutyrylase;BK, butyrate kinase, AAD, alcohol / aldehyde dehydrogenase( DH );BDHA and BDHB, butanol DHG isozymes A and B. Reactions involved with the intracellular fluxes ( e.g., rBIO and rHYD ) examinedhere are indicated by dashed boxes. Further details on these metabolic fluxes can be found in Refs. [ 2,3,7,17 ].
solR inactivation alone and in combination with increased aad gene
formation pathways ), rACUP, rBYUP ( acid re - utilization ), and
rBUOH ( butanol formation ). Strain SolRH exhibited a consistentelevation in rGLY1 from T N = 0 to 5 h ( Figure 2A ). Thisincreased flux of glucose utilization was also reflected by
Fermentation and flux analysis of solRH versus wild
elevated acid formation fluxes ( rPTAAK and rPTBBK ) from
T N = 0 to 5 h compared to the WT strain ( Figure 2B and C ). In
Fermentation characterizations showed that inactivation of solR in
addition, strain SolRH exhibited an earlier reversal ( indicated by
strain solRH resulted in altered product concentration profiles
negative flux values ) of flux through the butyrate formation
compared to the WT organism, C. acetobutylicum ATCC 824
pathway ( rPTBBK ). The most dramatic effect of the solR
( Table 2 ). Although peak butyrate levels did not differ significantly
inactivation was on solvent formation fluxes. Acetone formation
between strains SolRH and WT, final SolRH butyrate concen-
flux is the sum of the acetate re - utilization flux ( rACUP ) and the
trations were 68% lower than WT values ( 13 vs. 41 mM ). Both
butyrate re - utilization flux ( rBYUP ) and showed significantly
maximum and final acetate levels were slightly higher in SolRH
higher peak levels compared to the WT strain. The acetate re -
compared to WT. solR inactivation had a profound effect on solvent
utilization flux, rACUP, in strain SolRH reached a peak value
formation, resulting in final butanol, acetone, and ethanol
twice that of the WT strain ( Figure 2D ). The butyrate re -
concentrations that were 25%, 14%, and 81% higher, respectively,
utilization flux, rBYUP, in strain SolRH reached a peak value up
to 60% higher than in the WT ( Figure 2E ). In addition, the peak
Metabolic flux analysis of the fermentation data was used to
in rBYUP was 5 h earlier in strain SolRH than in the WT strain.
further characterize strain SolRH. The key pathway fluxes ( i.e.,
However, the elevated acetone formation fluxes in strain SolRH
specific intracellular reaction rates ) discussed ( Figure 1 ) are
were sustained for a shorter period of time than in the WT strain.
rGLY1 ( glucose utilization ), rPTAAK and rPTBBK ( acid
The butanol formation flux reached a peak up to 30% higher ( but
Analysis of C. acetobutylicum solR inactivation strainsLM Harris et al
Table 2 Product formation in fermentation experiments at pH 5.0
Data are shown as mean ± SEM values from two experiments. Glucose was fed in strain SolRH( pTAAD ) fermentations to prevent glucose depletion andpremature termination of the fermentation.
was sustained for a shorter period of time ) in strain SolRH than
pathways fluxes ( rGLY1, rGLY2, rTHL, and rBYCA ) were twice
as high, and acid formation fluxes, rPTAAK and rPTBBK, were
In addition to the time profiles, an integral flux analysis ( see
120% and 840% higher, respectively, in strain SolRH than in the
Materials and Methods; data not shown ) further reinforced the
WT strain. Solvent formation fluxes, rACUP, rBUOH, and rETOH,
observations discussed above, but also showed significant differ-
were ca. 130%, 150%, and 400%, respectively, higher in strain
ences that are not apparent from the kinetic analysis: the central
SolRH. While acetone formation via butyrate re - utilization
Figure 2 Time course profiles of metabolic fluxes in cultures of strain SolRH ( solid symbols ) and of the WT ( open symbols ). Different symbolsrepresent data from the two replicate experiments of Table 2.
Analysis of C. acetobutylicum solR inactivation strains
( rBYUP ) was elevated by ca. 60%, this pathway is responsible for
When tetracycline and clarithromycin were used in combina-
only a small amount of the carbon reutilization in strain SolRH. The
tion, acetate and butyrate levels were both significantly elevated.
calculated metabolic pathway fluxes were used to estimate changes
Final butanol, acetone, and ethanol concentrations were all reduced
in the split ratio at the butyryl – CoA branchpoint. During the
by ca. 80%, and cell densities significantly decreased ( Table 3 ).
stationary phase of the WT strain, the central pathway ( rBYCA )
Patterns of product formation when clarithromycin alone was
provided 82% of the carbon used in butanol formation, while
used during the SolR( pTAAD ) fermentation were similar to the
rBYUP and rPTBBK provided 10% and 8%, respectively. In
control fermentation with no selective pressure ( Table 3 ).
contrast, in strain SolRH, rBYCA provided 65% of the carbon,
These results indicate that the use of clarithromycin to select
while rPTBBK provided 28% of the carbon used for butanol
for SolRH( pTAAD ) does not alter product formation patterns,
formation. In fact, this reversal of the butyrate formation pathway is
growth rates, or cell densities in this organism. In contrast, the
largely responsible for the 150% increase in rBUOH, while glucose
data show that the use of tetracycline in SolRH( pTAAD )
fermentations, alone and in combination with clarithromycin,inhibits solvent production and enhances acidogenesis. Althoughtetracycline is useful for selection of plasmid - carrying strains, use
Effects of tetracycline and clarithromycin on product
of this antibiotic drastically reduces solvent production and is
therefore not appropriate for use in fermentations for solvent
Initially, two antibiotics were used for maintenance of C. aceto-
production. Loss of this family of plasmids during batch
butylicum strains SolRH( pTLH1 ): the control strain carrying a
fermentations of recombinant C. acetobutylicum strains in the
plasmid without the cloned aad gene and SolRH( pTAAD ).
absence of antibiotics as a selection pressure is known to be
Clarithromycin ( 100 g / ml ), a stable derivative of erythromycin,
was used to select for the host strain SolRH, and tetracycline use
Another example of inhibition of product formation in the
( 10 g / ml ) assured presence of the replicative plasmids pTLH1
presence of tetracycline involves the use of strain 34, a derivative
and PTAAD. Clarithromycin resistance was conferred by the
of C. acetobutylicum ATCC 824 with a single chromosomal
presence of the non - replicative plasmid integrated into the genomic
insertion of the tetracycline resistance conjugative transposon
solR [ 16 ], while tetracycline resistance was conferred by the tetM
Tn916. When tetracycline was used to select for strain 34, no
gene on the multicopy vectors pTLH1 and pTAAD. During
butyraldehyde dehydrogenase activity was detected and no
fermentations, the use of two antibiotics coincided with a drastic
butanol was produced ( C. Cass, unpublished results ). Tn916
decrease in growth and solvent production. Thus, experiments were
has been used extensively for generating clostridial strain
designed to determine the effects of tetracycline and clarithromycin
mutants, including solvent formation mutants ( e.g., Ref. [ 12 ] ).
use separately and together ( Table 3 ).
Our data suggest that the use of tetracycline - resistant Tn916 in
When tetracycline was used during SolRH( pTAAD ) fermen-
generating solvent formation mutants may be inappropriate. Use
tations, cell densities were decreased, peak butyrate concen-
of tetracycline and its derivatives in various microbial systems
trations were increased by over 35%, while final butyrate values
inhibits several types of enzymes, including some dehydrogenase
exceeded the control values by over 1000% ( Table 3 ). Peak and
systems [ 4 ]. While the mechanism by which tetracycline affects
final acetate concentrations were not significantly affected.
product formation is not proven, it is possible that tetracycline
Tetracycline inhibition of solvent production was severe: butanol,
interferes with the dehydrogenase activity required for solvent
acetone, and ethanol concentrations were decreased by 92%,
production in recombinant strains of C. acetobutylicum such as
94%, and 90%, respectively, compared to the no - antibiotic
Fermentation and flux analysis of strains solRH
Table 3 Tetracycline ( Tc ) and clarithromycin ( Clt ) effects on strain
( pTAAD ) versus solRH( pTLH1 ): effect of
plasmid - encoded aldehyde alcohol dehydrogenaseStrain SolRH( pTAAD ) was characterized in order to determine the
effect of pTAAD - encoded aldehyde alcohol dehydrogenase [ 5 ] onproduct formation in strain SolRH ( Table 2 ). No antibiotics were
used during SolRH( pTLH1 ) fermentations. However, selectivepressure was applied for maintenance of strains SolRH( pTAAD )
and SolRH( pTLH1 ) until the time that reactors were inoculated at
the start of each fermentation. Comparative plating studies ( data
not shown ) on selective and non - selective media indicated that the
plasmid - carrying strains were stable throughout the course of
fermentations without selective pressure.
Fermentations of strain SolRH( pTAAD ) were performed at pH
5.0 based on other studies showing that pH = 5.0 resulted in higher
solvent production than either pH 4.7 or 5.5 ( data not shown ).
These SolRH( pTAAD ) cultures were supplemented with glucose
during mid to late exponential growth to avoid prematuretermination of solvent production due to glucose exhaustion.
Fermentation experiments were carried out at pH 5.0. There was no glucose
Concentrated glucose ( 3.8 M ) was added when the residual glucose
fed into these fermentations. For the duplicate experiment, values arereported as mean ± SEM.
concentration dropped below 150 mM in order to keep glucose
Analysis of C. acetobutylicum solR inactivation strainsLM Harris et al
concentration above 100 mM until solvent production began to
[ SolRH( pTLH1 ) ] fermentations. Final butanol, acetone, and
plateau. Equivalent glucose supplementation of either the WT or
ethanol concentrations were elevated by 22%, 42%, and 161%,
SolRH( pTLH1 ) strains did not affect solvent production ( data not
respectively. While the SolRH( pTAAD ) fermentation doubling
shown ). The results of the SolRH( pTAAD ) fermentations were
time was 16% lower than the doubling time of the control, maximal
compared to the control strain SolRH( pTLH1 ), which was
optical densities did not vary significantly.
developed to account for host – plasmid effects [ 21 ] using a
Kinetic metabolic flux analysis ( Figure 3 ) indicated noticeable
plasmid without a cloned clostridial gene. Inactivation of solR,
differences between strains SolRH( pTAAD ) and SolRH( pTLH1 ).
combined with increased aad gene dosage, resulted in a strain
Glucose utilization in SolRH( pTAAD ) was transiently elevated at
[ SolRH( pTAAD ) ] with improved solvent - producing ability. At
the shift to the stationary phase ( Figure 3A ). A similar trend was
final concentrations of 248 mM ( 17.6 g / l ) butanol, 141 mM
observed in the other central pathway fluxes ( data not shown ). A
( 8.2 g / l ) acetone, and 47 mM ( 2.2 g / l ) ethanol, total solvent
stationary phase elevation was also observed in rPTAAK fluxes,
production ( 28 g / l ) by SolRH( pTAAD ) far exceeded the 19 g / l
but not in rPTBBK fluxes ( Figure 3B and C ). Instead, the butyrate
solvents produced by the WT. It should be noted that aad
formation pathway supported increased butyrate re - utilization. As
overexpression in the WT strain ( ATCC 824 ) did not result in
with strain SolRH, solvent formation fluxes showed the most
increased solvent formation [ 14 ]. This suggests that the inactiva-
dramatic differences. The peak acetone formation fluxes were
tion of solR is necessary for the positive effect of aad over-
evaluated in strain SolRH( pTAAD ) by up to 200% and 35% for
Maximum and final acetate concentrations produced by
SolRH( pTLH1 ) ( Figure 3D and E ). The peak butanol formation
SolRH( pTAAD ) were 30% higher than control values. Peak
flux, rBUOH, was elevated by up to 200% during the stationary
butyrate levels were 23% higher in SolRH( pTAAD ) than in the
phase ( Figure 3F ), and the ethanol formation flux was
control; however, final butyrate concentrations were 40% lower.
significantly elevated ( data not shown ). It should also be noted
This implies better re - utilization of butyrate by strain Sol-
that while strain SolRH( pTAAD ) exhibited higher peak fluxes,
RH( pTAAD ). Solvent production was significantly increased in
strain SolRH( pTLH1 ) sustained the fluxes for a longer period of
Figure 3 Time course profiles of metabolic fluxes in strains SolRH( pTAAD ) ( solid symbols ) and SolRH( pTLH1 ) ( open symbols ). Differentsymbols represent data from the two replicate experiments of Table 2. For strain SolRH( pTAAD ), data from two additional replicates ( not shownon Table 2 ) are also plotted.
Analysis of C. acetobutylicum solR inactivation strains
As before, integral flux analysis ( data not shown ) was used to
6 Green EM, ZL Boynton, LM Harris, FB Rudolph, ET Papoutsakis and
detect differences between SolRH( pTAAD ) and SolRH( pTLH1 )
GN Bennett. 1996. Genetic manipulation of acid formation pathwaysby gene inactivation in Clostridium acetobutylicum ATCC 824.
that are less noticeable from the kinetic flux analysis. After the shift
to stationary phase, The differences between strains Sol-
7 Harris LM, RP Desai, NE Welker and ET Papoutsakis. 2000.
RH( pTAAD ) and SolRH( pTLH1 ) were most pronounced: glucose
Characterization of recombinant strains of the Clostridium acetobuty-
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licum butyrate kinase inactivation mutant: need for new physiologicalmodels for solventogenesis and butanol inhibition? Biotechnol Bioeng
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