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Physiology, Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans


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Novel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacterium Clostridium phytofermentans was adapted to increased ethanol concentrations. The resulting ethanol-tolerant strain (ET strain) has nearly doubled ethanol tolerance relative to wild-type, but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bi-functional acetaldehyde CoA/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Physiology, Genomics, and Pathway Engineering of an Ethanol-
Tolerant Strain of Clostridium phytofermentans
Andrew C. Tolonen,
Trevor R. Zuroff,
*Mohandass Ramya,
Magali Boutard,
Tristan Cerisy,
Wayne R. Curtis
Genoscope-CEA, CNRS-UMR8030, Université d’Évry, Évry, France
; Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania,
; Department of Genetic Engineering, SRM University, Kattankulathur, India
Novel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential
cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the
presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacterium Clostridium phytofermen-
tans was adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tol-
erance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the
ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regula-
tion, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol
dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression of
Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and
restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations
incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for im-
proving microbial solvent tolerance.
The conversion of lignocellulosic biomass to fuels and com-
modities represents a large-scale, renewable alternative to pe-
troleum. This multistep bioconversion is traditionally performed
in a series of independent processes, but consolidated bioprocess-
ing (CBP) is an alternative paradigm with potential economic ad-
vantages (1). In CBP, enzyme production, hydrolysis, and fer-
mentation occur in a single reactor, leading to savings in capital
and operating costs as well as increased efficiencies due to system
synergies (2). Here we studied Clostridium phytofermentans,a
promising CBP candidate that ferments plant biomass primarily
to ethanol (3,4). C. phytofermentans hydrolyzes pretreated corn
stover (both glucans and xylans) with efficiencies similar to those
seen with simultaneous saccharification and cofermentation
(SSCF) using commercial enzymes and xylose-fermenting yeast
(Saccharomyces cerevisiae)(
5). Fermentation of pretreated corn
stover by C. phytofermentans reaches a titer of 7 g liter
(6), and stable cocultures of C. phytofermentans and S. cerevisiae
cdt-1 ferment 70 g liter
cellulose to 22 g liter
ethanol (7),
which is an ethanol concentration that reduces C. phytofermentans
growth. Thus, application of CBP bacteria such as C. phytofermen-
tans will likely require improving their solvent tolerances without
compromising enzyme production or fermentation of soluble
carbohydrates to ethanol.
Considerable effort has focused on adapting clostridia to
increased solvent levels and investigating the genetic and phys-
iological changes associated with adaptation to solvents (8–
13). Other studies have shown increased ethanol production in
clostridia that primarily produce fermentation products other
than ethanol. C. cellulolyticum expressing pyruvate decarboxylase
and alcohol dehydrogenase (ADH) overcame pyruvate accumu-
lation and shifted fermentation products from lactate to acetate
and ethanol (14). In C. thermocellum, redirection of carbon flow
through pyruvate kinase (15), inactivation of lactate dehydroge-
nase and phosphotransacetylase (16), and deletion of hydroge-
nases (17) all improve ethanol production. These results demon-
strate that, although the genetic tools are being developed only
now, engineering improved ethanol production in cellulolytic
clostridia is possible. However, development of strains that are
ethanol tolerant (ET) and that also produce ethanol in high titers
remains a significant challenge.
Here we sought to develop a strain of C. phytofermentans with
both improved resistance and production of ethanol, particularly
from cellulose. We isolated an ethanol-tolerant (ET) C. phytofer-
mentans strain by serial transfer into increasing ethanol levels and
characterized its growth and fermentation properties. We se-
quenced the ET strain genome to reveal genomic mutations that
arose during adaptation and overcame reduced ethanol yield in
the ET strain by heterologous expression of an alternative ethanol
formation pathway. We discuss how the findings from this study
improve our understanding of how microbes adapt to elevated
concentrations of solvents such as ethanol.
Received 23 February 2015 Accepted 27 May 2015
Accepted manuscript posted online 5 June 2015
Citation Tolonen AC, Zuroff TR, Ramya M, Boutard M, Cerisy T, Curtis WR. 2015.
Physiology, genomics, and pathway engineering of an ethanol-tolerant strain of
Clostridium phytofermentans. Appl Environ Microbiol 81:5440 –5448.
Editor: J. L. Schottel
Address correspondence to Andrew C. Tolonen,
*Present address: Trevor R. Zuroff, Biodomain, Shell Technology Center, Houston,
Texas, USA.
A.C.T. and T.R.Z. contributed equally to this article.
Supplemental material for this article may be found at
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
5440 August 2015 Volume 81 Number 16Applied and Environmental Microbiology
on August 13, 2015 by guest from
Culturing. C. phytofermentans ISDg (ATCC 700394) was grown anaero-
bically by preparing cultures in a Coy anaerobic chamber with a 1.5%
/98.5% N
atmosphere. Cultures were incubated without shaking at
30°C in GS2 medium (18) adjusted to a pH of 7 and supplemented with
carbon sources as described elsewhere in the text. Growth kinetics were
monitored by optical density at 600 nm (OD
) in sealed 100-well mi-
crotiter plates (Bioscreen 9502550) as previously described (19); cultures
were briefly shaken to resuspend cells before each optical density mea-
surement. Cellulose, cellobiose, and glucose cultures for substrate con-
sumption and fermentation product analysis were grown in 100-ml se-
rum bottles, which were sealed with butyl rubber stoppers after degassing.
The C. phytofermentans ethanol-tolerant (ET) strain was selected by
serial transfer (1:50 dilution) into culture tubes containing 10 ml of GS2
medium supplemented with increasing ethanol concentrations. Starting
with cultures in 4% (vol/vol) (31.5 g liter
) ethanol, cultures were trans-
ferred weekly to fresh medium containing the same ethanol concentration
and to medium with a 1%-higher ethanol concentration. If no growth was
observed at the higher concentration after 1 week, cultures were retrans-
ferred to the same ethanol concentrations. If a culture grew at a higher
ethanol concentration, this culture was transferred again to that ethanol
concentration and to a 1%-greater ethanol concentration. Growth was
observed at 5% ethanol after 7 weekly transfers, 6% after 13 transfers, and
7% after 19 transfers. Each time the ethanol tolerance improved, cells were
TABLE 1 Bacterial strains, primers, and plasmids used in this study
Strain name Genotype or description
Source or sequence
phytofermentans ISDg
ATCC type strain 700394 Susan Leschine Laboratory, University of Massachusetts, Amherst, Amherst,
E. coli Top 10 hsdR mcrA endA1 recA1 rpsL (Str
(cloning strain)
Invitrogen Corporation
E. coli S17-1 RP4-2 (Km::Tn7Tc::Mu-1) recA1
endA1 (conjugal strain)
Yale E. coli Stock Center
pdcAdhB_F Forward primer for amplification of pdc
and adhB from pES120
pdcAdhB_R Reverse primer for amplification of pdc
and adhB from pES120
pQexp_F Forward primer to confirm pdc-adhB
insertion in pQexp
pQexp_R Reverse primer to confirm pdc-adhB
insertion in pQexp
cphy3925F Forward primer to amplify Cphy3925
coding sequence
cphy3925R Reverse primer to amplify Cphy3925
coding sequence
pES120 Source of Z. mobilis pdc and adhB genes Jay Keasling Laboratory, University of California, Berkeley, Berkeley, CA, USA
pQexp Replicating plasmid for C.
Andrew Tolonen Laboratory, Genoscope-CEA, Évry, France
pQexpE pQexp with Z. mobilis pdc and adhB
cloned into the unique BamHI and
PvuI sites
This study
Str, streptomycin, Km, kanamycin; Tc, tetracycline.
FIG 1 Growth of wild-type (WT) (A) and ethanol-tolerant (ET) (B) C. phytofermentans strains at 30°C in GS2 medium with 3 g liter
glucose supplemented
with ethanol at the following levels (vol/vol): 0% (red squares), 2% (green circles), 4% (blue triangles), 6% (yellow diamonds), and 7% (black X’s). Data points
of growth (OD
) represent means of results from triplicate 400-l cultures in sealed microtiter plates; error bars represent 1 standard deviation.
C. phytofermentans Ethanol Tolerance and Production
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plated, individual colonies were picked, and liquid cultures were reinoc-
ulated to ensure that selection was based on a specific strain with increased
ethanol tolerance and not on a consortium of strains that collectively
survived the increased ethanol concentration. The ET strain is thus a
colony-purified isolate from a mother culture that grew in GS2 medium
supplemented with 7% (vol/vol) ethanol. After colony purification, it was
confirmed the ET strain has ethanol resistance similar to that of the
mother culture.
Cellulose and fermentation analysis. The level of cellulose remaining
in the culture was measured by taking a 1-ml sample from a 10-ml culture
tube with a sterile syringe, placing it in a preweighed 1.7-ml microcentri-
fuge tube, and centrifuging at 13,000 gfor 10 min. The supernatant was
removed, and the cellulose pellet was washed and centrifuged again at
13,000 gfor 10 min. The rinsed pellet was placed at 70°C to dry until a
constant mass was reached. The contribution of cellular biomass to total
cellulose weight was not accounted for and was assumed to be minimal
due to low anaerobic biomass yields.
Fermentation product concentrations were measured in 0.22-m-pore-
size-filtered culture supernatant using an Agilent 1100 high-performance liq-
uid chromatograph (HPLC) with a Jasco RI-1531 refractive index detector
(RID) and an Aminex HPX-87H cation exchange column (Bio-Rad). The
HPLC was run using a 0.01 M sulfuric acid mobile phase, 65°C column tem-
perature, 30°C RID temperature, 25 l sample volume, and 0.6 ml/min op-
erating flow rate. Product formation is reported relative to the concentration
in the medium at the point of inoculation. Gas phase measurements were
made by removing 1 ml of headspace and injecting 100 l into a gas chro-
matograph (Model 8610C multiple-gas analyzer; SRI Instruments). Argon
was used as a carrier gas and was adjusted to 30 lb/in
gauge pressure. A
stainless steel molecular sieve (13) and silica gel-packed columns were used
for sample separation, and the components were detected using a thermal
conductivity detector (TCD). The column compartment temperature was
held initially at 40°C for 3.5 min and then ramped to 160°C for 2 min and to
300°C for 10 min, after which the column was allowed to cool to 40°C for the
remainder of the sample run.
Genome sequencing and variant analysis of the ET strain. A total of
12 g of genomic DNA was extracted from a 4-ml ET strain culture using
a Sigma GenElute bacterial genomic DNA kit (NA2110). DNA was se-
quenced on an Illumina MiSeq instrument with an insertion size of 795 bp
FIG 2 C. phytofermentans yields (mole of product per mole of glucose equivalent consumed) of ethanol from WT (dark red) and ET (light red) strains (A) and
acetate from WT (dark blue) and ET (light blue) strains (B). Statistical differences between WT and ET yield averages are indicated at Pvalues of 0.01 (*), 0.05
(**), and 0.1 (***) using Student’s ttest. (C) C. phytofermentans WT and ET cellobiose consumption and fermentation products in GS2 cellobiose medium with
0, 3.75, or 7% (vol/vol) added ethanol. Bars show concentrations (in millimoles) of cellobiose consumption (gray) and production of ethanol (red), acetate
(blue), and formate (yellow) by WT (dark bars) and ET (light bars) strains. All cultures were grown in serum bottles containing GS2 medium at 30°C with 30g
cellulose, 10 g liter
cellobiose, or 30 g liter
glucose and measured after 14 days. Bars represent averages of the results from 4 cultures; error bars
represent 1 standard deviation.
Tolonen et al.
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and 300-bp paired-end reads. A total of 5,077,282 reads passed quality
filtering and mapping by Picard Tools (
/picard), yielding approximately 250-fold genome coverage. Sequence
variants (single nucleotide polymorphisms [SNPs] and indels) in the ET
genome relative to the reference strain genome (NCBI accession no.
NC_010001.1) were identified using the Genome Analysis Toolkit
(GATK) (20) (see the supplemental material for detailed descriptions of
the filtering and variant-calling methods).
ADH purification and activity measurements. The Cphy3925-en-
coding genes from the wild-type (WT) and ET strains were cloned by
ligation-independent cloning (21) into pET-22B()as previously de-
scribed (19). Genes were cloned with C-terminal His tags using primer
pair cphy3925F/cphy3925R (Table 1) and confirmed by sequencing. Plas-
mids were transformed into Escherichia coli BL21(DE3) (Novagen 70235)
and grown in 50 ml TB medium (12 g liter
tryptone, 24 g liter
extract, 4 ml liter
glycerol) to an OD
of 1, and expression was in-
duced by adding 500 M IPTG (isopropyl--D-thiogalactopyranoside)
and incubating overnight at 20°C. Cells were pelleted, resuspended in lysis
buffer (50 mM phosphate buffer [pH 8], 0.5 M NaCl, 10 mM imidazole,
15% glycerol, 1 mM Pefabloc [Sigma 76307]), and lysed by sonication
(Cole-Parmer Vibracell CV33) with lysozyme (Novagen 71230). His-
tagged proteins were purified from 50 ml culture on nickel-nitrilotriacetic
acid (Ni-NTA) spin columns (Qiagen 31014) and visualized on 12% SDS-
PAGE gels (Novex 12% Bis-Tris gel NP0342BOX). Enzyme activities were
measured as described in reference 22 in 100 lof 100 mM Tris-HCl (pH
8) containing cofactor [0.2 mM NADH(P)H or 2 mM NAD(P)
] and
substrate (300 M acetyl coenzyme A [acetyl-CoA] or CoA, 18 mM acet-
aldehyde, 2 M ethanol) as appropriate. NAD(P)H was measured as
340-nm absorbance (extinction coefficient, 6.22 mM
) using a
SAFAS UVmc2 spectrophotometer at room temperature.
Plasmid construction and transformations. pQexpE is derived from
pQexp (23), a plasmid that replicates stably with erythromycin selection
in E. coli (200 gml
erythromycin) and C. phytofermentans (40 g
erythromycin on plates and 200 gml
erythromycin in liquid
culture). To construct pQexpE, the Z. mobilis pdc and adhB genes were
PCR amplified from pES120 (24) using primer pair pdcAdhB_F/dcAd-
hB_R (Table 1) and cloned into the unique BamHI and PvuI sites of
pQexp. The insertion was confirmed by sequencing using primer pair
pQexp_F/pQexp_R. pQexpE was conjugally transferred to C. phytofer-
mentans using donor strain E. coli S17-1. Conjugation was performed as
described in references 23 and 25, except polyethersulfone membranes
were used to support 50-l culture mixtures and only nalidixic acid with-
out trimethoprim was used to select against E. coli following mating. Pos-
itive C. phytofermentans transconjugants containing pQexpE were con-
firmed by colony PCR using primer pair pQexp_F/pQexp_R to amplify
the 2.9-kb pdc-adhB operon.
Nucleotide sequence accession number. The FASTQ-formatted
DNA sequencing files for the ET genome were submitted to the European
Nucleotide Archive under primary accession no. PRJEB7255.
Isolation and physiology of an ethanol-tolerant C. phytofer-
mentans strain. The growth of wild-type (WT) C. phytofermen-
TABLE 2 Genomic DNA variants in the ET strain
(amino acids) Amino acid variant
Confidence (Q)
value Annotation
Energy and metabolism
Cphy3925 (AdhE) 872 G609D 8,929 Fe-dependent bifunctional acetaldehyde-CoA/alcohol dehydrogenase
Cphy0215 (RnfA) 191 C26S 5,569 Ferredoxin:NAD
oxidoreductase (Fno); couples electron transfer
from reduced ferredoxin to NAD
with cation transport out of the
cell to create an electrochemical gradient
Cphy3255 259 20-bp insertion at
15,938 FMN-dependent nitroreductase
Cphy0543 (MgtA/MgtB) 920 K417N 8,661 P-type ATPase for Mg
uptake transporter or Ca
Cphy3778 258 1-bp deletion, Y239
11,687 Na
efflux transporter, ABC-2 transmembrane permease (PFAM
accession no. PF06182)
Membrane and cell wall
Cphy0233 (PlsD) 237 D80N 9,246 Membrane synthesis: glycerol-3-phosphate acyltransferase; transfers
a fatty acid to the 1 position of glycerol-3-phosphate
Cphy0107 (MurC) 469 G115D 8,841 Peptidoglycan synthesis: ATP-dependent ligation of L-alanine and
UDP-N-acetylmuramic acid to form
Gene regulation
Cphy3040 301 D182N 8,214 LysR transcriptional activator/repressor
Cphy3687 (PolB) 1,278 D189Y 8,824 -Subunit of DNA-directed RNA polymerase
Noncoding changes
Cphy0267 524 Q65Q (synonymous) 7,470 Modification methyltransferase
Intergenic G¡T
None 52 Transversion between 2 convergently transcribed genes, encoding
Cphy1313 and Cphy1314
Cphy3036 (ApbE) 381 L244L (synonymous) 9,011 Thiamine biosynthesis: membrane-associated lipoprotein
Data include the gene name, encoded protein length, amino acid (if coding) or DNA variant, confidence value (Q) of the variant call, and annotation of the mutated protein. The
probability (P) that a variant exists in the genome is reported as a Phred-scaled probability, i.e., Q⫽⫺10 log
(1 P), meaning that a Qvalue of 100 indicates an error
probability (1 P)of10
(see GATK reports in the supplemental material for more information). FMN, flavin mononucleotide.
C. phytofermentans Ethanol Tolerance and Production
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tans was monitored in cultures supplemented with 0, 2, 4, 6, or 7%
(vol/vol) ethanol (Fig. 1A), and generation times (hours) and
maximum cell densities (OD
) were calculated (see Table S1 in
the supplemental material). The WT strain cultures grew similarly
at 0% ethanol and 2% ethanol but growth was significantly inhib-
ited at 4% ethanol, and very little growth was observed at 6 and 7%
ethanol. In contrast, the ET strain grew at 4, 6, and 7% ethanol,
with a maximum cell density (OD
) at 7% ethanol similar to that
of the WT strain at 4% ethanol (Fig. 1B; see also Table S1).
While the ET strain was more ethanol resistant, it grew more
slowly than the WT strain and grew at reduced cell yields in cul-
tures without added ethanol (Fig. 1). Glucose consumption by the
ET strain was also slower than glucose consumption by the WT
strain, reflecting the reduced growth rate. When grown on 30 g
glucose, the ET strain consumed only 20 g liter
in 200 h, while the WT strain completely exhausted the glucose in
less than 150 h (see Fig. S1 in the supplemental material). Thus,
the enhanced growth of the ET strain at a high ethanol concentra-
tion is accompanied by reduced growth under standard condi-
tions, supporting the idea that the ET strain has altered physiol-
ogy, with results similar to those seen with C. thermocellum
adapted to 5% ethanol (26,27). However, we observed no mor-
phological differences between ET and WT colonies grown on
GS2 agar plates or cells grown in liquid GS2 medium (see Fig. S2).
The ET strain also produces less ethanol per unit of sugar con-
sumption than the WT strain. For example, ethanol yield (moles
of product per mole of glucose equivalent consumed) by the ET
strain decreased 25 to 50% relative to that seen with the WT strain
when growing on cellulose, cellobiose, and glucose (Fig. 2A),
whereas the acetate yields of the strains were similar (Fig. 2B). We
also measured cellobiose consumption and formation of the pri-
mary fermentation products ethanol, acetate, and formate in WT
and ET cultures grown in cellobiose medium supplemented with
0, 3.75, or 7% ethanol (Fig. 2C). Ethanol production by the WT
strain was significantly lower at 3.75% ethanol, and cellobiose
consumption and fermentation ceased at 7% ethanol. In contrast,
cellobiose consumption and ethanol production by the ET strain
decreased only slightly with increased ethanol supplementation,
demonstrating the robustness of the ethanol resistance pheno-
type. In all, these results highlight the phenotypic advantages of
the ET strain with respect to metabolizing and producing ethanol
at elevated ethanol concentrations but also indicate that those
advantages occur at the expense of lower ethanol yields and slower
growth at low ambient concentrations of ethanol.
Genome sequence of the ET strain. The C. phytofermentans ET
strain genome contains 12 variants relative to the WT strain (Ta-
ble 2), many fewer than a C. thermocellum ET genome with similar
ethanol resistance that had 200 to 500 changes (9). While ethanol
resistance is likely a complex, multigenic trait, the small number of
changes in the ET strain genome shed light on DNA variants that
could have functional roles in ethanol tolerance. Two of the 12
mutations were in genes encoding transcriptional regulators that
could effectuate broad gene expression changes: PolB, the -sub-
unit of DNA-directed RNA polymerase, and a LysR regulator,
Cphy3040. LysR-type regulators often colocalize in the genome
with their targets (28) and the gene encoding Cphy3040 is adja-
cent to a gene encoding a NAD-dependent aldehyde dehydroge-
nase, suggesting that this regulator is related to alcohol formation.
Ethanol increases the permeability of the cell membrane, re-
sulting in toxic leakage of metabolites out of the cell (29). Ethanol
resistance thus often involves membrane modifications such as
altered protein content (8) or longer chain fatty acids and more
plasmalogen lipids (30) that increase membrane rigidity to miti-
gate the fluidizing effect of ethanol. The ET strain has a D80N
change in the putative acyl-acceptor binding pocket (NCBI acces-
sion no. cd07989) of Cphy0233, a homolog of C. butyricum PlsD
that transfers a fatty acyl group to the sn-1 position of glycerol-3-
phosphate in phospholipid biosynthesis (31). The D80N
FIG 3 Comparison of activities of purified Cphy3925 AdhE from wild-type and ET strains. (A) SDS-PAGE gel of purified Cphy3925 from the wild-type (WT)
and ET (G609D) strains showing single bands of the expected 95-kDa molecular mass. (B to E) Reactions for the two-step, bidirectional interconversionof
acetyl-CoA acetaldehyde and ethanol: reduction of 300 M acetyl-CoA to acetaldehyde (red) (B), reduction of 18 mM acetaldehyde to ethanol (green) (C),
oxidation of 2 M ethanol to acetaldehyde (purple) (D), and oxidation of 18 mM acetaldehyde to acetyl-CoA (blue) (E). Enzyme activities are shown in millimoles
of NAD(P)H per micromole of enzyme per second measured using NADH(P)H or NAD(P)
cofactors. Bar heights represent averages of duplicate activity
measurements, and error bars represent 1 standard deviation.
Tolonen et al.
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Cphy0233 mutation may thus enable synthesis of a more rigid,
ethanol-resistant cell membrane by altering which fatty acids are
incorporated into phospholipids.
The ET strain has a C26S mutation in the RnfA subunit of the
membrane-bound Rnf complex that couples efflux of H
(33) with electron transfer from reduced ferredoxin to
(34). The resulting electrochemical gradient is harnessed
by an F
ATPase for ATP synthesis. The C. phytofermentans Rnf
complex (Cphy0211 to Cphy0216) and F
ATPase (Cphy3735
to Cphy3742) are highly expressed on all tested carbon sources
(19) and may be important for energy conservation, similarly to C.
ljungdahlii (32). However, Rnf generates NADH, which may not
be tolerated by the ET strain that cannot reoxidize NADH by
AdhE-mediated ethanol formation (see below). The C26S RnfA
variant may thus cripple the Rnf complex, which sacrifices ATP
production, but may benefit the ET strain by balancing cellular
The ET strain also has mutations in two transporters putatively
involved in cation homeostasis. Cphy0543 is homologous to
MgtA, a P-type ATPase upregulated at low ambient Mg
trations (35) to mediate Mg
uptake (36)orCa
(37). Cphy3778, the membrane component of an ABC trans-
porter (PFAM accession no. PF06182), appears to be cotrans-
cribed with Cphy3780, an ABC-type Na
efflux protein (NCBI
accession no. cd03267). In Bacillus subtilis, this Na
efflux system
is induced by ethanol and is proposed to compensate for an influx
of extracellular Na
resulting from a weakened membrane barrier
(38). The variants in these cation transporters may increase their
activities to alleviate cation leakage due to ethanol stress.
AdhE activities. The ET strain has a G609D variant in
Cphy3925 AdhE, a putative acetaldehyde-CoA dehydrogenase
and alcohol dehydrogenase (ADH). The G609D mutation is in a
conserved position in the active site of the C-terminal ADH do-
main (NCBI accession no. cd08178). A previous study reported an
ethanol-tolerant C. thermocellum strain with AdhE mutations
(P704L and H735R) that shifted the cofactor specificity from
FIG 4 (A) Diagram of C. phytofermentans carbon metabolism showing insertion of the Z. mobilis pdc-adhB alternative ethanol formation pathway. Enzymatic
steps: {a}, lactate dehydrogenase (Cphy1117); {b}, pyruvate formate lyase (Cphy1174); {c}, pyruvate ferredoxin oxidoreductase (Cphy3558); {d}, Rnf ferredoxin:
oxidoreductase complex (Cphy0211 to Cphy0216); {e}, phosphate acetyltransferase (Cphy1326); {f}, acetate kinase (Cphy1327); {g}, acetaldehyde
dehydrogenase (Cphy1428 or Cphy3925); {h}, alcohol dehydrogenase (Cphy3925 or Cphy1029); {i}, Zymomonas mobilis pyruvate decarboxylase (pdc); {j},
alcohol dehydrogenase (adhB). Dashed lines represent multienzyme reactions where all enzymes are not listed. (B) Plasmid map of pQexpE for pdc-adhB
expression in C. phytofermentans. Plasmid features: Gram-negative pUC origin of replication (oriR pUC), C. phytofermentans pyruvate ferredoxin oxidoreduc-
tase promoter (P3558) to express the Z. mobilis pdc-adhB genes, the RP4 conjugal origin of transfer (oriT), the Gram-negative/Gram-positive erythromycin
resistance gene from TN1545 (ermR), the Gram-positive pAMB1 origin (oriR pAMB1), and repE-encoded protein.
FIG 5 Cellulose consumption (A) and ethanol production (B) by non-plas-
mid-bearing () and pdc-adhB-containing () WT and ET cultures at 30°C in
GS2 medium with 3 g liter
-cellulose after 30 days. Bars represent averages
of the results from duplicate cultures in serum bottles from three independent
experiments, and error bars represent 1 standard deviation. Statistical differ-
ences between treatments are indicated at Pvalues of 0.01 (*) and 0.05 (**)
using Student’s ttest.
C. phytofermentans Ethanol Tolerance and Production
August 2015 Volume 81 Number 16 5445Applied and Environmental Microbiology
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NADH to NADPH, which was proposed to confer ethanol re-
sistance by altering the internal redox balance (9). To deter-
mine the effect of the G609D mutation on Cphy3925 enzyme
activity, we purified WT and ET versions of the enzyme (Fig.
3A) and tested their in vitro catalysis of the two-step, bidirec-
tional reactions converting acetyl-CoA to ethanol using either
NADH or NADPH cofactors.
The mutated Cphy3925 lost NAD(H)-dependent activities,
but, unlike the mutated AdhE in C. thermocellum, the G609D
mutation did not result in NADPH-dependent ADH activity (Fig.
3B to E). Instead, our results support the notion that the ET strain
arrested AdhE-mediated interconversion of acetyl-CoA, acetalde-
hyde, and ethanol, which helps explain why the C. phytofermen-
tans ET strain had lower ethanol yield. AdhE loss of function could
mitigate ethanol stress by reducing intracellular levels of ethanol
and its highly toxic precursor, acetaldehyde. C. phytofermentans
encodes four Fe-dependent ADHs in addition to Cphy3925 as well
as a Zn-dependent ADH. All 6 ADHs are expressed, and
Cphy3925 and Cphy1029 are among the most highly expressed
proteins on all tested carbon sources (19,39). C. phytofermentans
thus likely produces ethanol by the concerted action of multiple
ADHs, and these other ADHs, especially Cphy1029, are responsi-
ble for ethanol produced by the ET strain.
Ethanol pathway engineering. To augment ethanol produc-
tion by the ET strain, an alternative ethanol production path-
way comprised of pyruvate decarboxylase (Pdc) and alcohol
dehydrogenase (AdhB) from Zymomonas mobilis (Fig. 4A) was
transferred into C. phytofermentans on the replicating pQexpE
plasmid (Fig. 4B). Together, these enzymes couple decarboxyl-
ation of pyruvate to ethanol with the oxidation of NADH and thus
represent an alternative to the AdhE ethanol formation pathway.
We chose to express foreign enzymes rather than a WT copy of
Cphy3925 because AdhE multimerizes (40) such that the mutant
AdhE could have a dominant-negative effect in a merodiploid.
Expression of pQexpE increased cellulolysis by 30% in both
the WT and ET strains (Fig. 5A) and boosted ethanol production
by 70% relative to the ET strain (P0.01), thereby restoring
ethanol yields to WT levels (Fig. 5B). CO
production increased
disproportionately relative to H
production in WT and ET
strains expressing pQexpE (Table 3). Elevated CO
synthesis is
likely due to increased pyruvate decarboxylation by the Pdc en-
zyme. Previous results showed that Pdc/AdhB expression en-
hanced cellulolysis and ethanol production in WT C. cellulolyti-
cum, which was proposed to result from consumption of excess
pyruvate that otherwise leads to metabolic arrest (14). Increased
metabolism (cellulolysis and production of CO
and ethanol) by
C. phytofermentans expressing Pdc/AdhB might be due to allevia-
tion of inhibition by excess pyruvate. Alternatively, expression or
activity of glycolytic enzymes might be regulated by NADH levels
such that NADH reoxidation by Pdc/AdhB stimulates glycolysis,
which results in increased substrate utilization.
Conclusions. In this study, we investigated the genetic basis
and phenotypic consequences of microbial ethanol tolerance by
isolating, characterizing, and engineering an ethanol-resistant
(ET) strain of Clostridium phytofermentans. The ET strain grows at
higher ethanol concentrations than the wild-type strain (Fig. 1)
and continues to produce ethanol at a 7% ambient ethanol
concentration (Fig. 2C) but has impaired growth (Fig. 1) and
ethanol yield (Fig. 2A) relative to the wild type. The genome
sequence of the ET strain revealed 12 mutations in genes in-
volved in diverse aspects of metabolism (Table 2), including a
G609D variant in the bifunctional acetaldehyde CoA/alcohol
dehydrogenase AdhE that abolishes its activity (Fig. 3). We
complemented the AdhE mutation in the ET strain by express-
ing pyruvate decarboxylase (Pdc) and alcohol dehydrogenase B
(AdhB) from Zymomonas mobilis on the pQexpE plasmid (Fig.
4), which boosted substrate conversion (Fig. 5A) and restored
ethanol production (Fig. 5B).
Additional work is needed to enhance C. phytofermentans
plant biomass degradation and ethanol formation rates and prod-
uct titers. Recently, improvement of C. phytofermentans’ growth
on cellobiose, cellulose, and xylan by experimental evolution
yielded strains that also produced ethanol more quickly (41). The
genome sequence of the ET strain presented here suggests other
novel approaches to potentially improve ethanol resistance and
production. For example, our results suggest that further studies
on ethanol resistance should focus on PlsD-mediated fatty acid
incorporation into phospholipids, LysR-regulated gene expres-
sion patterns, overexpression of the Rnf complex to stimulate
AdhE-mediated ethanol production, and prevention of cation
C. phytofermentans ISDg was kindly provided by Susan Leschine (Univer-
sity of Massachusetts, Amherst).
A.C.T. was supported by a CNRS Chaire d’Excellence and the Geno-
scope-CEA. T.R.Z. was supported by a GRFP Fellowship from the Na-
tional Science foundation (grant no. DGE1255832) and a John and Jean-
nette McWhirter Fellowship from The Pennsylvania State University.
M.R. was supported by the Faculty Abroad Program, SRM University.
Anaerobic growth capabilities for this work were financed by the Geno-
scope-CEA and a grant from the Department of Energy Advanced Re-
search Project Agency—Energy (ARPA-e; no. DE-AR0000092) to W.R.C.
We thank Alex Rajangam for facilitating M.R.’s international ex-
change, Sergio Florez for useful feedback, Patrick Hillery and Sam
Bjork for experimental assistance, and Nymul Khan for supporting GC
analysis. We acknowledge the Huck Institute of the Life Sciences
Shared Fermentation Facility for providing HPLC analytical capabili-
ties and the Genoscope-CEA Sequencing Platform for sequencing the
ET strain. Finally, we thank George Church and Noel Goddard for
facilitating a sabbatical leave for W.R.C. at Harvard, where these stud-
ies were initiated.
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(ml) H
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ET 25.2 4.7 3.47 0.12 4.46 0.19 0.78
ET (pdc-adhB) 31.0 2.3 4.41 0.15 7.20 0.13 0.61
Data represent averages standard deviations of the results from four replicates.
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... Evolution in continuous culture setups was applied to obtain biotechnological platform strains like Saccharomyces cerevisiae, Escherichia coli and Clostridium phytofermentans with enhanced ethanol tolerance (Ma and Liu, 2010;Tolonen et al., 2015;Yomano et al., 1998). ...
The objective of this project was the development of enhanced methylotrophic chassis strains capable of converting methanol as carbon and energy source into biomass and ultimately into commodity chemicals under industrial conditions. Methanol is an alternative to carbohydrates as feedstock in industrial biotechnology as its use does not interfere with food supply and its production can start from CO2.A prerequisite for an efficient and large scale industrial fermentation is stable growth of the methylotrophic producer strain on high methanol concentrations. For this purpose, two closely related methylotrophic strains, Methylobacterium extorquens AM1 and TK 0001, which both have a growth optimum at about 1% methanol, were adapted in continuous culture to proliferate stably in the presence of methanol of up to 10% (v/v). The adaptations were conducted using GM3 devices enabling automated long term cultivation of microorganisms.Growth curves recorded for isolates obtained from evolved populations showed enhanced proliferation in the presence of methanol at 5% as compared with wild type cells. The isolates showed comparable albeit not identical growth pointing to heterogeneity among the adapted cells in the population.Genomic sequencing of isolated clones at different steps of the adaptation revealed differences in their mutation profiles. The gene metY coding for O-acetyl-L-homoserine sulfhydrylase was found to be mutated in all isolates. This enzyme undergoes a side reaction with methanol leading to the production of the methionine analogue methoxinine known to be toxic through incorporation into proteins.Enzymatic tests conducted with these mutants showed an almost complete loss of activity even with their natural substrates, validating the involvement of MetY in methanol toxicity.Transcriptomic analysis was performed to study the gene expression response of an evolved derivative of M. extorquens TK 0001 to short and long term exposure to high methanol and compared with the response of the ancestor strain. Genes implicated in cell division, ribosomal and flagellar structures, protein stability and iron uptake showed differences in expression patterns between the strains.The M. extorquens TK 0001 cells adapted to high methanol produced more biomass from methanol than the wildtype cells. This suggests that a compound synthesized through a pathway branching from the central metabolism would be produced in higher yield from methanol by the adapted cells compared to the wildtype cells. The production of D-lactate was tested for wildtype and evolved cells both overexpressing native lactate dehydrogenase. The evolved cells produced more lactate than the control cells, confirming the interest of this methanol adaptation.
... C. phytofermentans is remarkable among the Clostridium genus due to its ability to catabolize a broad range of substrates. Its genome encodes over 169 carbohydrate-active enzymes, the largest number among sequenced clostridia, and its efficient ethanol production makes it a model system for cellulosic biofuel production 21,23,[34][35][36][37] . E. coli is a well studied, facultative anaerobe capable of fermenting a broad range of substrates including glucose and glycerol which is a widely available waste product from biodiesel production 21,23,38 . ...
Full-text available
Planktonic cultures, of a rationally designed consortium, demonstrated emergent properties that exceeded the sums of monoculture properties, including a >200% increase in cellobiose catabolism, a >100% increase in glycerol catabolism, a >800% increase in ethanol production, and a >120% increase in biomass productivity. The consortium was designed to have a primary and secondary-resource specialist that used crossfeeding with a positive feedback mechanism, division of labor, and nutrient and energy transfer via necromass catabolism. The primary resource specialist was Clostridium phytofermentans ( a.k.a. Lachnoclostridium phytofermentans ), a cellulolytic, obligate anaerobe. The secondary-resource specialist was Escherichia coli , a versatile, facultative anaerobe, which can ferment glycerol and byproducts of cellobiose catabolism. The consortium also demonstrated emergent properties of enhanced biomass accumulation when grown as biofilms, which created high cell density communities with gradients of species along the vertical axis. Consortium biofilms were robust to oxic perturbations with E. coli consuming O 2 , creating an anoxic environment for C. phytofermentans . Anoxic/oxic cycling further enhanced biomass productivity of the biofilm consortium, increasing biomass accumulation ~250% over the sum of the monoculture biofilms. Consortium emergent properties were credited to several synergistic mechanisms. E. coli consumed inhibitory byproducts from cellobiose catabolism, driving higher C. phytofermentans growth and higher cellulolytic enzyme production, which in turn provided more substrate for E. coli . E. coli necromass enhanced C. phytofermentans growth while C. phytofermentans necromass aided E. coli growth via the release of peptides and amino acids, respectively. In aggregate, temporal cycling of necromass constituents increased flux of cellulose-derived resources through the consortium. The study establishes a consortia-based, bioprocessing strategy built on naturally occurring interactions for improved conversion of cellulose-derived sugars into bioproducts.
... In 2012 Li et al. inactivated lactate and malate dehydrogenase in C. cellulolyticum resulting in a titer of 2.7 g/L ethanol from cellulose [41]. Tolonen et al. increased the ethanol tolerance of Clostridium phytofermentans by serial transfer and followed an approach similar to Guedon et al., leading to an ethanol titer of around 0.9 g/L [42]. Chung et al. engineered the hyperthermophile Caldicellulosiruptor bescii to produce 0.6 g/L ethanol from switchgrass by heterologously expressing adhE from C. thermocellum [43]. ...
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Background: Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains. Results: Contrary to lactate dehydrogenase, deleting phosphotransacetylase (pta, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A, ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively. Conclusions: Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB. Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.
... The targeted intron was PCR amplified and inserted between the NdeI and BsrGI sites of pQint (15). Targeted pQint plasmids were transferred into C. phytofermentans by conjugation with Escherichia coli under anaerobic conditions (15,36,37). Colonies on GS2 plates containing the 40 g ml Ϫ1 erythromycin were picked, and the genomic intron insertion was confirmed by PCR and sequencing using primers in Table S3. ...
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Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organism Clostridium phytofermentans . We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.
... Although ethanol tolerance has often been studied as a proxy for ethanol production, many studies have found that increases in ethanol tolerance have no effect on ethanol production [26][27][28], including studies of C. thermocellum [29]. Furthermore, in cases where ethanol tolerance has been improved by selection, many of the improvements appear to be due to idiosyncratic mutations whose effects are not generalizable to other strain backgrounds or growth conditions [17,26]. ...
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Background Clostridium thermocellum is a promising microorganism for conversion of cellulosic biomass to biofuel, without added enzymes; however, the low ethanol titer produced by strains developed thus far is an obstacle to industrial application. Results Here, we analyzed changes in the relative concentration of intracellular metabolites in response to gradual addition of ethanol to growing cultures. For C. thermocellum, we observed that ethanol tolerance, in experiments with gradual ethanol addition, was twofold higher than previously observed in response to a stepwise increase in the ethanol concentration, and appears to be due to a mechanism other than mutation. As ethanol concentrations increased, we found accumulation of metabolites upstream of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction and depletion of metabolites downstream of that reaction. This pattern was not observed in the more ethanol-tolerant organism Thermoanaerobacterium saccharolyticum. We hypothesize that the Gapdh enzyme may have different properties in the two organisms. Our hypothesis is supported by enzyme assays showing greater sensitivity of the C. thermocellum enzyme to high levels of NADH, and by the increase in ethanol tolerance and production when the T. saccharolyticum gapdh was expressed in C. thermocellum. Conclusions We have demonstrated that a metabolic bottleneck occurs at the GAPDH reaction when the growth of C. thermocellum is inhibited by high levels of ethanol. We then showed that this bottleneck could be relieved by expression of the gapdh gene from T. saccharolyticum. This enzyme is a promising target for future metabolic engineering work. Electronic supplementary material The online version of this article (10.1186/s13068-017-0961-3) contains supplementary material, which is available to authorized users.
Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.
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Short- and medium-chain volatile esters with flavors and fruity fragrances, such as ethyl acetate, butyl acetate, and butyl butyrate, are usually value-added in brewing, food, and pharmacy. The esters can be naturally produced by some microorganisms. As ester-forming reactions are increasingly deeply understood, it is possible to produce esters in non-natural but more potential hosts. Clostridia are a group of important industrial microorganisms since they can produce a variety of volatile organic acids and alcohols with high titers, especially butanol and butyric acid through the CoA-dependent carbon chain elongation pathway. This implies sufficient supplies of acyl-CoA, organic acids, and alcohols in cells, which are precursors for ester production. Besides, some Clostridia could utilize lignocellulosic biomass, industrial off-gas, or crude glycerol to produce other branched or straight-chain alcohols and acids. Therefore, Clostridia offer great potential to be engineered to produce short- and medium-chain volatile esters. In the review, the efforts to produce esters from Clostridia via in vitro lipase-mediated catalysis and in vivo alcohol acyltransferase (AAT)-mediated reaction are comprehensively revisited. Besides, the advantageous characteristics of several Clostridia and clostridial consortia for bio-ester production and the driving force of synthetic biology to clostridial chassis development are also discussed. It is believed that synthetic biotechnology should enable the future development of more effective Clostridia for ester production.
Consolidated bioprocessing (CBP) by micro-organisms is desired for efficient conversion of lignocellulosic biomass to bioethanol fuels. Potential candidates have been discovered, including cellulolytic bacteria and filamentous fungi. Genetic and metabolic manipulation of these organisms further promotes their fermentation capacities and the ethanol tolerance. In addition, Saccharomyces cerevisiae and several other yeasts were genetically modified to express recombinant cellulases in media or display them on the cell surface for CBP of cellulose. To compensate the insufficient capacity of a single strain, various microbial consortia have also been developed. In this article, we reviewed the recent advances in CBP microbes and focused on the efforts in strain improvement employing genetic engineering.
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The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol. H2 production in C. thermocellum is encoded by four hydrogenases. Rather than delete each individually, we targeted hydrogenase maturase gene hydG, involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe] hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in ∆hydG∆ech was undetectable, and the ethanol yield nearly doubled to 64% of the maximum theoretical yield. Genomic analysis of ∆hydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation was found in ethanol-tolerant C. thermocellum strain E50C, ∆hydG and ∆hydG∆ech are not more ethanol tolerant than the wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum. The dramatic increase in ethanol production suggests that targeting protein post-translational modification is a promising new approach for simultaneous inactivation of multiple enzymes.
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Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.
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7.1 Introduction Dwindling oil reserves and climate change have made the development of renewable, carbon-balanced energy resources a global necessity. As cellulosic biomass is the world's most abundant biological energy source, 1 fermentation of inedible, cellulosic feedstocks such as prairie grasses, corn stover, wood chips and agricultural waste represents one of the most promising avenues for replacing petroleum-based fuels and products. Indeed, over 1.3 billion metric tons of cellulosic biomass could be used annually to produce RSC Energy and Environment Series No. 10 Biological Conversion of Biomass for Fuels and Chemicals: Explorations from Natural Utilization Systems Edited by Jianzhong Sun, Shi-You Ding and Joy Doran-Peterson r The Royal Society of Chemistry 2014 Published by the Royal Society of Chemistry,
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Recycling of plant biomass by a community of bacteria and fungi is fundamental to carbon flow in terrestrial ecosystems. Here we report how the plant fermenting, soil bacterium Clostridium phytofermentans enhances growth on cellulose by simultaneously lysing and consuming model fungi from soil. We investigate the mechanism of fungal lysis to show that among the dozens of different glycoside hydrolases C. phytofermentans secretes on cellulose, the most highly expressed enzymes degrade fungi rather than plant substrates. These enzymes, the GH18 Cphy1799 and Cphy1800, synergize to hydrolyze chitin, a main component of the fungal cell wall. Purified enzymes inhibit fungal growth and mutants lacking either GH18 grow normally on cellulose and other plant substrates, but have a reduced ability to hydrolyze chitinous substrates and fungal hyphae. Thus, C. phytofermentans boosts growth on cellulose by lysing fungi with its most highly expressed hydrolases, highlighting the importance of fungal interactions to the ecology of cellulolytic bacteria.
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Large-scale production of lignocellulosic biofuel is a potential solution to sustainably meet global energy needs. One-step consolidated bioprocessing (CBP) is a potentially advantageous approach for the production of biofuels, but requires an organism capable of hydrolyzing biomass to sugars and fermenting the sugars to ethanol at commercially viable titers and yields. Clostridium thermocellum, a thermophilic anaerobe, can ferment cellulosic biomass to ethanol and organic acids, but low yield, low titer, and ethanol sensitivity remain barriers to industrial production. Here, we deleted the hypoxanthine phosphoribosyltransferase gene in ethanol tolerant strain of C. thermocellum adhE*(EA) in order to allow use of previously developed gene deletion tools, then deleted lactate dehydrogenase (ldh) to redirect carbon flux towards ethanol. Upon deletion of ldh, the adhE*(EA) Δldh strain produced 30% more ethanol than wild type on minimal medium. The adhE*(EA) Δldh strain retained tolerance to 5% v/v ethanol, resulting in an ethanol tolerant platform strain of C. thermocellum for future metabolic engineering efforts.
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The complexity of plant cell walls creates many challenges for microbial decomposition. Clostridium phytofermentans, an anaerobic bacterium isolated from forest soil, directly breaks down and utilizes many plant cell wall carbohydrates. The objective of this research is to understand constraints on rates of plant decomposition by Clostridium phytofermentans and identify molecular mechanisms that may overcome these limitations. Experimental evolution via repeated serial transfers during exponential growth was used to select for C. phytofermentans genotypes that grow more rapidly on cellobiose, cellulose and xylan. To identify the underlying mutations an average of 13,600,000 paired-end reads were generated per population resulting in ∼300 fold coverage of each site in the genome. Mutations with allele frequencies of 5% or greater could be identified with statistical confidence. Many mutations are in carbohydrate-related genes including the promoter regions of glycoside hydrolases and amino acid substitutions in ABC transport proteins involved in carbohydrate uptake, signal transduction sensors that detect specific carbohydrates, proteins that affect the export of extracellular enzymes, and regulators of unknown specificity. Structural modeling of the ABC transporter complex proteins suggests that mutations in these genes may alter the recognition of carbohydrates by substrate-binding proteins and communication between the intercellular face of the transmembrane and the ATPase binding proteins. Experimental evolution was effective in identifying molecular constraints on the rate of hemicellulose and cellulose fermentation and selected for putative gain of function mutations that do not typically appear in traditional molecular genetic screens. The results reveal new strategies for evolving and engineering microorganisms for faster growth on plant carbohydrates.
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Clostridium thermocellum is a major candidate for bioethanol production via consolidated bioprocessing. However, the low ethanol tolerance of the organism dramatically impedes its usage in industry. To explore the mechanism of ethanol tolerance in this microorganism, systematic metabolomics was adopted to analyse the metabolic phenotypes of a C. thermocellum wild-type (WT) strain and an ethanol-tolerant strain cultivated without (ET0) or with (ET3) 3% (v/v) exogenous ethanol. Metabolomics analysis elucidated that the levels of numerous metabolites in different pathways were changed for the metabolic adaption of ethanol-tolerant C. thermocellum. The most interesting phenomenon was that cellodextrin was significantly more accumulated in the ethanol-tolerant strain compared with the WT strain, although cellobiose was completely consumed in both the ethanol-tolerant and wild-type strains. These results suggest that the cellodextrin synthesis was active, which might be a potential mechanism for stress resistance. Moreover, the overflow of many intermediate metabolites, which indicates the metabolic imbalance, in the ET0 cultivation was more significant than in the WT and ET3 cultivations. This indicates that the metabolic balance of the ethanol-tolerant strain was adapted better to the condition of ethanol stress. This study provides additional insight into the mechanism of ethanol tolerance and is valuable for further metabolic engineering aimed at higher bioethanol production.
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Background Lignocellulosic ethanol is a viable alternative to petroleum-based fuels with the added benefit of potentially lower greenhouse gas emissions. Consolidated bioprocessing (simultaneous enzyme production, hydrolysis and fermentation; CBP) is thought to be a low-cost processing scheme for lignocellulosic ethanol production. However, no single organism has been developed which is capable of high productivity, yield and titer ethanol production directly from lignocellulose. Consortia of cellulolytic and ethanologenic organisms could be an attractive alternate to the typical single organism approaches but implementation of consortia has a number of challenges (e.g., control, stability, productivity). Results Ethanol is produced from α-cellulose using a consortium of C. phytofermentans and yeast that is maintained by controlled oxygen transport. Both Saccharomyces cerevisiae cdt-1 and Candida molischiana “protect” C. phytofermentans from introduced oxygen in return for soluble sugars released by C. phytofermentans hydrolysis. Only co-cultures were able to degrade filter paper when mono- and co-cultures were incubated at 30°C under semi-aerobic conditions. Using controlled oxygen delivery by diffusion through neoprene tubing at a calculated rate of approximately 8 μmol/L hour, we demonstrate establishment of the symbiotic relationship between C. phytofermentans and S. cerevisiae cdt-1 and maintenance of populations of 105 to 106 CFU/mL for 50 days. Comparable symbiotic population dynamics were observed in scaled up 500 mL bioreactors as those in 50 mL shake cultures. The conversion of α-cellulose to ethanol was shown to improve with additional cellulase indicating a limitation in hydrolysis rate. A co-culture of C. phytofermentans and S. cerevisiae cdt-1 with added endoglucanase produced approximately 22 g/L ethanol from 100 g/L α-cellulose compared to C. phytofermentans and S. cerevisiae cdt-1 mono-cultures which produced approximately 6 and 9 g/L, respectively. Conclusion This work represents a significant step toward developing consortia-based bioprocessing systems for lignocellulosic biofuels production which utilize scalable, environmentally-mediated symbiosis mechanisms to provide consortium stability.
The anaerobic acetogenic bacterium Acetobacterium woodii carries out a unique type of Na⁺-motive, anaerobic respiration with caffeate as electron acceptor, termed “caffeate respiration.” Central, and so far the only identified membrane-bound reaction in this respiration pathway, is a ferredoxin:NAD⁺ oxidoreductase (Fno) activity. Here we show that inverted membrane vesicles of A. woodii couple electron transfer from reduced ferredoxin to NAD⁺ with the transport of Na⁺ from the outside into the lumen of the vesicles. Na⁺ transport was electrogenic, and accumulation was inhibited by sodium ionophores but not protonophores, demonstrating a direct coupling of Fno activity to Na⁺ transport. Results from inhibitor studies are consistent with the hypothesis that Fno activity coupled to Na⁺ translocation is catalyzed by the Rnf complex, a membrane-bound, iron–sulfur and flavin-containing electron transport complex encoded by many bacterial and some archaeal genomes. Fno is a unique type of primary Na⁺ pump and represents an early evolutionary mechanism of energy conservation that expands the redox range known to support life. In addition, it explains the lifestyle of many anaerobic bacteria and gives a mechanistic explanation for the enigma of the energetic driving force for the endergonic reduction of ferredoxin with NADH plus H⁺ as reductant in a number of aerobic bacteria.