Redesigning Escherichia coli metabolism for · pathways that synthesize advanced biofuels are not...
Transcript of Redesigning Escherichia coli metabolism for · pathways that synthesize advanced biofuels are not...
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Redesigning Escherichia coli metabolism for 1
anaerobic production of isobutanol 2
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Cong T. Trinh1, Johnny Li, Harvey W. Blanch*, and Douglas S. Clark*
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Energy Biosciences Institute 7
and 8
Department of Chemical and Biomolecular Engineering 9
University of California, Berkeley, CA 94720 10
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Keywords: elementary mode analysis; metabolic pathway analysis; metabolic pathway design; 12
metabolic pathway evolution; fermentation; biofuels; isobutanol; ethanol; minimal cell; cell 13
design 14
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* To whom correspondence should be addressed 16
1Current address: Department of Chemical and Biomolecular Engineering, University of 17
Tennessee, Knoxville, TN 37996 18
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Revised for Applied and Environmental Microbiology 20
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00382-11 AEM Accepts, published online ahead of print on 3 June 2011
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ABSTRACT 21
Fermentation enables the production of reduced metabolites, such as the biofuels ethanol and 22
butanol, from fermentable sugars. This work demonstrates a general approach for designing and 23
constructing a production host that uses a heterologous pathway as an obligately fermentative 24
pathway to produce reduced metabolites, specifically the biofuel isobutanol. Elementary mode 25
analysis was applied to design an Escherichia coli strain optimized for isobutanol production 26
under strictly anaerobic conditions. The central metabolism of E. coli was decomposed into 27
38,219 functional, unique, and elementary modes (EMs). The model predictions revealed that 28
during anaerobic growth E. coli cannot produce isobutanol as the sole fermentative product. By 29
deleting 7 chromosomal genes, the total 38,219 EMs was constrained to 12 EMs, 6 of which can 30
produce high yields of isobutanol in a range from 0.29 to 0.41 g isobutanol/g glucose under 31
anaerobic conditions. The remaining 6 EMs rely primarily on the pyruvate dehydrogenase 32
enzyme complex (PDHC) and are typically inhibited under anaerobic conditions. The re-33
designed E. coli strain was constrained to employ the anaerobic isobutanol pathways through 34
deletion of 7 chromosomal genes, addition of 2 heterologous genes, and over-expression of 5 35
genes. Here we present the design, construction, and characterization of an isobutanol-producing 36
E. coli strain to illustrate the approach. The model predictions are evaluated in relation to 37
experimental data and strategies proposed to improve anaerobic isobutanol production. We also 38
show that the endogenous alcohol/aldehyde dehydrogenase, AdhE, is the key enzyme 39
responsible for the production of isobutanol and ethanol under anaerobic conditions. The 40
glycolytic flux can be controlled to regulate the ratio of isobutanol to ethanol production. 41
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INTRODUCTION 42
Society currently relies on the use of fossil fuels to meet its growing energy demands (16). 43
Oil, natural gas, and coal contribute about 84% of the world’s energy consumption (International 44
Energy Outlook 2010, U.S. Energy Information Administration, USA). There is thus a pressing 45
need to develop alternative technologies to reduce reliance on fossil fuels (26). One such 46
alternative is the biological conversion of lignocellulosic biomass into biofuels, which has 47
emerged as a promising route to sustainable energy (1, 9, 24). 48
Recent efforts have focused on engineering S. cerevisiae to ferment sugars derived from 49
lignocellulosic biomass hydrolysates to produce ethanol as a biofuel (17). Other microorganisms 50
such as Zymomonas mobilis (39) and E. coli (20) have also been engineered to produce ethanol. 51
However, ethanol is not an ideal biofuel due to its miscibility in water and low energy content 52
relative to gasoline. Recently, metabolic engineering and synthetic biology have been applied to 53
engineer recombinant microorganisms to produce biofuels such as butanol, isobutanol, 54
isopentanol, fatty alcohols, biodiesel, and isoprenoid-derived biofuels that have similar 55
properties to gasoline or jet fuel (3, 10, 18, 25, 31). However, most of these biofuels are 56
typically produced at low titers and with poor yields, in part because the producing organism 57
must be cultivated under aerobic or oxygen-limited conditions to address a redox imbalance 58
inherent in the production pathways employed. 59
Fermentation is the anaerobic metabolic conversion of sugars to products that involves 60
endogeneous electron acceptors and produces reducing equivalents such as NADH. 61
Fermentation is the most efficient route to synthesize biofuels that are reduced relative to the 62
sugar source. Relevant examples are ethanol production from S. cerevisiae and butanol 63
production from C. acetobutylicum (17, 30). Under anaerobic conditions, the NADH generated 64
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from sugar metabolism is recycled to NAD+ by the formation of reduced metabolites such as 65
alcohols, thus helping to maintain the appropriate redox balance within the cell. However, most 66
pathways that synthesize advanced biofuels are not obligately anaerobic and are active under 67
aerobic conditions even when the host is a facultative anaerobe. For example, E. coli is able to 68
produce biodiesel and certain hydrocarbons by employing fatty acid biosynthesis pathways only 69
under aerobic conditions (25, 31). 70
Under aerobic conditions, reducing equivalents in the form of NADH are most efficiently 71
regenerated by oxidation, directly generating ATP for cell synthesis and maintenance, rather than 72
by the production of reduced metabolites such as biofuels (37). To direct cellular metabolism 73
toward the formation of reduced products, cell cultivation is often carried out under oxygen-74
limited conditions (3). Precise control of the dissolved oxygen concentration is required to 75
partition carbon flow between the synthesis of biomass and reduced metabolites. Therefore, 76
converting biofuel-producing pathways to obligate anaerobic pathways would result in higher 77
yields of reduced metabolites and be advantageous for large-scale production because oxygen 78
supply and control are not required. 79
Isobutanol formation provides an example of a non-fermentative biofuel-producing pathway. 80
Isobutanol is produced via the valine biosynthesis pathway, which is not an obligately 81
fermentative pathway. Saccharomyces cerevisiae was the first microorganism reported to be 82
able to produce isobutanol from 13
C-labeled valine as a substrate (14). Recently, E. coli JCL260 83
pSA55/pSA69 has been engineered to produce isobutanol directly from glucose by using the 84
valine biosynthesis pathway and two additional exogeneous enzymes: α-ketoacid decarboxylase 85
(Kivd from Lactobacillus lactis) and alcohol dehydrogenase (Adh2 from Saccharomyces 86
cerevisiae) (3). However, this recombinant isobutanol producing E. coli was reported to grow 87
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and produce isobutanol only in the presence of oxygen even though E. coli is a facultative 88
anaerobe. The engineered strain could not consume glucose anaerobically because the deletion 89
of the competing pathways resulted in a redox imbalance. This behavior of this phenotype will 90
be clarified by the model predictions and experimental results in the present work. 91
In the present study, we demonstrate as a proof-of-concept that it is possible to “retrofit” E. 92
coli to produce isobutanol through a non-native obligate anaerobic pathway. Elementary mode 93
(EM) analysis was employed to redesign E. coli metabolism. This approach deconstructs the 94
complex metabolic network of E. coli into a complete set of EMs. Each EM contains a minimum 95
and unique set of enzymes that cells can use to function under quasi steady-state conditions (23, 96
27-29). Knowledge of the complete set of EMs allows the selection of routes that enable cell 97
growth and synthesis of isobutanol at high yields under anaerobic conditions (32, 33, 35, 36, 38). 98
Herein we illustrate why the former engineered E. coli JCL260 pSA55/pSA69 could not convert 99
glucose to isobutanol anaerobically. We provide details of how the re-engineered strain was 100
designed, constructed, and characterized to employ a heterologous, obligate anaerobic 101
isobutanol-producing pathway. Our goals are (1) to demonstrate how E. coli metabolism can be 102
retrofitted for anaerobic isobutanol production and (2) to identify what rate-limiting steps must 103
be overcome to improve anaerobic isobutanol production. This approach will also guide 104
strategies to rationally design microbes that can synthesize reduced metabolites by retrofitting 105
them with heterologous pathways that function as obligate anaerobic pathways. This paves the 106
way for more efficient and cost-effective routes to industrial biotechnological products that are 107
reduced with respect to their more oxidized carbohydrate feedstocks. 108
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MATERIALS AND METHODS 111
Organisms and plasmids 112
Table 1 shows a list of strains and plasmids used in the study. 113
Strain construction. All mutants with single deleted genes were obtained from the single-114
gene knockout library, the Keio collection (6). These mutants were derived from BW25113, a 115
derivative of E. coli MG1655 and constructed using the technique of one-step disruption of 116
chromosomal genes (12). Mutants with multiple deleted genes were created by multiple steps of 117
P1 transduction from strains with a single deleted gene (33). At each step, the recipient strain 118
that contained one or more deleted genes had the kanamycin cassette removed by using the 119
temperature-sensitive helper plasmid pCP20 (11). Donor strains used to prepare P1 lysate had a 120
single deleted gene with an intact kanamycin cassette. The polymerase chain reaction (PCR) test 121
was designed to detect complete gene disruption by using primers located outside of the 122
undeleted portion of the structural gene (Supplementary Table S1). To construct a mutant with 123
the T7 polymerase gene, the prophage λDE3 (Novagen, λDE3 lysogenization kit, catalog #: 124
69734-3, U.S.A.) was used to insert the gene into the specific site of the mutant chromosome. 125
Plasmid construction. To construct pCT01, the operon alsS::ilvC:ilvD was amplified from 126
the plasmid pSA69 with the primers alsS_pCOLA_1f: 5’-AAA AAA GGA TCC GTT GAC 127
AAA AGC AAC AAA AGA ACA AAA ATC CCT TGT G-3’ and ilvD_pCOLA_1r: 5’-AAA 128
AAA GCG GCC GCT TAA CCC CCC AGT TTC GAT TTA TCG CGC ACC-3’. The operon 129
amplification was carried out by PCR with a high fidelity Pfu polymerase (Agilent Technologies, 130
catalog #: 600674, USA). The PCR product was doubly digested with BamH1 and NotI, and 131
ligated into the backbone vector pCOLADuet (Novagen, U.S.A). 132
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To construct pCT02, the operon kivd::adh2 was amplified from the plasmid pSA55 with the 133
primers kivd_pCOLA_1f_d: 5’-AAA AAA CAT ATG TAT ACA GTA GGA GAT TAC-3’ and 134
adh2_pCOLA_1r _d: 5’-AAA AAA CTC GAG TTA TTT AGA AGT GTC AAC-3’. The PCR 135
product was doubly digested with NdeI and XhoI, and ligated into the backbone vector 136
pCOLADuet. To construct pCT03, the operon alsS::ilvC:ilvD was doubly digested with BamH1 137
and NotI, and ligated into the vector pCT01. 138
The operon kivd::adhE was constructed using the overlapping PCR extension technique. 139
The gene kivd was amplified from the plasmid pSA55 by using the primers kivd_adhE_1f_1: 5’-140
AAA AAA CAT ATG TAT ACA GTA GGA GAT TAC-3’ and kivd_adhE_1r_1: 5’-TTC AGC 141
GAC ATT AGT AAC AGC CAT TAT ATC TCC TTT TAT GAT TTA TTT TGT TC-3’, and 142
the gene adhE amplified from the genomic DNA of E. coli MG1655 by using the primers 143
kivd_adhE_1f_2: 5’-TTT GCT GAA CAA AAT AAA TCA TAA AAG GAG ATA TAA TGG 144
CTG TTA CTA ATG TC-3’ and kivd_adhE_1r_2: 5’-AAA AAA CTC GAG TTA AGC GGA 145
TTT TTT CGC-3’. The operon kivd::adhE was constructed by amplifying both the amplified 146
genes kivd and adhE with the primers kivd_adhE_1f_1 and kivd_adhE_1r_2. The operon 147
kivd::adhE was digested with NdeI and XhoI, and ligated into the backbone vector pCOLADuet 148
to create the vector pCT13A. To construct the vector pCT13, the operon alsS::ilvC:ilvD was 149
doubly digested with BamH1 and NotI, and ligated into the vector pCT13A. All constructed 150
vectors were confirmed by gel electrophoresis after being digested with either BamH1/NotI or 151
NdeI/XhoI and by sequencing the inserts. 152
To construct the plasmid pET24b adhE, the gene adhE was amplified from the genomic 153
DNA of E. coli MG1655 by using the primers adhE_pET24b_1f: 5’-AAA AAA CAT ATG GCT 154
GTT ACT AAT GTC GCT-3’ and adhE_pET24b_1r: 5’-AAA AAA CTC GAG AGC GGA TTT 155
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TTT CGC TTT-3’. The PCR product was doubly digested with NdeI and XhoI, and ligated into 156
the vector pET24b to create the vector pET24b adhE. This gene also contained an additional 18 157
nucleotide sequence from the pET24b to encode the His-tagged sequence at the C-terminus of 158
the enzyme AdhE. 159
Cell cultivation 160
Medium. Strains were characterized in a medium containing 6.8 g/L Na2HPO4, 3.0 g/L 161
KH2PO4, 1.0 g/L NH4Cl, 2 mL/L 1M MgSO4, 100 µL/L CaCl2, 1 mL/L stock trace metals 162
solution, 5 g/L yeast extract, 100 µg/mL kanamycin, and 20 g/L glucose (unless otherwise 163
specified). The stock trace metals solution contained 0.15 g/L H3BO4, 0.065 g/L CoSO4, 0.05 164
g/L ZnSO4.7H2O, 0.015 g/L MnCl2.4H2O, 0.015 g/L NaMo4.2H2O, 0.01 g/L NiCl2.6H2O, 0.005 165
g/L CuSO4.5H2O, 3 g/L Fe(NH4) citrate. A stock of 1M IPTG was added to a cell culture with 166
an amount of 1mL/L as the OD600nm of the cell culture reached about 0.5. Antifoam 204 (Sigma-167
Aldrich, cat# A6426) was added to the medium with an amount of 100 µL/L before cell 168
inoculation. 169
Fermentation. Batch fermentations were conducted in 3L bioreactors (Bioengineering AG, 170
Switzerland) with a working volume of 2L, under anaerobic growth conditions. The operating 171
conditions were 200 rpm, pH 7 (unless otherwise specified), and 37oC. Anaerobic conditions 172
were maintained by continuously sparging the bioreactor with N2 at a volumetric flow rate of 173
100 mL/min throughout the fermentation. Dissolved oxygen was measured by a polarographic 174
dissolved oxygen probe and found to be 0%, confirming that the bioreactor was anaerobic. 175
Exponentially grown cell cultures (OD600nm= 0.5~1) were used for inoculation, and the OD600nm 176
of initial cultures in the bioreactors following inoculation was about 0.05. Optical density was 177
measured at 600nm using a spectrophotometer (Model: Genesys 20, Thermo Scientific, USA). 178
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A correlation between OD600nm and dry cell weight (DCW) was used to calculate the biomass 179
concentration during fermentation (1 OD600nm = 0.5 g DCW/L). All batch fermentations were 180
performed at least twice in independent experiments, with three samples taken at each time point. 181
Data analysis. Product yields were calculated and a carbon balance was determined as 182
previously described (34, 36). Statistical analysis was performed using SigmaPlot (Systat 183
Software, Inc., USA). 184
Protein expression and purification 185
From the petri dish, a single colony of BL21(DE3) pET24b adhE was picked and grown 186
overnight in a shake tube containing Luria-Bertani (LB) medium plus 10 g/L glucose and 50 187
µg/mL kanamycin. The overnight culture was transferred into a 1L shake flask containing 500 188
mL of the identical medium with the inoculum ratio of 1:100. After the OD600nm reached 0.5, 189
IPTG was added to a final concentration of 0.5 mM to induce the expression of the His-tagged 190
AdhE. The cell pellet was collected after 4 hours of induction by centrifugation at 5000xg for 10 191
min and at 4oC. Cells were lysed by the French press cell operated at a constant pressure of 192
21,300 psi. The lysed cell was used to purify the His-tagged AdhE by using the Ni-NTA agarose 193
bead. The purification procedure followed protocols 9 and 12 in the QIAexpressionist manual 194
(Qiagen Inc., CA, USA). The protein concentration was measured by the Bradford method 195
(Biorad, cat no: 500-0006, Hercules, CA, USA). 196
Enzyme assay 197
The enzyme activity of AdhE was measured based on the oxidation of NADH by different 198
aldehydes including acetaldehyde, isobutyraldehyde, and butyraldehyde at the wavelength of 340 199
nm. A 1mL reaction mixture was carried out in the MOPS buffer (pH 7.0) containing 10 µg/mL 200
of the purified protein AdhE, 5 mM aldehyde, and various concentrations of NADH (40-150 201
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µM) at 37oC. The reaction was initiated by adding the aldehyde last and the OD340nm was 202
measured and recorded for every 10 seconds during the course of 180-360 seconds. 203
Analytical techniques 204
Metabolites such as glucose, isobutanol, succinate, lactate, acetate, and formate were 205
measured using the HPLC Prominence system (Shimadzu10A, Shimadzu, Columbia, MD) as 206
described previously (34). 207
Metabolic network reconstruction and analysis 208
The metabolic network of E. coli central metabolism was constructed as previously reported 209
(36). In this work, the isobutanol-producing pathway consisting of 5 enzymatic reactions that 210
convert pyruvate into isobutanol (Figure 1) was also added. They are IBUT1 [AlsS, acetolactate 211
synthase]: 2 pyruvate = acetolactate + CO2; IBUT2 [IlvC, 2,3-dihydroxy isovalerate 212
oxidoreductase]: acetolactate + NADPH + H+
= 2,3-dihydroxy isovalerate + NADP+; IBUT3 213
[IlvD, 2,3-dihydroxy isovalerate dehydratase]: 2,3-dihydroxy isovalerate = isovalerate + 214
H2O; IBUT4 [Kivd/AlsS, ketoacid decarboxylase]: isovalerate = isobutanol + CO2; IBUT5 [Adh, 215
alcohol dehydrogenase]: isobutanal + NADH + H+ = isobutanol + NAD
+. EM analysis was used 216
as a metabolic pathway analysis tool to study the metabolic network structure (27) and to 217
rationally design optimal cells with efficient minimized metabolic functionalities (32). EMs of 218
the metabolic network were calculated using the publically available software program 219
METATOOL 5.0. 220
RESULTS 221
Metabolic network analysis 222
Metabolic network structure. EM analysis identified a total of 38,219 EMs that used glucose 223
as a carbon source, 5,621 of which operated under anaerobic conditions. These anaerobic EMs 224
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included 1,899 isobutanol-producing EMs, only 1,458 of which could co-produce isobutanol and 225
cell biomass (Figure 2A). The solution space of all admissible EMs was used to study anaerobic 226
isobutanol metabolism in E. coli and to identify the most efficient pathways for anaerobic 227
isobutanol production. 228
Infeasibility of E. coli fermentation for isobutanol production as the sole product. To 229
examine whether fermentation for isobutanol production as the sole product is feasible in E. coli, 230
we searched for EMs that support both cell biomass and isobutanol production under anaerobic 231
growth conditions. By eliminating all fermentative pathways that produced succinate, lactate, 232
acetate, and ethanol (∆frd, ∆ldhA, ∆pta, ∆poxB, ∆adhE), we found no existing EMs that could 233
co-produce cell biomass and isobutanol under anaerobic growth conditions (Table 2). All 14 234
remaining EMs produced isobutanol as the sole product at a theoretical yield of 0.41 g 235
isobutanol/g glucose. A similar result was obtained if only the anaerobic pathways that produced 236
succinate and ethanol, but neither lactate nor acetate, were eliminated. However, if the 237
isobutanol producing pathway was forced to couple with one of the anaerobic pathways that 238
could oxidize NADH, there existed EMs that can co-produce both cell biomass and isobutanol. 239
For instance, if the isobutanol-producing pathway was coupled with the ethanol-producing 240
pathway, then 814 out of 1150 EMs could produce isobutanol. Among these isobutanol-241
producing pathways, 282 EMs could co-produce cell biomass and isobutanol (Table 2). This 242
result indicates that anaerobic production of isobutanol as the sole product is infeasible due to an 243
imbalance of the reducing equivalents. Isobutanol must be co-produced with other reduced 244
metabolites, such as succinate and ethanol, to balance the reducing equivalents by recycling 245
NADH. Our strategy was thus directed toward production of biofuels by coupling the 246
biosynthesis of isobutanol and ethanol. 247
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Rational design of the most efficient isobutanol producing E. coli strain 249
Strain design strategy. The optimal pathways for supporting cell growth and converting 250
glucose into isobutanol can be selected and strictly enforced by removing the inefficient 251
pathways from the metabolic network. The search algorithm to remove the inefficient pathways 252
has been developed and reported in previous publications (15, 32, 33, 35, 36, 38). Briefly, the 253
algorithm identifies the minimal set of reactions that should be deleted from the metabolic 254
network in order (1) to remove as many inefficient pathways for isobutanol production as 255
possible, (2) to retain a small subset of efficient isobutanol producing pathways that can achieve 256
maximal isobutanol yield, and (3) to tightly couple isobutanol and biomass synthesis during cell 257
growth. 258
Strain design implementation. In order to achieve maximum efficiency of isobutanol 259
production, cellular metabolism must be redirected to use only the optimal anaerobic isobutanol-260
producing pathways. By deleting 6 reactions (∆PPP1, ∆TCA9r, ∆TRA5, ∆OPM4r, ∆TRA2, 261
∆TRA4) corresponding to 7 knockout genes (∆zwf, ∆mdh, ∆frdA, ∆ndh, ∆pta, ∆poxB, ∆ldhA), 262
the total numbers of EMs, biomass producing EMs, isobutanol producing EMs, and biomass and 263
isobutanol co-producing EMs were reduced from 5621, 4500, 1899, 1458 to 12, 4, 6, 2, 264
respectively (Figure 2A). After removing the 6 reactions, half of the total 12 EMs produced 265
solely isobutanol while the other half made only ethanol. Interestingly, all 6 EMs that produced 266
solely ethanol used the pyruvate dehydrogenase complex (PDHC) enzyme (the reaction GG13) 267
to convert pyruvate into acetyl CoA. This enzyme is known to be down-regulated by a high 268
intracellular redox state (NADH/NAD+ ratio) under anaerobic conditions (13). In principle, 269
these 6 EMs should not be functional under anaerobic conditions. 270
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If the additional reaction GG13 was deleted by knocking out the gene aceF, the total number 271
of EMs, biomass producing EMs, isobutanol producing EMs, and biomass and isobutanol co-272
producing EMs could be reduced from 12, 4, 6, 2 to 6, 2, 6, 2, respectively (Figure 2A). With 273
the deletion of the complete set of seven reactions, the total number of EMs was equal to that of 274
the isobutanol producing EMs, which emphasizes that the cell was designed to obligately utilize 275
the isobutanol-producing pathway for redox balancing. In addition, the number of the biomass 276
producing EMs was equal to that of the biomass and isobutanol co-producing EMs, which 277
indicates that cell growth and isobutanol production were coupled during the growth-associated 278
phase. In this phase, the designed E. coli strain could produce isobutanol with a yield of 0.29 (g 279
isobutanol/g glucose). In the no-growth phase, all 4 EMs could produce isobutanol at the 280
maximum theoretical yield of 0.41 (g isobutanol/g glucose) (Figure 2B). 281
From the analysis, the optimal designed E. coli strain that can efficiently convert glucose into 282
isobutanol by retrofitting the heterologous isobutanol-producing pathway as an obligately 283
anaerobic pathway has the following deleted genes: ∆zwf, ∆mdh, ∆frdA, ∆ndh, ∆pta, ∆poxB, 284
∆ldhA, ∆aceF*. The asterisk on the deleted gene aceF implies that if the negative regulation of 285
the high intracellular redox state NADH/NAD+ on the PDHC enzyme is strongly exerted under 286
anaerobic conditions, the PDHC enzyme will be completely down-regulated and replaced by 287
pyruvate formate lyase (PFL) to convert pyruvate to acetyl CoA. Thus, the gene aceF may not 288
need to be deleted. 289
Phenotypic operating space of the designed isobutanol-producing E. coli strain. From the 290
stoichiometry reactions of the 12 remaining EMs of the designed isobutanol producing strain 291
(Supplementary Table S2), the yields of ethanol and isobutanol on glucose were calculated and 292
mapped on the 2-D phenotypic space (Figure 3). This phenotypic space is shown by the bold 293
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trapezoid and constructed by connecting the four “star” symbols located at the edge of the 294
trapezoid. These symbols represent four groups of EMs. The group G1 is comprised of EMs 295
that can produce isobutanol at the theoretical yield of 0.41 (g/g) without biomass production 296
while the group G2 co-produces isobutanol and biomass with an isobutanol yield of 0.29 (g/g). 297
It should be emphasized that both groups G1 and G2 contain the 6 optimal isobutanol producing 298
pathways that do not use the pyruvate dehydrogenase enzyme complex (PDHC). This group of 299
EMs is also constrained to produce a small amount of ethanol 0.0012 (g/g) to maintain the redox 300
balance. The isobutanol to ethanol mass ratio in this group is 236:1. 301
The group G3 produces only ethanol at the theoretical yield of 0.51 (g/g) whereas the group 302
G4 co-produces ethanol and biomass with a high ethanol yield of 0.36 (g/g). The EMs of both 303
G3 and G4 use the PDHC complex enzyme to convert pyruvate into ethanol, which is typically 304
inhibited under anaerobic conditions. The designed isobutanol producing strain can operate 305
anywhere in the trapezoidal phenotypic space (Figure 3). For isobutanol production, it is 306
desirable to have the designed strain operate close to groups G1 and G2. 307
Strain comparison 308
Other E. coli strains have been engineered to produce isobutanol such as JCL260 pSA55 309
pSA69 (3). The host JCL260 has the following deleted genes: ∆adhE, ∆fnr, ∆pflB, ∆frdBC, 310
∆pta, ∆ldhA::kan-. According to EM analysis, deletion of these genes disrupts cell metabolism 311
required to support cell growth since no existing EMs can produce cell biomass (Table 3). 312
Construction of the designed E. coli strain 313
Host strain construction. The designed host E. coli strain BFA7.001(DE3) was constructed 314
with seven gene knockouts (∆zwf, ∆ldhA, ∆pta, ∆poxB, ∆mdh, ∆ndh, ∆frdA, ∆aceF*) according 315
to the model prediction for anaerobic conversion of glucose to isobutanol. In this study, we did 316
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not delete the gene aceF to remove the PDHC reaction GG13, operating under the assumption 317
that the PDHC enzyme is down-regulated and the PFL enzyme is solely responsible for 318
converting pyruvate to acetyl CoA. In addition, we were interested in exploring how the level of 319
down-regulation of the PDHC enzyme affects the production of isobutanol and ethanol. The 320
knockout genes in the designed strain were confirmed by PCR. The designed strain also had the 321
T7 polymerase gene inserted into the chromosome (Supplementary Figure S1). 322
Construction of the isobutanol producing pathway (Supplementary Figure S2, Figure 1). 323
The isobutanol producing pathway was constructed by using a single plasmid pCT03 that had a 324
low copy number, a strong T7 promoter, and a kanamycin selection. The plasmid pCT03 325
derived from pCOLADuet contains two operons that included a complete set of five genes for 326
converting pyruvate to isobutanol. The first operon pT7::alsS::ilvC::ilvD contained 3 genes 327
(alsSB. subtilis, ilvC, and ilvD) under the T7 promoter that were used to amplify fluxes from 328
pyruvate to α-ketoisovalerate. It should be noted that the native E. coli has these endogeneous 329
enzymes to generate the precursor α-ketoisovalerate for L-valine biosynthesis. The second 330
operon (pT7::kivd::adh2) contained two genes (kivdL.lactis and adh2S.cerevisiase) under the T7 331
promoter that were used to convert α-ketoisovalerate to isobutanol. Native E. coli does not have 332
α-ketoacid decarboxylase (encoded by KivdL.lactis), an important enzyme for converting α-333
ketoisovalerate to isobutyraldehyde. 334
Besides pCT03, we also constructed different incomplete parts of the isobutanol-producing 335
pathway and expressed different alcohol dehydrogenases such as AdhE (encoded by adhEE.coli). 336
Specifically, the plasmid pCT01 contained the first operon, pT7::alsS::ilvC::ilvD. The plasmid 337
pCT02 contained the second operon, pT7::kivd::adh2. The plasmid pCT13 contained both the 338
first operon, pT7::alsS::ilvC::ilvD, and the second operon, pT7::kivd::adhE. It should be noted 339
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that pCT03 had the alcohol dehydrogenase Adh2 from S. cerevisiae while pCT13 had the alcohol 340
dehydrogenase AdhE from E. coli. 341
Characterization of the designed isobutanol producing strains 342
Anaerobic growth of isobutanol producing E. coli strains. The designed isobutanol-343
producing E. coli strain BFA7.001(DE3) pCT03 was first tested for its ability to grow 344
anaerobically under defined cultivation conditions (pH 7, 37oC, 200 rpm, 100 mL/min nitrogen). 345
Consistent with the model prediction, the designed strain was able to completely ferment 20 g/L 346
glucose after 36 hrs and had a specific growth rate of 0.37±0.03 (hr-1
) (Figure 4). 347
To confirm that the ethanol producing pathway is required to couple with the isobutanol 348
producing pathway in order to support cell growth, we constructed a variant E. coli strain 349
BFA8.001(DE3) that was derived from BFA7.001(DE3) and had the endogeneous alcohol 350
dehydrogenase gene adhE deleted. Strains BFA8.001(DE3) pCT01, BFA8.001(DE3) pCT02, 351
and BFA8.001(DE3) pCT03 that contained different portions of the isobutanol-producing 352
pathway were also constructed and tested for their fermentation capabilities. The result shows 353
that none of these strains was able to grow anaerobically (Figure 5), presumably due to the 354
reducing equivalent (NADH) imbalance. However, when the ethanol producing pathway 355
containing the endogeneous enzyme AdhE was restored, the resulting E. coli strain 356
BFA8.001(DE3) pCT06 was able to grow even though it exhibited a long lag phase (Figure 5). 357
Here the plasmid pCT06 contained two operons including pT7::alsS::ilvC::ilvD and pT7::adhE. 358
We also tested the fermentation capability of JCL260 pSA55 pSA69 (3). The result shows 359
that the strain was unable to consume glucose anaerobically as a result of a genetic manipulation 360
that led to a redox imbalance, as predicted by EM analysis. The strain was only able to grow 361
aerobically with a supply of 100 mL/min air instead of nitrogen, with other operating conditions 362
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remaining the same (Figure 6). In the experiment, the oxygen transfer coefficient (kLa) was set 363
at 1.2 (hr-1
) to test the growth sensitivity of JCL260 pSA55 pSA69 under oxygen-limited growth 364
condition. Within three hours after inoculation, the dissolved oxygen in the bioreactor was 365
reduced to zero, indicating that oxygen became limiting. The strain had an oxygen consumption 366
rate of 8.16 mg/L/hr. During the oxygen-limited condition, the growth of JCL260 pSA55 pSA69 367
was dramatically affected, and exhibited a specific growth rate of 0.08±0.00 (hr-1
) (Figure 6). 368
This result demonstrates the challenge of precisely supplying oxygen to support cell growth and 369
isobutanol production under oxygen-limited conditions, which becomes especially difficult for 370
commercial production of isobutanol. 371
Metabolite production of the designed isobutanol-producing strain. Figure 4 shows the 372
distribution of the fermentative products that BFA7.001(DE3) pCT03 produced during 373
fermentation. The fraction of carbon in the products that were derived from glucose was 374
0.83±0.02. Ethanol and isobutanol were produced as the main alcohols with a mass ratio of 375
0.26±0.01:1. The designed strain was able to produce isobutanol during the growth-associated 376
(7-24 hrs) and no-growth (>24 hrs) phases and to recycle the reducing equivalent NADH. 377
Ethanol was produced as the major product, indicating that the isobutanol-producing pathway 378
was limiting and the ethanol-producing pathway contributed more in recycling NADH. The low 379
mass ratio of isobutanol to ethanol implies that pyruvate dehydrogenase could have been active 380
in converting pyruvate to acetyl CoA, as suggested by the EM analysis. 381
Formate was also produced as an unavoidable fermentative product (Figure 4). From the 382
stoichiometry of E. coli fermentative metabolism, for every mole of ethanol (MW = 46 g/g-mole) 383
produced from pyruvate via the PFL enzyme, one mole of formate (MW = 46 g/g-mole) is made 384
together with one mole of acetyl CoA, which is a precursor for cell biomass. During the first 12 385
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hr of fermentation, formate production paralleled ethanol production, indicating the major role of 386
the PFL enzyme. After 12 hr, formate production decreased as a result of either the oxidation of 387
formate to CO2 by formate hydrogenlyase (FHL) or the dehydrogenation of pyruvate directly to 388
CO2 and acetyl CoA by the PDHC enzyme. 389
Effect of over-expression of different portions of isobutanol producing pathways on 390
isobutanol production and cell physiology. Different portions of the isobutanol producing 391
pathways were over-expressed and characterized in the designed host strain BFA7.001(DE3) 392
under identical anaerobic conditions. Figure 7 compares fermentations of all strains including 393
BFA7.001(DE3) pCT01, BFA7.001(DE3) pCT02, and BFA7.001(DE3) pCT03. Both 394
BFA7.001(DE) pCT01 (µ=0.45±0.02 hr-1
) and BFA7.001(DE3) pCT02 (µ=0.49±0.01 hr-1
) grew 395
faster than BFA7.001(DE) pCT03 (µ=0.37±0.03 hr-1
), which probably suffered from the 396
significant metabolic burden of carrying a larger plasmid with the complete isobutanol-397
producing pathway. Similarly, both BFA7.001(DE) pCT01 (4.71±0.20 g glucose/g dcw/hr) and 398
BFA7.001(DE3) pCT02 (4.43±0.15 g glucose/g dcw/hr) had higher specific glucose uptake rates 399
than BFA7.001(DE3) pCT03 (4.20±0.39 g glucose/g dcw/hr) (Figure 7A). 400
The strain BFA7.001(DE3) pCT01 overexpressed the first portion of the isobutanol-401
producing pathway to convert pyruvate to α-ketoisovalerate. It was not expected to produce 402
isobutanol because the native E. coli is not known to have an α-ketoacid decarboxylase to 403
convert α-ketoisovalerate into isobutyraldehyde. However, the strain was unexpectedly capable 404
of producing isobutanol under anaerobic conditions (Figure 7C). This result implies that 405
AlsSB.subtilis has the dual function of not only a known acetotalactate synthase, converting 406
pyruvate into 2-acetolactate, but also an α-ketoacid decarboxylase converting α-ketoisovalerate 407
into isobutyraldehyde. In addition, the result strongly suggests that the endogenous alcohol 408
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dehydrogenase(s) of E. coli converted isobutyraldehyde to isobutanol. These observations are 409
consistent with the recent reports of Atsumi et al. (4, 5). 410
In strain BFA7.001(DE3) pCT02 the second portion of the isobutanol-producing pathway 411
was amplified to convert α-ketoisovalerate into isobutanol. Nevertheless, BFA7.001(DE3) 412
pCT02 could not produce isobutanol (Figure 7C) but did secrete pyruvate, a precursor metabolite 413
for isobutanol production that was not detected in the fermentation of either BFA7.001(DE3) 414
pCT01 and BFA7.001(DE3) pCT03 (data not shown). This result implies that the first section of 415
the isobutanol-producing pathway was limiting. Because BFA7.001(DE3) pCT02 could not 416
synthesize isobutanol, it produced ethanol at a higher rate and titer than BFA7.001(DE3) pCT01 417
and BFA7.001(DE3) pCT03 in order to recycle the reducing equivalent of NADH (Figure 7B). 418
Strain BFA7.001(DE3) pCT03 contained not only the overexpressed α-ketoacid 419
decarboxylase from KivdL.lactis and AlsSS.subtilis but also the amplified alcohol dehydrogenase 420
from Adh2S.cerevisiase. However, the strain produced isobutanol almost at the same titer as 421
BFA7.001(DE3) (Figure 7C). This result strongly implies that Adh2S.cerevisiase had either low or 422
no activity for isobutyraldehyde, at least under anaerobic conditions, in sharp contrast to the 423
report by Atsumi et al. (3), and that alcohol/aldehyde dehydrogenase was a rate-limiting step. 424
Recently, Atsumi et al. (5) demonstrated that the endogeneous alcohol/aldehyde dehydrogenase 425
YqhD was responsible for aerobic isobutanol production, not the exogeneous enzyme 426
Adh2S.cerevisiae as originally reported (3). Because the enzyme YqhD is NADPH-dependent, and 427
the source of NADPH produced by the glucose-6-phosphate dehydrogenase (Zwf) from the 428
oxidative pentose phosphate pathway was knocked out in the designed isobutanol-producing 429
strain, it is unlikely that YqhD played a role in anaerobic isobutanol production. 430
431
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Role of the endogeneous AdhE in the isobutanol producing pathway. BFA7.001(DE3) pCT01 432
produced isobutanol without the exogeneous alcohol dehydrogenase Adh2S.cerevisiase, indicating that 433
the endogeneous alcohol dehydrogenase(s) of E. coli was responsible for anaerobic isobutanol 434
production. BFA7.001(DE3) pCT01 also produced isobutanol at the same titer as BFA7.001(DE3) 435
pCT03, indicating that Adh2S.cerevisiase was not important in anaerobic isobutanol production. In 436
addition, BFA8.001(DE3), which was derived from the parent strain BFA7.001(DE3) and had the 437
gene adhE deleted, could not consume glucose anaerobically when carrying the plasmids pCT01, 438
pCT02, and pCT03 (Figure 5). Together, these results suggest that the endogenous enzyme AdhE 439
was involved in anaerobic isobutanol production. To confirm the role of AdhE in anaerobic 440
isobutanol production, his-tagged AdhE was produced in BL21(DE3) pET24b adhE, purified 441
using Ni-NTA agarose beads (Supplementary Figure S3) and assayed for activity. The activity 442
assay shows that AdhE was NADH-dependent and had activity not only for acetaldehyde but also 443
for isobutyraldehyde and butyraldehyde. It is interesting to observe that AdhE had the highest 444
enzymatic activity for butyraldehyde under the assay conditions employed (Table 4). 445
Alleviating the alcohol dehydrogenase rate-limiting step. To alleviate the alcohol 446
dehydrogenase rate-limitation, we constructed strain BFA7.001(DE3) pCT13 to overexpress the 447
entire isobutanol producing pathway and to amplify the endogeneous alcohol dehydrogenase 448
AdhE instead of Adh2S.cerevisiae. Even though BFA7.001(DE3) pCT13 (0.42±0.01 hr-1
) carried a 449
larger plasmid than BFA7.001(DE3) pCT03 (Supplementary Figure S2), it was still able to grow 450
at a faster rate under identical anaerobic conditions (pH 7, 200 rpm, 37oC, 100 mL/min nitrogen), 451
further supporting the unimportant role of Adh2S.cerevisiae (Figure 8A). BFA7.001(DE3) pCT13 452
produced isobutanol at a higher titer than either BFA7.001(DE3) pCT01 (66% higher) or 453
BFA7.001(DE3) pCT03 (28% higher) (Table 5). 454
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Effect of the glycolytic flux on the isobutanol production. Our recent study of E. coli KO11 455
in both batch and continuous fermentation shows that an increase in glycolytic flux led to an 456
increasing specific glucose uptake rate. This increased uptake rate of glucose resulted in rapid 457
cell growth and production of lactate from pyruvate as a route to balance the redox potential 458
(34). However, since the designed strain BFA7.001(DE3) pCT13 had the lactate dehydrogenase 459
LdhA deleted and the endogeneous alcohol/aldehyde dehydrogenase AdhE had higher specific 460
activity toward acetaldehyde than isobutyraldehyde (Table 4), it is hypothesized that the 461
reducing equivalent NADH was more quickly and efficiently recycled for faster cell growth by 462
using the ethanol producing pathway. This phenotype might explain the higher production of 463
ethanol than isobutanol with a mass ratio of isobutanol to ethanol of 0.31±0.02:1 (Table 5). 464
To test this hypothesis, BFA7.001(DE3) pCT13 was grown at pH 6 and 7 in order to change 465
both the cell growth and specific glucose uptake rates that regulate the glycolytic fluxes. The 466
result from Figure 8A shows that when pH was reduced from 7 to 6, the specific growth rate 467
decreased by 16% from 0.42±0.01 (1/hr) to 0.35±0.01 (1/hr), and the specific glucose uptake rate 468
dropped by 21% from 5.23±0.32 to 4.13±0.65 (g glucose/g DCW/hr). However, the isobutanol 469
production increased by 28% from 1.34±0.05 to 1.74±0.22 g/L and the ethanol production 470
decreased by 12% from 4.71±0.20 to 4.17±0.05 g/L (Figure 8B,C). The mass ratio of isobutanol 471
to ethanol shifted from 0.31±0.02:1 to 0.53±0.08:1 (Table 5). It should be noted that each value 472
represents the mean ± the standard deviation (n≥6). The t-test statistical analysis using 473
SigmaPlot gave p-values < 0.05 for comparing the respective concentrations, indicating that 474
these results are statistically significant with greater than 95% confidence. 475
One interesting observation during fermentation at low pH is that formate production was 476
significantly reduced, and was lower than that of ethanol (Figure 8B). The lower production was 477
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due to the conversion of formate into CO2 and H2 via formate hydrogenlyase, which is known to 478
be overexpressed in acidic environments (8, 22, 40). This phenotype is useful for the cell 479
because the accumulation of formate is toxic to cell growth and hence increases cell 480
maintenance. 481
Model validation 482
Experimental data on the physiological states of all isobutanol-producing strains, including 483
BFA7.001(DE3) pCT01, BFA7.001(DE3) pCT02, BFA7.001(DE3) pCT03, and 484
BFA7.001(DE3) pCT13, were projected on the 2-D phenotypic space shown in Figure 3. The 485
physiological states of BFA7.001(DE3) pCT13 fit very well on the predicted phenotypic space 486
when operated at both pH 6 and pH 7. As the pH was reduced from 7 to 6, the designed strain 487
was able to shift from a lower isobutanol- and higher ethanol-producing phenotype to a higher 488
isobutanol- and lower ethanol-producing phenotype. Strain optimization can be employed to 489
improve the isobutanol to ethanol selectivity, but was not an objective of the present work. 490
DISCUSSION 491
In this proof-of-concept study, the central metabolism of E. coli was rewired to convert the 492
heterologous isobutanol-producing pathway to an obligately anaerobic pathway. The engineered 493
E. coli strain was rationally designed based on EMA in order to identify the optimal pathways 494
for the strain to grow and produce isobutanol anaerobically. The designed strain was then 495
constructed and its performance characterized under controlled anaerobic conditions. 496
It is noteworthy that the designed isobutanol-producing strain BFA7.001(DE3) pCT13 could 497
operate in the predicted phenotypic space (Figure 3). The designed strain was able to grow and 498
produced isobutanol anaerobically. Due to the infeasibility of solely producing isobutanol, 499
ethanol must be co-produced with isobutanol to balance the high intracellular redox state under 500
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anaerobic conditions. Thus, the designed strain BFA7.001(DE3) pCT13 produced mixed 501
alcohols, with isobutanol and ethanol as the major fermentative products. The adhE-deficient 502
strain derived from the host BFA7.001(DE3) could not grow anaerobically unless complemented 503
with the gene adhE. 504
In a previous study, JCL260 pSA55/pSA69 was reported to produce isobutanol under 505
oxygen-limited conditions (3). The strain could not consume glucose anaerobically because 506
multiple gene were inactivated to remove competitive pathways, resulting in a redox imbalance. 507
This was predicted by EM analysis and experimentally verified in the controlled bioreactor 508
experiments performed in the present study. In particular, EM analysis showed that deleting only 509
fumarate reductase (frdABC) and alcohol/aldehyde dehydrogenase (adhE) was sufficient to 510
inactivate anaerobic growth in JCL260 pSA55/pSA69 (Table 2). Even though JCL260 511
pSA55/pSA69 could convert glucose to isobutanol micro-aerobically, the precise control of 512
oxygen to support cell growth and enable isobutanol production is a difficult challenge. 513
Both the isobutanol yield (~20% of the theoretical yield) and mass ratio of isobutanol to 514
ethanol (0.52) were lower than the predicted values derived from the 6 most efficient isobutanol 515
producing pathways (Figure 2 and Supplementary Table S2), which are located on the line 516
connecting groups G1 and G2 in Figure 3. The key reason is that the PDHC enzyme that was 517
assumed to be strongly inhibited under anaerobic conditions could be unexpectedly employed for 518
anaerobic isobutanol production. The PDHC enzyme is typically present at low levels under 519
anaerobic conditions due to the inhibition of NADH on the E2 subunit of the enzyme, reducing 520
the production of NADH. Its anaerobic function is replaced by the PFL enzyme (13), but this 521
does not imply that the PDHC enzyme is not expressed and produced (19). In the designed 522
isobutanol producing strains, the PDHC enzyme was likely activated because over-expression of 523
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the endogeneous alcohol/aldehyde dehydrogenase AdhE could help the cell to quickly recycle 524
NADH and alleviate NADH inhibition of the PDHC enzyme. In addition, the low or non-specific 525
activity of the isobutyraldehyde dehydrogenase Adh2S.cerevisiae contributed significantly to the low 526
isobutanol and high ethanol production. Thus, the isobutanol-producing pathway is limiting at 527
the final conversion step. The observed phenotype provides valuable insights into the 528
metabolism of the designed isobutanol-producing strain and suggests the next step of strain 529
optimization. Anaerobic isobutanol production can be improved by (1) using a more specific 530
isobutyraldehyde dehydrogenase in place of either AdhEE.coli or Adh2S.cerevisiae (potential 531
candidates are AdhE2 from C. acetobutylicum and engineered AdhA from L. lactis (7)) and (2) 532
completely inactivating the PDHC enzyme in BFA7.001(DE3) by deleting aceF. 533
An important finding of this study is that the endogeneous NADH-dependent 534
alcohol/aldehyde dehydrogenase AdhE plays a key role in anaerobic isobutanol production by 535
converting isobutyraldehyde to isobutanol. Its unique role is supported by several lines of 536
evidence. First, the designed strains BFA8.001(DE3) pCT01, BFA8.001(DE3) pCT02, and 537
BFA8.001(DE3) pCT03 that had the gene adhE deleted, were unable to grow. However, the 538
complement strain BFA8.001(DE3) pCT06 was able to grow when the AdhE function was 539
restored. In addition, overexpression of the gene adhE in the designed isobutanol-producing 540
strain BFA7.001(DE3) pCT13 increased isobutanol production. Finally, the role of AdhE role 541
was confirmed by the in vitro enzyme assay of the purified His-tagged AdhE. Interestingly, the 542
enzyme AdhE had the highest activity on butyraldehyde, and its activity on acetaldehyde was 543
about 3-fold higher than on isobutyraldehyde. This result explains why overexpression of adhE 544
in the designed strain did not significantly improve isobutanol production. 545
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Control of the glycolytic flux plays a significant role in improving isobutanol production and 546
adjusting the isobutanol to ethanol mass ratio in the designed isobutanol producing strain. The 547
high glycolytic flux causes a high intracellular redox state (ratio of NADH/NAD+) that requires 548
the strain to recycle NADH as quickly as possible from the ethanol-producing pathway. Thus, 549
the strain will make more ethanol and less isobutanol. However, if the glycolytic flux is 550
adjusted, the isobutanol to ethanol mass ratio can be controlled. We have demonstrated this by 551
carrying out controlled batch fermentations at pH 6 and 7. Lowering the pH decreases the 552
glycolytic flux, reduces the high intracellular redox state, and hence increases the isobutanol to 553
ethanol mass ratio. Other cultivation techniques such as chemostat operation, can also be applied 554
to demonstrate the observed phenotype. 555
In summary, we have demonstrated a powerful approach to rationally design an E. coli strain 556
that employs the heterologous isobutanol-producing pathway as an obligately anaerobic 557
pathway. Metabolic pathway analysis was used to dissect the E. coli cellular metabolism and 558
identify the most efficient pathways for anaerobic isobutanol production. The designed strain 559
functioned in accordance with these selected optimal pathways, and its metabolism closely 560
matches the model predictions. With the anaerobic isobutanol metabolism of the designed strain 561
characterized, strain optimization to improve the isobutanol production and control the 562
isobutanol to ethanol mass ratio can be addressed. The present approach has thus proven to be 563
useful in rational strain development for conversion of renewable and sustainable biomass 564
feedstocks into biofuels and biochemicals. 565
ACKNOWLEDGEMENTS 566
We would like to thank Prof. James C. Liao at UCLA for kindly providing the strain JCL260 and 567
the plasmids pSA55 and pSA69. 568
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Figure legends 681
Figure 1. The simplified metabolic network of the designed isobutanol-producing cell 682
optimized for anaerobic isobutanol synthesis. Pfl (encoded by the gene pflB): pyruvate formate 683
lyase; PDHC (pdh): pyruvate dehydrogenase complex (PDHC) enzyme; AlsSB.subtilis 684
(alsSB.subtilis): acetolactate synthase from B. subtilis. It should be noted that E. coli has the 685
endogeneous acetolactate synthase enzyme encoded by ilvHI, ilvGM, or ilvNB 686
(http://www.ecocyc.com). Here AlsSB.subtilis is used to bypass the allosteric inhibition by valine 687
(2, 21); IlvC (ilvC): 2,3-dihydroxy isovalerate oxidoreductase; IlvD (ilvD): 2,3-dihydroxy 688
isovalerate dehydratase; KivdL.lactis (kivdL.lactis): α-ketoacid decarboxylase from L. lactis; 689
Adh2S.cerevisiae: alcohol/aldehyde dehydrogenase from S. cerevisiae; AdhE: aldehyde/alcohol 690
dehydrogenase from E. coli. 691
Figure 2. Identification of multiple deleted genes in the central metabolism of E. coli to design 692
the most efficient fermentative pathways for anaerobic isobutanol production. A. Effect of 693
multiple deleted gene on the remaining total EMs, biomass producing EMs, isobutanol-694
producing EMs, and biomass and isobutanol co-producing EMs. B. Effect of multiple deleted 695
genes on minimal and maximal isobutanol yields under the anaerobic condition. The presented 696
yields are calculated from the model prediction. 697
Figure 3. Phenotypic operating space of the designed isobutanol producing strain and model 698
validation for isobutanol production by strains BFA7.001(DE3) pCT01, BFA7.001(DE3) pCT02, 699
BFA7.001(DE3) pCT03, and BFA7.001(DE3) pCT13. 700
Figure 4. Fermentation of the designed isobutanol-producing E. coli BFA7.001(DE3) pCT03. 701
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Figure 5. Cell growth of BFA8.001(DE3) containing plasmids (○) pCT01, (∆) pCT02, (□) 702
pCT03, and (∇) pCT06 under identical anaerobic growth conditions. The medium contained 40 703
g/L of glucose as the carbon source. 704
Figure 6. Cell growth of JCL260 pSA55 pSA69 under anaerobic and oxygen-limited 705
conditions. The medium contained 40 g/L of glucose as the carbon source. 706
Figure 7. Effect of overexpressing different portions of the isobutanol-producing pathway on 707
the designed host BFA7.001(DE3) including (A) glucose consumption and biomass production, 708
(B) ethanol production, and (C) isobutanol production. The characterized strains were (○) 709
BFA7.001(DE3) pCT01, (∆) BFA7.001(DE3) pCT02, and (□) BFA7.001(DE3) pCT03. In the 710
Figure 7A, the filled symbols are referred to glucose consumption while the open symbols 711
represent the biomass production. 712
Figure 8. Fermentation of the designed isobutanol-producing E. coli BFA7.001(DE3) pCT13 at 713
pH 6 and pH 7. Kinetic profiles include (A) glucose consumption and biomass production, (B) 714
ethanol and formate production, and (C) isobutanol production. 715
716
717
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Table 1. List of strains and plasmids used in this study 718
Strains/Plasmids Description Source
Strains
BW25113 E. coli K12, λ-, F
-, lacI
q rrnBT14 ∆lacZWJ16
hsdR514 ∆araBADAH33∆rhaBADLD78
(12)
JW0855 BW25113, poxB::kanR (6)
JW2294 BW25113, pta::kanR (6)
JW1841 BW25113, zwf::kanR (6)
JW3205 BW25113, mdh::kanR (6)
JW1375 BW25113, ldhA::kanR (6)
JW4115 BW25113, frdA::kanR (6)
JW1095 BW25113, ndh::kanR (6)
JW1228 BW25113, adhE::kanR (6)
BFA7.001(λDE3) BW25113, λDE3 ∆zwf ∆mdh ∆frdA ∆ndh
∆pta ∆poxB ∆ldhA::kan-
This study
BFA8.001(λDE3) BW25113, λDE3 ∆zwf ∆mdh ∆frdA ∆ndh
∆pta ∆poxB ∆ldhA ∆adhE::kan-
This study
BL21(DE3) E. coli F- ompT hsdSB (rB
-mB
-) gal dcm
(λDE3)
Invitrogen (cat # C600003)
Plasmids
pSA55 colE1 ori, plac::kivd::adh2, ampR (3)
pSA69 colE1 ori, plac::alsS::ilvC::ilvD, kanR (3)
pCOLADuet colA ori, kanR Novagen (cat # 71406-3)
pET24b f1 ori, kanR Novagen (cat # 69750-3)
pCT01 pCOLA pT7::alsS::ilvC::ilvD, inserted at
BamH1 and NotI restriction sites, kanR
This study
pCT02 pCOLA pT7::kivd::adh2, inserted at NdeI
and XhoI restriction sites, kanR
This study
pCT03 pCOLA pT7::alsS::ilvC::ilvD::
pT7::kivd::adh2, kanR
This study
pCT06 pCOLA pT7::alsS::ilvC::ilvD::
pT7:: adhE, kanR
This study
pCT13 pCOLA pT7::alsS::ilvC::ilvD::
pT7::kivd::adhE, kanR
This study
pET24b adhE pET24b pT7::adhE, kanR This study
pPC20 FLP+, λ cI857+, λ pR Rep
ts, amp
R, Cm
R (11)
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Table 2. Effect of deleting anaerobic pathways on the feasibility of producing isobutanol as the 719
sole product 720
Deleted genes# Total
EMs
Biomass
EMs
iBuOH
EMs
Biomass
& iBuOH
EMs
Range of iBuOH yield
on glucose
(g /g)
min max
∆frd ∆ldhA ∆pta ∆poxB ∆adhE 14 0 14 0 0.41 0.41
∆frd ∆ldhA ∆pta ∆poxB 1,150 900 814 282 0.04 0.41
∆frd ∆ldhA ∆adhE 14 0 14 0 0.41 0.41
∆frd ∆pta ∆poxB ∆adhE 28 0 14 0 0.41 0.41
∆ldhA ∆pta ∆poxB ∆adhE 427 251 427 251 0.06 0.41
∆frd ∆adhE 28 0 14 0 0.41 0.41
#∆frd: removal of succinate producing pathway; ∆ldhA: removal of lactate producing 721
pathway; ∆pta ∆poxB: removal of acetate producing pathway; ∆adhE: removal of ethanol 722
producing pathway. Abbreviation: iBuOH, isobutanol. 723
724
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Table 3. Elementary mode analysis for the effect of multiple deleted genes applied to different 725
designed E. coli strains including JCL260 pSA55 pSA69 (3) and BFA7.001(DE3) pCT03 or 726
BFA7.001(DE3) pCT13 (this study) under anaerobic conditions. 727
728
Strains
JCL260
pSA55 pSA69
BFA7.001(DE3)
pCT03
Deleted genes
∆adhE +
∆fnr +
∆pflB +
∆frdBC + +
∆pta + +
∆ldhA + +
∆ndh +
∆poxB +
∆zwf +
∆mdh +
∆aceF +
Total anaerobic EMs 14 6
Biomass producing EMs 0 2
Isobutanol producing EMs 14 6
Range of isobutanol yields
(g/g)
0.41-0.41 0.29-0.41
729
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Table 4. Kinetic parameters for the endogeneous alcohol/aldehyde dehydrogenase AdhE 730
reacting with acetaldehyde, isobutyraldehyde, and butyraldehyde. Each value is a mean ± average 731
deviation from two independent measurements. Non-linear regression (SigmaPlot) was used to determine 732
Km and kcat with p-values < 0.05. The R2 coefficients for non-linear regression were greater than 0.99 for 733
all cases. 734
Substrate Km
(µM)
kcat
(sec-1
)
kcat/Km x 103
(µΜ−1
sec-1
)
Acetaldehyde 48.6±3.5 5.72±0.07 118±7
Isobutyraldehyde 54.3±5.1 2.13±0.05 39.5 ±2.8
Butyraldehyde 196±38 59.2±8.1 305±17.1
735
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Table 5. Specific growth rates, isobutanol concentrations, and mass ratios of isobutanol to 736
ethanol of the strains BFA7.001(DE3) pCT01, BFA7.001(DE3) pCT02, BFA7.001(DE3) 737
pCT03, and BFA7.001(DE3) pCT13 characterized in batch culture under anaerobic conditions. 738
739
Strains pH Specific growth rate
(hr-1
)
Isobutanol
(g/L)
IBUT:ETOH ratio
(g/g)
BFA7.001(DE3) pCT01 7 0.45±0.02 0.81±0.13 0.28±0.04
BFA7.001(DE3) pCT02 7 0.49±0.01 0.00±0.00 0.00±0.00
BFA7.001(DE3) pCT03 7 0.37±0.03 1.11±0.07 0.26±0.01
BFA7.001(DE3) pCT13 7 0.42±0.01 1.34±0.05 0.31±0.02
BFA7.001(DE3) pCT13 6 0.35±0.01 1.72±0.22 0.52±0.08
740
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741
Figure 1 742
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743
744
Figure 2 745
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746
747
Figure 3 748
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749
750
Figure 4 751
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752
753
Figure 5 754
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755
756
Figure 6 757
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758
759
Figure 7 760
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761
762
Figure 8 763
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