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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248

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


pT7:: adhE, kanR

This study


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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redesigning escherichia coli metabolism for · pathways that synthesize advanced biofuels are not...

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