Butanol Production in - ACS Publications - American Chemical Society

KEYWORDS: Escherichia coli, CRISPR/Cas9, genome editing, 5′-untranslated region, n-butanol. Biobutanol is a good alternative to fossil fuels because...
4 downloads 0 Views 1MB Size
Subscriber access provided by Northern Illinois University

Letter

Controlling Citrate Synthase Expression by CRISPR/Cas9 Genome Editing for n-Butanol Production in Escherichia coli Min-Ji Heo, Hwi-Min Jung, Jaeyong Um, Sang-Woo Lee, and Min-Kyu Oh ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00134 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 6, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Synthetic Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

1

Controlling Citrate Synthase Expression by CRISPR/Cas9 Genome Editing

2

for n-Butanol Production in Escherichia coli

3

Min-Ji Heoa,#, Hwi-Min Junga,#, Jaeyong Uma, Sang-Woo Leea, and Min-Kyu Oha,*

4 5 6 7

a

Department of Chemical & Biological Engineering Korea University, 5-1 Anam-dong, Seongbuk-gu, Seoul ,136-713, South Korea

8 9 10

Graphical Table of Contents

11

12 13 14

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

15

ABSTRACT

16 17

Genome editing using CRISPR/Cas9 was successfully demonstrated in Esherichia coli to effectively

18

produce n-butanol in a defined medium under micro-aerobic condition. The butanol synthetic pathway

19

genes including those encoding oxygen-tolerant alcohol dehydrogenase were overexpressed in

20

metabolically engineered E. coli, resulting in 0.82 g/L butanol production. To increase butanol

21

production, carbon flux from acetyl-CoA to citric acid cycle should be redirected to acetoacetyl-CoA.

22

For this purpose, the 5′-untranslated region sequence of gltA encoding citrate synthase was designed

23

using an expression prediction program, UTR designer, and modified using the CRISPR/Cas9

24

genome editing method to reduce its expression level. E. coli strains with decreased citrate synthase

25

expression produced more butanol and the citrate synthase activity was correlated with butanol

26

production. These results demonstrate that redistributing carbon flux using genome editing is an

27

efficient engineering tool for metabolite overproduction.

28 29

Keywords: Escherichia coli; CRISPR/Cas9; genome editing; 5’-untranslated region; n-butanol

30 31

ACS Paragon Plus Environment

Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

32

Biobutanol is a good alternative to fossil fuels because its energy density (29.2 MJ/L) is 90%

33

of that of gasoline (32 MJ/L)1. n-Butanol is naturally produced by Clostridium species by acetone–

34

butanol–ethanol fermentation, which has been intensively studied and developed2,3. Although

35

Clostridium species are good host strains for producing n-butanol, they have shortcomings due to

36

limited genetic engineering tools and complex physiology. Therefore, other strains, such as

37

Escherichia coli, have been developed as hosts to produce n-butanol4. As E. coli is not a natural

38

producer, the n-butanol pathway must be introduced into the strain. The coenzyme A (CoA)

39

dependent pathway or a modified pathway have been introduced from Clostridium5,6. Other pathways,

40

such as the reverse β-oxidative7, and ACS-dependent pathway8, etc. have been successfully used to

41

produce butanol. Meanwhile, metabolic engineering of the host strain is also required. For example,

42

E. coli with deleted metabolic pathways to by-products, including acetate, lactate, ethanol, and

43

succinate, was used to achieve 30 g/L butanol production with gas-stripping method9. Rebalancing

44

intracellular redox state has also been proven as an efficient method to achieve high butanol

45

productivity10,11.

46

The expression levels of multiple genes must be controlled for the host strain to efficiently

47

produce biofuel12. Knockout and knockin of multiple genes in the E. coli genome have been utilized

48

to produce butanol7-11. However, recent advances in metabolic engineering have provided more

49

diverse tools to search for optimal gene expression levels. For example, knockdown of single or

50

multiple gene(s) using sRNA and Hfq protein allowed to optimize expression levels of metabolic

51

genes to overproduce of tyrosine and cadaverine13. Another synthetic biology tool for regulating gene

52

expression level, dCas914, has been used in metabolic engineering of Corynebactrium glutamicum15.

53

Although these techniques for modulating gene expression are very useful in metabolic engineering,

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

54

the expression level of the target gene is difficult to estimate and engineered strains must express

55

additional protein components, such as Hfq or dCas9. Therefore, optimizing gene expression level by

56

modifying the 5′- untranslated region (UTR) is a good alternative to control gene expression because

57

efficient genome editing methods and gene expression predicting tools have been developed. A

58

specific sequence on the E. coli genome can be precisely changed for gene expression control using

59

the RNA-guided genome editing tool CRISPR/Cas system16,17. On the other hand, the correlation

60

between expression level of a certain gene and the 5′-UTR sequence was evaluated using recently

61

developed computational tools18,19. Combining these methods allows for more precise control of the

62

expression level of a specific gene in the host genome.

63

In preliminary experiments, we engineered E. coli to produce n-butanol using glucose

64

minimal media. A butyrate-producing strain previously developed in our laboratory20 was modified.

65

Briefly, the N-terminals of three enzymes that convert acetoacetyl-CoA to butyryl-CoA, such as 3-

66

hydroxybutyryl-CoA dehydrogenase (Hbd), 3-hydroxybutyryl-CoA dehydratase (Crt), and trans-

67

enoyl-coenzyme A reductase (Ter), were modified to attach to a scaffold protein. For this purpose, the

68

GBD, SH3, and PDZ ligands were translationally fused to Hbd, Crt, and Ter, respectively, and

69

expressed with the pCDF-HCT vector (Table 1). The scaffold protein with GBD, SH3, and PDZ

70

ligand binding sites at a 1:1:2 ratio was overexpressed by the pJD758 vector21. Endogenous

71

acetoacetyl-CoA thiolase (atoB), alcohol dehydrogenase (adhE2) from C. acetobutylicun and formate

72

dehydrogenase (fdh1) from C. boidinii22 were overexpressed by the pACYC-AEF vector (Figure 1).

73

The three plasmids were transformed to the DSM01 strain to construct EMJ40. After a 96 h

74

incubation in a flask under anaerobic conditions with M9G defined medium, 0.052 g/L butanol was

75

synthesized. Most butanol production experiments have been performed with rich media supplied

ACS Paragon Plus Environment

Page 4 of 24

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

76

with glucose4-11. However, to understand carbon flux and calculate carbon yield exactly, producing

77

butanol in a defined medium is necessary. The strain developed here produced only a noticeable

78

amount of butanol in a defined medium with a minimum amount of yeast extract (0.5 g/l) needed for

79

initial growth of the strain. This must be because the optical density of this culture was very low under

80

anaerobic condition, which was suspected to be the main limitation to butanol production in minimal

81

medium. When EMJ40 was cultured under micro-aerobic condition, butanol production decreased,

82

but optical density increased significantly (Figure 2a). It was inferred that significant energy was

83

required for microbial growth with expression of butanol pathway enzymes and scaffolding protein,

84

which was not supplied enough in a defined medium in anaerobic condition. The energy limitation

85

might be overcome by cultivating strains in microaerobic or aerobic condition. However, further

86

engineering strategies were needed to improve n-butanol in aerobic condition.

87

The CoA-dependent pathway of Clostridium species introduced in this strain is oxygen

88

sensitive. In particular, aldehyde/alcohol dehydrogenase (AdhE2) from C. acetobutylicum loses

89

activity when exposed to oxygen23. An oxygen-tolerant pathway from butyryl-CoA to butanol was

90

demonstrated with CoA-acylating propionaldehyde dehydrogenase (PduP) from S. enterica and

91

alcohol dehydrogenase (AdhA) from L. lactis24. Therefore, the pACYC-APAF plasmid to expressing

92

atoB, pduP, adhA, and fdh1 was constructed and transformed with pCDF-HCT and pJD758 into

93

DSM01, and the resulting strain was named EMJ50 (Table 1). After 96 h of culture under micro-

94

aerobic conditions with M9G medium, 0.82 g/l butanol was synthesized by strain EMJ50, which was

95

much higher than the 0.014 g/l from EMJ40. Butanol yield also increased from 0.0029 to 0.068 g/g

96

glucose (Figure 2b), indicating that oxygen-tolerant alcohol dehydrogenase was very helpful to

97

increase butanol synthesis under micro-aerobic conditions. Although the butanol titer obtained under

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

98

micro-aerobic conditions increased after using oxygen-tolerant PduP and AdhA, the carbon yield (g

99

butanol/g glucose) was still slightly lower than that of anaerobic culture using AdhE2 (0.082 g/g

100

glucose). This may have been caused by loss of acetyl-CoA to the citric acid cycle under micro-

101

aerobic condition because acetyl-CoA is a precursor for both butanol and the citric acid cycle.

102

Therefore, carbon flux to the citric acid cycle must be reduced to increase butanol yield.

103

For this purpose, we knocked down the expression level of gltA by editing its 5′-UTR

104

sequence (Figure 3). UTR Designer19, which predicts protein expression levels using the 5′-UTR

105

sequence, has been used in metabolic engineering25. Therefore, four different gltA 5′-UTR sequences

106

predicted to have 50, 30, 10, and 1% of the expression levels of wild-type gltA were designed with the

107

program (Table 2 and Figure 3). CRISPR/Cas9 experiments were conducted to edit the gltA 5′-UTR

108

sequence on the DSM01 genome, using a slightly modified experimental method developed by Jiang

109

et al16. Four 5` UTR- engineered strains were constructed and transformed with three plasmids

110

expressing butanol synthesis pathway genes and named EMJ51–EMJ54 (Table 1). We also

111

constructed a gltA deleted mutant and transformed it with the same set of plasmids (EMJ55).

112

Under micro-aerobic condition, the gltA deletion mutant grew very poorly. All the genome

113

edited mutants did as well as the wild type except EMJ54 (Figure 4a). EMJ54 showed 24% reduced

114

growth at the late exponential phase (48 h). Modifying the gltA 5′-UTR sequences was supposed to

115

reduce the gltA expression level and activity of its product. We measured enzyme activity of each

116

strain using a citrate synthase assay kit. The gradual decrease in specific citrate synthase activity was

117

in accordance with the prediction, but the magnitude of the decrease was much less than that the

118

predicted gene expression (Figure 4b). EMJ54 had 67% reduced citrate synthase activity compared to

119

EMJ50. The growth pattern suggested that significant reduction of microbial growth was recognized

ACS Paragon Plus Environment

Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

120

ACS Synthetic Biology

in a defined medium when the citrate synthase activity was reduced more than 50%.

121

After culturing the strains in M9G medium, titers and yields of butanol were measured. Clear

122

correlations were found between citrate synthase activity and butanol titer/yield. As citrate synthase

123

activity decreased, the n-butanol titer increased up to 1.3-fold in EMJ52 (Figure 4c). The maximal

124

butanol titer was achieved in EMJ52, which has 55% citrate synthase activity compared to parental

125

strain. However, butanol titer deteriorated when citrate synthase activity was below 46% (EMJ53,

126

EMJ54, EMJ55). Also as the citrate synthase activity decreased, ethanol yield increased significantly

127

(Table S1). It is expected that as the expression of citrate synthase diminished, availability of acetyl-

128

CoA increased. Although major ethanol production pathway was removed, other alcohol

129

dehydrogenase can convert acetyl-CoA to ethanol. Furthermore, introduced alcohol dehydrogenase,

130

adhA, has substrate specificity not only to butyraldehyde but also to acetaldehyde26. Therefore, more

131

ethanol can be formed as aceyl-CoA accumulated in cytosol.

132

A linear relationship was observed between citrate synthase activity and butanol yield, but

133

the relationship between citrate synthase activity and butanol titer was more complex due to cell

134

growth. Although the highest yield was obtained from EMJ55, in which gltA was deleted, a low

135

butanol titer was observed due to the low growth rate and EMJ55 hardly consumed glucose for 72 h

136

cultivation (Figure 4c, Table S1). Therefore, gltA knockdown was chosen as a better strategy11.27. We

137

attempted gltA knockdown using the genome editing method with CRISPR/Cas9, and the necessary

138

sequences for genome editing of the 5′-UTR region were designed using a bioinformatics tool, UTR

139

designer.

140

Both the genome editing and 5′-UTR design methods worked effectively. The components

141

developed for genome editing, such as CRISPR, tracrRNA, the crRNA targeted gltA 5′-UTR, and the

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

142

141 bp double-stranded rescue DNA, effectively changed the genome sequence as designed after

143

transformation. More than 10 colonies were obtained after each experiment and at least 30% of them

144

provided properly edited ones. Because the sacB gene was added to the plasmids for the

145

CRISPR/Cas9 experiment, the plasmids were eliminated after an overnight cultivation with sucrose28.

146

The experiment to modify the host genome took less than 1 week. On the other hand, designing the 5′-

147

UTR sequence was effective but should be improved. All of the designed gltA 5′-UTR sequences

148

provided reduced citrate synthase activity after genome editing. However, UTR Designer expected

149

those sequences to reduce gltA expression levels to 50, 25, 10, and 1% of that of the wild type, but

150

citrate synthase activity was actually reduced to 75, 55, 46, and 34%, respectively, possibly because

151

of the complex interactions among various factors at the levels of transcription and translation.

152

Nevertheless, our results demonstrate that combining these tools was quite useful to modulate the

153

expression levels of a specific enzyme, which is very useful to balance metabolic flux between the

154

product and cell growth or host maintenance to overproduce a metabolite.

155

In summary, expression of citrate synthase, the first enzyme in the citric acid cycle, was

156

controlled by modifying the 5′-UTR sequence of gltA on the genome. The citric acid cycle competes

157

with butanol pathway as they both use acetyl-CoA as a precursor. We designed four different 5′-UTR

158

sequences with different predicted gltA expression levels and edited them on the genome using the

159

CRISPR/Cas9 system. These modifications improved the butanol titer and yield, which were

160

correlated with citrate synthase activity.

161 162

Methods

163

ACS Paragon Plus Environment

Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

164

ACS Synthetic Biology

Strains, plasmids, and primers

165 166

The strains, plasmids, and primers used in this study are listed in Tables 1 and 2. E. coli

167

DH5α was used to construct plasmids. Escherichia coli MG1655 integrated with λDE3 was

168

genetically modified to produce butanol. Four genes, such as frdA, ldhA, pta, and adhE, were deleted

169

from MG1655(DE3) using P1 phage transduction and used as the host strain (DSM01)20,29.

170

Additionally, gltA was deleted in the same manner.

171 172

Genome editing experiment

173 174

Four

different

gltA

5′-UTR

sequences

were

designed

using

UTR

designer

175

(http://sbi.postech.ac.kr/utr_designer) to modulate the gltA expression level19. A protocol modified

176

from a previous report was used for the genome editing experiment with CRISPR/Cas916. Briefly,

177

cas9, crRNA, and the tracrRNA region were amplified by polymerase chain reaction (PCR) using the

178

primers listed in Table 2 with the pCas9 plasmid (Addgene, Cambridge, MA, USA)16. sacB was

179

amplified using genomic Bacillus subtilis DNA. The cas9 and sacB PCR products were cloned into

180

pZA31MCS using Gibson assembly (New England Biolabs, Ipswich, MA, USA), and named pZA-

181

Cas9 (Table 1). Similarly, crRNA, tracrRNA, and sacB were cloned into pZS21MCS and named pZS-

182

CRISPR (Table 1). Both strands of the gltA 5'-UTR sequence (gltA crRNA-S and gltA crRNA-A,

183

Table 2) were synthesized by Bioneer Inc. (Daejeon, Korea) to insert the gltA targeting crRNA

184

sequence. Then, the synthesized DNA fragments were slowly annealed and ligated into pZS-CRISPR

185

digested with BsaI, resulting in pZS-CRISPRgltA containing crRNA targeting gltA-5′-UTR. The

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

186

rescue DNAs for homologous recombination after Cas9 nuclease digestion were prepared from pairs

187

of synthesized oligomer DNAs with 22 bp overlapping sequence (Table 2). The oligomer pairs, such

188

as gltA Rescue F and 0.6R, gltA Rescue F and 0.3R, gltA Rescue F2 and 0.5R, and gltA Rescue F2 and

189

0.4R, respectively, were denatured at 96°C and annealed by slow cooling, followed by extended DNA

190

synthesis using the Klenow fragment (Takara Bio, Shiga, Japan). We conducted a genome editing

191

experiment using DSM01 as the host. First, pKD4630 and pZA-Cas9 was sequentially transformed

192

into DSM01 using electroporation. Then, gltA Rescue DNA and pZS-CRISPRgltA were co-

193

transformed into the strain and the transformants were screened on a LB plate containing ampicillin

194

(50 µg/ml), kanamycin (50 µg/ml), and chloramphenicol (50 µg/ml).

195

Mutant strains harboring the plasmids for CRISPR/Cas9 editing were incubated overnight at

196

37°C in liquid LB medium containing 40 g/l sucrose and streaked on an LB agar plate. The colonies

197

that lost all antibiotics resistance were selected on an agar plate. The genome-editing results were

198

confirmed by PCR of the gltA 5′-UTR region using the gltA-5′-F and gltA-5′-R primers, followed by

199

sequencing of the PCR products by CosmoGenetech (Seoul, Korea) using the gltA-5′-seq primer

200

(Table 2).

201 202

Plasmid construction for butanol production

203 204

The pCDF-HCT vector constructed by Beak et al. (2013)20 was used to convert acetoactyl-

205

CoA to butyryl-CoA (Figure 1). The genes of three enzymes, such as Hbd, Crt, and Ter, attached with

206

the GBD, SH3, and PDZ ligands, respectively, were expressed using the pCDF-HCT vector3. The

207

scaffold protein (GBD1SH31PDZ2) was expressed by pJD75821 to increase pathway efficiency. Other

ACS Paragon Plus Environment

Page 10 of 24

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

208

enzymes, such as atoB, adhA, adhE2, and fdh1, were amplified from genomic DNAs of E. coli

209

MG1655, Lactococcus lactis, Clostridium acetobutylicum, and Candida boidinii, respectively, using

210

the primers listed in Table 2. The codon-optimized pduP gene of Salmonella enterica was synthesized

211

by Bioneer Inc. and amplified using the primers listed in Table 2. Four genes (atoB, pduP, adhA, and

212

fdh1) were ligated into the corresponding sites of the pACYCDuet vector and named pACYC-APAF

213

(Table 1). Similarly, three genes (atoB, adhE2, fdh1) were ligated into pACYCDuet and named

214

pACYC-AEF. Either pACYC-APAF or pACYC-AEF was co-transformed with pCDF-HCT and the

215

pJD758 using electroporation to produce butanol.

216 217

Media and culture conditions

218 219

Strains were selected and cultured on LB medium (10 g/l tryptone, 5 g/l yeast extract, and 10

220

g/l NaCl) supplied with 50 µg/ml ampicillin, 50 µg/ml chloramphenicol, 50 µg/ml kanamycin, and

221

100 µg/ml spectinomycin. Engineered E. coli was cultured in M9G medium (12.8 g/l Na2HPO4, 3 g/l

222

KH2PO4, 0.5 g/l NaCl, 1 g/l NH4Cl, 1 mM MgSO4, 0.1 mM CaCl2, 10 mg/l thiamine, 25 g/l glucose,

223

0.5 g/l yeast extract, and 1,000× trace elements (27 g/l FeCl3·6H2O, 2 g/l ZnCl2·4H2O, 2 g/l

224

CaCl2·2H2O, 2 g/l Na2MoO4·2H2O, 1.9 g/l CuSO4·5H2O, and 0.5 g/l H3BO3)) supplemented with 50

225

µg/ml ampicillin, 50 µg/ml chloramphenicol, and 100 µg/ml spectinomycin5. E. coli was pre-cultured

226

in 5 ml M9G medium overnight with appropriate antibiotics to produce butanol. The culture broth

227

was transferred to 50 ml M9G medium in 250 ml flasks. All flasks were sealed with rubber stoppers to

228

create micro-aerobic conditions and incubated at 37°C with shaking at 250 rpm, and 0.05 mM IPTG

229

and 108 nM anhydrotetracycline (aTc) were added as inducers at 6 h.

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

230 231

Citrate synthase activity assay

232 233

E. coli was cultured in M9G minimal medium for 32 h to measure citrate synthase activity.

234

Strains from 50 mL culture broth were harvested and washed twice by centrifugation at 35,000 × g for

235

10 min followed by resuspension. The pellet was suspended in 10 mL of 120 mM Tris HCl (pH 8.0),

236

containing 10 mM MgCl2, 0.1 M KCl, and 1 mM EDTA. The cells were mechanically disrupted by

237

sonication on ice for 30 min. After centrifugation at 35,000 × g for 10 min, the supernatant was used

238

to measure enzyme activity31. Citrate synthase activity was measured with a citrate synthase assay kit

239

(Sigma-Aldrich, St. Louis, MO, USA). The enzyme reaction occurred with 30 mM acetyl-CoA, 10

240

mM oxaloacetate, and 10 mM 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) in citrate synthase assay

241

buffer. Enzyme activity was determined by conversion of CoA-SH with DTNB to form

242

thionitrobenzoic acid (yellow) per min in a 1 ml cuvette.

243

Total protein concentration in cell lysates was determined by the Bradford based Bio-Rad

244

Protein Assay Dye Reagent Concentrate with bovine serum albumin as the standard (Bio-Rad,

245

Hercules, CA, USA). Citrate synthase activity was normalized to total protein in cell lysates to obtain

246

specific citrate synthase activity (U/mg protein).

247 248

Analytical methods

249 250

Cell growth (OD600) and protein concentration (595 nm) were monitored with a UV-visibility

251

spectrophotometer (DU Series 700; Beckman Coulter, Inc., Brea, CA, USA). Citrate synthase activity

ACS Paragon Plus Environment

Page 12 of 24

Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

252

was measured at 412 nm using a multimode microplate reader (InfiniteⓇ-200PRO; Tecan, Hõganãs,

253

Sweden). Glucose and butanol were analyzed by high-performance liquid chromatography (ACME-

254

9000; Younglin Instrument, Seoul, Korea) with a Sugar SH1011 column (Shodex, Tokyo, Japan). The

255

Column temperature was 60°C, and 5 mM sulfuric acid was used as the mobile phase at a flow rate of

256

0.6 ml/min.

257 258

Author Information

259 260

Corresponding Author

261

*E-mail: [email protected]

262 263

Author Contributions

264

#

265

wrote the manuscript. M.J.H., H.M.J. J.Y.U. and S.W.L. performed experiments.

M.J.H., H.M.J. contributed equally to this work. M.J.H., H.M.J. and M.K.O. conceived the idea and

266 267

Notes

268

The authors declare no financial conflicts of interest.

269 270

Acknowledgement

271 272

This study was supported by a National Research Foundation of Korea funded by the Korean

273

Government (2012M1A2A2026560 and 2014R1A2A2A03007094) and New & Renewable Energy

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

274

Program of the Korea Institute of Energy Technology Evaluation and Planning (No.

275

20133030000300).

276 277

References

278 279

1. Durre, P. (2007) Biobutanol: An attractive biofuel. Biotechnol. J. 2, 1252-1534

280 281

2. Jang, Y. S., Malaviya, A.,Cho, C,. Lee, J., and Lee, S. Y. (2012) Butanol production from renewable

282

biomass by clostridia. Bioresour. Technol. 123, 653-663

283 284

3. Xue, C., Zhao, X.Q., Liu, C. G., Chen, L. J., and Bai, F. W. (2013) Prospective and development of

285

butanol as an advanced biofuel. Biotechnol. Adv. 31, 1575-1584

286 287

4. Lan, E. I. and Liao, J. C. (2013) Microbial synthesis of n-butanol, isobutanol, and other higher

288

alcohols from diverse resources. Bioresour. Technol. 135, 339-349

289 290

5. Atsumi, S., Cann, A.F., Connor, M. R., Shen, C. R., Smith, K. M., Brynildsen, M. P., Chou K. J. Y.,

291

Hanai, T., and Liao, J. C. (2008) Metabolic engineering of Escherichia coli for 1-butanol production.

292

Metab. Eng. 10, 305-311

293 294

6.. Bond-Watts, B. B., Bellerose, R. J., and Chang, M. C. Y. (2011) Enzyme mechanism as a kinetic

295

control element for designing synthetic biofuel pathways. Nat. Chem. Biol. 7, 222-227

296 297

7. Dellomonaco, C., Clomburg, J. M., Miller, E. N., and Gonzalez, R. (2011) Engineered reversal of

298

the β-oxidation cycle for the synthesis of fuels and chemicals. Nature 476, 355-359.

299 300

8. Pásztor, A., Kallio, P., Malatinszky, D., Akhtar, M. K., and Jones, P. R. (2015) A synthetic O2‐

301

tolerant butanol pathway exploiting native fatty acid biosynthesis in Escherichia coli. Biotechnol.

302

Bioeng. 112, 120-128.

303 304

9. Shen, C. R., Lan, E. I., Dekishima, Y., Baez, A., Cho, K. M., and Liao, J. C. (2011) Driving forces

305

enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl. Environ. Microbiol. 77,

306

2905-2915.

ACS Paragon Plus Environment

Page 14 of 24

Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

307 308

10. Lim, J. H., Seo, S. W., Kim, S. Y., and Jung, G. Y. (2013) Model-driven rebalancing of the

309

intracellular redox state for optimization of a heterologous n-butanol pathway in Escherichia coli.

310

Metab. Eng. 20, 56-62.

311 312

11. Sani, M., Li, S. Y., Wang, Z. W., Chiang, C. J., and Chao, Y. P. (2016) Systematic engineering of

313

the central metabolism in Esherichia coli for effective production of n-butanol. Biotechnol. Bofuel. 9,

314

69

315 316

12. Song, C.W., Lee, J., and Lee, S. Y. (2015) Genome engineering and gene expression control for

317

bacterial strain development. Biotechnol. J. 10, 56-68

318 319

13. Na, D.K., Yoo, S. M., Chung, H., Park, H., and Lee, S. Y. (2013) Metabolic engineering of

320

Escherichia coli using small regulatory RNAs. Nat. Biotechnol.31, 179-174

321 322

14. Bikard, D., Jiang, W. Y., Samai, P., Hochschild, A., Zhang, F., and Marraffini, L. A. (2013)

323

Programmable repression and activation of bacterial gene expression using an engineered CRISPR-

324

Cas system. Nucleic Acids Res. 41, 7429-7437

325 326

15. Cleto, S., Jensen, J. V. K., Wendisch, V. F., and Lu T. K. (2016) Corynebacterium glutamicum

327

metabolic

328

10.1021/acssynbio.5b00216

engineering

with

CRISPR

interference

(CRISPRi).

ACS

Syn.

Biol.

DOI:

329 330

16. Jiang, W. Y., Bikard, D., Cox, D., Zhang, F., and Marraffini, L. A. (2013) RNA-guided editing of

331

bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233-239

332 333

17. Qi, L. S., Larson, M. H., Gilbert, L., Doudna, J. A., Weissman, J. S., Arkin, A. P., and Lim, W. A.

334

(2013) Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene

335

Expression. Cell 152. 1173-1183

336 337

18. Salis, H.M., Mirsky, E. A., and Voigt, C. A. (2009) Automated design of synthetic ribosome

338

binding sites to control protein expression. Nat. Biotechnol. 27, 946-950

339 340

19. Seo, S. W., Yang, J. S., Kim, I., Yang, J., Min, B. E., Kim, S., and Jung, G. Y. (2014) Predictive

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

341

design of mRNA translation initiation region to control prokaryotic translation efficiency. Metab. Eng.

342

15, 67-74

343 344

20. Baek, J. M., Mazumdar, S., Lee, S. W., Jung, M. Y., Jung, G. Y., Lim, J. H., Seo, S. W., and Oh, M.

345

K. (2013) Butyrate production in engineered Escherichia coli with synthetic scaffolds. Biotechnol

346

Bioeng. 110, 2790-2794.

347 348

21. Dueber, J. E., Wu, G. C., Malmirchegini, G. R., Moon, T. S., Petzold, C. J., Ullal, A. V., and

349

Keasling, J. D. (2009) Synthetic protein scaffolds provide modular control over metabolic flux. Nat.

350

Biotechnol. 27, 753-759.

351 352

22. Berrı́, S. J., Bennett, G. N., and San, K. Y. (2002) Metabolic engineering of Escherichia coli:

353

increase of NADH availability by overexpressing an NAD+-dependent formate dehydrogenase.

354

Metab. Eng. 4, 217-229.

355 356

23. Fontaine, L., Meynial-Salles, I., Girbal, L., Yang, X., Croux, C., and Soucaille, P. (2002)

357

Molecular characterization and transcriptional analysis of adhE2, the gene encoding the NADH-

358

dependent aldehyde/alcohol dehydrogenase responsible for butanol production in alcohologenic

359

cultures of Clostridium acetobutylicum ATCC 824. J. Bacteriol. 184, 821-830.

360 361

24. Bogorad, I. W., Chen, C. T., Theisen, M. K., Wu, T. Y., Schlenz, A. R., Lam, A. T., and Liao, J. C.

362

(2014) Building carbon–carbon bonds using a biocatalytic methanol condensation cycle. Proc. Natl.

363

Acad. Sci. USA 111, 15928-15933.

364 365

25. Lim, H. G., Lim, J. H., and Jung, G. Y. (2015) Modular design of metabolic network for robust

366

production of n-butanol from galactose–glucose mixtures. Biotechnol. Biofuel. 8, 137

367 368

26. Atsumi S., Wu T.Y., Eckl E.M., Hawkins S.D., Buelter T., Liao J.C. (2010) Engineering the

369

isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde

370

reductase/alcohol dehydrogenase genes. Appl Microbiol Biotechnol. 85, 651-657

371 372

27. van Ooyen, J., Noack, S., Bott, M., Reth, A., & Eggeling, L. (2012) Improved L‐lysine production

373

with Corynebacterium glutamicum and systemic insight into citrate synthase flux and activity.

374

Biotechnol. Bioeng. 109, 2070-2081.

ACS Paragon Plus Environment

Page 16 of 24

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

375 376

28. Xin-tian Li, Lynn C. Thomason, James A. Sawitzke, Nina Costantino and Donald L. Court. (2013).

377

Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in

378

Escherichia coli. Nucleic Acids Res. 41, e204

379 380

29. Mazumdar, S., Lee, J., and Oh, M. K. (2013) Microbial production of 2, 3 butanediol from

381

seaweed hydrolysate using metabolically engineered Escherichia coli. Bioresour. Technol. 136, 329-

382

336.

383 384

30. Datsenko, K. A. and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in

385

Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640-6645.

386 387

31. Bloxham, D. P., Herbert, C. J., Ner, S. S., and Drabble, W. T. (1983) Citrate synthase activity in

388

Escherichia coli harbouring hybrid plasmids containing the gltA gene. Microbiology 129, 1889-1897.

389

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

390

Figure legends

391 392

Figure 1. Butanol synthetic pathway map constructed in EMJ50. Red crosses on the arrow denote

393

deleted pathway genes in the host genome. Blue bold characters are overexpressed pathway genes,

394

and their sources are presented in parenthesis. EC: Escherichia coli, CA: Clostridium acetobutylicum,

395

TD: Treponema denticola, CB: Candida boidinii, SE: Salmonella enterica, and LL: Lactococcus

396

lactis

397 398

Figure 2. Butanol production related to oxygen sensitive and tolerant pathways. (a) Optical

399

density (gray bar) and butanol titer (white bar) of EMJ40 after 96 h cultivation in M9G medium. (b)

400

Butanol production (gray bar) and butanol yield (black circle) in EMJ50 and EMJ40. Error bars

401

represent standard deviations of three independent experiments.

402 403

Figure 3. Scheme for the change in E. coli metabolic flux after editing the gltA 5′-untranslated

404

region (UTR) sequence. The sequences include the PAM sequence (red), the gltA 5′-UTR sequence

405

(underlined), the ribosome binding site (bold), and the translational start codon (blue).

406 407

Figure 4. Results of citrate synthase activity and butanol production (a) Optical density of

408

engineered E. coli cultivating for 72 h in M9G medium. Symbols represent EMJ50 (●), EMJ51 (○),

409

EMJ52 (▲), EMJ53 (△), EMJ54 (■) and EMJ55 (□). (b) Citrate synthase activity of the genome-

410

edited E. coli strains. (c) Correlations between citrate synthase activities and butanol titer (●) or yield

411

(□) of the engineered E. coli after cultivating for 72 h in M9G medium. Error bars and dashed lines

ACS Paragon Plus Environment

Page 18 of 24

Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

412

represent standard deviations of three independent experiments and regression results, respectively.

413 414

Table 1 Strains and plasmids used in this study

Name

Relevant characteristics

Source

DSM01

MG1655(DE3)△frdA::FRT△pta::FRT△ldhA::FRT△adhE::FRT

Baek et al. (2013)20

EMJ30

DSM01 △gltA

This work

EMJ31

DSM01, but GltA activity adjusted to 75%

This work

EMJ32

DSM01, but GltA activity adjusted to 55%

This work

EMJ33

DSM01, but GltA activity adjusted to 46%

This work

EMJ34

DSM01, but GlA activity adjusted to 34%

This work

EMJ40

DSM01/pJD758, pCDF-HCT, pACYC-AEF

This work

EMJ50

DSM01/pJD758, pCDF-HCT, pACYC-APAF

This work

EMJ51

EMJ31/pJD758, pCDF-HCT, pACYC-APAF

This work

EMJ52

EMJ32/pJD758, pCDF-HCT, pACYC-APAF

This work

EMJ53

EMJ33/pJD758, pCDF-HCT, pACYC-APAF

This work

EMJ54

EMJ34/pJD758, pCDF-HCT, pACYC-APAF

This work

EMJ55

EMJ30/pJD758, pCDF-HCT, pACYC-APAF

This work

Expression vector, CmR, p15A ori

EXPRESSYS

Strains

Plasmids pZA31MCS pZS21MCS pACYCDuet

R

Expression vector, Km , pSC101 ori

EXPRESSYS

R

Expression vector, Cm , p15A ori

pKD46

Red recombinase expression vector, Amp

pZA-Cas9

pZA31MCS, but PLtetO-1::cas9-PsacB::sacB

Novagen Datsenko and Wanner et al. (2000)32 This work

pZS-CRISPR

pZS21MCS, but TracerRNA-crRNA-PsacB::sacB

This work

R

pZS-CRISPRgltA pZS21MCS, but TracerRNA-crRNA(gltA)-PsacB::sacB

415 416 417 418 419

R

This work

pJD758

ColE1 ori, Amp , Ptet::scaffold protein

Dueber et al. (2009)27

pCDF-HCT

CDF ori, SmR, Plac::hbdGBDL-crtSH3L-terPDZL

Beak et al. (2013)20

pACYC-AEF

pACYCDuet, but Plac::atoB-adhE2-fdh1

This work

pACYC-APAF

pACYCDuet, but Plac::atoB-pduP-adhA-fdh1

This work

AmpR, ampicillin; CmR, chloramphenicol; KmR, kanamycin; SmR, spectromycin resistance, respectively.

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

420

Table 2 Oligonucleotide sequences used in this study Name

Sequence (5’-3’)

cas9 F cas9 R tracrRNA F tracrRNA R crRNA F crRNA R

aaaagtcgacATGGATAAGAAATACTCAATAGGCT aaaactgcagTCAGTCACCTCCTAGCTGAC ataaaagcttTTACGAAATCATCCTGTGGAG taatggatccTTTTGCCTCCTAAAATAAAAAGTT aattggtaccAGTATATTTTAGATGAAGATTATTTCTTA attaaagcttATCACACTACTCTTCTTTTGCCTA

gltA crRNA S

aaacAGGTTGATGTGCGAAGGCAAATTTAAGTTCg

gltA crRNA A

aaaacGAACTTAAATTTGCCTTCGCACATCAACCT

sacB F

ataagcagcatcgcctgtTACCTGCCGTTCACTATTATTTAG

sacB R

cacatagacagcctgaATCGGCATTTTCTTTTGC

gltA Rescue Uni F

ccaaataacaaacgggtaaagccaggttgatgtgcgaaggcaaatttaagttcccgcagtcttacgctgtaggttaaaag gagcat

gltA Rescue 0.6 R

tcaacagctgtgtccccgttgagggtgagttttgcttttgtatcagccatctctgatgctccttttaacctacagcg

gltA Rescue 0.3 R

421 422 423 424

tcaacagctgtgtccccgttgagggtgagttttgcttttgtatcagccatcaacgatgctccttttaacctacagcg ccaaataacaaacgggtaaagccaggttgatgtgcgaaggcaaatttaagttcccgcagtcttacgcggctggtgtaaa gltA Rescue Uni F2 ggagcat gltA Rescue 0.5 R tcaacagctgtgtccccgttgagggtgagttttgcttttgtatcagccatggccgatgctcctttacaccagccgcg gltA Rescue 0.4 R tcaacagctgtgtccccgttgagggtgagttttgcttttgtatcagccatctcagatgctcctttacaccagccgcg gltA-5’-F AAAGTTGTTACAAACATTACCAGGAA gltA-5’-R TTCACCATTCAGCAGGATGTA gltA-5’-seq TACCCAGGTTTTCCCCTCTT atoB-F tatagtcgacaaggagatataATGAAAAATTGTGTCATCGTC atoB-R tatagcggccgcTTAATTCAACCGTTCAATCA adhE2-F ggagatatacatatggcaATGAAAGTTACAAATCAAAAAGAAC adhE2-R atatctccttTTAAAATGATTTTATATAGATATCCTTAAG fdh1-F aatcattttaaaaggagatataATGAAGATCGTTTTAGTCTTATATG fdh1-R cggtttctttaccagacTTATTTCTTATCGTGTTTACCG adhA-F tataagaaggagatatacaATGAAAGCAGCAGTAGTAAGAC adhA-R gatcttcattatatctccttTTATTTAGTAAAATCAATGACCATTC pduP-F tataggatccaaggagatataATGAATACTTCTGAACTCGAAACC pduP-R tatagagctcTTAGCGAATAGAAAAGCCGTTG Underlined letters indicate sequences used to increase efficiency of homologous recombination with E. coli MG1655 genomic DNA. Overlapping sequence for annealing two oligomers are shown in italic. Sequence which is binding to DNA template are shown in capital.

425 426 427 428

ACS Paragon Plus Environment

Page 20 of 24

Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

429 430 431

Figure 1.

432 433

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

434

(a)

435 436

(b)

437 438

Figure 2.

439 440

ACS Paragon Plus Environment

Page 22 of 24

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

441 442 443

Figure 3.

444 445 446 447 448 449 450 451 452 453 454 455 456

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

457

(a)

458 459

(b)

460 461

(c)

462 463

Figure 4.

ACS Paragon Plus Environment

Page 24 of 24