Sortase A-Mediated Metabolic Enzyme Ligation in - ACS Publications

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Sortase A-Mediated Metabolic Enzyme Ligation in Escherichia coli Takuya Matsumoto, Kou Furuta, Tsutomu Tanaka, and Akihiko Kondo ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00194 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 5, 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

Sortase A-Mediated Metabolic Enzyme Ligation in

2

Escherichia coli

3

Takuya Matsumoto,† Kou Furuta,‡ Tsutomu Tanaka,‡ and Akihiko Kondo†,‡,*

4

†

5

Nada, Kobe 657-8501, Japan

6

‡

7

University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan

8

*

9

email: [email protected]

10

Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodaicho,

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe

Corresponding author;

tel:+81-78-803-6196

11

ACS Paragon Plus Environment

1


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

Page 2 of 24

12

ABSTRACT

13

We demonstrate metabolic enzyme ligation using a transpeptidase (Staphylococcal sortase A) in

14

the microbial cytoplasm for the redirection of metabolic flux through metabolic channeling.

15

Here, sortase A expression was controlled by the lac promoter to trigger metabolic channeling by

16

the addition of isopropyl-β-D-thiogalactopyranoside (IPTG). We tested covalent linking of

17

pyruvate-formate lyase and phosphate acetyltransferase by sortase A-mediated ligation and

18

evaluated the production of acetate. The time point of addition of IPTG was not critical for

19

facilitating metabolic enzyme ligation, and acetate production increased upon expression of

20

sortase A. These results show that sortase A-mediated enzyme ligation enhances an

21

acetate-producing flux in E. coli. We have validated that sortase A-mediated enzyme ligation

22

offers a metabolic channeling approach to redirect a central flux to a desired flux.

23 24

Keywords: Sortase A, Escherichia coli, Enzyme ligation, Metabolic engineering

25


2

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


26

Microbial bioproduction exploits sustainable natural resources, such as lignocellulosic biomass,

27

to produce target compounds. Escherichia coli (E. coli) is a well-studied microorganism that has

28

significant potential to convert biomass to valuable compounds, including fuels, building-block

29

chemicals and pharmaceuticals. The use of strain engineering to achieve sufficient titers, yields

30

and productivities through E. coli-based bioproduction has been an important focus for several

31

decades.1,2 Enhancing the metabolic flux from substrate to product through pathway engineering

32

is an important strategy. Some common methods include the introduction of heterologous

33

pathways, overexpression of an endogenous gene and deletion of competing pathways. These

34

methods have been successfully applied for the design of desired strains. However, the

35

traditional gene deletion technique is limited because it cannot be used for essential genes, which

36

are often involved in cell growth or maintenance. To overcome this limitation, more complex

37

and systematic methods have been reported in recent years. Several genetic tools are more useful

38

than these standard methods and have a number of advantages, such as portability, conditionality

39

and tunability.3 For example, the noncoding small RNAs-mediated tool,4,5 riboswitch6 and

40

synthetic promoter/terminator7,8 approaches have been employed for metabolic engineering. In

41

addition, biosensor-assisted methods have been proposed.9,10 Recently, dynamic metabolic

42

engineering has attracted much attention as an approach to redirect metabolic pathways from

43

endogenous pathways to targeted pathways. The chemical additive- or stress-induced1 promoter

44

is a simple tool for dynamic metabolic engineering, which can redirect fluxes by the addition of

45

an exogenous factor.11 Dynamic knockdown of a pathway based on controlled protein

46

degradation has also been reported. To dynamically alleviate the central metabolic flux, the

47

native Pfk-I coding gene was replaced with the degradation-tag modified Pfk-I. The half-life of

48

Pfk-I was controlled by inducing the expression of SspB, which is an adapter protein tethered to


3


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

Page 4 of 24

49

ClpXP.12 Using this system, E. coli-based myo-inositol production was successfully increased in

50

both titer and yield.13 Several synthetic gene circuits have also been demonstrated for the

51

redirection of an endogenous pathway into a heterologous pathway. These redirections have been

52

elaborately designed for improving bioproduction of compounds, including fatty acids,14

53

gluconate,15 isopropanol,16,17 and 3-hydroxypropionic acid.18

54

Metabolic channeling is another approach to ameliorate a strain for bioproduction.

55

Generally, small metabolites are either processed by metabolic enzymes or by substrate

56

channeling involving multi-enzyme complexes that carry out a series of reactions in a

57

step-by-step process. Substrate channeling facilitates the transfer of an intermediate between two

58

enzymes, which catalyze sequential metabolic reactions in cells. Substrate channeling prevents

59

the diffusion of an intermediate and improves the efficiency of a cascade metabolic reaction.

60

Naturally, some multi-enzyme complexes, such as carbamoyl-phosphate synthase and

61

phosphoribosylpyrophosphate amidotransferase have been suggested as enzyme complexes

62

suitable for substrate channeling.19,20 In recent years, designed metabolic channeling has been

63

reported for synthetic proteins or RNA scaffolds, or enzyme clusters that facilitate metabolite

64

processing on a metabolic pathway in vivo and in vitro.21-24 These studies indicate that placing

65

metabolic enzymes in proximity to each other will achieve substrate channeling of a metabolic

66

cascade, which enhances microbial bioproduction of desired chemicals. For instance, Lewicka

67

and coworkers demonstrated the metabolic channeling that pyruvate decarboxylase (Pdc) and

68

alcohol dehydrogenase (AdhB) were fused in E. coli. The strain expressing the fused Pdc-AdhB

69

produced more ethanol than strains co-expressing Pdc and AdhB, suggesting that processing of

70

acetaldehyde to ethanol were enhanced by metabolic channeling.25 Tippmann and coworkers

71

reported also about the metabolic channeling with affibody-based scaffold. Two metabolic


4

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


72

enzymes were assembled on anti- idiotypic affibody pair in Saccharomyces cerevisiae for the

73

production of farnesene, and three metabolic enzymes were assembled on affibody-scaffold in

74

E.coli for the production of polyhydroxybutyrate (PHB). A two- or three-component scaffold

75

improved farnesene productino or PHB production, respectively.26 Furthermore, designed

76

metabolic channeling has the added advantage of directing metabolic fluxes to desired pathways

77

without gene disruption or manipulation.

78

Here, we demonstrate the redirection of metabolic flux with metabolic channeling based on

79

protein ligation. Staphylococcal sortase A (SrtA) is used for ligation of metabolic enzymes. SrtA

80

is a transpeptidase that recognizes Leu-Pro-Xaa-Thr-Gly sequences (LP tag) and cleaves

81

between Thr and Gly, and subsequently links amino groups of oligoglycine sequences (G tag) by

82

formation of a native peptide bond. SrtA-mediated ligation enables the conjugation of a protein

83

with other molecules in a site-specific manner. Minimal modification of the protein with the

84

short LP and G tags is required for site-specific ligation. Hence, SrtA-mediated protein ligation

85

has been used to prepare a variety of bioconjugations in vitro and in vivo.27 In this study, we

86

hypothesize that SrtA-mediated metabolic enzyme ligation in the cytoplasm of E. coli facilitates

87

the processing of a metabolic intermediate, and redirects metabolic fluxes to a desired pathway.

88

As proof of concept, we have constructed an acetate producing E. coli with an engineered

89

endogenous metabolic pathway, which redirects central metabolic fluxes to an acetate producing

90

flux by metabolic enzyme ligation (Figure 1A, B and C). The expression of SrtA was controlled

91

by the lac promoter. Acetyl-CoA was chosen as the intermediate model because acetyl-CoA is

92

one of the most important central metabolic intermediates, which is converted to alcohols, fatty

93

acids and mevalonate derivatives.1,2 In this study, pyruvate-formate lyase (PFL) and phosphate

94

acetyltransferase (PTA) were successfully linked by SrtA-mediated ligation, and this linking of


5


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

Page 6 of 24

95

two enzymes increased the accumulation of acetate. Our results demonstrate that SrtA-mediated

96

metabolic enzyme ligation enables metabolic channeling to redirect a central flux to a desired

97

flux.

98

RESULTS AND DISCUSSION

99

SrtA-Mediated Metabolic Enzyme Ligation in vivo. Disruption of an endogenous gene

100

that regulates cell growth or cell maintenance dramatically decreases cell viability and growth.

101

To solve this limitation the redirection of metabolic fluxes has been reported.11-18 Similarly, the

102

goal of our concept is based on the redirection method with SrtA representing a protein stapler

103

for ligating metabolic enzymes. An additional goal of this study is metabolic flux control using

104

only metabolic enzyme ligation. Recently, scaffold-based metabolic enzyme assemblies have

105

been reported that facilitate the processing of metabolic intermediates, giving rise to metabolic

106

channeling.21,22,24 Here, we hypothesize that simply tethering of metabolic enzymes should also

107

achieve metabolic channeling. To tether metabolic enzymes in the cytosol, SrtA-mediated

108

protein ligation was employed. SrtA from S. aureus has been used as a protein stapler for linking

109

proteins.29 We also successfully demonstrated protein-protein ligation28 or protein assembly29 in

110

vitro. In this work, SrtA and SrtA-recognition sequence modified metabolic enzymes were

111

expressed under inducible- or constitutive-promoters in E. coli. Acetyl-CoA is one of the most

112

important intermediates in central metabolism, which is converted to the variety of useful

113

compounds by endogenous or exogenous pathways. Acetyl-CoA is converted to ethanol, acetate

114

or citrate through endogenous pathways in E. coli. As proof of concept, we constructed an

115

acetate-producing strain with an engineered endogenous metabolic pathway. Pyruvate is

116

converted to acetyl-CoA by pyruvate-formate lyase (PFL) under anaerobic (or microaerobic)


6

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


117

conditions, and subsequently acetyl-CoA is converted to acetate by phosphate acetyltransferase

118

and acetate kinase (PTA-ACK) (Figure 1A).

119

We constructed the plasmid for the expression of SrtA under the control of the lac

120

promoter (PLac) and the plasmid for the expression of metabolic enzymes under the constitutive

121

promoter (PHCE). The expression of SrtA was controlled by PLac, thereby the addition of IPTG

122

induced metabolic enzyme ligation. PFL encoded by the pflB gene and PTA encoded by the pta

123

gene were selected as the model of metabolic enzyme ligation. The LP tag was C-terminally

124

appended to PFL, whereas the G tag was N-terminally placed to PTA. Protein expression was

125

complemented in native pflB- and pta-deficient E. coli BW25113 (KT19) cells following

126

transformation of the pHLA-PFL-lp_g-PTA plasmid under the control of PHCE. In the absence of

127

SrtA, all of expressed PFL was not linked to PTA, as observed by the band of unconjugated

128

g-PTA (ca. 80 kDa) using SDS-PAGE and western blotting analysis. Only cells expressing SrtA

129

led to the formation of the PFL-PTA conjugate at ca. 150 kDa (Figure 2A and B). This result

130

indicates that PFL-lp and g-PTA were successfully conjugated by SrtA-mediated ligation in E.

131

coli. However, SrtA-mediated ligation between PFL-lp and g-PTA also occurred without the

132

addition of IPTG. This result suggests that the expression of SrtA was not strictly controlled by

133

the lac operator. In order to redirect metabolic flux with the addition of chemical additives, a

134

more strictly controllable system than the lac operator system for the expression of SrtA is

135

required in future work, e.g., tetracycline, arabinose or rhamnose-inducible promoters.30-32

136

The Effect on Acetate Production by SrtA-Mediated Metabolic Enzyme Ligation. To

137

further investigate the effect on metabolic enzyme ligation in E. coli, engineered strains were

138

cultivated with 5 g L−1 glucose containing LB medium under microaerobic conditions and

139

particular organic acids were analyzed by HPLC. We hypothesized that the conjugation of PFL


7


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

Page 8 of 24

140

and PTA would lead to metabolic channeling and facilitate the enhancement of a PFL-PTA flux

141

(Figure 1B). In the current study, the POX enzyme encoded by the poxB gene was deficient

142

(KT19) to minimize acetate production via another pathway (Figure 1A). We examined the

143

accumulation of acetate in three strains: a pflB-complemented strain (KT190); a pflB- and

144

pta-complemented strain (KT193) and a pflB- and pta- complemented and SrtA-expressing strain

145

(KT195). IPTG was added to KT195 cells after 6 h cultivation to induce the expression of SrtA.

146

As expected, the KT190 cells showed minimal accumulation of acetate (Figure 3B), whereas

147

both KT193 and KT195 cells were found to accumulate acetate. There were a number of

148

observable differences in the accumulation of acetate after the addition of IPTG to KT193 and

149

KT195 cells. KT193 no longer accumulated acetate after 12 h cultivation. Conversely, the

150

accumulation of acetate using KT195 cells increased after the addition of IPTG at 6 h cultivation.

151

These results suggest that the induction of the expression of SrtA at 6 h cultivation facilitated an

152

enhancement of the PFL-PTA flux. Contrastingly, KT190 and KT193 accumulated lactate during

153

the early phase (Figure 3C). With regard to pyruvate accumulation, only KT190 and KT193 cells

154

released this metabolite into the culture supernatant (Figure 3D) because neither cells was able to

155

convert pyruvate to acetate. On the other hand, KT195 cells efficiently processed pyruvate to

156

acetate through acetyl-CoA. Hence, KT190 and KT193 cells were likely to convert pyruvate into

157

lactate during the early phase; however, both cells steadily accumulated and effused pyruvate

158

because the strains could not efficiently process pyruvate and acetyl-CoA. In addition, although

159

KT195 grew significantly slower but exhibited higher glucose uptake rate than KT190 and

160

KT193 cells (Figure 3A, E). This result complements the observation that KT195 cells did not

161

accumulate pyruvate and produced more acetate than KT193 cells. We also evaluated the effect

162

on an acetate producing flux by the burden of the expression of SrtA using a pflB- and pta-


8

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


163

complemented and deactivated SrtA mutant-expressing strain (KT198). SrtA was deactivated by

164

the replacement of active cysteine in the active site of sortase A to alanine. From the metabolite

165

analysis results (Figure S2), the KT198 exhibited closely similar performance with KT193.

166

These results suggest that PFL-PTA ligation catalyzed by active sortase A was required for

167

enhancing acetate-producing flux. As shown in Figure 2, PFL-lp and g-PTA were conjugated

168

even without the addition of IPTG and the time point of IPTG addition has no effect on the

169

efficiency of PFL-lp and g-PTA ligation. The time point of addition of IPTG also had minimal

170

effect on the accumulation of organic acids (Supporting Information Figure 1). In addition,

171

western blotting analysis showed that PFL-lp and g-PTA were not completely conjugated by

172

SrtA-mediated ligation (Figure 2). Therefore, insufficient metabolic ligation might have

173

decreased the effect of metabolic channeling. Previous results have shown that the insertion of a

174

proper linker facilitated the efficiency of SrtA-mediated protein ligation.33 In addition, other

175

works have shown that the concentration of Ca2+ was insufficient for SrtA-mediated protein

176

ligation in the E. coli cytosol, and have also demonstrated that a mutation of SrtA dramatically

177

improved its catalytic activity under conditions of depleted Ca2+.34,35 Thus, more effective

178

metabolic channeling with our system may be achieved by considering these results.

179

CONCLUSIONS

180

We succeeded in metabolic channeling triggered by enzyme mediated-transpeptidation in E. coli

181

cells. An acetate-producing strain was successfully produced by the metabolic enzyme ligation.

182

IPTG-induced expression of SrtA facilitated the conjugation of PFL-lp and g-PTA in E. coli,

183

leading to an enhancement in acetate production. These results imply that SrtA-mediated

184

metabolic ligation facilitated an enhancement of the PFL-PTA flux. To harness this system,

185

microbial bioproduction of chemicals can be improved without disrupting genes.


9


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

Page 10 of 24

METHODS

187

Bacterial Strains and Growth Conditions. The bacterial strains used in this study are

188

listed in Table 1. E. coli NovaBlue cells (Novagen Inc., Madison, WI, USA) were used for DNA

189

manipulations. E. coli BW25113 cells were used as the base strain for acetate production. Cells

190

were pre-cultivated with 100 µg mL−1 ampicillin and 20 µg mL−1 kanamycin containing 5 mL

191

Luria-Bertani (LB) medium in a test tube overnight. Cells were grown in 10 mL 0.5% glucose

192

containing LB medium at 37 °C, 190 rpm in 15 mL sealed test tubes (initial optical density at

193

600 nm = 0.1). SrtA expression was induced by the addition of IPTG (final conc. 0.5 mM).

194

Plasmid Construction and Gene Disruption. All of the plasmids and primers used in this

195

study are listed in Table 1 and Supplementary Table 1, respectively. The polymerase chain

196

reaction (PCR) was performed with KOD plus polymerase (TOYOBO CO., LTD., Osaka, Japan).

197

Vectors and inserts were ligated with the In-Fusion® HD Cloning Kit following the

198

manufacturer’s protocol (TAKARA BIO INC., Shiga, Japan). The pHLA vector36 was employed

199

as the base vector for the expression of tagged metabolic enzymes. Each amplified fragment was

200

inserted into the BglII/XhoI restriction sites of the pHLA vector. The resulting plasmids were

201

pHLA-pflBlp and pHLA-pflBlp-gpta, and named pHKT1 and pHKT2, respectively. To confirm

202

the expression of PFL and PTA, the FLAG peptide and cmyc peptide were genetically

203

introduced at the C-terminus, respectively. To construct vectors expressing SrtA, the

204

pZA23MCS (Expressys, Bammental, Germany) was employed as the backbone plasmid. To

205

suppress the expression of genes under the lac promoter, the lacI gene was inserted under the

206

constitutive promoter of pZA23MCS. The resulting plasmid was named pZA23LMCS. The gene

207

of sortase A from Staphylococcus aureus was amplified using pET30b-SrtA37 as the template.


10

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


208

The amplified sortase A gene was inserted into pZA23MCSL, and the resulting plasmid was

209

named pZA23LSrtA. In addition, pZA23LSrtAc184a was constructed by quick-change PCR. 38

210

E. coli BW25113 genes, pflB, pta and poxB were deleted, as described in a previous

211

report.39 Confirmation of gene deletions was carried out by colony PCR. The resulting strain was

212

BW25113 ∆pflB, ∆pta and ∆poxB, and was named KT19. pHKT1 and pZA23LMCS, pHKT2

213

and pZA23LMCS, pHKT2 and pZALSrtA, pHKT2 and pZA23LSrtAc184a, were transformed

214

into KT19 cells, which were named KT190, KT193, KT195 and KT198, respectively.

215

SDS-PAGE Analysis and Western Blotting Analysis. To evaluate the ligation between

216

PFL and PTA using SrtA in vivo, extracts of cells were analyzed by SDS-PAGE or western

217

blotting analysis. After cultivation for 24 h at 37 °C, samples were centrifuged and the

218

supernatant removed. Cells were gently mixed with B-PER™ Bacterial Protein Extraction

219

Reagent (Thermo Fisher Scientific Inc., Kanagawa, Japan) at RT for 30 min. The reaction

220

mixture was then mixed with the SDS-PAGE sample buffer (50 mM Tris-HCl, 2% SDS, 6%

221

2-mercaptoethanol) followed by boiling. The samples were then subjected to SDS-PAGE and the

222

gels were stained with Coomassie brilliant blue R-250, or analyzed by western blotting with

223

rabbit anti-c-myc (Bethyl Laboratories, Inc., Montgomery, TX) and anti-rabbit IgG (Fc) AP

224

conjugate (Promega KK, Tokyo, Japan) and BCIP/NBT, according to the manufacturer’s

225

procedure.

226

Metabolite Analysis. The concentrations of acetic acid, pyruvate and lactic acid were

227

determined by high-performance liquid chromatography (Shimadzu Co., Kyoto, Japan; solvent

228

delivery system, LC-10ADvp; column, Shim-pack SPR-H; column temperature, 50 °C; detector,

229

CDD-10A). A 5 mM concentration of p-toluenesulfonic acid was used as the mobile phase, and


11


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

Page 12 of 24

230

20 mM bis-Tris containing 5 mM p-toluenesulfonic acid and 100 µM EDTA was mixed just

231

before detection to enhance sensitivity. Chromatography was carried out at 50 °C at a flow rate

232

of 1.4 mL/min.

233 234

ASSOCIATED CONTENT

235

Supporting Information

236

The Supporting Information is available free of charge on the ACS Publications website

237

・Supporting figure, primer list

238

AUTHOR INFORMATION

239

Corresponding Author

240

Akihiko Kondo

241

[email protected]

242

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe

243

University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan

244 245

Notes

246

The authors declare no competing financial interest.

247

ACKNOWLEDGMENTS


12

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


248

This work was supported in part by a Grant-in-Aid for Young Scientists B (15K18276) from the

249

Japan Society for the Promotion of Science (JSPS), and by Special Coordination Funds for

250

Promoting Science and Technology, Creation of Innovation Centers for Advanced

251

Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan

252


13


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

Page 14 of 24

253

REFERENCES

254

1. Becker, J., Wittmann, C. (2015) Advanced Biotechnoology: Metabolically Engineered Cells

255

for the Bio-Based Production of Chemicals and Fuels, Materials, and Health-Care Products.

256

Angew. Chem. Int. Ed. Engl. 54, 3328−3350.

257

2.

258

(2015) Bio-based production of monomers and polymers by metabolically engineered

259

microorganisms. Curr. Opin. Biotechnol. 36, 73−84.

260

3. McNerney, M.-P., Watstein, D.-M., Styczynski, M.-P. (2015) Precision metabolic

261

engineering: The design of responsive, selective, and controllable metabolic systems. Metab.

262

Eng. 31, 123−131.

263

4. Na, D., Yoo, S.-M., Chung, H., Park, H., Park, J.-H., Lee, S.-Y. (2013) Metabolic engineering

264

of Escherichia coli using synthetic small regulatory RNAs. Nat. Biotechnol. 31, 170−174.

265

5. Chae, T.-U., Kim, W.-J., Choi, S., Park, S.-J., Lee, S.-Y. (2015) Metabolic engineering of

266

Escherichia coli for the production of 1,3-diaminopropane, a three carbon diamine. Sci. Rep. 5,

267

13040.

268

6. Zhou, L.-B., Zeng, A.-P. (2015) Exploring lysine riboswitch for metabolic flux control and

269

improvement of L-lysine synthesis in Corynebacterium glutamicum. ACS Synth. Biol. 4, 729−

270

734.

271

7. Song, C.-W., Lee, J., Ko, Y.-S., Lee, S.-Y. (2015) Metabolic engineering of Escherichia coli

272

for the production of 3-aminopropionic acid. Metab. Eng. 30, 121−129.

Chung, H., Yang, J.-E., Ha, J.-Y., Chae, T.-U., Shin, J.-H., Gustavsson, M., Lee, S.-Y.


14

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


273

8. Tong, Y.-J., Ji, X.-J., Shen, M.-Q., Liu, L.-G., Nie, Z.-K., Huang, H. (2016) Constructing a

274

synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol

275

production. Appl. Microbiol. Biotechnol. 100, 637−647.

276

9. Li, S., Si, T., Wang, M., Zhao, H. (2015) Development of a Synthetic Malonyl-CoA Sensor in

277

Saccharomyces cerevisiae for Intracellular Metabolite Monitoring and Genetic Screening. ACS

278

Synth. Biol. 4, 1308−1315.

279

10. Fang, M., Wang, T., Zhang, C., Bai, J., Zheng, X., Zhao, X., Lou, C., Xing, X.-H. (2016)

280

Intermediate-sensor

281

deoxyviolacein production in Escherichia coli. Metab. Eng. 33, 41−51.

282

11. Kang, Z., Wang, Q., Zhang, H., Qi, Q. (2008) Construction of a stress-induced system in

283

Escherichia coli for efficient polyhydroxyalkanoates production. Appl. Microbiol. Biotechnol.

284

79, 203−208.

285

12. McGinness, K.-E., Baker, T.-A., Sauer, R.-T. (2006) Engineering controllable protein

286

degradation. Mol. Cell. 22, 701−707.

287

13. Brockman, I.-M., Prather, K.-L. (2015) Dynamic knockdown of E. coli central metabolism

288

for redirecting fluxes of primary metabolites. Metab. Eng. 28, 104−113.

289

14. Torella, J.-P., Ford, T.-J., Kim, S.-N., Chen, A.-M., Way, J.-C., Silver, P.-A. (2013) Tailored

290

fatty acid synthesis via dynamic control of fatty acid elongation. Proc. Natl. Acad. Sci. U S A.

291

110, 11290−11295.

assisted

push-pull

strategy

and

its

application

in

heterologous


15


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

Page 16 of 24

292

15. Solomon, K.-V., Sanders, T.-M., Prather, K.-L. (2012) A dynamic metabolite valve for the

293

control of central carbon metabolism. Metab. Eng. 14, 661−671.

294

16. Soma, Y., Tsuruno, K., Wada, M., Yokota, A., Hanai, T. (2014) Metabolic flux redirection

295

from a central metabolic pathway toward a synthetic pathway using a metabolic toggle switch.

296

Metab. Eng. 23, 175−184.

297

17. Soma, Y., Hanai, T. (2015) Self-induced metabolic state switching by a tunable cell density

298

sensor for microbial isopropanol production. Metab. Eng. 30, 7−15.

299

18. Tsuruno, K., Honjo, H., Hanai, T. (2015) Enhancement of 3-hydroxypropionic acid

300

production from glycerol by using a metabolic toggle switch. Microb. Cell. Fact. 14, 155.

301

19. Miles, E.-W.,

302

J. Biol. Chem. 274, 12193−12196.

303

20. Huang, X., Holdem, H.-M., Raushel, F.-M. (2001) Channeling of substrates and

304

intermediates in enzyme-catalyzed reactions. Annu. Rev. Biochem. 70, 149−180.

305

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

306

Prather, K.-L., Keasling, J.-D. (2009) Synthetic protein scaffolds provide modular control over

307

metabolic flux. Nat. Biotechnol. 27, 753−759.

308

22. Sachdeva, G., Garg, A., Godding, D., Way, J.-C., Silver, P.-A. (2014) In vivo co-localization

309

of enzymes on RNA scaffolds increases metabolic production in a geometrically dependent

310

manner. Nucleic. Acids Res. 42, 9493−9503.

Rhee, S., Davies, D.-R. (1998) The molecular basis of substrate channeling.


16

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


311

23. Castellana, M., Wilson, M.-Z., Xu, Y., Joshi, P., Cristea, I.-M., Rabinowitz, J.-D., Gitai, Z.,

312

Wingreen, N.-S. (2014) Enzyme clustering accelerates processing of intermediates through

313

metabolic channeling. Nat. Biotechnol. 32, 1011−1018.

314

24. Zhang, Y.-H. (2011) Substrate channeling and enzyme complexes for biotechnological

315

applications. Biotechnol. Adv. 29, 715−725.

316

25. Lewicka, A.-J., Lyczakowski, J.-J., Blackhurst, G., Pashkuleva, C., Rothschild-Mancinelli,

317

K., Tautvaišas, D., Thornton, H., Villanueva, H., Xiao, W., Slikas, J., Horsfall, L., Elfick, A.,

318

French C. (2014) Fusion of pyruvate decarboxylase and alcohol dehydrogenase increases ethanol

319

production in Escherichia coli. ACS Synth. Biol. 3, 976-978.

320

26. Tippmann, S., Anfelt, J., David, F., Rand, J.-M., Siewers, V., Uhlén, M., Nielsen, J., Hudson,

321

E.-P. (2016) Affibody Scaffolds Improve Sesquiterpene Production in Saccharomyces cerevisiae.

322

ACS Synth. Biol. in press. DOI: 10.1021/acssynbio.6b00109

323

27. Schmohl, L., Schwarzer, D. (2014) Sortase-mediated ligations for the site-specific

324

modification of proteins. Curr. Opin. Chem. Biol. 22, 122−128.

325

28. Matsumoto, T., Sawamoto, S., Sakamoto, T., Tanaka, T., Fukuda, H., Kondo, A. (2011)

326

Site-specific tetrameric streptavidin-protein conjugation using sortase A. J. Biotechnol. 152, 37−

327

42.

328

29. Matsumoto, T., Isogawa, Y., Minamihata, K., Tanaka, T., Kondo, A. (2016) Twigged

329

streptavidin polymer as a scaffold for protein assembly. J. Biotechnol. 225, 61−66.


17


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

Page 18 of 24

330

30. Skerra, A. (1994) Use of the tetracycline promoter for the tightly regulated production of a

331

murine antibody fragment in Escherichia coli. Gene. 151, 131−135.

332

31. Guzman, L.-M., Belin, D., Carson, M.-J., Beckwith, J. (1995) Tight regulation, modulation,

333

and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177,

334

4121−4130.

335

32. Egan, S.-M., Schleif, R.-F. (1993) A regulatory cascade in the induction of rhaBAD. J. Mol.

336

Biol. 234, 87−98.

337

33. Yamamura, Y., Hirakawa, H., Yamaguchi, S., Nagamune, T. (2011) Enhancement of sortase

338

A-mediated protein ligation by inducing a β-hairpin structure around the ligation site. Chem.

339

Commun. (Camb). 47, 4742−4744.

340

34. Hirakawa, H., Ishikawa, S., Nagamune, T. (2012) Design of Ca2+-independent

341

Staphylococcus aureus sortase A mutants. Biotechnol. Bioeng. 109, 2955−2961.

342

35. Glasgow, J.-E., Salit, M.-L., Cochran, J.-R. (2016) In Vivo Site-Specific Protein Tagging

343

with Diverse Amines Using an Engineered Sortase Variant. J. Am. Chem. Soc. 138, 7496−7499.

344

36.

345

cellooligosaccharide-assimilating Escherichia coli strain by displaying active beta-glucosidase on

346

the cell surface via a novel anchor protein. Appl. Environ. Microbiol. 77, 6265−6270.

347

37. Tanaka, T., Yamamoto, T., Tsukiji, S., Nagamune, T. (2008) Site-specific protein

348

modification on living cells catalyzed by Sortase. Chembiochem 9, 802−807.

Tanaka,

T.,

Kawabata,

H.,

Ogino,

C.,

Kondo,

A.

(2011)

Creation

of

a


18

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


349

38. Matsumoto, T., Takase, R., Tanaka, T., Fukuda, H., Kondo, A. (2012) Site-specific protein

350

labeling with amine-containing molecules using Lactobacillus plantarum sortase. Biotechnol. J.

351

7, 642-648.

352

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

353

Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U S A. 97, 6640−6645.

354


19


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

Page 20 of 24

355

Figure Captions

356

Figure 1. An integrated shceme of sortase A-mediated metabolic enzyme ligation in E. coli. (A)

357

Engineered E. coli metabolism of acetate producing flux used in this study. (B) Schematic

358

illustration of metabolic enzyme ligation between PFL and PTA by SrtA as a stapler for enzyme

359

conjugation. (C) The strategy for PFL-PTA ligation catalyzed by SrtA. The expression of SrtA,

360

PFL and PTA were controlled under Lac promoter and constitutive HCE promoter, respectively.

361

PFL-PTA ligation was triggered by the expression of SrtA.

362

Figure 2. SDS-PAGE analysis (A) or Western blotting analysis (B) of metabolic enzyme ligation

363

in E. coli extracts after 24 h cultivation. Lane 1, KT193; Lane 2, KT195 without the addition of

364

IPTG; Lanes 3−6, KT195 with the addition of IPTG at 0, 3, 6, 9 h cultivation.

365

Figure 3. The cell growth (optical density at 600 nm) (A), the production of acetate (B), lactate

366

(C), pyruvate (D) and the consumption of glucose (E). The cells were inoculated at initial OD600

367

= 0.1 and IPTG was added after 6 h cultivation. Symbols represent: KT190: black; KT193: blue;

368

and KT195: red. Data are presented as the average of three independent experiments, and error

369

bars represent the standard deviation.

370


20

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

371


Table 1. Strains and plasmids used in this study

Strain

Characteristics

Source

Nova blue

endA1 hsdR17(rK12− mK12+) supE44 thi-1 recA1 gyrA96 relA1 lac[F′ proAB+ lacIqZΔ M15::Tn10 (Tetr)]; host for DNA manipulation

Novagen

BW25113

Δ(araD-araB)567 ΔlacZ4787(::rrnB-3) λ− rph-1 Δ(rhaD-rhaB)568 hsdR514

National Institute of Genetics, Japan

KT19

As BW25113, but ΔpflB, ΔpoxB, Δpta

This study

KT190

KT19 harboring pHKT1 and pZA23LMCS

This study

KT193

KT19 harboring pHKT2 and pZA23LMCS

This study

KT195

KT19 harboring pHKT2 and pZA23LSrtA

This study

KT198

KT19 harboring pHKT2 and pZA23LSrtAc184a

This study

Plasmid

Characteristics

Source

pHLA

ColE1 ori; AmpR; PHCE::pgsA

36

pHKT1

ColE1 ori; AmpR; PHCE::pflBlp

This study

pHKT2

ColE1 ori; AmpR; PHCE::pflBlp-gpta

This study

pZA23MCS

p15A ori; KanR; PA1lacO-1::MCS1

Expressys, Bammental, Germany

pZA23LMCS

p15A ori; KanR; Placiq-::lacI; PA1lacO-1::MCS1

This study

pZA23LSrtA

p15A ori; KanR; Placiq-::lacI; PA1lacO-1::SrtA

This study

pZA23LSrtAc184a

p15A ori; KanR; Placiq-::lacI; PA1lacO-1::SrtAc184a

This study


21


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

372

Figures

373

Figure 1

Page 22 of 24

374 375 376

Figure 2

377 378


22

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

379


Figure 3

380 381 382 383


23


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

384

Page 24 of 24

TOC

385


24

Sortase A-Mediated Metabolic Enzyme Ligation in - ACS Publications

Recommend Documents