A Glucose-Sensing Toggle Switch for Autonomous, High Productivity

Subscriber access provided by University of Newcastle, Australia

Article

A glucose-sensing toggle switch for autonomous, high productivity genetic control William Henry Bothfeld, Grace Kapov, and Keith Tyo ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00257 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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 31

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

2 3 4 5 6 7 8

A glucose-sensing toggle switch for autonomous, high productivity genetic control

William Bothfeld, Grace Kapov, Keith E.J. Tyo*

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Department of Chemical and Biological Engineering E-136, Northwestern University, 2145 Sheridan road, Evanston, IL, USA

Manuscript in preparation for: ACS SynBio

*Corresponding author: Telephone: +1 847 868 0319 Fax: +1 847 491 3728 email: [email protected]

1

ACS Paragon Plus Environment


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

28

Page 2 of 31

ABSTRACT

29

Many biosynthetic strategies are coupled to growth, which is inherently limited, as (1)

30

excess feedstock (e.g., sugar) may be converted to biomass, not product, (2) essential genes must be

31

maintained and (3) growth toxicity must be managed. A decoupled growth and production phase

32

strategy could avoid these issues. We have developed a toggle switch that uses glucose sensing to

33

enable this two-phase strategy. Temporary glucose starvation precisely and autonomously activates

34

product expression in rich or minimal media, obviating the requirement for expensive inducers. The

35

switch remains stably in the new state even after reintroduction of glucose. In the context of

36

polyhydroxybutyrate (PHB) biosynthesis, our system enables shorter growth phases and comparable

37

titers to a constitutively expressing PHB strain. This two-phase production strategy, and specifically

38

the glucose toggle switch, should be broadly useful to initiate many types of genetic program for

39

metabolic engineering applications.

40

41 42

KEY WORDS [Glucose sensing, genetic toggle switch, PHB production]

43 44

2


Page 3 of 31

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

45


INTRODUCTION

46

Great progress has been made in expanding the portfolio of chemicals that can be produced

47

biologically1. However, efficient, economical bio-production of these chemicals remains a challenging

48

barrier to shifting from a fossil-fuel economy to a renewable one. Currently, many bio-production

49

strategies rely on growth-coupled production, because constitutive expression of the product

50

pathway is more feasible than inducing production after growth.

51

production limits product yield and productivity. Biomass can consume 20-60% of the carbon

52

source2 across different cultivation techniques. This biomass consumption could be minimized if an

53

extended production phase was maintained after cells reached a target biomass level. Furthermore,

54

engineered pathways can cause cell-toxicity slowing growth, and limiting yields, titers and types of

55

chemicals considered for production3. Because strains containing these toxic pathways have

56

suboptimal growth, escape mutants with mutations that reduce productivity have a growth

57

advantage, leading to overall lower batch yields4 (figure 1a).

However, growth-coupled

58

Decoupling growth and production phases can address these limitations by preventing

59

selection on burdensome pathways and enhancing growth rates in the growth phase, while allowing

60

pathway expression levels in the non-growing, production phase that would otherwise inhibit

61

growth. Essential enzymes can be eliminated in the production phase, greatly expanding the possible

62

engineering targets. Chemicals toxic to certain growth-associated processes of the host could be

63

produced using this two-phase strategy. A separate production phase could also alleviate limits of

64

oxygen and nutrient uptake as biomass accumulates in growth-coupled platforms. Batch and fed-

65

batch cultivations are discontinuous processes that involve repeated reactor prep and growth,

66

followed by reactor breakdown and cleaning. A strategy that maintains an extended production

67

phase could lessen reactor down-time by enabling longer, robust production within reasonable

68

biomass and process parameters.

69

A useful tool for decoupling growth and production would, upon transition to production

70

phase, precisely activate a designed genetic program to express pathway enzymes and activate or 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 31

71

inactivate genomic targets. On an industrial scale, expensive inducers such as Isopropyl β-D-1-

72

thiogalactopyranoside (IPTG) and anhydrotetracycline (aTc) are infeasible. Instead, pathways are

73

either (1) constitutive or (2) initiated by nutrient-limitation sensing. Constitutive expression of a

74

product pathway causes metabolic burden, slowing growth. This is demonstrated in previous PHB

75

studies4. Nutrient-limitation eliminates the slow growth by not expressing the product pathway until

76

late in the culture. However, nutrient limitations may require the culture to be maintained in

77

suboptimal conditions. For example, nutrient limitation strategies lower glucose uptake rate (GUR)

78

by ~ 16, 10, 6, and 2 fold for nitrogen, phosphorus, sulfur and magnesium limitation, respectively5.

79

Therefore, production strategies dependent on induction in these limiting regimes will limit carbon

80

flux to desired enzyme pathways.

81

rapidly switched to an ‘on’ state with complex regulation present6,7.

Finally induction in these systems is often graded, rather than

82

Unlike simple induction, a genetic toggle switch could enable complete activation, to a new

83

stable state, that would eliminate graded induction. The culture could stay in the production state

84

even after the activating condition is gone due to the memory capacity encoded by the switch. The

85

“new” switch state could be maintained stably during the entire production phase. A functional

86

genetic toggle switch has been demonstrated8 and improved by Collins9, using inducers that are not

87

appropriate for industrial scale (IPTG, aTc, arabinose or heat shock). A metabolic switch requiring

88

IPTG to induce has been demonstrated using this toggle-switch framework for isopropanol

89

production with success10. In production phase, expression of an essential gene (citrate synthase)

90

was limited and pathway enzymes were induced to increase titer and yield 3.7 and 3.1 fold to

91

~50mM isopropanol10. The same group has subsequently developed an auto-inducible metabolic

92

switch using quorum sensing eliminating the need for IPTG in fermentations at industrial scales11.

93

This approach is autonomous, but is not flexible. Significant effort would be required to tune the

94

auto-inducer system to engage at different cell densities. Furthermore, the inducer was never fully

95

removed from the system.

96

Glucose levels may be a particularly useful inducer for such a toggle switch. Specifically,

97

glucose starvation (1) causes robust gene activation12 (2) has a mode of action that is well studied13 4


Page 5 of 31

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

(3) is compatible with common glucose fed-batch systems14 and (4) occurs after a precise amount of

99

biomass has been made in a wide range of media types (simple and complex). The glucose-starvation

100

promoter would only toggle a switch event, so glucose could be subsequently reintroduced for

101

production. This would be an improvement on current strategies that use native nutrient-sensing for

102

induction but also drastically reduce metabolic rates.

103

The basic architecture of the toggle switch includes regulatory genes and their

104

corresponding target promoters, arranged to be mutually inhibitory (figure 1b). The version used in

105

this work contains a LacI responsive promoter-(trcp) that expresses TetR and a TetR responsive

106

promoter –(tetOp) promoter that expresses LacI; each repressor transcription factor (TF) is under

107

control of the opposite promoter9. Reporter proteins mCherry and GFP signal which state the circuit

108

is in. Each reporter has a degradation tag to allow observation of dynamic switching events upon

109

addition of the chemical inducers IPTG or aTc through fluorescence. For the purposes of the specific

110

way we cloned our pathway expression, we refer to LacI/GFP expression as ON and TetR/mCherry

111

expression as OFF.

112

Functionally, the encoded logic of native transcriptional regulation, such as glucose

113

starvation, can be integrated into the toggle switch. Glucose starvation activates the catabolic

114

response using cyclic-AMP (cAMP) receptor protein (CRP), one of the best-studied TF’s. Low glucose

115

environments cause the cAMP-CRP complex to bind target DNA and regulate hundreds of gene

116

targets15. There are 3 classes of CRP promoters, categorized by the number and placement of CRP

117

binding sites within each. Class I promoters have one CRP binding site at various distances upstream

118

of the -35 box which interact with the α-C Terminal Domain (α-CTD) of RNA Polymerase (RNAP)16.

119

Class II promoters have CRP encompassing the -35 box, and interact with α-CTD and α-NTD of RNAP

120

for promoter recruiting17.

121

multiple interactions with cAMP-CRP are made. Class III promoters contain a mix of multiple Class I

122

and II CRP sites16.

This site partially occludes the σ70 subunit of RNAP, and is likely why

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 31

123

These CRP/glucose-sensitive promoters could be used to switch states in the synthetic

124

genetic toggle switch. The switch occurs by using the transient glucose-starvation signal to express

125

one of the toggle TF’s (LacI), resulting in a stable output from the genetic switch. The starvation

126

event is encoded in the “memory” of the switch and is stable during the desired production phase,

127

even after reintroduction of glucose. Importantly, this separates the sensing promoter from the

128

production promoter, which isn’t possible using only nutrient-limitation induction of product

129

pathways. A separate sensor and production promoter increases flexibility to design different

130

expression levels of pathway enzymes without being constrained by the native capacity and kinetics

131

of a nutrient sensing induction strategy.

132

In this study, we have developed an industrially relevant auto-inducible genetic switch that

133

responds to glucose availability to precisely time the expression of burdensome pathway enzymes

134

for enhanced bio-production. We characterized the dynamics of a variety of glucose sensitive

135

promoters. Select promoters were then integrated into a bi-stable toggle switch to utilize the cell’s

136

native capacity to sense and respond to glucose starvation to activate a switching event. The

137

resulting state was stable upon re-introduction of glucose (see figure 1c for genetic program). This

138

glucose-sensitive switch was then used to autonomously express PHB pathway enzymes at levels

139

that would severely limit growth, but when coordinated with initial glucose concentrations designed

140

to induce PHB expression at a particular biomass level, improved growth by two-fold with

141

comparable PHB production yields to a constitutively expressing system.

142

143

MATERIALS AND METHODS

144

STRAINS AND MEDIA

145

Cloning was carried out in DH5α Escherichia coli and all experiments were conducted in

146

wild-type MG1655 or MG1655 ΔlacI strains as indicated. pSB3C5 from the 2012 iGEM kit was used

6


Page 7 of 31

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


147

as a source for the p15A origin and chloramphenicol acetyl transferase (CAT). pKDL071 (a gift from

148

the Collins Lab) was the source for gfpmut3b and lacI used in cloning and was the basis for the

149

genetic toggle switch circuitry9. CRP promoters were from MG1655 gDNA and pAGL20 was the

150

source of the phaECAB (PHB) operon18.

151

Luria-Bertani Broth (LB)(Difco, New Jersey, USA) was used for cultivation during cloning.

152

MOPS minimal (MOPS min) media was prepared according to Neihardt’s recipe19. Supplements for

153

EZ rich defined MOPS media were purchased from Teknova (California, USA). Working

154

concentrations of antibiotics and inducers used are as follows: kanamycin (25 mg/mL) and

155

chloramphenicol (17 mg/L) from Sigma Aldrich, (St Louis, MO); IPTG (1 mM) and aTc (100 μg/L)

156

from Promega (Madison, WI). All cloning enzymes were purchased from New England Biolabs

157

(Medford, MA).

158

CLONING

159

The pWB8 base plasmid was created to facilitate swapping in select CRP promoters

160

upstream of GFP to assay expression dynamics in response to glucose starvation. PCR products of

161

the p15A origin, CAT, RBS:gfpmut3b and a variation of the synthetic promoter CC(-41.5)α(-63)20

162

were generated using primers and source DNA as indicated in Table 1 and combined using Gibson

163

assembly21. Primers for Gibson assembly were designed using J5 online software22.

164

pWB8-crp variants were created using restriction-ligation cloning to insert CRP promoters

165

amplified by PCR from MG1655 gDNA, using primers indicated in Table 1. Three versions of each

166

promoter were generated: (1) “full length” promoters (crp) starting ~ 500 bp upstream of the open

167

reading frame and ending at the transcription start site (2) minimal “truncated” promoters (T-crp)

168

containing ~ 50 bp upstream of the transcription start site with care taken to include the full CRP

169

binding site, and (3) enhanced truncated promoters (T-αcrp) which include a modified enhancer

170

element from the CC(-41.5)α(-63) synthetic elements20 (see supplementary Table 1 for sequence)

171

upstream of the T-crp promoters. pWB8-crp and T-crp variants were cloned using HindIII-BamHI

172

digestion of the appropriate PCR products and pWB8 backbone, while pWB8Tα-crp variants were 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 31

173

generated using EcoRI HindIII digests of the T-crp PCR product, and ligated into the pWB8 backbone

174

cut with the same restriction enzymes.

175

Two strategies were pursued to integrate glucose sensing into the toggle switch. First,

176

pWB9-crp variants were created to express LacI from crpp promoters on a separate plasmid to

177

enable glucose-limitation switching of the toggle switch. The RBS:lacI from pKDL071 was amplified

178

by PCR and cloned into select pWB8 variants using HindII-PstI digestion. The resulting pWB9-crp

179

variants were co-transformed with plasmid pKDL071 to create the “two plasmid” system. Secondly,

180

pWB9 variants were digested with BamHI-SphI and this crpp:RBS:lacI fragment was ligated in place

181

of the original RBS:lacI in the toggle switch. This resulted in the tetOp and crpp promoters in series,

182

upstream of lacI, to create the “tandem promoter” test system (pKDL071-crp).

183

A heterologous pathway under control of the toggle switch was created by inserting the

184

phaECAB operon into pKDL071 in place of gfpmut3b. phaECAB was amplified by PCR with SphI-PstI

185

overhangs and ligated into the pKDL071 backbone to create pKDL071-phaECAB. The resulting

186

plasmid was co-transformed with pWB9 variants to create the two plasmid production system.

187

Select pKDL071-crp variants were digested using BamHI-SphI and the crpp:lacI fragment was ligated

188

into the pKDL071-phaECAB backbone downstream of tetOp, in place of the existing lacI to create the

189

tetOp:crpp:lacI control element in the pKDL071-phaECAB production plasmid, resulting in the

190

tandem promoter production system.

191

CULTURE CONDITIONS

192

Prior to inoculation of all experiments, all strains were streaked out on LB + antibiotic plates

193

and single colonies were inoculated into an initial pre-culture of 5 mL of media (type is noted for

194

each experiment) in 15 mL culture tubes, and incubated using a New Brunswick Scientific Innova44

195

incubator (New Jersey, USA) maintained at 37°C and 250 rpm in a rack angled at 45°. The pre-

196

culture was incubated for 12-14 hours and then diluted into a second pre-culture at 1/100 dilution

197

into fresh media to return cells to exponential growth. The second pre-culture was then inoculated

198

into the experimental culture (LB, LB + glucose, MOPS min + glucose, MOPS min + glucose and 8


Page 9 of 31

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


199

glycerol, MOPS EZ rich + glucose, MOPS EZ rich + glycerol or MOPS EZ rich + gluconate) as indicated.

200

Additional culturing steps, as necessary, are described for each experiment.

201

CRP-PROMOTER CHARACTERIZATION

202

Promoter characterization was conducted in LB media with 0.0 to 0.4% glucose (w/v) as a

203

carbon source, and cells were grown in a 96-well plate-reader. Plates with lids were maintained at

204

37° C with 300 rpm agitation in an incubator, then manually transferred to the plate reader for

205

OD600 and fluorescent measurements. Growth and fluorescence were measured every 30 minutes

206

for up to 10 hours.

207

TOGGLE SWITCH STABILITY TRIALS

208

The stability of the OFF state of glucose toggle switch variants (two promoter and tandem

209

promoter) was tested (supplemental figure 1, figure 3). Stable candidates were then tested for OFF

210

and ON state stability (figure 3). First, colonies were inoculated in 3 mL of MOPS EZ rich + 0.2 %

211

glucose with inducer (1mM IPTG for experimental strains and the OFF control, aTc for the ON

212

control). These pre-cultures were incubated at 4° C, then warmed to 37° C and shaken at 250 rpm for

213

6 hours to set the OFF state of the genetic switch and generate biomass. Pre-cultures were diluted

214

1/100 into fresh media with 0.1 mM IPTG for ~ 2 hours (OFF and ON controls were cultured inducer-

215

free from this step on), and these secondary pre-cultures were diluted 1/10 fresh media for another

216

2 hours (tertiary pre-cultures) to maintain a glucose-rich environment. Finally, the tertiary pre-

217

culture was diluted 1/100 in fresh media lacking inducer, and cells were considered inducer free

218

(0.0001 mM IPTG shows no response in a dose-response curve8). This “set” the toggle-state to OFF in

219

exponentially growing cultures. Culture was repeatedly transferred into fresh media for 6 hours to

220

maintain exponential growth in a glucose rich environment in the absence of inducers. Cultures

221

were then spun down at 4000 x g for 5 minutes, resuspended, and grown in LB for 1 hour to test

222

glucose-limitation induced toggling of the switch. Culture was then diluted 1/100 in the original

223

0.2% MOPS EZ rich media for 10 hours (with re-dilutions) to monitor the transition from the OFF to

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

Page 10 of 31

224

the ON state. Finally, culture was diluted 1/100 into MOPS EZ rich + 0.2 % glucose + 1mM IPTG to

225

monitor the switch back to the OFF state (figure 3).

226

Other media conditions that initiate a switch from the ON to the OFF state were tested for

227

the TaraF tandem promoter glucose toggle variant. Colonies were inoculated in a 96-well plate with

228

MOPS EZ rich + 0.1% glucose and 0.1 mM IPTG (aTc only for the ON control) for 3 hours. Culture was

229

re-diluted into media without inducers for an additional 4 hours. Cultures were then diluted 1/100 in

230

MOPS EZ rich with no extra carbon, 0.1% gluconate, or 0.1% glycerol, grown for 1 hour, then 1 µL of

231

40% glucose was added. Cells were then grown for 6 hours in a plate reader to monitor the transition

232

from the OFF to the ON state (figure 4).

233

PHB PRODUCTION

234

PHB production trials were conducted in 50 mL MOPS min with 0.2% or 0.4% glucose in 250

235

mL baffled shake-flasks with foam tops for oxygen transfer, and were pre-cultured as described

236

previously. 1-2 mL of 400 g/L glucose stock was added to the remaining culture volume at the onset

237

of stationary phase (8-10 hours after inoculation of experimental culture, ~42 mL of culture

238

remained due to volume lost from sampling and evaporation). Glycerol was added at 0.1% (v/v) to

239

the starting media before inoculation, in a select few experiments to optimize PHB production, with

240

either 0.05 or 0.1% starting glucose, as noted.

241

Chemically induced PHB production experiments were inoculated as described with

242

additional steps: 1 mM IPTG was added to the pre-culture to set the OFF state. The secondary pre-

243

culture was grown for ~ 2 hours to return cells to exponential phase, then spun down at 4000 x g for

244

5 minutes and re-suspended in inducer-free media, and inoculated into 50 mL of media at OD600 =

245

0.01 to start each experimental culture. Varied PHB-pathway induction times were investigated:

246

induction with aTc upon inoculation (0 h) and at the end of exponential phase (10 h). Un-induced

247

controls (always off) and pre-culture induced controls (-14 h, no IPTG in pre-culture, induced with

248

aTc) were included for comparison. A genomically integrated, 29-operon copy strain (KS29)23 was

10


Page 11 of 31

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


249

tested to bench-mark a constitutively expressing strain with near maximal pathway copy number;

250

this represents a traditional growth-coupled metabolic engineering design approach.

251

Autonomous PHB production experiments were cultured as described above, without

252

addition of aTc for induction to express the PHB pathway. To mirror the chemical-induction trials, 2

253

mL of stock glucose solution was added for the PHB production phase, at ~ 10, 9, or 5 hours

254

(depending on cessation of growth in glycerol trials). In all trials, OD600/fluorescence, dry cell

255

weight (DCW), PHB production and organic acid/ glucose consumption were monitored.

256

ASSAYS

257

OPTICAL DENSITY AND FLUORESCENCE

258

OD600 and fluorescence were monitored using a Synergy HI Microplate Reader from Biotek

259

using Gen5 V 2.04 software. OD600 was monitored at 600 nm, and GFP and mCHERRY fluorescence

260

were monitored at 485nm:525nm and 585nm:615nm excitation:emission, respectively. 200 μL of

261

culture was measured for plate-reader growth experiments and 100 μL was measured in triplicate

262

for shake-flask cultures in 96-well black-walled Greiner plates (Sigma-Aldrich, St Louis, MO).

263

GLUCOSE AND ORGANIC ACID ASSAYS

264

Samples for organic acid analysis were collected and centrifuged for 10 minutes at 17000 x

265

g. Sodium azide (Sigma-Aldrich, St. Louis, MO) was added at a final concentration of 0.1 mg/L to

266

prevent subsequent cell growth. The supernatant was transferred into 1.5 mL auto-sampler vials

267

and stored at -20 °C until HPLC analysis. Organic acids and glucose were measured on an HPLC

268

Agilent 1200 series with: a Binary pumper with Degasser (G1379B), an 1290 Thermostat (G1330B),

269

an hiP-ALS SL+ autosampler (G1367D) maintained at 4 °C, TCC SL (G1316B), DAD SL (G1315C), and

270

1260 RID (G1362A). An isocratic method was used with 5mM filtered H2S04 at a 0.6 ml/min flow

271

rate through a Biorad Aminex 87-H column maintained at 65 °C. 10 μL of sample was injected.

272

Results were monitored and analyzed on Chemstation for LC 3D system Rev B.04.03[16] software.

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 31

273

Glucose, citrate, pyruvate, succinate, lactate, formate, glycerol and acetate standards (Sigma-Aldrich,

274

St. Louis, MO) ranging from 0.05-20 g/L were measured at the beginning and end of each run, and

275

used to construct a calibration curve for quantification of organic acids and sugars.

276

PHB ANALYSIS

277

5-10 mL of culture was collected at indicated times for DCW and PHB quantification. The

278

culture was centrifuged at 4000 x g for 10 minutes, washed twice with 1 mL of ddH20 and dried for

279

24 hours in an 80 °C oven. PHB was hydrolyzed with 1 mL 95-98% pure H2SO4 (Sigma Aldrich, St

280

Louis, MO) and boiled at 95 °C for one hour24. Samples were diluted to 5 mL with water, spun at

281

15000 x g for 10 minutes and supernatant was diluted 1/10 or 1/100 in water for HPLC analysis.

282

Approximately 5 mg of pure PHB (Sigma Aldrich, St Louis, MO) was processed in parallel, serially

283

diluted to precise concentrations and used to construct a standard curve to quantify PHB.

284

RESULTS & DISCUSSION

285

SELECTING GLUCOSE RESPONSIVE PROMOTERS FOR EVALUATION

286

To use glucose as a trigger for the proposed toggle switch, we first needed to identify

287

promoters with strong activation due to glucose depletion.

CRP-promoters likely have varied

288

dynamic responses to glucose limitation and may be under alternative forms of regulation as well. A

289

range of CRP-responsive promoters was chosen to screen for dynamic activity. Candidate promoters

290

were identified using the Ecocyc database and were chosen if there was evidence for CRP binding.

291

Due to the large abundance of possible CRP promoters, we focused on Class II promoters, which limit

292

the necessary size of the promoter, and promoters with few or no known binding sites for other TF’s

293

to reduce complexity from other regulation. Each promoter was generated by PCR in three

294

variations: (crp contains 500 upstream of the ORF; T-crp contains the CRP binding site ~50 bp

295

upstream of the transcription start site; and Tα-crp combines an upstream enhancer element with T-

296

crp). These promoters were cloned into the pWB8 plasmid to investigate the effects of limiting

297

upstream elements (T-crp) or artificially enhancing expression (Tα-crp). 12


Page 13 of 31

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


298

DIFFERENT GLUCOSE-RESPONSIVE PROMOTERS HAVE DIFFERING ACTIVATATION

299

CHARACTERISTICS

300

A promoter with both a tight OFF-state and an ON-state with adequate expression during

301

glucose-starvation is desirable to integrate sensing with the toggle switch to prevent early switching

302

and avoid wasted translation resources, respectively. Therefore, time course trials were carried out

303

to assay dynamic GFP activation upon glucose starvation for a variety of CRP-promoters. Strains

304

with the different CRP promoters were cultivated in LB + 0.0, 0.05, and 0.4% glucose and GFP

305

expression was monitored in the transition from growth to stationary phase (figure 2). Expression

306

varied within ~1 order of magnitude for most of the variants. The amplitude of activated CRP

307

promoters (by GFP expression) varied, ~ 15-fold between low and high expressing promoters (araF

308

vs. TychH), indicating a wide-range of expression capacity among the pool of constructed promoters.

309

Generally, T-crp and Tα-crp variants enhanced GFP expression, though sometimes increased baseline

310

levels (araF variants) and/or disrupted the dynamic range compared to wildtype (T-csiE and Tα-araF

311

promoters). There weren’t any discernable trends across all truncated or all full-length promoters,

312

likely due to complexity in the form of unknown regulation mechanisms. The promoter enhanced Tα-

313

crp variants had variable expression patterns compared to the crp and T-crp promoters, and may be

314

useful to test if trying to enhance promoter activity without altering regulation sites. This promoter

315

characterization should be a useful resource for stationary-phase induction applications.

316

GFP expression is due to glucose limitation and not a general stationary phase response, as

317

the 0.4% glucose condition was chosen to maintain excess glucose throughout the trial. (figure 2).

318

GFP expression is detectable for cultures lacking glucose (LB condition) at ~1.5 hours after

319

inoculation, while there is a lag in GFP expression in LB + 0.05% glucose trials until ~3- 4 hours.

320

Importantly, there is little GFP expression in the LB + 0.4% glucose condition beyond the baseline

321

level, even after cultures reach a stationary phase (growth curves not shown). Notably, growth rates

322

are comparable in all cases, which indicates GFP expression is due to glucose starvation, rather than

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 31

323

a general stationary phase response or accumulation of GFP due to slow down of growth. This

324

confirms the role of glucose-starvation in activation of GFP expression.

325

SELECT CRP PROMOTERS ENABLE TOGGLE BEHAVIOR

326

Promoters found to successfully activate GFP production upon glucose limitation were

327

incorporated into the original toggle switch design (the trcp promoter producing mCHERRY is “OFF”

328

and the tetOp producing GFPmut3b is “ON”, Figure 1B). This language mirrors future insertion of

329

metabolic pathways in place of gfpmut3b, so that pathway expression is defined as the ON state. This

330

orientation was chosen because tetOp promoters have a stronger OFF (TetR/mCherry) state, and

331

constitutive LacZ expression would allow the OFF state to be set with lactose rather than IPTG, to

332

further lower process costs. Glucose-sensitive promoters may enable autonomous switching of the

333

circuit with glucose starvation, in a manner analogous to adding aTc. Due to the acceptable dynamic

334

range of the constructed promoters, crp and T-crp variants were both carried forward. crp promoters

335

were placed upstream of LacI in either the two plasmid or tandem promoter system to enable

336

glucose limitation induced switching of the toggle state from OFF to ON (LacI/GFPmut3b) (figure 1b).

337

The stability of the circuit containing glucose-starvation sensing was investigated to ensure

338

switching was due solely to glucose starvation rather than nonspecific LacI expression or circuit

339

imbalance. The addition of new copies of LacI activation may bias the circuit towards the ON state.

340

Only two promoter systems demonstrate a stable OFF state after IPTG removal in a glucose-rich

341

environment (tandem promoter araF and TaraF systems, figure 3 and supplemental figure 1). None

342

of the two plasmid systems could maintain a stable OFF state, despite a wide range of dynamic

343

activity. The tandem TaraF promoter was the only system with a stable OFF state AND a comparable

344

ON state to the control (pKDL071), depicted in figure 3. Furthermore, the full transition to the ON

345

state after induction took ~6-8 hours, while chemical induction takes ~2 hours (figure 3 and 4).

346

Since chemical inducers immediately inhibit their targets, there is immediate repression of the

347

opposing TF. Whereas increased expression of a TF (LacI) needs to be above the stability point of the

348

opposing TF (TetR) to cause a switch in toggle states, and there is an inherent time delay needed for

14


Page 15 of 31

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

transcription and translation of the TF. Since the toggle switch TF’s do not have degradation tags like

350

the fluorescent reporters, dilution by growth is speculated to be an important mechanism to switch

351

the state of the toggle once newly expressed TF levels are above the threshold for a switching event,

352

ultimately leading to longer switching times.

353

The T-araF tandem glucose toggle switch was able to change states through 4 mechanisms,

354

growth in LB (figure 3), glucose starvation in MOPS EZ rich, and growth in MOPS EZ rich glycerol and

355

gluconate (figure 4). Secondary carbon sources are a cheap way to prevent cells from going into

356

stationary phase, while still enabling a change in the switch states. This strategy proved useful for

357

MOPS min PHB production, described below.

358

THE ORIGINAL GENETIC SWITCH EFFICIENTLY PRODUCES PHB

359

In order to verify the original toggle switch can be used for PHB production and demonstrate

360

the utility of splitting growth and production regimes using a toggle switch, gfpmut3b was replaced

361

by the phaECAB operon for the production of PHB in the toggle switch. Constitutive overexpression

362

of the pathway (stable 29 genomic copies of the PHB pathway in E. coli) leads to cell-burden and

363

reduced growth rates23 18. Decoupling growth and production using a toggle switch could enhance

364

productivity significantly by reducing the burden during growth, allow higher cell densities, suppress

365

low-productivity escape mutants, and allow interrogation of pathway enzyme levels beyond those

366

possible if simultaneous growth is also required.

367

Early induction of the PHB pathway in the cultivation leads to highly variable PHB

368

production titers and reduced growth rates; -14 h induced experiments produced titers from 0.5 g/L

369

to 1.5 g/L of PHB, with growth rates ranging from 0.1-.45 /hour. The relative standard deviation

370

(RSD) for the growth rate among these trials is 38% and for %PHB titer is 30% (figure 5b, green

371

circles). Separate growth and production strategies generally lead to comparable, more consistent

372

titers, and faster growth. Induction time resulted in a range of PHB titers. 0 h and 10 h induced

373

cultures (i.e. at inoculation and 10 h after inoculation, respectively) grew at similar rates (0.55-0.65

374

/h) and produced similar titers (0.8-1.4 g/L PHB)- (figure 5b orange and yellow circles). In

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

375

comparison to -14 h pre-culture induced experiments, 0 and 10 h induction cultures had faster

376

growth (up to 5 x faster, 6.5% RSD) and more consistent PHB production (60.9 %PHB, RSD 9%).

377

Non-induced cultures remained tightly off with no detectable PHB production. These results

378

highlight the challenges associated with early induction (-14 h), and the benefits of controlled

379

induction (0 or 10 h).

380

Page 16 of 31

Constitutive, stable expression of PHB is consistent, but at the expense of titer. A strain

381

constitutively expressing 29 copies of the PHB operon (KS29-consitutive) was benchmarked. It grew

382

slightly slower (0.50-0.56) and produced lower titers (0.7-1.0 g/L of PHB) than the induced PHB

383

cultures. While the plasmid induction strategy allows high copy25 (colE1 origin, 50-70 copies) that is

384

induced after growth is complete, the KS29-constitutive strain (29 copies) was developed by

385

selecting for the highest pathway levels compatible with growth4. Therefore, splitting growth and

386

production using induction control appears to enable higher pathway flux in the production phase,

387

while allowing a fast growth phase, which may enable higher biomass-dense cultures to be reached.

388

These results indicate separating growth and production phases using a toggle switch is a promising

389

production strategy.

390

391

A GLUCOSE INDUCED TOGGLE SWITCH ENABLES AUTONOMOUS PHB PRODUCTION

392

In the previous study, the toggle switch was switched to PHB production (ON) by aTc, which

393

was present throughout the production phase. For a successful glucose toggle, the switch to ON

394

should be induced by glucose starvation and should be stably ON after glucose is re-introduced. To

395

test this, successful autonomous, glucose-based switch plasmids were paired with the PHB toggle

396

system. The only stable glucose toggle out of 20 tested was the tandem promoter TaraF system.

397

(Figure 3 and 4). The TaraF tandem system allowed growth rates that were nearly double that of -14

398

h induction trials (0.30 vs. 0.65 h-1), likely due to no growth burden from the PHB pathway.

399

However, this candidate performed poorly from a production standpoint, with %PHB titer at ~ 10%.

16


Page 17 of 31

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


400

One distinction between the original toggle and our glucose toggle is the original toggle can

401

continuously grow through the toggling (which dilutes existing TF), while the glucose toggle pauses.

402

This, along with the immediate inhibition of the TF due to inducing chemicals, means the original

403

toggle has a much shorter transition. This was indeed the case, as the time for switching using

404

glucose starvation was ~6-8 hours, while the IPTG induction was closer to two hours (figure 3). To

405

improve the glucose toggle performance, we included a second carbon source, glycerol that would

406

allow the glucose toggle to maintain growth during the transition. Upon glucose exhaustion, glycerol

407

is consumed, allowing growth while not repressing CRP, like glucose. This growth allows more rapid

408

dilution of existing LacI, compared to glucose starved cells that stop growing. We used growth

409

conditions with less glucose (0.05% or 0.1%) and the addition of glycerol 0.1%. %PHB increased 4-

410

fold in these conditions, to ~40%, while maintaining fast growth (.65 h-1) (see figure 5 b and c). We

411

imagine for larger bioprocesses over longer production times, the difference between the chemical

412

and auto induction switch times (2 vs. 6-8 hours) should be less important than the conditions tested

413

in this study (with a maximal growth phase of ~10 hours).

414

Tandem promoters in series (rather than two promoters expressing two different copies of a

415

gene) to encode regulatory function in a genetic circuit proved useful. Previous studies have used

416

repeated tandem promoters to increase pathway expression,26, 27 but we are aware of only one that

417

order promoters in tandem to enable logic functions28 (the repression promoter is upstream of the

418

sensing-promoter to allow response to a transient signal), and it was described as a suboptimal

419

strategy. The benefits of the tandem system is that it requires only one plasmid, was the only stable

420

system, and led to improvements in growth rate even compared to the two plasmid production

421

system. In future studies, mRNA modelling may enhance the design phase, and it may be useful to

422

include mRNA processing in tandem promoter systems to standardize the mRNA resulting from

423

either of the tandem promoters (tetOp or crp)29.

424

This system may be amenable to other transient signals, whether that be other nutrient-

425

limitation, stressors, oxidative, pH stress, etc. However, these other systems may face additional

426

challenges. Sugar consumption is very hierarchical in E. coli due to the overwhelming preference for 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 31

427

glucose as the sole carbon source12. Due to this overwhelming preference, the CRP system allows

428

sharp repression and induction. Other nutrients like phosphate, nitrogen, and redox state, and even

429

secondary carbon sources are part of a complex regulatory network, and are often controlled at a

430

level relative to other nutrient cues (i.e. nitrogen sensing is tied to α-ketoglutarate which is a

431

measure of the carbon and nitrogen state of the cell30), rather than an absolute level like glucose.

432

Glucose is convenient, as it is an important energy and biomass constituent.

433

Beyond using the glucose toggle in large scale production, toggles could significantly enable

434

the strain engineering process.

Most strain engineering strategies sequentially require (1)

435

introducing genetic changes (2) growing the cells (typically to a colony and then many generations in

436

liquid culture) and (3) measuring the productivity.

437

opportunities for productive genetic changes to be lost due to slow growth or toxicity and low

438

productivity variants with growth fitness advantages may overtake the culture. This means useful

439

genetic modifications may be passed over and may confound the knowledge-base by implying those

440

are the wrong type of modifications. By generating new genetic constructs with pathways off, then

441

turning them on for the product screening process, it could be possible to recover mutants that

442

would not be viable during constitutive expression of the product pathway.

During the growth phase, there is ample

443

We have developed an autonomous genetic switch that has demonstrated utility in

444

improving growth rates compared to a constitutively expressing system, with a PHB pathway known

445

to cause burden at high enzyme expression levels. We have implemented a tandem promoter system

446

that allows logic to be encoded using different promoters in control of a single-copy of a regulatory

447

gene. Across experiments, regardless of media composition (defined rich, rich, or minimal) the

448

glucose level was the ultimate determination of gene expression, whether it was simple gene

449

expression assays, the stability tests, or the application to PHB production. This glucose sensitive

450

toggle circuit is flexible; it can be used for chemical production in minimal media or protein

451

production in rich media- and is a generally useful synthetic biology tool. This tandem promoter

452

strategy and the glucose-sensitive toggle system are generally useful additions to the field of

18


Page 19 of 31

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


453

synthetic biology, and we envision that they can be rapidly implemented into a variety of platforms

454

to push the boundaries of industrially relevant processes.

455

ACKNOWLEDGEMENTS

456

We would like to thank the Collins lab for providing plasmid pKDL071. We thank Karthik

457

Sekar and Jennifer Greene for helpful discussion regarding the design and analysis of experiments.

458

We thank the Northwestern Recombinant Protein Production Core (rPPC) for use of plate readers

459

and the NUSeq core for Sanger sequencing services.

460

SUPPORTING INFORMATION

461

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

462

Supplementary Table 1

463

Supplementary figure 1.

464

AUTHOR INFORMATION

465

Corresponding Author

466

* E-mail Keith E.J. Tyo: [email protected]

467

Department of Chemical and Biological Engineering E-136, Northwestern University, 2145 Sheridan

468

road, Evanston, IL, USA

469

Author Contributions

470

WHB and GK carried out experiments and WHB designed experiments. WHB and KEJT wrote the

471

manuscript.

472

Notes

473

The authors declare no competing conflicts of interest.

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

474

Page 20 of 31

FUNDING

475

This work was funded by the National Science Foundation CAREER (1452549), the Institute

476

for Sustainability and Energy at Northwestern (ISEN) Early Career Investigator Award, and funds

477

from the McCormick School of Engineering and Applied Science, Northwestern University.

478

20


Page 21 of 31

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

479


REFERENCES

480

(1) Cheong, S., Clomburg, J. M., and Gonzalez, R. (2016) Energy- and carbon-efficient

481

synthesis of functionalized small molecules in bacteria using non-decarboxylative Claisen

482

condensation reactions. Nat. Biotechnol. In press, 1–8.

483

(2) Soini, J., Ukkonen, K., and Neubauer, P. (2008) High cell density media for Escherichia coli

484

are generally designed for aerobic cultivations - consequences for large-scale bioprocesses and shake

485

flask cultures. Microb. Cell Fact. 7, 26.

486

(3) Chubukov, V., Mukhopadhyay, A., Petzold, C. J., Keasling, J. D., and García Martín, H.

487

(2016) Synthetic and systems biology for microbial production of commodity chemicals : from target

488

selection to scale-up. Npj Syst. Biol. Appl. 2, 16009.

489 490

491 492

(4) Tyo, K. E. J., Ajikumar, P. K., and Stephanopoulos, G. (2009) Stabilized gene duplication enables long-term selection-free heterologous pathway expression. Nat. Biotechnol. 27, 760–767.

(5) Chubukov, V., and Sauer, U. (2014) Environmental dependence of stationary-phase metabolism in Bacillus subtilis and Escherichia coli. Appl. Environ. Microbiol. 80, 2901–9.

493

(6) Hua, Q., Yang, C., Oshima, T., Mori, H., and Shimizu, K. (2004) Analysis of Gene Expression

494

in Escherichia coli in Response to Changes of Growth-Limiting Nutrient in Chemostat Cultures. Appl.

495

Environ. Microbiol. 70, 2354–2366.

496

(7) Kumar, R., and Shimizu, K. (2011) Transcriptional regulation of main metabolic pathways

497

of cyoA, cydB, fnr, and fur gene knockout Escherichia coli in C-limited and N-limited aerobic

498

continuous cultures. Microb. Cell Fact. 10, 3.

499 500

(8) Gardner, T. S., Cantor, C. R., and Collins, J. J. (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342. 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

Page 22 of 31

501

(9) Litcofsky, K. D., Afeyan, R. B., Krom, R. J., Khalil, A. S., and Collins, J. J. (2012) Iterative

502

plug-and- play methodology for constructing and modifying synthetic gene networks. Nat. Methods 9,

503

1077–1081.

504

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

505

redirection from a central metabolic pathway toward a synthetic pathway using a metabolic toggle

506

switch. Metab. Eng. 23, 175–184.

507 508

(11) Soma, Y., and Hanai, T. (2015) Self-induced metabolic state switching by a tunable cell density sensor for microbial isopropanol production. Metab. Eng. 30, 7–15.

509 510

(12) Aidelberg, G., Towbin, B. D., Rothschild, D., Dekel, E., Bren, A., and Alon, U. (2014) Hierarchy of non-glucose sugars in Escherichia coli. BMC Syst. Biol. 8, 133.

511 512

(13) Shimizu, K. (2014) Regulation Systems of Bacteria such as Escherichia coli in Response to Nutrient Limitation and Environmental Stresses 1–35.

513

(14) Lin, H. Y., Mathiszik, B., Xu, B., Enfors, S., and Neubauer, P. (2001) Determination of the

514

Maximum Specific Uptake Capacities for Glucose and Oxygen in Glucose-Limited Fed-Batch

515

Cultivations of Escherichia coli.

516

(15) Shimada, T., Fujita, N., Yamamoto, K., and Ishihama, A. (2011) Novel Roles of cAMP

517

Receptor Protein ( CRP ) in Regulation of Transport and Metabolism of Carbon Sources. PLoS One 6,

518

e20081.

519 520

(16) Busby, S., and Ebright, R. H. (1999) Transcription Activation by Catabolite Activator Protein (CAP). J. Mol. Biol. 293, 199–213.

521

(17) Savery, N. J., Lloyd, G. S., Kainz, M., Gaal, T., Ross, W., Ebright, R. H., Gourse, R. L., and

522

Busby, S. J. W. (1998) Transcription activation at Class II CRP-dependent promoters: identification of

523

determinants in the C-terminal domain of the RNA polymerase α subunit. EMBO J. 17, 3439–3447. 22


Page 23 of 31

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


524

(18) Tyo, K. E. J., Fischer, C. R., Simeon, F., and Stephanopoulos, G. (2010) Analysis of

525

polyhydroxybutyrate flux limitations by systematic genetic and metabolic perturbations. Metab. Eng.

526

12, 187–195.

527 528

(19) Neidhardt, F. C., Bloch, P. L., and Smith, D. F. (1974) Culture Medium for Enterobacteria. J. Bacteriol. 119, 736–747.

529

(20) Lloyd, G. S., Busby, S. J. W., and Savery, N. J. (1998) Spacing Requirements for

530

interactions between the C-terminal domain of α subunit of Escherichia coli RNA polymerase and the

531

cAMP receptor protein. Biochem. J. 420, 413–420.

532

(21) Gibson, D. G., Young, L., Chuang, R., Venter, J. C., Hutchison III, C. A., and Smith, H. O.

533

(2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 12–

534

16.

535 536

537 538

(22) Hillson, N. J., Rosengarten, R. D., and Keasling, J. D. (2014) J5 DNA Assembly Design Automation Software. ACS Synth. Biol. 1, 14–21.

(23) Sekar, K., and Tyo, K. E. J. (2015) Regulatory effects on central carbon metabolism from poly-3-hydroxybutryate synthesis. Metab. Eng. 28, 180–9.

539

(24) Tyo, K. E., Zhou, H., and Stephanopoulos, G. N. (2006) High-Throughput Screen for Poly-

540

3-Hydroxybutyrate in Escherichia coli and Synechocystis sp. Strain PCC6803. Appl. Environ. Microbiol.

541

72, 3412–3417.

542

(25) Lutz, R., and Bujard, H. (1997) Independent and tight regulation of transcriptional units

543

in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res.

544

25, 1203–1210.

545 546

(26) Blazeck, J., Liu, L., Redden, H., and Alper, H. (2011) Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Appl. Environ. Microbiol. 77, 7905–7914. 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

547 548

549 550

551 552

553 554

555

Page 24 of 31

(27) Li, M., Wang, J., Geng, Y., Li, Y., Wang, Q., Liang, Q., and Qi, Q. (2012) A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli. Microb. Cell Fact. 11, 19.

(28) Tamsir, A., Tabor, J. J., and Voigt, C. A. (2011) Robust multicellular computing using genetically encoded NOR gates and chemical “wires”. Nature 469, 212–5.

(29) Qi, L., Haurwitz, R. E., Shao, W., Doudna, J. A., and Arkin, A. P. (2012) RNA processing enables predictable programming of gene expression. Nat. Biotechnol. 30, 1002–1006.

(30) Huergo, L. F., and Dixon, R. (2015) The Emergence of 2-Oxoglutarate as a Master Regulator Metabolite. Microbiol. Mol. Biol. Rev. 79, 419–435.

ABSTRACT FIGURE

556

557 558

559

560

24


Page 25 of 31

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


561

FIGURES

562

Figure 1: Strategies for Biosynthesis of Chemicals using Cell Factories

563

Idealized strategies for Biosynthesis of Chemicals using Cell Factories. (a) depicts common biomass

564

(solid line) and product (dashed line) profiles expected from a variety of biosynthesis strategies

565

(green: decoupled growth and production, yellow: constitutive growth and production, red:

566

constitutive pathway overexpression). (b) Shows the integration of glucose sensing into the toggle

567

switch architecture. (c) Depicts Relevant genetic programs followed in this study

568

a.

Growth and Product Profiles for Biosynthesis Strategies

b.

Toggle Switch Architecture with integrated Glucose sensing

569 570

571

25



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

572

c.

Page 26 of 31

Program for the glucose-sensing genetic switch

573 574 575 576

26


Page 27 of 31

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


577

Figure 2: Glucose-sensitive promoters activate expression over a wide dynamic range only in

578

glucose limited conditions

579

crpp promoters were grown in LB + 0.1% glucose, then transition into three different types of media

580

(LB, LB + 0.1% and 0.4% glucose). GFP expression was due to glucose availability rather than

581

cessation of growth. GFP expression initiation could be titrated depending on the glucose

582

concentration in the media. Red vertical lines indicate time points beyond which fluorescence was

583

above the detection limit of the plate reader.

584 585 586 587

27



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 28 of 31

588

Figure 3: Glucose Sensing Autonomously Switches Toggle State from OFF (RFP) to ON (GFP)

589

Successful crpp glucose-starvation sensing promoters were integrated into the toggle switch and

590

tested for the ability to switch the toggle state. After pre-culture (described in the text), each variant

591

was sequentially grown in media conditions and monitored for autonomous toggling of genetic

592

switch states. The araF and TaraF tandem promoter toggle switches were tested. Only the TaraF

593

system was able to remain stably OFF, and activate a switch from the OFF to the ON state upon

594

glucose starvation, while remaining stable upon reintroduction of glucose. The system then returned

595

to the OFF state after IPTG addition.

596 28


Page 29 of 31

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


597

Figure 4: The glucose toggle switch can be autonomously activated in varied media conditions

598

The ability of the glucose-sensing toggle switch to activate in a variety of conditions was tested. After

599

pre-culturing to set the switch to the OFF state (described in the text), cells were transferred to 3

600

conditions without glucose (1) no additional carbon- starvation (2) gluconate and (3) glycerol. All

601

three conditions successfully switch the toggle state from OFF to ON. The timescale represents time

602

after starvation and the first time point recorded is after glucose was re-added to the media.

603 604

Figure 5 Production phase glucose consumption and organic acid/PHB production profiles

605

(a) The timeline of chemical and autonomous PHB production is depicted (b) A variety of toggle

606

switch systems for PHB production including pre-culture, chemical and autonomous production. (c)

607

Representative plots of carbon consumption and secretion over time for the glycerol optimized trials.

608

a.

Experimental Timeline

29



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 30 of 31

609 610

b.

Growth vs. Production phase for PHB trials

a.

Carbon consumption and secretion over time for glycerol optimized trials

611 612

30


Page 31 of 31

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


613

31


A Glucose-Sensing Toggle Switch for Autonomous, High Productivity

Recommend Documents