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A physiology-oriented engineering strategy to improve gamma-aminobutyrate production in Lactobacillus brevis Changjiang Lyu, Weirui Zhao, Sheng Hu, Jun Huang, Tao Lu, Zhihua Jin, Lehe Mei, and Shan-Jing Yao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04442 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 14, 2017

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Journal of Agricultural and Food Chemistry

A physiology-oriented engineering strategy to improve gamma-aminobutyrate production in Lactobacillus brevis Chang-jiang Lyu1,2, Wei-rui Zhao2, Sheng Hu2, Jun Huang3, Tao Lu1, Zhi-hua Jin2, Le-he Mei1,2*, Shan-jing Yao1* 1

College of Chemical and Biological Engineering, Zhejiang University, Hangzhou

310027, China 2

School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology,

Zhejiang University, Ningbo 315100, China 3

School of Biological and Chemical Engineering, Zhejiang University of Science and

Technology, Hangzhou 310023, China

Corresponding authors: Professor Le-He Mei and Professor Shan-Jing Yao Tel./Fax: +86 571 87951982 E-mail addresses: [email protected] (Prof. L.-H. Mei), and [email protected] (Prof. S.-J. Yao).

ACS Paragon Plus Environment


1

ABSTRACT: Gamma-aminobutyrate (GABA) is an important chemical in

2

pharmaceutical field. GABA-producing lactic acid bacteria (LAB) offer the

3

opportunity of developing this health-oriented product. In this study, the gadA, gadB,

4

gadC, gadCB and gadCA gene segments of Lactobacillus brevis were cloned into

5

pMG36e, and strain Lb. brevis/pMG36e-gadA was selected for thorough

6

characterization in terms of GABA production after analysis of GAD activities.

7

Subsequently, a physiology-oriented engineering strategy was adopted to construct an

8

FoF1-ATPase deficient strain NRA6 with higher GAD activity. As expected, strain

9

NRA6 could produce GABA at a concentration of 43.65 g/L with a 98.42% GABA

10

conversion rate in GYP fermentation medium, which is 1.22-fold higher than that

11

obtained by wild type strain in the same condition. This work demonstrates how the

12

acid stress response mechanisms of LAB can be employed to develop cell factories

13

with improved production efficiency, and contributes to research into the

14

development of the physiology-oriented engineering.

15

KEYWORDS: GABA; Lb. brevis; physiology-oriented engineering; GAD system;

16

FoF1-ATPase.

17


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INTRODUCTION

19

Lactic acid bacteria (LAB) constitute a heterogeneous group of non-sporulating

20

Gram-positive bacteria that are found in a variety of different environments. Most of

21

them are used for the production of the consumer-accepted fermented food products,

22

probiotic products and biologically active supplements.1 Recently, some new

23

applications for LAB have also been developed, including the production of

24

pharmaceutical agents and the delivery of oral vaccines.1-3 However, during the

25

process of production and application, LAB usually encounter various environmental

26

stresses, such as acid, cold, drying, oxidative, osmotic stresses, antibiotics, organic

27

solvents, and heat.4-8 To meet these challenges, LAB should exert strong

28

physiological robustness and fitness, in addition to excellent metabolic capabilities, to

29

enable them to work efficiently in actual bioprocesses.9,

30

physiology-oriented engineering has emerged as a discipline that focuses on the

31

rational improvement of physiological performances of industrial useful strains.11

10

Consequently,

32

Among different physiological features, adaptation and tolerance to acid stress

33

are important factors for LAB as lactic acid is the main catabolism product, which

34

acidifies the media and arrests cell multiplication.4, 5, 12 Therefore, it is important to

35

understand how LAB cells sense and subsequently adapt to the acidic environment

36

and what contributes to these adaptations. In recent years, several mechanisms have

37

been identified for acid resistance in LAB, including FoF1-ATPase proton pump,

38

glutamate decarboxylase system (GAD), ornithine decarboxylase system, tyrosine

39

decarboxylase system, biofilm formation, production of dextran, reuteran or levan,

40

protection or repair of macromolecules, as well as the alkali production through

41

urease, glutaminase, arginine deiminase and agmatine deiminase pathways.4,

42

Remarkably, FoF1-ATPase and GAD system have been regarded as two of the most


12, 13


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potent acid resistance systems. FoF1-ATPase is a common pathway that LAB employ

44

for protection against acidic conditions, which regulates cytoplasmic pH by expelling

45

protons out of the cell utilizing the energy released by ATP hydrolysis with the

46

concomitant generation of proton motive force (PMF).12 Similarly, GAD system

47

consumes intracellular protons through decarboxylation of glutamate in the cytoplasm

48

and exchange of the reaction product GABA with extracellular glutamate, which

49

efficiently works to protect cells from the acid stress that encountered during food

50

fermentation and gastrointestinal transit.14

51

Besides the contribution to acid resistance, the reaction product of GAD system

52

has enjoyed a welcome boost thanks to the discovery of its application as a bioactive

53

component in various functional foods and pharmaceuticals due to its potential in

54

controlling neurotransmitter signal and lowering blood pressure in human.15, 16 In

55

addition, several well-characterized physiological functions, such as secretagogue of

56

insulin, stimulation of immune cells, as well as diuretic, antidiabetic and tranquilizer

57

effects have also been related to the administration of GABA.17,

58

development of strategies for efficient and cost-effective production of GABA

59

becomes an important issue to meet its increasing commercial demand. Hitherto, the

60

most interesting and practical group of microorganism for GABA production is LAB,

61

some of which could catalyze the decarboxylation of glutamate in the

62

proton-consuming reaction, thus contributing to the pH homeostasis within the cells

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and resulting in a stoichiometric release of the functional product GABA.19,

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Meanwhile, the antiport of glutamate and GABA generates a ∆pH and ∆Ψ

65

contributing to a PMF for the ATP synthesis.21 In view of the evidence for PMF

66

generation by the decarboxylation of glutamate to GABA, it was of particular interest

67

to investigate the connection between FoF1-ATPase and GAD system and address an


18

Thus, the

20

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issue that whether the alteration of proton flux would be an explanation of the

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possible effects of changed FoF1-ATPase activity on the GABA production.

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Amongst a variety of the reviewed LAB species, Lb. brevis was the most

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frequently isolated species from traditional fermented products with the highest

72

GABA productivity.19, 20 Biotransformation of glutamate to GABA by growing and

73

resting cells systems of Lb. brevis have been extensively investigated in the past few

74

years.20, 22-26 However, almost all of the existing studies have focused on the optimal

75

medium and culture conditions, including temperature, pH, substrate and fermentation

76

model. Hence there has been an increasing interest to investigate the probability of

77

improving GAD activity of Lb. brevis through genetic engineering. Moreover,

78

engineering the performance of industrial microbes should not only rely on

79

strengthening the metabolic capability, as this is often affected by external

80

circumstances.4, 9-11, 27 In considering the physiological features of GAD system in

81

LAB,14, 28 more attention should also be paid to the possibility that whether the acid

82

stress response mechanisms of Lb. brevis could be employed to develop cell factories

83

with improved GABA production efficiency.

84

As mentioned above, consumption and expelling of intracellular protons are the

85

main themes of the acid resistance systems.4,

5

86

directional regulation of the proton homeostasis would be a prerequisite for exerting

87

the expected characteristics in GABA production. To address these issues, pioneering

88

efforts have been made currently to adopt an unconventional physiology-oriented

89

engineering strategy that based on the correlations among different acid resistance

90

systems for constructing an FoF1-ATPase deficient and glutamate decarboxylase

91

overexpressed Lb. brevis strain, with the aim of shifting the influx of protons toward

92

GAD system and then achieving enhanced production of GABA.

Therefore, effectual control and



93

MATERIALS AND METHODS

94

Bacterial strains, plasmids, and growth conditions

95

The bacterial strains and plasmids used in this study are listed in table 1. E. coli

96

strains were aerobically grown with shaking at 250 rpm in Luria‒Bertani (LB) broth

97

at 37 °C. The Lb. brevis strains were generally cultured in MRS medium at 37 °C

98

without agitation. Solid media for plating were prepared by adding 1.5% agar to the

99

appropriate liquid media. Selective media contained 7 or 200 µg/mL erythromycin for

100

selection of Lb. brevis or E. coli, respectively. In fermentation experiments, Lb. brevis

101

cells were grown in glucose yeast extract peptone (GYP) fermentation medium23 as

102

described previously but with minor modifications (g/L): glucose, 20; yeast extract,

103

15; peptone, 5; CH3COONa, 3; FeSO4·7H2O, 0.001; MgSO4·7H2O, 0.03; NaCl, 0.001;

104

MnSO4·4H2O, 0.02; L-monosodium glutamate (MSG), 72.75. Glucose was sterilized

105

separately at 115 ºC for 15 min. Under the pH-controlled batch fermentations, pH was

106

adjusted and maintained by automatic addition of 3 M NaOH or 3 M H2SO4. Table 1

107

108

DNA manipulations and reagents

109

Chromosomal DNA of Lb. brevis CGMCC1306 was extracted using a kit from

110

Qiagen according to the manufacturer’s instructions. Plasmid DNA of E. coli was

111

purified with the miniprep plasmid purification kit (Sangon, Shanghai, China). For Lb.

112

brevis, cells were treated with lysozyme first. Restriction enzymes and T4 DNA ligase

113

were purchased from Fermentas and used according to the instructions of the supplier.

114

When required, DNA fragments were purified from PCR or isolated from agarose

115

gels by using the PCR Purification Kit (Qiagen) or Gel Extraction Kit (Qiagen),

116

respectively. PCR amplifications, using Taq DNA polymerase (Takara), were

117

performed in a Veriti96-well thermal cycler (Applied Bio-systems).


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Electrotransformation of Lb. brevis

119

Electroporation of Lb. brevis CGMCC1306 was performed as previously

120

described with some modifications.29 Briefly, a 2% inoculum of an overnight culture

121

was grown in MRS medium supplemented with 1% glycine at 37 ºC until the OD600

122

reached 0.4. The cells were harvested and washed twice with cold buffer I (0.5 M

123

sucrose, 5 mM potassium phosphate pH 7.4 and 0.5 M MgCl2). The cells were then

124

washed once and resuspended to 1% of the original culture volume in a cold buffer II

125

(1 M sucrose, 3 mM MgCl2). For electroporation, 50 µL of the cell suspension was

126

mixed with 400 ng of plasmid DNA and subjected to electroporation at the field

127

strength of 10 kV/cm (pulse duration, 3.5 ms). After the pulse, 450 µL of cold MRS

128

with 0.3 M sucrose was immediately added to the cell suspension and the sample

129

mixture was incubated for 10 min on ice. After further incubation of the sample for 3

130

h at 37 °C, the transformants were plated onto MRS agar plates containing the

131

appropriate antibiotic and incubated for 48 to 72 h.

132

Construction of expressing plasmids and strains

133

For expression of gad genes in Lb. brevis, gadA, gadB, gadC and gadCB

134

segments were amplified from the genome of strain CGMCC1306 with primers

135

gadA-F1 and gadA-R, gadB-F and gadB-R, gadC-F and gadC-R1, and with gadC-F

136

and gadB-R, digested with SalI and HindIII, and then ligated into the constitutive

137

expression vector pMG36e, respectively. Information on the primers is listed in table

138

2. E. coli DH5α was used as the intermediate host and transformed with this ligate.

139

For recombinant vector pMG36e-gadCA, gadA and gadC fragments were amplified

140

with primers gadA-F2 and gadA-R and with gadC-F and gadC-R2. Primers gadC-R2

141

and gadA-F2 are designed such that amplification produces fragments that have tails

142

(table 2; double underscore) with identity to the ends of a cassette that, in this case,



143

contains the gadC and gadA genes. Subsequently, the fragment of gadCA was

144

constructed using fusion PCR approach,30 and then cloned into pMG36e plasmid31 to

145

obtain pMG36e-gadCA. The recombinant vectors were confirmed by restriction

146

enzyme analysis and DNA sequencing, and then transformed into the Lb. brevis

147

CGMCC1306 by electroporation as described above. The sequences of gadR, gadCB

148

operon and gadA locus were deposited in GenBank database under accession number

149

KU759571, JQ246952 and GU987102.1, respectively. Table 2

150

151

Derivation of FoF1-ATPase-defective mutants from Lb. brevis/pMG36e-gadA

152

Lb. brevis cells were grown in MRS medium until an OD600 of ∼1.0 was reached.

153

Cells were then harvested by centrifugation at 5000 g, for 10 min at 4 °C. The cell

154

pellet was washed twice in sterile saline (0.85% NaCl) solution and suspended in

155

saline to an OD600 of about 0.5. Then 0.1 mL aliquots of 10-fold dilution of the

156

suspended cells were spread onto half strength MRS plates containing 800 µg/mL

157

neomycin sulfate hydrate (Sangon, Shanghai, China) for FoF1-ATPase-defective

158

mutants generation and incubated at 37 °C for 2 days. Neomycin-resistant colonies

159

were selected and streaked on fresh MRS agar plates and incubated as described

160

above. This procedure was repeated at least two times in order to obtain purified

161

colonies.

162

Measurement of intracellular pH

163

The internal pH was measured with 5 (and-6)-carboxyfluorescein diacetate

164

succinimidyl ester (CFSE; Eugene, Oregon, USA) as described previously32 with

165

minor modifications. In brief, cells were cultured until OD600 of 0.5, harvested and

166

washed twice in GCPK buffer (glycine 50 mM, citric acid 50 mM, disodium

167

phosphate 50 mM, potassium chloride 50 mM) at pH 7.0. Cells were resuspended in


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GCPK buffer at different pH values (5.0, 6.0 and 7.0) and incubated at 37 °C for 30

169

min in the presence of the membrane permeable precursor probe cFSE, washed twice,

170

resuspended in GCPK buffer at the corresponding pH and incubated for another 30

171

min with 10 mM glucose to eliminate non-conjugated CFSE. The cells were

172

subsequently washed and resuspended in GCPK buffer, pH 5 to 7, and placed on ice

173

until pH measurements were carried out. Fluorescence intensities were measured at

174

excitation wavelengths of 490 nm (pH sensitive) and 440 nm (pH insensitive) by

175

rapidly alternating the monochromator (Hitachi, F4500, Japan) between the two

176

wavelengths. The emission was determined at 525 nm, and the excitation and

177

emission slit widths were set at 5 and 10 nm, respectively. At the end of each assay,

178

the extracellular fluorescence signal (background) was determined by filtration of the

179

cell suspension through a 0.22-µm-pore-size membrane filter and measurement of the

180

filtrate. Previously, calibration curves were made for each strain in buffers with pH

181

values between 4.0 and 8.0. The pH was adjusted with either NaOH or HCl. The pHin

182

and pHout were equilibrated by addition of valinomycin (potassium ionophore; 1 µM)

183

and nigericin (protonophore; 1 µM) and the internal pH values were calculated from

184

the ratio of the fluorescent signal obtained at 490/440 nm as described previously.

185

Isolation of membrane vesicles

186

Membrane vesicles were obtained essentially as indicated in a previous work.33

187

Cells of Lb. brevis were harvested by centrifugation at 6000 g at 4 °C for 10 min and

188

washed twice in 50 mM HEPES-potassium (pH 7.4) containing 10 mM MgSO4. The

189

cells, suspended in the same buffer, were lysed at 37 °C by treatment for 60 min with

190

10 mg/mL lysozyme and 50 µg/mL mutanolysin in the presence of a cocktail of

191

proteinase inhibitors (PMSF, Bestatin, Pepstatin A and E-64; Sangon) with constant

192

stirring. After addition of DNase I (10 U/mL, Sigma) and RNase (1 µg/mL, Sigma),



193

the suspension was passed three times through a precooled French pressure cell

194

(Constant Systems TS1.1KW, England) operated at 20,000 psi. Unbroken cells were

195

subsequently removed by centrifugation at 15,000 g for 15 min at room temperature.

196

The supernatant was centrifuged at 300,000 g for 1 h at 4 °C (Beckman Optima

197

Max-XP, America), and the pellet was suspended in the same buffer plus 10%

198

glycerol, frozen in liquid nitrogen and stored at -80 °C until use. This membrane

199

fraction was used for ATPase assays. The concentration of the membrane proteins was

200

determined by the Bradford method.34

201

Assay of ATPase activity

202

Activity of ATPase was assessed in terms of the release of inorganic phosphate

203

(Pi) from ATP basically as described by the method of Driessen et al.35 Two

204

micrograms of membrane protein was incubated at 37 °C for 10 min in 50 mM

205

MES-potassium buffer (pH 5.5) containing 5 mM MgCl2. ATP (disodium salt, Sangon)

206

was added at a final concentration of 2 mM to initiate the reaction. The reaction (total

207

volume of 40 µL) was stopped after 5 min by immediately cooling the test tubes on

208

ice. Malachite green solution (200 µL of 0.034%) was added, and after 40 min color

209

development was terminated by adding 30 µL of citric acid solution [34% (w/v)].

210

Immediately, the absorbance at 660 nm was measured. ATPase activity was defined as

211

the release of 1 µM of inorganic phosphate in 1 min. For the determination of pH

212

dependency of the ATPase activity, membranes were incubated for 60 min on ice in

213

50 mM MES-potassium buffer, adjusted to various pH values. The ATPase activity

214

was assayed at the different pH values as described above.

215

Colorimetric screening assay for GAD activity of Lb. brevis mutants

216

To obtain high-activity strains capable of producing GABA, the cell-associated

217

GAD activities of Lb. brevis mutants were detected using a pH-sensitive


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218

high-throughput colorimetric assay method in a 96-well microplate format. The assay

219

is based on detecting the pH increase that occurs as the GAD catalyzed reaction

220

proceeds due to the consumption of protons.36, 37 Briefly, individual mutant colonies

221

were picked into 96-deep-well plates containing 1.5 mL of GYP medium

222

supplemented with 20 g/L MSG in each well. Plates were incubated for 36 h at 37 ºC.

223

Aliquots of 50 µL of the culture were transferred into a new 96-deep-well plate with

224

1.5 mL of GYP medium supplemented with 20 g/L MSG in each well. Plates were

225

then incubated for an additional 36 h at 37 ºC. The plates were centrifuged at 6000 g

226

for 20 min at 4 ºC. The supernatants were removed, and the pellets were resuspended

227

in 300 µL of 20 mM sodium acetate buffer (pH 4.8) containing 60 mM MSG. After

228

incubation for 15 min at 37 ºC on an orbital shaker at 150 rpm, the plates were

229

centrifuged with the same parameters as above. Then 190 µL cell-free supernatant of

230

each mutant and 10 µL 1.4 mM BCG (bromocresol green sodium salt dissolved in

231

water; pH 4.8) were added into the into 96-well standard microplate; after shaking for

232

10 s to ensure complete mixing, the colors were identified and the absorbances were

233

measured at 620 nm using a microplate reader (BMG Labtech, Ortenberg, Germany).

234

Cell-bound GAD activity determination

235

Cell-bound GAD activity was determined by measuring the amount of GABA

236

formed at 37 ºC in a reaction mixture containing 0.1 mg (dry cell weight)/mL cell

237

biomass, 0.2 M sodiumacetate buffer (pH 4.8), 60 mM MSG.38 The concentrations of

238

glutamate and GABA were determined by reversed phase high-performance liquid

239

chromatography (RP-HPLC) as described by Marquez et al.39 One unit (U) of GAD

240

activity was defined as the amount of cells that produced 1 µM of GABA in 1 min

241

under the above conditions. In addition, the conversion rate of MSG to GABA was

242

calculated as follows:

243

Conversion rate=

() ()


× 100%


244

The effects of N,N'-dicyclohexylcarbodiimide on the GAD and ATPase activity

245

In order to investigate the effects of N,N'-dicyclohexylcarbodiimide (DCCD) on

246

GAD activities, Lb. brevis strains were cultured in GYP medium containing 20 g/L

247

MSG for 30 h (pH 5.2), harvested and washed twice with phosphate-buffered saline

248

(PBS, pH 7.2). Then, the experiments were performed in a reaction mixture

249

containing 0.1 mg/mL cell biomass, 0.2 M sodiumacetate buffer (pH 4.8), 60 mM

250

MSG, and various concentrations of DCCD for 30 min at 37 °C. After this treatment,

251

samples were centrifuged at 6000 g for 10 min at 4 °C and the GABA concentrations

252

in supernatants were determined by HPLC analysis.39 Furthermore, cells were

253

harvested and the membrane vesicles were obtained as described above.33 To measure

254

the effects of DCCD on the ATPase activities, the membranes were incubated with the

255

corresponding concentration of DCCD for 10 min at 37 °C, and subsequently for 60

256

min on ice. The activity of membrane samples without inhibitor was measured and

257

used as control.

258

RESULTS AND DISUSSION

259

Comparison of GAD activities in wild type and recombinant Lb. brevis strains

260

Owing to the great promise potential in large-scale fermentation for the

261

production food-grade GABA, biotransformation of glutamate to GABA by Lb. brevis

262

has been extensively investigated during the past few years.19, 20, 23 Previously, we

263

isolated a high GABA-producing strain, Lb. brevis CGMCC 1306, which was a

264

potential candidate for the food-grade GABA industrial production.40-42 Subsequent

265

studies have revealed the existence of two different glutamate decarboxylase encoding

266

genes (gadA and gadB) in this strain (Figure 1a). The gadB is linked to the

267

glutamate-GABA antiporter gene (gadC) and forms an operon (gadCB), and gadA

268

gene is located separately from the other gad genes. To achieve the high-level


Page 12 of 32

Page 13 of 32


269

production of GABA in Lb. brevis, it is one of the important issues to construct the

270

gene expression system which expresses the enzymes responsible for the production

271

of this target product with high solubility and functionality. Thus, the gadA, gadB,

272

gadC and gadCB segments were cloned and ligated into a constitutive expression

273

vector pMG36e, respectively (Figure 1b, 1c, 1d and 1e). In considering the functional

274

role of gadA, the gadCA segment was also obtained using the fusion PCR-based

275

approach and then ligated into pMG36e (Figure 1f). Subsequently, Lb. brevis

276

harbouring these recombinant expression plasmids were further constructed.

277

Lb. brevis strains were grown in GYP fermentation media under acidic conditions

278

(pH 5.2). The samples were collected at exponential phase (EXP.; 24 h) and stationary

279

phase (STAT.; 36 h), and the cytoplasmic as well as cell-bound GAD activities were

280

determined by measuring the amount of GABA formed at 37oC in a reaction mixture

281

as described in materials and methods. Under the strong and constitutive P32 promoter,

282

gadA, gadB and gadC were expressed well and the proteins could be produced with

283

high solubility (supplementary data Figure S1). After 24 h of incubation, the

284

cytoplasmic GAD activities (U/mg protein) of Lb. brevis/pMG36e-gadA (4.455

285

±0.163), Lb. brevis/pMG36e-gadB (3.875 ±0.078), Lb. brevis/pMG36e-gadCA (3.715

286

±0.106) and Lb. brevis/pMG36e-gadCB (3.241 ±0.099) were obviously higher than

287

those of Lb. brevis/pMG36e-gadC (1.254 ±0.052) and Lb. brevis/pMG36e (1.305

288

±0.092) (Figure 2). Equally remarkable was the fact that the cell-bound GAD

289

activities

290

brevis/pMG36e-gadC, Lb. brevis/pMG36e-gadCA and Lb. brevis/pMG36e-gadCB

291

strains were 0.811 ±0.047, 0.795 ±0.023, 0.746 ±0.042, 0.872 ±0.025 and 0.845

292

±0.012 U/mg DCW, respectively. As expected, all of the five recombinant strains

293

revealed higher cell-bound GAD activities than that of wild type strain (0.717 ±0.023

for

Lb.

brevis/pMG36e-gadA,

Lb.

brevis/pMG36e-gadB,


Lb.


Page 14 of 32

294

U/mg DCW), however, the increased values were relatively smaller. The same trends

295

were also observed in stationary phase cells, although the cell-bound GAD activities

296

of

297

brevis/pMG36e-gadCB were decreased in stationary phase compared to the

298

exponential phase (Table 3). Moreover, our data also demonstrated that cell-bound

299

GAD activities in wild-type strain differ substantially during different growth phases

300

because the GAD is an induced protein present in cytoplasm to provide resistance to

301

acid stress, as was the case in Shigella flexneri, Lc. lactis and Listeria

302

monocytogenes.14,

303

complex plays a major role in the overall GAD activity of Lb. brevis,45 however, the

304

strain containing pMG36e-gadA rather than pMG36e-gadB, pMG36e-gadCA or

305

pMG36e-gadCB achieves a preferable result in the increased cell-bound GAD activity,

306

at least under the conditions tested in this work. Therefore, the recombinant strain Lb.

307

brevis/pMG36e-gadA was selected for thorough characterization in terms of GABA

308

production.

Lb.

brevis/pMG36e-gadC,

43, 44

Lb.

brevis/pMG36e-gadCA

Lb.

Interestingly, previous work has shown that the GadCB

309

Figure 1, Figure 2 and Figure S1

310

Table 3

311

and

Effects of reduced FoF1-ATPase activity on the cell-bound GAD activity

312

FoF1-ATPase is a multiple-subunit enzyme consisting of a catalytic portion (F1)

313

incorporating α, β, γ, δ and ε subunits for ATP hydrolysis and an integral membrane

314

portion (Fo) including a, b, and c subunits, which function as a membranous channel

315

for proton translocation.46, 47 Previous work has shown that ATP synthesis can be

316

inhibited by DCCD, a relatively specific inhibitor of the Fo part of the FoF1-ATPase,

317

without inhibition of the glutamate decarboxylation.21 In order to investigate whether

318

there would be corresponding changes in GAD activity in response to the reduced


Page 15 of 32


319

FoF1-ATPase activity, DCCD was employed as the inhibitor and its effects on the

320

ATPase and GAD activity of Lb. brevis/pMG36e-gadA were determined. As shown in

321

Figure 3, addition of DCCD resulted in a decline in membrane ATPase activity. Once

322

the cells suffered over 0.05 mM of DCCD, the ATPase activities decreased rapidly.

323

Moreover, the deduced FoF1-ATPase activity, calculated as the difference between the

324

ATPase activity in the absence and presence of DCCD (1 mM), accounted for more

325

than 82.06% of the total ATPase activity, indicating that the FoF1-ATPase is

326

responsible for the majority of the ATPase activity found in the membranes of Lb.

327

brevis/pMG36e-gadA, as indicated for Bifidobacterium animalis, Lb. delbrueckii

328

subsp. bulgaricus and Lb. plantarum.48-50 In contrast, beneficial effects of DCCD on

329

GAD activity were distinct at concentrations of 0.2 mM and below, albeit the

330

inhibitory effects were observed as the concentration was further increased. In

331

particular, the cells incubated with 0.1 mM of DCCD had the highest GAD activity,

332

which was about 1.28-fold greater than the activity measured in untreated cells. In

333

addition, similar phenomena have also been observed in the control strain (Lb.

334

brevis/pMG36e), although there was a little difference in the increased levels of GAD

335

activities between the two strains (supplementary data Figure S2). It is noteworthy

336

that the DCCD has no effect on the activity of purified recombinant GAD (Figure S2),

337

which means that the cell-bound GAD activity is associated with the FoF1-ATPase

338

activity. According to the activity measurements of ATPase (Figure 3), the cells

339

exposed to high concentrations of DCCD showed less GAD activity than the control

340

group which probably owing to less available cellular energy for glutamate transport

341

in this strain.48 On the other hand, the cells incubated with low concentrations of

342

DCCD had higher GAD activity, which might be due to the accelerated shift of

343

protons consumption from FoF1-ATPase to GAD pathway as a compensatory response



344

to their acid stress. These results extend earlier observations with Lb. plantarum that

345

there existed a close correlation between FoF1-ATPase and GAD system in acidic

346

conditions48 and shed a new light on the issue that enhanced production of GABA in

347

Lb. brevis by weakening the ATPase activity is possible. In other words, the aim of

348

enhanced production of GABA in Lb. brevis by adopting the physiology-oriented

349

engineering strategy is expected.

350

Figure 3 and Figure S2

351

Isolation of FoF1-ATPase-defective mutants of Lb. brevis/pMG36e-gadA with

352

higher GAD activities

353

According to the contribution of reduced FoF1-ATPase activity to the cell-bound

354

GAD activity, there was an increasing interest to develop a procedure for effectively

355

screening the FoF1-ATPase-defective mutants of Lb. brevis/pMG36e-gadA with higher

356

GABA productivity. Previously, selection for resistance to neomycin has been used in

357

the isolation of strains defective in FoF1-ATPase activity due to the inability of these

358

mutants to generate sufficient energy to uptake the antibiotic.48, 51 Therefore, the key

359

obstacle remaining to be solved is how to rapidly select the desirable strains from the

360

mutants libraries caused by neomycin. In light of our previous work,37 a pH-sensitive

361

colorimetric assay based on detecting the pH increase that occurs as the cell catalyzed

362

proton consumption reaction proceeds has been established to measure the cell-bound

363

GAD activity using a 96-well microplate format. As described in Figure 4, the pH

364

increase is reflected by the color change of the indicator BCG whose color profile

365

falls into the pH range of GAD activity. The color change can be detected by a

366

spectrophotometer (Figure 5), and thus enables the GAD activity measurements to be

367

made more effectively. Using the microplate format, the absorbance at 620 nm could

368

be monitored, allowing the qualitative or quantitative determination of GAD activity.


Page 16 of 32

Page 17 of 32


369

As expected, after treatment with 800 mg/mL neomycin sulfate hydrate,

370

approximately 1300 spontaneous neomycin-resistant mutants were obtained with a

371

frequency of 5.53×10-6. Subsequently, 95 colonies of different sizes and the parent

372

strain were meticulously picked into a 96-deep-well multiwell plate and then

373

underwent the optimized procedure as described in materials and methods. Among the

374

tested mutants, strain numbers A6 and D10 exhibited higher absorbance at 620 nm

375

(Figure 5), which was in line with the obvious color change (Figure S3). Thus, the

376

two mutants identified as NRA6 and NRD10 that met the selection criteria were used

377

for further trials. Figure 4, Figure S3 and Figure 5

378 379

Membrane

ATPase

activities

and

internal

pH

380

FoF1-ATPase-defective mutations under acidic conditions

of

the

representative

381

The beneficial effects of reduced ATPase activity on GAD activity open a new

382

avenue for improved GABA productivity of Lb. brevis. To get an insight into the

383

cause of enhanced GAD activity, the ATPase activities in membranes of the

384

representative FoF1-ATPase-defective mutations and parent strain were determined at

385

pH values ranging from 4.5 to 7.0. The result was shown in Figure 6A. Obviously, the

386

ATPase activities of all mutant strains were significantly lower than that of parent

387

strain at all conditions and at pH 4.5 the ATPase activity of all mutants decreased

388

below the values at other pHs, as was the case of Lb. plantarum.48 However, in

389

contrast to Lb. helveticus,51 there was no difference in the optimal pH of ATPase

390

among the strains examined. All membranes of the three strains showed maximum

391

ATPase activity at around pH 5.5. And at pH 5.5 the ATPase activity of membranes

392

from the parent strain was about 1.26-fold and 1.35-fold greater than that of strains

393

NRA6 and NRD10, respectively. In addition, measurements of intracellular pH (pHin)



394

values at three different external pHs (pHout) were also recorded for the

395

FoF1-ATPase-defective mutants and the parent strain. As expected, the higher pHout,

396

the higher pHin was observed (Figure 6B). The largest differences among the three

397

strains were observed at pHout 5.0, for which the parent was able to maintain a pHin

398

much higher than the mutants NRA6 and NRD10. Remarkably, the parent was able to

399

maintain the pHin above 6 when the pHout was 5.0, whereas NRA6 and NRD10 could

400

maintain a ∆pH of only 0.41 and 0.67, and it is likely that acidification of the

401

cytoplasm under acidic condition affects the physiology of the mutant strains

402

considerably.48, 50 Moreover, the results not only correlated well with the notion that

403

relatively lower ATPase activity might be beneficial for GABA production by Lb.

404

brevis, but also agreed with the conventional view that the FoF1-ATPase plays an

405

essential role in the regulation of intracellular pH in LAB.5 Particularly, as a result of

406

the higher absorbance at 620 nm (Figure 5), it is worthy to pay more attention to

407

investigate the characterization of GABA production in strain NRA6.

408

Figure 6 A and B

409

Enhanced production of GABA in the FoF1-ATPase defective mutant of Lb. brevis

410

NRA6

411

To confirm that gadA of Lb. brevis encoded glutamate decarboxylase and

412

weakened ATPase activity contributes to GAD activity, the GABA production of Lb.

413

brevis/pMG36e-gadA and mutant NRA6 in batch culture were compared to the wild

414

type strain. As described above, samples of the Lb. brevis that grown in GYP

415

fermentation media under acidic conditions (pH 5.2) were collected at different

416

growth phases, and then the corresponding cell-bound GAD activities as well as the

417

amount of GABA were determined. As shown in Figure 7A and 7B, the recombinant

418

strain Lb. brevis/pMG36e-gadA grew as well as the wild strain after inoculation,


Page 18 of 32

Page 19 of 32


419

although the slight decrease in cell densities were observed during different growth

420

phases. In contrast, there was a significant difference in the cell densities of the

421

cultures between the wild strain and the FoF1-ATPase-defective mutant NRA6 after

422

incubation. The cell density of wild strain was about 1.30-fold higher than the mutant

423

strain in the GYP fermentation media after 36 h incubation. Moreover, deprivation of

424

MSG from the GYP broth resulted in a further decrease in the rate and extent of

425

growth for strain NRA6 (Figure 7C). Similar to previous work that has shown the

426

mutants defective in FoF1-ATPase were impaired for survival at low extracellular

427

pH,47 our results also demonstrated that the expulsion of protons mediated by

428

FoF1-ATPase is indispensable for acid stress defense in Lb. brevis, although a

429

compensatory response to acid stress by the GAD pathway was observed.

430

As for the activities of GAD, the recombinant strain Lb. brevis/pMG36e-gadA

431

(Figure 7B) and mutant NRA6 (Figure 7C) showed the similar trends with that of wild

432

strain (Figure 7A) during the course of batch fermentation. Increased cell-bound GAD

433

activity was observed throughout the exponential phase. The GAD activity of Lb.

434

brevis was the highest in the late exponential growth phase, and then a continuous

435

decrease was found from the start of the stationary. Remarkably, strain NRA6

436

revealed higher GAD activities than that of wild strain and Lb. brevis/pMG36e-gadA

437

during the exponential and stationary phase of growth. Also, volumetric productivity

438

of 1.70 g/L/h obtained in cultivation at pH 5.2 for NRA6 was higher than those

439

obtained for Lb. brevis/pMG36e-gadA (1.61 g/L/h) and the wild strain (1.39 g/L/h)

440

during exponential growth phase. As expected, relatively higher GABA concentration

441

in the culture medium and conversion rate of Lb. brevis NRA6 (43.65 g/L, 98.42%)

442

than Lb. brevis/pMG36e-gadA (41.49 g/L, 93.55%) and wild type strain (35.81 g/L,

443

80.74%) was also observed at 48 h of cultivation, although the difference in the



444

GABA concentration is small during the batch fermentation process. In consideration

445

of the conversion rate of MSG to GABA, we think that maintaining the substrate

446

concentration in certain desired ranges during the fermentation process would be a

447

key parameter for effective GABA production by strain Lb. brevis NRA6. Therefore,

448

there is still a great potential for improving the GABA production of this mutant strain

449

by optimizing fermentation conditions, such as fed-batch fermentation. Moreover,

450

diminished cell viability usually results in negative consequences, for example,

451

sluggish fermentation and lower productivity.9 However, it is noteworthy that our aim

452

of enhanced production of GABA in Lb. brevis was achieved by constructing a

453

pertinent FoF1-ATPase deficient mutant strain, which not only demonstrates how the

454

acid stress response mechanisms of LAB can be employed to develop cell factories

455

with improved GABA production efficiency, but also contributes to research into the

456

development of the physiology-oriented engineering. Figure 7 A, B and C

457 458

ASSOCIATED CONTENT

459

Supporting Information

460 461

The Supporting Information is available free of charge via the internet at http://pubs.acs.org.

462

Figure S1: SDS-PAGE analysis of production of GAD proteins. Figure S2:

463

Effects of DCCD on the cell-bound GAD activity of Lb. brevis/pMG36e (control

464

strain) and the relative activity of recombinant GAD that purified from IPTG-induced

465

E. coli BL21/pET28a-gadA. Figure S3: Different isolated FoF1-ATPase-defective

466

mutants of Lb. brevis/pMG36e-gadA checked for their GABA synthesis capability.

467


Page 20 of 32

Page 21 of 32


468

AUTHOR INFORMATION

469

Corresponding Author

470

*(L.-H. Mei and Prof. S.-J. Yao) Phone/Fax: +86 571 87951982.

471

E-mail: [email protected], and [email protected].

472

Funding

473

This work was supported by National Natural Science Foundation of China (Nos.

474

21176220, 31470793, 31670804) and Natural Science Foundation of Zhejiang

475

Province (No. Z13B060008).

476

Notes

477

The authors declare no competing financial interest.



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acid and gamma-aminobutyric acid. Process Biochem 2015, 50, 1523-1527. 39. Marquez, F. J.; Quesada, A. R.; Sanchezjimenez, F.; Decastro, I. N., Determination of 27 dansyl amino acid derivatives in biological fluids by reversed-phase

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Page 24 of 32

Page 25 of 32


Figure captions Figure 1. Genetic organization of the gad gene cluster in the genome of Lb. brevis CGMCC1306 (a), and the construction of recombinant plasmids based on the lactobacilli expression vector pMG36e containing the gadB gene (b), gadA gene (c), gadC gene (d), gadCB segment (e) and gadCA segment (f). Figure 2. The cytoplasmic GAD activity of Lb. brevis. Cells were collected at exponential phase (EXP.; 24 h), and then disintegrated by passing them three times through a French pressure cell at 20,000 psi. Subsequently, the supernatant fraction was subjected to the determination of GAD activity. One unit (U) of GAD activity was defined as the amount of enzyme that produced 1 µM of GABA in 1min. The specific activity is expressed as U/mg of protein. Figure 3. Effects of DCCD on the cell-bound GAD activity and membrane ATPase activity of Lb. brevis/pMG36e-gadA. The GAD and ATPase activity was measured at pH 4.8 and pH 5.5 in the absence or presence of DCCD, respectively. One unit (U) of GAD activity was defined as the amount of cells that produced 1 µM of GABA in 1 min at pH 4.8. Specific activity of GAD was defined as U/mg dry cell weight (DCW) cells. The ATPase activity is expressed as units per mg of protein. Error bars represent standard deviations experiments with three different batches of cells or membrane vesicles. Figure 4. BCG-based colorimetric method for the determination of the cell-bound GAD activity of Lb. brevis. BCG, which has a pKa value of 4.7, changes from yellow to blue within the pH range 3.8 to 5.4. Figure 5. Absorption spectra of protonated (d) and deprotonated forms of BCG. Increased absorbance at 620 nm is due to the deprotonation of indicator via the glutamate decarboxylation reaction. The mutants NRA6 (a) and NRD10 (b) exhibited higher absorbance than the parent strain (c) at 620 nm. Figure 6. Effects of pH on the membrance ATPase activities of Lb. brevis/pMG36e-gadA and its mutants NRA6 and NRD10 (A), and the internal pH (pHin) at extracellular pH (pHout) 7.0, 6.0 and 5.0 (B). Figure 7. Growth, GABA production and GAD activity of Lb. brevis during fermentation with pH controlled at 5.2. (A): Lb. brevis/pMG36e; (B): Lb. brevis/pMG36e-gadA; (C): Lb. brevis NRA6/pMG36e-gadA.



Page 26 of 32

Tables Table 1. Bacterial strains and plasmids. Strain/plasmid

Characteristics

Source/reference

E. coli DH5α

Transformation host

Invitrogen

E. coli DH5α/pMG36e-gadA

E. coli DH5α harbouring pMG36e-gadA

This work

E. coli DH5α/pMG36e-gadB

E. coli DH5α harbouring pMG36e-gadB

This work

E. coli DH5α/pMG36e-gadC

E. coli DH5α harbouring pMG36e-gadC

This work

Strains

E. coli DH5α/pMG36e-gadCA

E. coli DH5α harbouring pMG36e-gadCA

This work

E. coli DH5α/pMG36e-gadCB

E. coli DH5α harbouring pMG36e-gadCB

This work

E. coli BL21/pET28a-gadA

E. coli BL21 harbouring pET28a-gadA

Lb. brevis CGMCC1306

Wild-type strain, originally isolated from raw milk

Lb. brevis/pMG36e

Control strain; Lb. brevis harbouring pMG36e

This work

Lb. brevis/pMG36e-gadA

Lb. brevis harbouring pMG36e-gadA

This work

Lb. brevis/pMG36e-gadB

Lb. brevis harbouring pMG36e-gadB

This work

Lb. brevis/pMG36e-gadC

Lb. brevis harbouring pMG36e-gadC

This work

Lb. brevis/pMG36e-gadCA

Lb. brevis harbouring pMG36e-gadCA

This work

Lb. brevis/pMG36e-gadCB

Lb. brevis harbouring pMG36e-gadCB

This work

Lb. brevis NRA6/pMG36e-gadA

Lb. brevis NRD10/pMG36e-gadA

FoF1-ATPase deficient Lb. brevis strain harbouring pMG36e-gadA FoF1-ATPase deficient Lb. brevis strain harbouring pMG36e-gadA

41 40

This work

This work

Plasmids pET28a-gadA

Kanr, gadA gene was cloned into pET28a

41

pMG36e

Emr, constitutive expression vector

31

r

pMG36e-gadA

Em , gadA gene was cloned into pMG36e

This work

pMG36e-gadB

Emr, gadB gene was cloned into pMG36e

This work

pMG36e-gadC

r

This work

r

Em , gadC gene was cloned into pMG36e

pMG36e-gadCA

Em , gadCA segment was cloned into pMG36e

This work

pMG36e-gadCB

Emr, gadCB segment was cloned into pMG36e

This work


Page 27 of 32


Table 2. Primers used for PCR amplification. Primer name

Primer sequence (5′ to 3′)

Restriction site

gadA-F1

ACGCGTCGACCATGGCTATGTTATATGGTAAAC

SalI

gadA-F2

GGATATGACGACTATATGGCTATGTTATATGGTAAAC

gadA-R

CCCAAGCTTTTAGTGAGTGAATCCGTATT

HindIII

gadB-F

ACGCGTCGACCATGAATAAAAACGATCAGGAAAC

SalI

gadB-R

CCCAAGCTTTTAACTTCGAACGGTGGTC

HindIII

gadC-F

ACGCGTCGACCATGGATGAAAATAAGTCTGAAC

SalI

gadC-R1

CCCAAGCTTCTACTTGGTTTCTTTTTCCAAC

HindIII

gadC-R2

CATATAACATAGCCATATAGTCGTCATATCCGTATTGC

Table 3. Cell-bound GAD activitiesa. Lb. brevis/pMG36e

Lb.

Lb.

Lb.

Lb.

Lb.

brevis/pMG36e-

brevis/pMG3

brevis/pMG36e-

brevis/pMG36e-g

brevis/pMG36e-

gadA

6e-gadB

gadC

adCB

gadCA

EXP.

0.717±0.023

0.811±0.047

0.795±0.023

0.746±0.042

0.845±0.012

0.872±0.025

STAT.

0.485±0.024

0.913±0.052

0.890±0.049

0.505±0.033

0.834±0.020

0.825±0.039

a.

One unit (U) of GAD activity was defined as the amount of cells that produced 1 µM of GABA in 1min. Specific activity was defined as U/mg dry cell weight (DCW) cells.



Page 28 of 32

Figure 1

Figure 2

GAD activity (U/mg protein)

7

Lb. brevis/pMG36e Lb. brevis/pMG36e-gadA Lb. brevis/pMG36e-gadB Lb. brevis/pMG36e-gadC Lb. brevis/pMG36e-gadCA Lb. brevis/pMG36e-gadCB

6 5 4 3 2 1 0

Strains Figure 3

0.20 0.16

1.0

0.12 0.8 0.08 0.6

Cell-bound GAD activity

0.04

ATPase activity

0.4

0.00 0 0.010.030.050.07 0.1 0.2 0.3 0.5 0.7 1 DCCD (mM)


ATPase activity Pi µmol/min/mg protein

Cell-bound GAD activity (U/mg DCW)

1.2

Page 29 of 32


Figure 4

Figure 5

3.0

Absorbance

2.5 2.0

(a) NRA6 (b) NRD10 (c) Parent strain (d) Protonated

1.5 1.0 0.5 0.0 400 450 500 550 600 650 700 750 800 Wavelength (nm)

Figure 6

ATPase activity Pi µmol/min/mg protein

(A)

0.25

Parent strain NRA6 NRD10

0.20 0.15 0.10 0.05 0.00 4.5

5.0

5.5

6.0

6.5

pH


7.0


(B) 8

Parent strain NRD10 NRA6

7 pHin

Page 30 of 32

6 5 4

7

6 pHout

5

Figure 7 1.5

80

OD600

60

OD600 GABA MSG GAD activity

6

50 40

4

30 20

2

1.0

0.5

10

0 6

(B)

MSG/GABA (g/L)

70 8

12

18

24 30 36 Time (h)

42

0

0.0

80

1.5

48

10

OD600

6

OD600

60

GABA MSG GAD activity

50 40

4

30 20

2

MSG/GABA (g/L)

70 8

1.0

0.5

10 0

0 6

12

18

24 30 36 Time (h)

42

48


0.0


10


(A)

(C)

10

GABA MSG GAD activity

OD600

8

OD600 with MSG OD600 without MSG

80 70 60 50

6

40 4

30 20

2

1.5

1.0

0.5

10 0

0 6

12

18

24 30 36 Time (h)

42

48


0.0



MSG/GABA (g/L)

Page 31 of 32


TOC Graphic


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