Malate Translocator (OMT1) in

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The Role of Glucose and 2-Oxoglutarate/malate Translocator (OMT1) in the Production of Phenyllactic Acid and pHydroxyphenyllactic Acid, Two Food-borne Pathogen Inhibitors Ya Dao, Ke Zhang, Xiafei Lu, Zebao Lu, Chenjian Liu, Min Liu, and Yi-Yong Luo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01444 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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Journal of Agricultural and Food Chemistry

1

The Role of Glucose and 2-Oxoglutarate/malate Translocator (OMT1)

2

in the Production of Phenyllactic Acid and p-Hydroxyphenyllactic

3

Acid, Two Food-borne Pathogen Inhibitors

4 †,║

5

Ya Dao,

Ke Zhang,

6

Yiyong Luo*,†

†,║

Xiafei Lu,

†

Zebao Lu,

§

Chenjian Liu,

†

Min Liu,

#

and

7 8

†Faculty

9

Technology, Kunming 650500, P. R. China

of Life Science and Technology, Kunming University of Science and

10

§Department

11

P. R. China

12

#Shandong

of Laboratory Medicine, Chuxiong Medical College, Chuxiong 675005,

Tobacco Monopoly Bureau (Company), Jinan 250101, P. R. China

13 14

║These

authors contributed equally to this work

15 16

*To

17

+86-871-65920759. E-mail: [email protected]

whom correspondence should be addressed. Phone: +86-871-65920759. Fax:

18 19 20 21 22 1

ACS Paragon Plus Environment


23

ABSTRACT: This paper aims to uncover how glucose affected the production of

24

phenyllactic acid (PLA) and p-hydroxyphenyllactic acid (p-OH-PLA). The highest

25

yields of PLA (68.53 mg/L) and p-OH-PLA (50.39 mg/L) were observed after

26

Lactobacillus plantarum strain YM-4-3 fermentation in media containing 30 and 10

27

g/L glucose, respectively. Additionally, the antimicrobial activity of YM-4-3 against

28

food-borne pathogens and the NADH/NAD+ ratio were positively correlated with the

29

production of PLA and p-OH-PLA, respectively. In addition, a 2-oxoglutarate/malate

30

translocator coding gene (Omt1) was selected based on the qPCR results, and its

31

knockout mutant, compared with the wild-type strain YM-4-3, showed that the PLA

32

and p-OH-PLA production was decreased by 1.37-6.99 and 1.53-1.59 times,

33

respectively. This result indicated that OMT1 was involved in the biosynthesis of

34

PLA and p-OH-PLA. To conclude, this study suggests that glucose, NADH/NAD+

35

ratio and/or the Omt1 gene, PLA and p-OH-PLA production, and antimicrobial

36

activity contribute to a cause-and-effect relationship.

37

KEYWORDS: Lactobacillus plantarum, phenyllactic acid, p-hydroxyphenyllactic

38

acid, NADH/NAD+ ratio, 2-oxoglutarate/malate translocator

39 40 41 42 43 44 2


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46

INTRODUCTION

47

Lactic acid bacteria (LAB) represent a group of gram-positive bacteria that have long

48

been used as natural or selected starter cultures for food fermentation because of their

49

GRAS (generally regarded as safe) status, their ability to produce a range of

50

functional metabolites and influence the food flavor, and their antagonistic properties

51

which offer protection against food spoilage bacteria and molds.

52

phenyllactic acid (PLA) and p-hydroxyphenyllactic acid (p-OH-PLA) have received

53

growing interest in recent years due to their effective antimicrobial activity. PLA

54

occurs in honey and some fermented foods

55

towards some bacterial pathogens, such as Listeria (L.) monocytogenes,

56

Staphylococcus (S.) aureus and Escherichia (E.) coli,

57

food-borne fungi including Aspergillus (A.) flavus, Penicillium (P.) verrucosum and P.

58

citrinum etc. 2, 5 p-OH-PLA is the 4-hydroxy derivative of PLA, which also shows a

59

broad inhibitory activity against both bacterial and fungal pathogens. 6 However, PLA

60

is more effective than p-OH-PLA and shows a synergistic effect that enhances the

61

antimicrobial potential of these compounds.

62

potential for practical application in the food industry as novel biopreservatives.

63

2

1

In this respect,

and has a broad-spectrum inhibition

2, 7

3, 4

and a wide range of

Thus, PLA and p-OH-PLA have

PLA and p-OH-PLA are produced by LAB strains through phenylalanine (Phe) and 8

64

tyrosine (Tyr) degradation, respectively.

Transamination reaction is the first

65

catabolic step undergone by Phe and Tyr to produce phenylpyruvic acid (PPA) and

66

p-hydroxyphenylpyruvic acid (p-OH-PPA), respectively, in which the α-amino group

67

is transferred to a suitable acceptor, such as 2-oxoglutarate, by an aminotransferase 3



8

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68

(ATase).

PPA and p-OH-PPA are further reduced to PLA and p-OH-PLA by

69

hydroxyl acid dehydrogenases, such as lactate dehydrogenase (LDH). 9, 10

70

Among the biocatalytic approaches, PLA production can be significantly improved

71

by the addition of precursors, such as Phe and PPA, to the growth medium, 9, 11, 12 and

72

the effect of PPA was remarkably better than that of Phe. 11, 12 In particular, PLA yield

73

increased 14-fold in Lactobacillus sp. SK007 upon the addition of PPA instead of Phe

74

as substrate.

75

SK007 fermentation could significantly enhance the yield of p-OH-PLA, and the

76

direct precursor p-OH-PPA had a much better effect than Tyr. 6 In general, glucose is

77

not only an essential nutrient for bacterial growth, but is also involved in PLA and

78

p-OH-PPA bioproduction. In E. coli, an expanded shikimate pathway allows PLA and

79

p-OH-PPA production from glucose. 10 With regard to Lactobacillus spp., Mu et al., 12

80

found that PLA yield first increased and then decreased with increasing glucose

81

concentration, and 30 g/L glucose was the optimized concentration.

82

11

Similarly, Tyr and p-OH-PPA supplements during Lactobacillus sp.

To improve PLA and p-OH-PLA production, some enzymes and genes involved in 8

83

Phe and Tyr metabolism were purified/cloned and characterized. Yvon et al.,

84

reported

85

5’-phosphate-dependent enzyme and initiated the conversion of Phe and Tyr to PPA

86

and p-OH-PPA, respectively. LDH is one of the key enzymes responsible for PLA

87

and p-OH-PLA biosynthesis. Zheng et al., 9 cloned and expressed two LDH encoding

88

genes (LdhL and LdhD) and found that both the recombinant LDHs (L-LDH and

89

D-LDH) converted PPA to PLA with a similar catalytic efficiency. Li et al., 13 found

that

ATase

from

Lactococcus

(La.)

4


lactis

was

a

pyridoxal

Page 5 of 32


90

that NADH was necessary for the enzymatic production of PLA from PPA. In

91

addition, the result from the heterologous coexpression of LDH and formate

92

dehydrogenase in E. coli indicated that PLA production using LDH coupled with

93

NADH regeneration system was significantly higher than that by a single-enzyme

94

reaction. 14, 15 Conceivably, some genes coding for dicarboxylates transporters were

95

crucial for the yield of extra-cellular PLA and p-OH-PLA. The citrate transporter

96

(CitP) of LAB is such a transporter that catalyzes the exchange of citrate versus

97

L-lactate/PLA.

98

(OMT), was characterized to accumulate organic acids. 17 Therefore, it is worthwhile

99

to uncover the relationship between OMTs and the production of PLA and

100

16

Another transporter, a pea 2-oxoglutarate/malate translocator

p-OH-PLA.

101

Recently, the canonical pathway of PLA and p-OH-PLA biosynthesis has become

102

well known. However, the regulation process of PLA and p-OH-PLA bioproduction

103

remains uninvestigated. To solve this issue, at least in part, the effect of glucose on

104

PLA and p-OH-PLA production was first determined. Then, the expression profiling

105

of six genes was evaluated to screen the associated factors between glucose and PLA

106

and p-OH-PLA formation. Finally, one gene coding for a 2-oxoglutarate/malate

107

translocator (Omt1) was chosen and its role on PLA and p-OH-PLA production was

108

investigated. The experiments will help us to more fully understand the biosynthesis

109

process of PLA and p-OH-PLA.

110 111

MATERIALS AND METHODS 5



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112

Chemicals, Strains and Growth Conditions. PLA and p-OH-PLA were

113

purchased from Sigma-Aldrich (St. Louis, MO). Chromatographic grade methanol

114

and trifluoroacetic acid (TFA) were obtained from Sangon Biothech Co., Ltd.

115

(Shanghai, China). All the strains used in this study are listed in Table S1.

116

Lactobacillus (Lb.) plantarum strain YM-4-3

117

cultivated in de Man, Rogosa and Sharpe medium (MRS) (Oxoid, Hamshire, UK) or

118

chemically defined medium (CDM) prepared according to Teusink et al.

119

food-borne pathogens A. fumigatus HH6, P. expansum BNCC146144, Botrytis (B.)

120

cinerea BNCC338228, Fusarium (F.) oxysporum DQL, E. coli O157:H7 ATCC43895,

121

S. aureus KM3 and L. monocytogenes JS2 were used as indicator strains. The molds

122

were cultivated in PDB media (Coolaber, Beijing, China). E. coli was cultured in LB

123

broth (HKM, Guangdong, China), and strain DH5α was the host cell for plasmid

124

construction. S. aureus and L. monocytogenes were grown in BHI broth (Oxoid,

125

Hamshire, UK). Solid media were prepared by adding agar (18 g/L) to the

126

corresponding broth. When required, erythromycin was used at a concentration of 500

127

μg/mL for E. coli and 5 μg/mL for YM-4-3.

18

(hereafter referred as YM-4-3) was

19

The

128

Preparation of YM-4-3 Cell-free Supernatant. The YM-4-3 cell suspension (1.5

129

× 106 CFU/mL) was inoculated (4‰, v/v) in 100 mL of modified MRS or CDM broth

130

where the glucose concentration was changed to 0, 10, 20, 30, 40, 50, 60 or 70 g/L.

131

After incubation at 37 °C for 48 h, the cell-free supernatant (CFS) was prepared by

132

centrifugation (10,000g for 10 min; 4 °C) and sterile filtration using a 0.45 μm filter

133

(Millipore, Billerica, MA). Subsequently, the CFS was used for further high 6


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134

performance liquid chromatography (HPLC) analysis and antimicrobial activity

135

investigation. To avoid the impact of H2O2 and bacteriocins on the antimicrobial

136

activity evaluation, the CFS, which was obtained from YM-4-3 cultivated in mMRS0,

137

mMRS30 and mMRS60 (the modified MRS whose glucose concentration was 0, 30 or

138

60 g/L, respectively), was treated in succession using 1 mg/mL proteinase K at 37 °C

139

for 2 h, 1 mg/mL catalase at 25 °C for 30 min, and heat to inactivate the residual

140

enzymes. Then, the treated CFS was centrifuged at 10,000g for 10 min and filtered

141

with a 0.45 μm filter. The filter liquor was designated as tCFS.

142

Determination of PLA and p-OH-PLA Production. The assessment of PLA and

143

p-OH-PLA production in CFS was determined by a HPLC system (Agilent

144

Technologies Inc, Palo Alto, CA). Aliquots of 10 μL were injected onto an Agilent

145

Eclipse DB-C18 column (4.6 × 250 μm). Linear gradient elution was used with

146

solvent A (water + 0.05% TFA) and solvent B (methanol + 0.05% TFA) at a

147

temperature of 30 °C and a flow rate of 1 mL/min. The gradient profile was as follows:

148

(1) 0–6 min, 72% A + 28% B; (2) 6–13 min, 62% A + 38% B; (3) 13–18 min, 62% A

149

+ 38% B; (4) 18–20 min, 100% B; (5) 20–30 min, 100% B; and (6) 30–40 min, 72%

150

A+ 28% B. PLA and p-OH-PLA were monitored at 210 nm and their concentrations

151

were determined by integrating the calibration curves obtained from the standards.

152

Evaluation of YM-4-3 Antimicrobial Activity in vitro. The microdilution method

153

was employed to determine the antimicrobial activity. For the antifungal activity test,

154

the fungal conidia were collected according to our previous study

155

day-old PDA (Coolaber, Beijing, China) cultures and prepared with sterile water to 7


20

from 7 to 14


156

produce 1.5 × 105 conidia/mL suspension. A total of 20 µL of the conidial suspension

157

and 130 µL YM-4-3 CFS or tCFS were added into a well of sterile, disposable,

158

multiwell microdilution plates (96 wells; Corning Incorporated, Corning, NY). For

159

evaluation of the antibacterial activity, E. coli O157:H7, S. aureus and L.

160

monocytogenes were cultivated overnight in LB, BHI and BHI media, respectively,

161

and bacterial culture with a density of 1.0 × 107 CFU/mL was obtained. The test

162

solution in a well of the microdilution plates contained 75 µL YM-4-3 CFS or tCFS,

163

1.5 µL bacterial culture and 73.5 µL LB (for E. coli O157:H7) or BHI (for S. aureus

164

and L. monocytogenes) media. The inoculated wells were prepared in triplicate. All

165

microdilution plates were incubated in a humid chamber at 28 °C for 72 h or at 37 °C

166

for 16 h for the antifungal and antibacterial activity test, respectively. The microbial

167

growth was recorded with photos and measured by determining the optical density at

168

600 nm with a microplate reader (BioTek, Winooski, VT). In each experiment, the

169

untreated control (fungal conidial suspension + MRS medium or bacterial cells +

170

MRS medium + LB/BHI medium) was included. Each experiment was repeated three

171

times. The antimicrobial activity was expressed as percentage of inhibition, which

172

was calculated as (Ac - At)/(Ac) × 100%, where Ac is the absorption value of the

173

untreated control and At is that of a treatment.

174

Determination of the NADH/NAD+ Ratio. YM-4-3 was grown in mMRS0,

175

mMRS30 and mMRS60 for 48 h, and cells from a 1 mL culture were harvested by

176

centrifugation at 10,000g for 1 min. The pellets were washed once with cold PBS and

177

resuspended with 800 μL of extraction buffer. The samples were homogenized with 8


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178

ultrasonication (Φ3, 45%, with sonication for 3 s and rest for 10 s, over a total

179

duration of 7 min) on ice. The homogenized samples were successively centrifuged at

180

4 °C at 10,000g for 5 min to remove insoluble material and to deproteinize with a 10

181

kDa cut-off spin column (Abcam, Cambridge, UK). The intracellular concentrations

182

of NADH and NAD+ were measured using the NAD/NADH Quantitation Kit

183

(Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. The

184

NADH and NAD+ levels were normalized to the protein concentrations.

185

RNA Extraction, cDNA Synthesis and Quantitative Real-time PCR (qPCR).

186

Six genes that were possibly involved in PLA and p-OH-PLA biosynthesis, two

187

aminotransferase (ATaseI and ATaseII), three lactate dehydrogenase (L1-LDH,

188

L2-LDH and D-LDH) and one 2-oxoglutarate/malate translocator (OMT1), were

189

selected, and their expression profiling was evaluated by qPCR. YM-4-3 was grown

190

in mMRS0, mMRS30 and mMRS60 for 16 h and then 5 mL of the culture was pelleted

191

down. The total RNA was extracted using Trizol reagent (Takara, Dalian, China), and

192

the first strand cDNA was synthesized using a Hiscript II Q RT SuperMix for qPCR

193

(+ gDNA wiper) Kit (Vazyme, Nanjing, China) as recommended by the manufacturer.

194

Further qPCR and calculations were essentially performed as previously described. 21

195

The 16S rRNA gene was used for an internal control and the primers are listed in

196

Table S1.

197

Cloning and Sequence Analysis of the Omt1 Gene. The total genomic DNA of

198

YM-4-3 was extracted by a DNAprep Pure Bacteria Kit (Bioteke, Beijing, China). An

199

Omt1 gene was amplified using the primer pair HC-F and HC-R (Table S1), and the 9



Page 10 of 32

200

PCR product was directly sequenced. The amino acid sequence was deduced using

201

DNAman software (Lynnon BioSoft, San Ramon, CA), and its theoretical isoelectric

202

point

203

(http://web.expasy.org/compute_pi/). The deduced amino acid sequence was

204

submitted to the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/BLAST) and

205

the search for homology was performed using the BLAST algorithms. The amino acid

206

sequences of OMT1 homologs from different bacteria and Spinacia (Sp.) oleraceae

207

chloroplasts were downloaded, and a neighbor-joining tree was constructed using the

208

MEGA 6.0 software package. 22 The transmembrane helices of OMT1 were detected

209

by TMHMM (v2.0c) (http://www.cbs.dtu.dk/services/).

210

and

molecular

weight

were

calculated

using

the

pI/MW

tool

Plasmid Construction and the Omt1 Gene Knockout. The gene knockout 23

211

plasmid was constructed similar to Mashburn-Warren et al.

212

approximately 1 kbp flanking Omt1 gene were amplified by PCR from the genome of

213

YM-4-3 using the primer pairs Up-F/Up-R and Dn-F/Dn-R (Table S1). The primer

214

Dn-F has a 5' tail homologous to the primer Up-R (Table S1). The flanking fragments

215

were fused together by overlapping PCR using primers Up-F and Dn-R with the

216

amplified fragments as the templates. The fused fragment was digested with SpeI and

217

EcoRI and subsequently ligated into thermosensitive plasmid pFED760 (a gift from

218

Michael J Federle, University of Illinois, USA, Table S1) that was digested with the

219

same restriction enzymes to create the Omt1 gene knockout construct, pKO. Then, the

220

plasmid pKO was transferred into YM-4-3 by electroporation, and the Omt1 gene

221

knockout strain was selected as described by Okano et al. 10


24

The fragments of

The resulting mutant,

Page 11 of 32


222

named ∆Omt1, was confirmed by PCR using the primer pairs V-F/V-R (Table S1)

223

and by sequencing the PCR products.

224

Cell Growth, Acid Production and Morphology Assays. The growth and acid

225

levels of ∆Omt1 and YM-4-3 cells in MRS and CDM broth were monitored by

226

measuring the OD600 and pH, respectively. A morphological analysis was investigated

227

using a scanning electron microscope (SEM) and transmission electron microscope

228

(TEM). Briefly, ∆Omt1 and YM-4-3 were grown in MRS broth at 37 °C for 16 h, and

229

the bacteria were harvested by centrifugation at 5,000g for 5 min at 4 °C. The cells, in

230

the following processes, were treated according to the method described by Wang et

231

al. 25 Finally, the bacteria were observed with a Hitachi S-3000N SEM (Tokyo, Japan)

232

at 10 kV in high-vacuum mode or JEOL JEM-1011 TEM (Tokyo, Japan) operated at

233

the 100 kV accelerating voltage.

234

Production of PLA and p-OH-PLA of the Omt1 Gene Knockout Strain. ∆Omt1

235

and YM-4-3 were cultivated in mMRS0, mMRS30 and mMRS60 at 37 °C for 48 h. For

236

the extra-cellular PLA and p-OH-PLA test, the CFS was prepared as described above.

237

For the intra- and extra-cellular PLA and p-OH-PLA tests, cell cultures of 10 mL

238

were disrupted by ultrasonication (Φ3, 45%, with sonication for 5 s and rest for 5 s,

239

over a total duration of 15 min) before centrifugation, and the CFS was then prepared

240

as described above. The PLA and p-OH-PLA levels in CFS were determined by the

241

HPLC method describe above.

242 243

RESULTS AND DISCUSSION 11



244

The PLA and p-OH-PLA Production are Affected by Glucose. Apparently,

245

glucose is the main carbon source for bacterial growth and should have a multifaceted

246

role on bacterial metabolites bioproduction. In this paper, the PLA and p-OH-PLA

247

yields of YM-4-3 responding to different glucose concentrations were investigated.

248

As shown in Figure 1, the yields of PLA and p-OH-PLA first increased and then

249

decreased with increasing glucose concentration when YM-4-3 was grown in the

250

modified MRS and CDM broth; glucose at 30 and 10 g/L was the optimal

251

concentration for PLA and p-OH-PLA production at the maximum output of 68.53

252

and 50.39 mg/L, respectively. These observations were consistent with a previous

253

report of Mu et al., 12 who demonstrated that glucose facilitated PLA production and

254

30 g/L was the optimal concentration. The significance of glucose for PLA and

255

p-OH-PLA production can be explained as follows. Generally, low glucose

256

concentration promotes bacterial growth, and increase in biomass is always in concert

257

with metabolites accumulation.

258

cofactor (NADH) regeneration from glycolysis, 9 and NADH provides the reducing

259

power for reductive product formation. For the yield decrease of PLA and p-OH-PLA

260

under > 30 and > 10 g/L glucose, respectively, the end-products inhibition and/or the

261

carbon metabolites (e.g., lactate, acetate and diacetyl) repression may be one of the

262

explanatory factors, as reported by de Felipe and Gaudu. 27

26

In addition, glucose is a good carbon source for

263

The Antimicrobial Activity is Affected by Glucose. As mentioned above, PLA

264

and p-OH-PLA are pathogen inhibitors whose production was affected by glucose.

265

This finding prompts us to investigate whether glucose accordingly will change the 12


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266

antimicrobial activity of YM-4-3. As shown in Figure 2 and Figure S1, the CFS and

267

tCFS from mMRS30, mMRS60 and mMRS0 constituted at descending order in

268

antimicrobial activity although the inhibition ratios were dependent on indicator

269

pathogens. This result indicates that glucose facilitates PLA and p-OH-PLA

270

production, which in turn affects YM-4-3 antimicrobial effect. In addition, the

271

inhibition ratios between CFS and tCFS were different in some conditions, such as

272

when CFS and tCFS were harvested from mMRS0 and B. cinerea, F. oxysporum and

273

E. coli O157:H7 worked as indicators (Figure 2), which indicated that hydroxyl acids

274

and other metabolites together play a key role in antimicrobial action. 5, 7

275

The NADH/NAD+ Ratio is Affected by Glucose. Using the methods described

276

above, the levels of NADH and NAD+ were measured. The concentrations of NADH

277

and NAD+ ranged from 1.43 to 220.68 nmole/mg/L, and with the increasing glucose

278

concentration, NADH showed a gradual increase, while NAD+ decreased first and

279

then increased, which made the largest ratio of NADH/NAD+ appear when YM-4-3

280

was grown in mMRS30 (Table 1). The fact of the NADH/NAD+ ratio being affected

281

by different carbon source was usually reported, which is the theoretical basis for

282

enhancing some microbial metabolites production. 28, 29 As glycolysis facilitates the

283

NADH regeneration and the conversion of α-keto acid and hydroxyl acid needs

284

NADH, 9, 10 it was speculated that glucose, the NADH/NAD+ ratio, and the production

285

of PLA and p-OH-PLA contributed to a cause-and-effect relationship.

286

The Expression of PLA and p-OH-PLA Biosynthetic-related Genes is

287

Regulated by Glucose. The result of qPCR showed that the expression levels of four 13



288

genes (AtaseI, LdhL1, LdhL2 and Omt1) increased as the glucose concentration

289

increased initially and decreased afterwards (Figure 3). Compared with AtaseI, LdhL1

290

and LdhL2, Omt1 showed the largest change rate of gene expression (Figure 3). When

291

YM-4-3 was grown in mMRS30, the expression levels were 44.3 and 4.51 times than

292

those in mMRS0 and mMRS60, respectively (Figure 3). The consistency of the

293

correlation of glucose concentrations and four genes expression levels with the

294

correlation of glucose concentrations and PLA and p-OH-PLA levels suggests that

295

four genes may be involved in PLA and p-OH-PLA production. As ATases and LDHs

296

were characterized in much of the literature,

297

biological functions were studied by gene knockout technology in the following

298

experiments.

8, 9, 14, 15, 30

Omt1 was chosen and its

299

Cloning, Sequencing and Phylogenetic Analysis of the Omt1 Gene. The

300

predicted open reading frame (ORF) of Omt1 is 1,419 bp, encoding a polypeptide of

301

472 amino acid residues, whose theoretic molecular weight and isoelectric point are

302

50.63 kDa and 9.14, respectively. The predicted polypeptide contains 14 putative

303

transmembrance segments in an α-helical conformation, suggesting that OMT1 is

304

located in the cell membrane. The result of homology search indicates that sequences

305

showing high homology with OMT1 are anion permease, 2-oxoglutarate/malate

306

translocator, and citrate transporter (data not shown). As the anion permease comes

307

from genome annotation data and no specific biological functions are characterized in

308

literature, the sequence similarity analysis of OMT1, Sp. oleraceae chloroplastic

309

2-oxoglutarate/malate translocator (SoOMT)

31

and La. lactis citrate transporter

14


Page 14 of 32

Page 15 of 32


310

(LlCitP) 32 was performed. The results showed that OMT1 was 35.73% and 12.78%

311

identical to SoOMT and LlCitP, respectively, suggesting that OMT1 was a

312

2-oxoglutarate/malate translocator. This conclusion was further supported by the

313

phylogenetic tree analysis (Figure S2). Interestingly, two other OMT1 homologs,

314

OMT2 and OMT3, were found in the YM-4-3 genome using a local BLAST search.

315

OMT1, OMT2 and OMT3 demonstrated 49.26%-69.98% homology with each other

316

and were clustered into three different subclades (Figure S2). The GenBank accession

317

numbers of Omt1, Omt2 and Omt3 were MH726204, MH726205 and MH726206,

318

respectively.

319

The Omt1-deficient Strain Construction and its Morphology Investigation.

320

After selection based on a temperature change according to Okano et al., 24 the mutant

321

(∆Omt1) losing a fragment in the middle of Omt1 ORF (Figure S3A) was obtained.

322

On agarose gel electrophoresis, the ∆Omt1 showed a smaller band compared with the

323

wild-type strain YM-4-3 (278 bp vs. 1424 bp) (Figure S3B), which corresponded with

324

the expected changes. These results and further DNA sequencing analyses (data not

325

shown) confirmed the deletion of the Omt1 gene. The strain growth ratio and acid

326

production ability had no obvious difference between ∆Omt1 and YM-4-3 (Figure S4).

327

For the morphological analysis, the SEM images revealed that the cell surface of

328

YM-4-3 was smooth and the cell size was uniform, while the ∆Omt1 cells showed

329

characteristics with different size and aggregation distribution and some with ruptured

330

cell walls, which lead to the cellular contents exudation (Figure 4A and B). The

331

phenomenon that the cell integrity of ∆Omt1 was damaged was further confirmed by 15



332

the TEM images (Figure 4C and D). These results suggest that the Omt1 gene is

333

involved in the cell morphogenesis of YM-4-3, which may be related to the identity of

334

the OMT1 membrane protein.

335

Production of PLA and p-OH-PLA in the Omt1-deficient Strain. To explore

336

whether OMT1 is related to the transport and biosynthesis of PLA and p-OH-PLA,

337

the intra- and/or extra-cellular PLA and p-OH-PLA levels were detected. As shown in

338

Figure 5, the intra- and extra-cellular production of PLA and p-OH-PLA was not

339

significantly different from the extra-cellular production when YM-4-3 or ΔOmt1 was

340

grown in the same media. The result indicated that the intra-cellular PLA and

341

p-OH-PLA was completely transported out of the cells once synthesized, and the

342

Omt1 gene disruption had no effect on the transport of PLA and p-OH-PLA. However,

343

the extra-cellular PLA production of YM-4-3 was increased to 1.37-6.99 times

344

compared with that of ΔOmt1 when the strains were cultivated in the same media

345

(Figure 5A). In comparison, the extra-cellular p-OH-PLA output of YM-4-3 was 1.53

346

and 1.59 times higher than that of ΔOmt1 when the strains were grown in mMRS30

347

and mMRS60, respectively (Figure 5B). These results together with the fact of the

348

Omt1 gene knockout without impacting the transport of PLA and p-OH-PLA

349

illustrated that OMT1 was involved in the biosynthesis of PLA and p-OH-PLA. As

350

mentioned above, OMT1 was identified as a 2-oxoglutarate/malate translocator. In

351

plant chloroplasts and plastids, the 2-oxoglutarate/malate translocator is responsible

352

for the importation of 2-oxoglutarate in exchange for stromal malate. 17, 31, 33 In Lb.

353

plantarum, OMTs may import 2-oxoglutarate into the cytosol and in counter 16


Page 16 of 32

Page 17 of 32


354

exchange with the export of malate, just like the citrate carrier CitT (an OMT1

355

homolog) works in E. coli.

356

intra-cellular 2-oxoglutarate level, which accordingly interferes with the conversion of

357

Phe and Tyr to PPA and p-OH-PPA, respectively, because 2-oxoglutarate is a suitable

358

and necessary α-amino group acceptor in amino acid metabolism. 8, 35 As a result of

359

the decreasing PPA and p-OH-PPA content, the production of PLA and p-OH-PLA

360

was consequentially reduced. This may be the reason that OMT1 affects the synthesis

361

of PLA and p-OH-PLA.

34

In this sense, Omt1 gene disruption will reduce the

362

In conclusion, glucose showed pleiotropic effects on the metabolism of YM-4-3,

363

including on the production of PLA and p-OH-PLA, the antimicrobial activity, the

364

NADH/NAD+ ratio and the expression of PLA and p-OH-PLA biosynthetic-related

365

genes. Among them, PLA and p-OH-PLA showed strong antimicrobial ability,

366

NADH supplied the reducing capability in the process of PLA and p-OH-PLA

367

biosynthesis, and OMT1 mediated the biosynthesis of PLA and p-OH-PLA and the

368

cell morphogenesis of YM-4-3. The data indicated that glucose, the NADH/NAD+

369

ratio and/or some genes (e.g., the Omt1 gene), the PLA and p-OH-PLA production,

370

and the antimicrobial activity were involved in the cause-and-effect relationship. To

371

our knowledge, this study reported for the first time the mechanism of glucose

372

affecting the production of PLA and p-OH-PLA from the perspectives of both

373

metabolites and genes.

374 375

ASSOCIATED CONTENT 17



Page 18 of 32

376

Supporting Information

377

The strains, plasmids and primers used in this study (Table S1), the growth of

378

food-borne pathogens in 96 well plate (Figure S1), the phylogenetic neighbor-joining

379

tree of OMTs (Figure S2), the schematic illustration of the Omt1 gene knockout

380

(Figure S3), and the growth and acid production curves of YM-4-3 and ∆Omt1

381

(Figure S4).

382 383

AUTHOR INFORMATION

384

Corresponding Author

385

*(Y.L.)

386

[email protected].

387

Author Contributions

388

║Y.

389

Funding

390

This work was supported by the National Natural Science Foundation of China (No.

391

31660451 and 31300068) and the Foundation of Key Scientific Research Project of

392

China Tobacco Yunnan Industrial Co., Ltd. (No. 2018XY03).

393

Notes

394

The authors declare no competing financial interest.

Phone:

+86-871-65920759.

Fax:

+86-871-65920759.

E-mail:

Dao and K. Zhang made equal contributions to this paper.

395 396 397 18


Page 19 of 32

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REFERENCES

399

(1) Crowley, S.; Mahony, J.; van Sinderen, D. Current perspectives on antifungal

400

lactic acid bacteria as natural bio-preservatives. Trends Food Sci. Tech. 2013, 33,

401

93–109.

402

(2)

Lavermicocca, P.; Valerio, F.; Visconti, A. Antifungal activity of phenyllactic

403

acid against molds isolated from bakery products. Appl. Environ. Microbiol. 2003, 69,

404

634–640.

405

(3) Ohhira, I.; Kuwaki, S.; Morita, H.; Suzuki, T.; Tomita, S.; Hisamatsu, S.;

406

Sonoki, S.; Shinoda, S. Identification of 3-phenyllactic acid as a possible antibacterial

407

substance produced by Enterococcus faecalis TH10. Biocontrol Sci. 2004, 9, 77–81.

408

(4) Ning, Y.; Yan, A.; Yang, K.; Wang, Z.; Li, X.; Jia, Y. Antibacterial activity of

409

phenyllactic acid against Listeria monocytogenes and Escherichia coli by dual

410

mechanisms. Food Chem. 2017, 228, 533–540.

411

(5) Cortes-Zavaleta, O.; Lopez-Malo, A.; Hernandez-Mendoza, A.; Garcia, H. S.

412

Antifungal activity of Lactobacilli and its relationship with 3-phenyllactic acid

413

production. Int. J. Food Microbiol. 2014, 173, 30–35.

414

(6) Mu, W.; Yang, Y.; Jia, J.; Zhang, T.; Jiang, B. Production of

415

4-hydroxyphenyllactic acid by Lactobacillus sp. SK007 fermentation. J. Biosci.

416

Bioeng. 2010, 109, 369–371.

417

(7) Axel, C.; Brosnan, B.; Zannini, E.; Peyer, L. C.; Furey, A.; Coffey, A.; Arendt,

418

E. K. Antifungal activities of three different Lactobacillus species and their

19



419

production of antifungal carboxylic acids in wheat sourdough. Appl. Microbiol.

420

Biotechnol. 2016, 100, 1701–1711.

421

(8) Yvon, M.; Thirouin, S.; Rijnen, L.; Fromentier, D.; Gripon, J. C. An

422

aminotransferase from Lactococcus lactis initiates conversion of amino acids to

423

cheese flavor compounds. Appl. Environ. Microbiol. 1997, 63, 414–419.

424

(9) Zheng, Z.; Ma, C.; Gao, C.; Li, F.; Qin, J.; Zhang, H.; Wang, K.; Xu, P.

425

Efficient conversion of phenylpyruvic acid to phenyllactic acid by using whole cells

426

of Bacillus coagulans SDM. PLoS One 2011, 6, e19030.

427

(10) Koma, D.; Yamanaka, H.; Moriyoshi, K.; Ohmoto, T.; Sakai, K. Production of

428

aromatic compounds by metabolically engineered Escherichia coli with an expanded

429

shikimate pathway. Appl. Environ. Microbiol. 2012, 78, 6203–6216.

430

(11) Li, X.; Jiang, B.; Pan, B. Biotransformation of phenylpyruvic acid to

431

phenyllactic acid by growing and resting cells of a Lactobacillus sp. Biotechnol. Lett.

432

2007, 29, 593–597.

433

(12) Mu, W.; Chen, C.; Li, X.; Zhang, T.; Jiang, B. Optimization of culture medium

434

for the production of phenyllactic acid by Lactobacillus sp. SK007. Bioresour

435

Technol. 2009, 100, 1366–1370.

436

(13) Li, X.; Jiang, B.; Pan, B.; Mu, W.; Zhang, T. Purification and partial

437

characterization of Lactobacillus species SK007 lactate dehydrogenase (LDH)

438

catalyzing phenylpyruvic acid (PPA) conversion into phenyllactic acid (PLA). J.

439

Agric. Food Chem. 2008, 56, 2392–2399.

20


Page 20 of 32

Page 21 of 32


440

(14) Yu, S.; Zhu, L.; Zhou, C.; An, T.; Jiang, B.; Mu, W. Enzymatic production of

441

D-3-phenyllactic acid by Pediococcus pentosaceus D-lactate dehydrogenase with

442

NADH regeneration by Ogataea parapolymorpha formate dehydrogenase. Biotechnol.

443

Lett. 2014, 36, 627–631.

444

(15) Zheng, Z.; Zhao, M.; Zang, Y.; Zhou, Y.; Ouyang, J. Production of optically

445

pure L-phenyllactic acid by using engineered Escherichia coli coexpressing L-lactate

446

dehydrogenase and formate dehydrogenase. J. Biotechnol. 2015, 207, 47–51.

447 448

(16) Pudlik, A. M.; Lolkema, J. S. Substrate specificity of the citrate transporter CitP of Lactococcus lactis. J. Bacteriol. 2012, 194, 3627–3635.

449

(17) Riebeseel, E.; Hausler, R. E.; Radchuk, R.; Meitzel, T.; Hajirezaei, M. R.;

450

Emery, R. J.; Kuster, H.; Nunes-Nesi, A.; Fernie, A. R.; Weschke, W.; Weber, H. The

451

2-oxoglutarate/malate translocator mediates amino acid and storage protein

452

biosynthesis in pea embryos. Plant J. 2010, 61, 350–363.

453

(18) Song, X. D.; Liu, C. J.; Huang, S. H.; Li, X. R.; Yang, E.; Luo, Y. Y.

454

Cloning, expression and characterization of two S-ribosylhomocysteine lyases from

455

Lactobacillus plantarum YM-4-3: implication of conserved and divergent roles in

456

quorum sensing. Protein Expr. Purif. 2018, 145, 32–38.

457

(19) Teusink, B.; van Enckevort, F. H.; Francke, C.; Wiersma, A.; Wegkamp, A.;

458

Smid, E. J.; Siezen, R. J. In silico reconstruction of the metabolic pathways of

459

Lactobacillus plantarum: comparing predictions of nutrient requirements with those

460

from growth experiments. Appl. Environ. Microbiol. 2005, 71, 7253–7262.

21



461

(20) Luo, Y.; Yang, J.; Zhu, M.; Yan, J.; Mo, M.; Zhang, K. Characterization of

462

mutations in AlHK1 gene from Alternaria longipes: implication of limited function of

463

two-component histidine kinase on conferring dicarboximide resistance. J. Microbiol.

464

Biotechnol. 2008, 18, 15–22.

465

(21) Yang, J.; Yin, Z. Q.; Kang, Z. T.; Liu, C. J.; Yang, J. K.; Yao, J. H.; Luo, Y.

466

Y. Transcriptomic profiling of Alternaria longipes invasion in tobacco reveals

467

pathogenesis regulated by AlHK1, a group III histidine kinase. Sci. Rep. 2017, 7,

468

16083.

469

(22) Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6:

470

molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 2013, 30,

471

2725–2729.

472

(23) Mashburn-Warren, L.; Morrison, D. A.; Federle, M. J. A novel

473

double-tryptophan peptide pheromone controls competence in Streptococcus spp. via

474

an Rgg regulator. Mol. Microbiol. 2010, 78, 589–606.

475

(24) Okano, K.; Zhang, Q.; Shinkawa, S.; Yoshida, S.; Tanaka, T.; Fukuda, H.;

476

Kondo, A. Efficient production of optically pure D-lactic acid from raw corn starch

477

by using a genetically modified L-lactate dehydrogenase gene-deficient and

478

alpha-amylase-secreting Lactobacillus plantarum strain. Appl. Environ. Microbiol.

479

2009, 75, 462–467.

480

(25) Wang, W.; He, J.; Pan, D.; Wu, Z.; Guo, Y.; Zeng, X.; Lian, L. Metabolomics

481

analysis of Lactobacillus plantarum ATCC 14917 adhesion activity under initial acid

482

and alkali stress. PLoS One 2018, 13, e0196231. 22


Page 22 of 32

Page 23 of 32


483

(26) Pinto, L.; Malfeito-Ferreira, M.; Quintieri, L.; Silva, A. C.; Baruzzi, F. Growth

484

and metabolite production of a grape sour rot yeast-bacterium consortium on different

485

carbon sources. Int. J. Food Microbiol. 2019, 296, 65–74.

486

(27) de Felipe, F. L.; Gaudu, P. Multiple control of the acetate pathway in

487

Lactococcus lactis under aeration by catabolite repression and metabolites. Appl.

488

Microbiol. Biotechnol. 2009, 82, 1115–1122.

489

(28) Bao, T.; Zhang, X.; Zhao, X.; Rao, Z.; Yang, T.; Yang, S. Regulation of the

490

NADH pool and NADH/NADPH ratio redistributes acetoin and 2,3-butanediol

491

proportion in Bacillus subtilis. Biotechnol. J. 2015, 10, 1298–1306.

492

(29) Zhang, J. H.; Zeng, X.; Chen, X. S.; Mao, Z. G. Metabolic analyses of the

493

improved epsilon-poly-L-lysine productivity using a glucose-glycerol mixed carbon

494

source in chemostat cultures. Bioprocess Biosyst. Eng. 2018, 41, 1143–1151.

495

(30) Zhu, Y.; Hu, F.; Zhu, Y.; Wang, L.; Qi, B. Enhancement of phenyllactic acid

496

biosynthesis by recognition site replacement of D-lactate dehydrogenase from

497

Lactobacillus pentosus. Biotechnol. Lett. 2015, 37, 1233–1241.

498

(31) Weber, A.; Menzlaff, E.; Arbinger, B.; Gutensohn, M.; Eckerskorn, C.;

499

Flugge, U. I. The 2-oxoglutarate/malate translocator of chloroplast envelope

500

membranes: molecular cloning of a transporter containing a 12-helix motif and

501

expression of the functional protein in yeast cells. Biochemistry 1995, 34, 2621–2627.

502

(32) Drici, H.; Gilbert, C.; Kihal, M.; Atlan, D. Atypical citrate-fermenting

503

Lactococcus lactis strains isolated from dromedary's milk. J. Appl. Microbiol. 2010,

504

108, 647–657. 23



505

(33) Kinoshita, H.; Nagasaki, J.; Yoshikawa, N.; Yamamoto, A.; Takito, S.;

506

Kawasaki, M.; Sugiyama, T.; Miyake, H.; Weber, A. P. M.; Taniguchi, M. The

507

chloroplastic 2-oxoglutarate/malate transporter has dual function as the malate valve

508

and in carbon/nitrogen metabolism. Plant J. 2011, 65, 15–26.

509

(34) Pos, K. M.; Dimroth, P.; Bott, M. The Escherichia coli citrate carrier CitT: a

510

member of a novel eubacterial transporter family related to the 2-oxoglutarate/malate

511

translocator from spinach chloroplasts. J. Bacteriol. 1998, 180, 4160–4165.

512

(35) Vermeulen, N.; Ganzle, M. G.; Vogel, R. F. Influence of peptide supply and

513

cosubstrates on phenylalanine metabolism of Lactobacillus sanfranciscensis

514

DSM20451 (T) and Lactobacillus plantarum TMW1.468. J. Agric. Food Chem. 2006,

515

54, 3832–3839.

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527


Figure Captions

528 529 530

Figure 1. The bioproduction yields of PLA and p-OH-PLA from YM-4-3 incubated in the modified MRS broth (A) and the modified CDM broth (B).

531

Figure 2. The inhibitory effects of YM-4-3 CFS and tCFS on food-borne pathogenic

532

fungi (A) and bacteria (B). * (P < 0.05), ** (P < 0.01) and *** (P < 0.001) are

533

considered significantly different from CFS or tCFS harvested from mMRS0.

534

Figure 3. The relative expression levels of PLA and p-OH-PLA biosynthetic-related

535

genes of YM-4-3. The expression level in mMRS0 was set as 1 and # denotes the

536

fold change relative to mMRS0 ≥ 2.

537

Figure 4. SEM (A and B) and TEM (C and D) micrographs of YM-4-3 (A and C) and

538

ΔOmt1 (B and D). The arrows in B and D indicate the leakage of cell contents

539

and cytolysis, respectively.

540

Figure 5. The intra- and extra-cellular PLA (A) and p-OH-PLA (B) levels of YM-4-3

541

and ΔOmt1. Significant differences are presented as *** (P < 0.001). Error bars

542

show standard deviations (SD) from three repeated experiments.

25



Page 26 of 32

Table 1. Determination of the Amount of NADH and NAD+ a

a

Media

NADH (nmol/mg/L)

NAD+ (nmol/mg/L)

NADH/NAD+ (1e-2)

mMRS0

1.43 ± 0.47

89.03 ± 0.61

1.61 ± 0.54

mMRS30

4.84 ± 0.34

45.94 ± 0.56

10.54 ± 0.88*

mMRS60

15.34 ± 0.27

220.68 ± 4.5

6.96 ± 0.27*

Each value is the mean of three parallel replicates ± standard deviation. Asterisks

indicate NADH/NAD+ ratios of YM-4-3 cultivated in mMRS30 and mMRS60 that are significantly different (P < 0.05) from YM-4-3 in mMRS0.

26


Page 27 of 32


Figure 1. The bioproduction yields of PLA and p-OH-PLA from YM-4-3 incubated in the modified MRS broth (A) and the modified CDM broth (B). 147x63mm (300 x 300 DPI)



Figure 2. The inhibitory effects of YM-4-3 CFS and tCFS on food-borne pathogenic fungi (A) and bacteria (B). * (P < 0.05), ** (P < 0.01) and *** (P < 0.001) are considered significantly different from CFS or tCFS harvested from mMRS0. 147x77mm (600 x 600 DPI)


Page 28 of 32

Page 29 of 32


Figure 3. The relative expression levels of PLA and p-OH-PLA biosynthetic-related genes of YM-4-3. The expression level in mMRS0 was set as 1 and # denotes the fold change relative to mMRS0 ≥ 2. 99x83mm (300 x 300 DPI)



Figure 4. SEM (A and B) and TEM (C and D) micrographs of YM-4-3 (A and C) and ΔOmt1 (B and D). The arrows in B and D indicate the leakage of cell contents and cytolysis, respectively. 119x96mm (300 x 300 DPI)


Page 30 of 32

Page 31 of 32


Figure 5. The intra- and extra-cellular PLA (A) and p-OH-PLA (B) levels of YM-4-3 and ΔOmt1. Significant differences are presented as *** (P < 0.001). Error bars show standard deviations (SD) from three repeated experiments. 177x58mm (300 x 300 DPI)



For Table of Contents Only 84x45mm (300 x 300 DPI)


Page 32 of 32

Malate Translocator (OMT1) in

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