Improving Expression of Bovine Lactoferrin N-Lobe by Promoter

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Agricultural and Environmental Chemistry

Improving Expression of Bovine Lactoferrin N-lobe by Promoter Optimization and Codon Engineering in Bacillus subtilis and its Antibacterial Activity Liang Jin, Rong-Zhen Zhang, Lixian Zhou, Lihong Li, Yan Xu, and Jiming Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02350 • Publication Date (Web): 15 Aug 2019 Downloaded from pubs.acs.org on August 15, 2019

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Journal of Agricultural and Food Chemistry

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Improving Expression of Bovine Lactoferrin N-lobe by

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Promoter Optimization and Codon Engineering in Bacillus

3

subtilis and its Antibacterial Activity

4

Liang Jin, Lihong Li, Lixian Zhou, Rongzhen Zhang*, Yan Xu, Jiming Li

5 6

1Key

7

of Biotechnology, Jiangnan University, Wuxi 214122, P. R. China

Laboratory of Industrial Biotechnology of Ministry of Education & School

8

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*Corresponding author: Rongzhen Zhang

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Tel: +86-510-85197660; Fax: +86-510-85864112

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Email address: Rongzhen Zhang, [email protected]

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Present address: School of Biotechnology, Jiangnan University, 1800

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Lihu Avenue, Wuxi City, China, 214122

14 15 16 17 18 19 20 21 22

1

ACS Paragon Plus Environment


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ABSTRACT

24

Bovine lactoferrin N-lobe plays an important key in the nonimmunological

25

defense system. In this work, the most suitable promoter Pveg was selected

26

and the fragment coding bovine lactoferrin N-lobe was optimized according to

27

codon bias of Bacillus. The recombinant plasmid pMA0911-Pveg-mBLF-N was

28

introduced

29

subtilis/pMA0911-Pveg-mBLF-N. The bovine lactoferrin N-lobe was highly

30

expressed at 28 oC for 15 h. Its purified protein was obtained with 16.5 mg/L

31

and a purity of 93.6% using ammonium sulfate precipitation, Ni-NTA and

32

molecular exclusion. About 200 ng/mL purified bovine lactoferrin N-lobe

33

completely inhibited cell-growth of Escherichia coli JM109 (DE3), 70.3% of

34

Pseudomonas aeruginosa CGMCC 1.6740 and 41.5% of Staphylococcus

35

aureus CGMCC 1.282. To our knowledge, this is the first report about active

36

expression, purification and characterization of bovine lactoferrin N-lobe in

37

safe bacterium B. subtilis, which opens an available application way in the

38

biomedical and food industries.

into

Baicillus

subtilis

168

to

create

B.

39 40

Keywords: Bovine lactoferrin N-lobe; Bacillus subtilis; expression; codon bias;

41

promoter optimization;

42 43 44 2


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INTRODUCTION

46

Lactoferrin (LF) has important functions related to its antimicrobial

47

activities which many scientists have confirmed in vivo and in vitro 1. It has

48

been named “nutraceutical protein” due to its remarkably important use as a

49

therapeutic agent in clinical settings 2. LF is capable of preventing

50

Gram-positive and -negative bacteria, and viruses such as HIV, parasites, and

51

fungi from their proliferation3. Very recently, LF was confirmed that it could

52

inhibit angiogenesis in a HT29 human colon tumor model to exert antitumor

53

effects 4. Lakshman et al. reported LF could resistant against plant pathogens

54

in transgenic plants 5.

55

LF is one of iron-binding proteins in transferrin family, which presents in

56

the whey protein fraction of milk 6, 7. It contains human lactoferrin (HLF), bovine

57

lactoferrin (BLF), camel lactoferrin and sheep lactoferrin etc. according to its

58

sources. The BLF protein backbone consists of 703 amino acids with a

59

theoretical molecular weight of about 80 kDa for its glycosylated protein. It

60

shared a high amino acid sequence identity of about 69% with HLF. However,

61

even though HLF and BLF showed the similar 3D structures, the fully folded

62

proteins are not entirely super-imposable7, 8. The BLF contains the N- and

63

C-lobes. Each lobe contains two domains, each domain has an Fe3+ binding

64

site. Lactoferricin is the antibacterial core of lactoferrin, which can be yielded

65

through pepsin-catalyzed cleavage. The N-lobe consists of the active domains

66

for exerting bactericidal function and heparin binding, while the C-lobe consists 3



67 68

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of a domain for performing hepatocyte binding and internalization functions. BLF is an important bioactive molecule in the nonimmunological defense

69

system of bovine 9. However, the concentration of BLF is very low. For

70

example, bovine colostrum has a highest production of 2–5 mg/mL BLF, while

71

normal milk only has 0.02 mg/mL 10. Among the main applications of BLF, it is

72

used as a nutritional additive for both animal and human consumption, which

73

was obtained naturally or as a recombinant additive, and other industrial

74

purpose including potentially in infant food. The development of preperation

75

strategies for recombinant BLF as a safe, effective drug and nutraceutical

76

protein attracted much interest in both research and industry. Therefore, the

77

development of strategies for the production of larger amounts of BLF was

78

required.

79

In recent years, genetic engineering technology has been rapidly

80

developed. Most commercia proteins are produced using the bioreactors

81

including the recombinant bacteria, yeast fungi, or animal cells. BLF is

82

considered as one of the most recombinantly produced proteins 11. BLF has

83

been expressed in several microscopic models. Kim et al. expressed the BLF

84

C-lobe in Rhodococcus erythropolis, purified and characterized the

85

recombinant BLF 12. Koo et al. expressed BLF N-lobe in a green alga of

86

Chlorella vulgaris 13. Rascón-Cruz’ group highly expressed BLF in Pichia

87

pastoris with antibacterial activity against Escherichia coli, Staphylococcus

88

aureus, and Pseudomonas aeruginosa in a small percentage 14.

4


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89

García-Montoya et al. used an Escherichia coli system for the expression of

90

BLF, and they obtained of BLF fractions with functional antibacterial activity 15.

91

However, for the application in food systems, it will be of more interest to

92

construct cell factories of heterologous genes in a safe strain, such as Bacillus

93

subtilis.

94

B. subtilis is the best studied GRAS (generally recognized as safe) strain,

95

which has been widely used for preparation of many enzymes of clinical or

96

industrial interest 16,17. It has prominent characteristics, such as

97

high-cell-density growth, well-established genetic manipulation, and available

98

large-scale preparation 18,19. Currently, many new expression toolboxs to tune

99

genetic expression have been broadened the applications of B. subtilis 20.

100

To realize high protein expression in B. subtilis, one of the major

101

strategies is the construction of expression systems under strong promoters.

102

Some promoters have proven to be highly successful for the over-expression

103

of proteins in B. subtilis 20. For example, Bongers et al. performed the high

104

expression under strictly control by subtilin 21. Bonnet’s group optimized a

105

genetic toolbox containing promoter libraries, including Pveg, PserA and PymdA

106

etc. to tune gene expression in B. subtilis 20. However, there is no any report

107

about the accommodation of BLF N-lobe to be highly expressed in B. subtilis

108

through promoter optimization. The other major strategy for high protein

109

expression is to select the preferred codons for microbials. Each

110

microorganism has its own favorable codons 22. Synonymous substitution of

5



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111

rare codons with those favorable ones can enhance target protein expression

112

in hosts 23 or increase the specific activity of enzyme 24. However, the BLF

113

N-lobe coding gene contains rare codons such as TTC, TCC, CCC etc 25. Thus,

114

both promoter and codon optimization could benefit BLF N-lobe expression in

115

B. subtilis.

116

In this study, five promoters on the plasmid pMA0911 were compared for

117

the expression of BLF N-lobe in B. subtilis system. And the coding fragment of

118

BLF N-lobe was optimized basing on the preferred codon of B. subtilis. The

119

high heterologous expression of BLF N-lobe was performed in B. subtilis. The

120

recombinant BLF N-lobe showed antimicrobial activity for the selected E. coli

121

JM109, Pseudomonas aeruginosa CGMCC 1.6740 and Staphylococcus

122

aureus CGMCC 1.282. To the best of our knowledge, this work reports the first

123

successful functional expression of BLF N-lobe in B. subtilis via

124

electrotransformation and shows antimicrobial activity for Gram-negative and

125

–positive bacteria.

126 127

128

METERIALS AND METHODS

129

Strains, Plasmids and Primers

130

Escherichia coli JM 109 (Invitrogen Co., Shanghai, China) was used as a

131

host for plasmid propagation. Bacillus subtilis 168 in our laboratory was used 6


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132

as the host for protein expression. Pseudomonas aeruginosa CGMCC 1.6740

133

and Staphylococcus aureus CGMCC 1.282 were purchased from China

134

General Microbiological Culture Collection Center (CGMCC, Beijing, China).

135

The two strains and E. coli JM109 (DE3) were used for the antimicrobial assay.

136

The plasmid pMA0911 with kanamycin resistance was used for expression

137

vector. All restriction enzymes were purchased from Takara Co. Ltd (Shanghai,

138

China). The recombinant strains, plasmids and primers used in this work were

139

summarized in Table 1.

140

Media and Growth Conditions

141

E. coli JM 109 was cultured at 37 °C in Luria–Bertani (LB) broth (10 g/L

142

tryptone, 5 g/L yeast extract, 10 g/L NaCl) and LB plates (20 mg/mL agar)

143

supplemented with kanamycin (50 μg/mL) as the selective marker. B. subtilis

144

168 was cultured at 37 °C and 180 rpm on LB broth and LB plates (20 mg/mL

145

agar) supplemented with 50 μg/mL kanamycin. E. coli BL21 (DE3) were

146

cultured at 37°C in LB broth. P. aeruginosa CGMCC 1.6740 and S. aureus

147

CGMCC 1.282 were cultured at 37°C using the following medium: 10 g/L

148

tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 5 g/L glucose.

149

Transformation of B. subtilis 168

150

Expression vector pMA0911 was propagated in E. coli JM109. The

151

plasmid was isolated from the positive clones. Linearized plasmid DNA of 1 μg

7



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152

was used to transform B. subtilis 168. The transformation was obtained by

153

electroporation using a MicroPulser BioRad® (BioRad, Hercules, CA, USA)

154

according to the manufacturer’s instructions. After 1 days of incubation in LB

155

broth with kanamycin, the positive clones were isolated for further

156

experiments.

157

Construction of BLF N-lobe Expression Vector with

158

Different Promoters

159

The fragment encoding BLF N-lobe and 5’-terminal promoters (Pcat, PsacB,

160

P43, Pveg and PserA) were chemically synthesized by the Takara Co., Ltd

161

(Shanghai, China). The promoter sequence was added at the 5’-terminal of

162

CAP binding site using overlap PCR technique. The synthesized DNA

163

fragment of BLF N-lobe was digested with BamH I and Mlu I and cloned into

164

the corresponding sites of an expression vector pMA0911. So the different

165

expression

166

pMA0911-P43-BLF-N, pMA0911-Pveg-BLF-N and pMA0911-PserA-BLF-N were

167

constructed. The five plasmids were transformed into the competent cells of B.

168

subtilis

169

subtilis/pMA0911-Pcat-BLF-N,

170

subtilis/pMA0911-P43-BLF-N, B. subtilis /pMA0911-Pveg-BLF-N and B. subtilis

171

/pMA0911-PserA-BLF-N.

plasmids

168

to

pMA0911-Pcat-BLF-N,

obtaining B.

the

pMA0911-PsacB-BLF-N,

recombinant

strains

B.

subtilis/pMA0911-PsacB-BLF-N,

B.

8


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172

173


Codon Optimization of BLF in B. subtilis Based

on

codon

usage 25,

bias

in

Bacillus

sp.

OY1-2

174

(http://www.kazusa.or.jp/codon)

the variant mBLF N-lobe was designed to

175

improve the protein production. In mBLF N-lobe, 16 types of rare codons (TTC,

176

TCC, TCA, TGC, TGG, CCC, CCG, CAG, ATC, ATA, ACC, AAG, GTC, GCC,

177

GAC and GGC) were substituted with the synonymous ones used at the

178

highest frequency which was referenced from the international DNA sequence

179

databases. The optimized codon sequence of mBLF N-lobe was chemically

180

synthesized. The expression plasmid pMA0911-Pveg-mBLF-N was constructed

181

and the recombinant strains B. subtilis /pMA0911-Pveg-mBLF-N were obtained

182

after nucleotide sequencing.

183

RNA Isolation and Real-Time Quantitative PCR

184

Cellular RNA was extracted and purified using a GeneMark kit (TaKaRa

185

Co., Ltd., Shanghai, China), and cDNA was synthesized with a GeneCopoeia

186

kit. Independent reaction mixtures were performed using the same cDNA for

187

both the target gene and internal control. After an initial denaturation (95°C for

188

10 min), 40 amplification cycles were performed, with each cycle including

189

denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and extension at

190

72°C for 45 s using a 7300 Real Time PCR System (Applied Biosystems,

191

Hercules,

192

5'-TCCAGACTCTGTGCCTTGTG-3'; R: 5'-TGTTCTCCCAGACTGTGTCG-3')

CA).

Oligonucleotide

primers

(F:

9



Page 10 of 41

193

were used for Real-Time Quantitative PCR to produce the sequence of 160 bp.

194

Relative gene expression levels were calculated by the 2−ΔΔCT method (where

195

CT = cycle threshold).

196

Batch Fermentation

197

To enhance the mBLF N-lobe production, batch fermentation was carried

198

out as follows: 200 mL of LB bloth supplemented with kanamycin (50 mg/L)

199

were inoculated with cells from a single transformant clone and incubated

200

overnight with shaking at 180 rpm. Then, the culture was added to a 5-L

201

fermentor, which contained 3000 mL of LB media supplemented with 50 mg/L

202

kanamycin. The culture was incubated at 37 °C on shaking for 3 h. Then it was

203

cultured for 15 h at 28 °C for the induction of BLF N-lobe expression.

204

Preparation of Crude Enzyme

205

The recombinant B. subtilis/pMA0911-BLF-N was cultured in LB medium

206

for 3 h at 37°C on a rotary shaker at 180 rpm, and was then cultured at 28 °C

207

on a rotary shaker at 180 rpm for 15 h. The cutures were collected by

208

centrifugation at 8000 ×g for 5 min at 4 °C. The cell pellets were suspended

209

and washed with 0.1 M potassium phosphate buffer (pH 7.0) for three times.

210

For crude enzyme preparation, the recombinant B. subtilis cells were

211

resuspended in 0.1 M potassium phosphate buffer containing 0.1 mM

212

β-mercaptoethanol and 2 μg/ml PMSF (pH 6.5). The collected cells were

10


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213

treated with 1 mg/mL lysozyme for 60 min at 4 °C , and was used for

214

sonication at 0 °C. The homogenate was centrifuged at 10,000×g for 40 min at

215

4 °C to obtain cell-free extracts. The obtained soluble extracts were stored at

216

-70 °C for further use.

217

Purification of Recombinant BLF-N and mBLF-N

218

The purification of the recombinant proteins were performed using the

219

following processes. The cell-free extracts were precipitated with 27% of

220

ammoniu sulfate resuspended and dialyzed against 10 vol of phosphate buffer.

221

The pooled extracts with a His6-tag at C-terminal were subjected to Ni-NTA

222

affinity chromatography (Pharmacia, Uppsala, Sweden) according to the

223

manufacture’s instructions. The pooled fractions were loaded on a SuperdexTM

224

200 (10/300 GL), which was equilibrated with the buffer (20 mM Tris-HCl, 150

225

mM NaCl, pH 7.5) using an ÄKTA Protein Purifier system (Pharmacia, Uppsala,

226

Sweden).

227

sulfate-polyacrylamide

228

concentration was measured by Bradford method

229

protein.

230

Measurement of the Antibacterial Activity

231 232

The

enzyme gel

was

assayed

electrophoresis

by

sodium

(SDS-PAGE). 26

The

dodecyl protein

using BSA as standard

The antibacterial activity of mBLF N-lobe was tested by the filter-disc plate assay method using three bacterial strains (E. coli JM109, P. aeruginosa

11



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233

CGMCC 1.6740 and S. aureus CGMCC 1.282) as the target cells. LB agar of 5

234

mL was cooled to 45 °C, mixed with 1 mL precultured cells, and then overlaid

235

on an LB plate. The total volume of plate was recorded as 6 mL. Sterile filter

236

discs were placed on the plate. The mBLF N-lobe in final concentrations of 100

237

ng/mL and 200 ng/mL were spotted on the filter disc. All plates were inverted

238

and incubated at an optimum temperature for 15 h. Antibacterial activity was

239

monitored by the size of growth inhibition zones. The antibacterial activity assay of mBLF N-lobe was carried out according

240 241

to the method described by Flores-Villaseñor et al 27 with minor modification.

242

Approximately 1×108 UFC per ml of each strain (E. coli JM109, P. aeruginosa

243

CGMCC 1.6740 and S. aureus CGMCC 1.282) was mixed with mBLF N-lobe

244

in a 96-well microplate containing LB broth. The mixture was incubated at 37

245

oC

246

concentration of mBLF-N in Tris-HCl buffer (pH 8.0) was 100 ng/ml and 200

247

ng/ml. The culture of E. coli JM109 without addition of mBLF N-lobe was used

248

as a control. Kanamycin was used at 60 ng/mL as a control of growth inhibition.

249

Cultures were incubated at 37 oC with a shaking of 200 rpm for 1 h and their

250

CFU was evaluated by serial 10-fold dilutions prepared in LB agar and

251

counting their clone number.

252

Statistical Analysis

253

for 2 h. And its OD600 value was recorded every 30 min. The final

Experimental data in triplicates or sextuplicates were analyzed using the

12


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254

Statistical Package for the Social Sciences v. 20.0 (SPSS/IBM Corp., Chicago,

255

IL). A one-way analysis of variance (ANOVA) was used to compare mean

256

values using a significance level of P < 0.05. The differences among the mean

257

values within groups were achieved using Tukey’s post hoc test.

258

259

RESULTS AND DISCUSSION

260

Choice of Five Constitutive Promoters for BLF N-lobe

261

Expression in B. subtilis

262

BLF has been called “nutraceutical protein” due to its remarkable

263

importance to have multiple properties and the potential use of therapeutic

264

protein 2, 3. With the increased need for larger amounts of BLF, it is urgent to

265

the develop strategies to improve BLF production through the heterologous

266

expression method. One of the major strategies is the construction of

267

expression systems with strong promoters 20, 21. The expression level of the

268

different enzymes can be tuned by promoter optimization 28. The BLF

269

(GenBank accession No. EU812318) molecule is proposed to contain N-lobe

270

and C-lobe. And the N-lobe contains the active domains with bactericidal

271

action and heparin binding function.

272

In this work, to improve the production of BLF N-lobe, we expressed BLF

273

N-lobe on vector pMA0911 with five different promoters in B. subtilis 168. The

274

coding gene of BLF N-lobe and the respective sequence of five promoters: Pcat, 13



Page 14 of 41

275

PsacB, P43, Pveg and PserA at its upstream were chemically synthesized. Then

276

the sequence was inserted into the vector pMA0911 with five constitutive

277

promoters Pcat, PsacB, P43, Pveg and PserA, resulting in five corresponding

278

plasmids. Then the recombinant plasmids were transformed into the

279

competent

280

subtilis/pMA0911-Pcat-BLF-N,

281

subtilis/pMA0911-P43-BLF-N, B. subtilis /pMA0911-Pveg-BLF-N and B. subtilis

282

/pMA0911-PserA-BLF-N were obtained after confirmed by DNA sequencing.

283

The changes in mRNA and protein expression of BLF N-lobe were

cells

of

B.

subtilis B.

168.

The

recombinant

strains

subtilis/pMA0911-PsacB-BLF-N,

B. B.

284

determined by RT-PCR technique. The mRNA and protein expression of BLF

285

N-lobe are shown in Figure 1. The promoters Pveg, P43 and PserA resulted in the

286

increases in mRNA of 240.2, 10.4, and 161.5%, and protein expression of 80.3,

287

3.5, and 55.4%, respectively, compared with Pcat as the control (P < 0.05). But

288

under the promoter PsacB, there were 48.2 and 25.4% decreases in mRNA and

289

protein expression compared with the control (P < 0.05). The protein

290

production of BLF N-lobe under the promoters P43, Pveg, PserA, Pcat and PsacB

291

were about 8.9, 15.5, 13.4, 8.6 and 6.4 mg per liter culture. It is important to

292

note that the promoter Pveg gave the highest increases in mRNA and protein

293

expression of BLF N-lobe. So the BLF N-lobe was expressed at the higher

294

level in B. subtilis /pMA0911-Pveg-BLF-N, B. subtilis /pMA0911-P43-BLF-N, B.

295

subtilis /pMA0911-PserA-BLF-N and B. subtilis /pMA0911-BLF-N than B. subtilis

296

/pMA0911-PsacB-BLF-N. And among the five types, the BLF N-lobe was

14


Page 15 of 41


297

expressed at the highest level in B. subtilis /pMA0911-Pveg-BLF-N (Figure 1).

298

Therefore, the recombinant B. subtilis /pMA0911-Pveg-BLF-N was used for

299

further codon optimization experiments.

300

Insert Figure 1

301

302

303

Codon Optimization of BLF-N in B. subtilis In some typical microorganisms, such as E. coli and Saccharomyces

304

cerevisiae, both synonymous and nonsynonymous substitution frequencies

305

correlate with expression levels 29, 30. The codon usage difference between the

306

target protein source and expression host has an affection on the protein

307

translation rate, frequently resulting in a low level of protein expression 30. So

308

codon engineering could change the protein expression components, modify

309

the translation frequency, and enhance the protein production.

310

To

further

improve

BLF

N-lobe

expression

through

the

gene

311

recombination method in B. subtilis, the other major strategy is to select the

312

preferred codons for the replacement of rare codons for microbials

313

analysis of the gene sequence of BLF N-lobe (the signal peptide was not

314

excluded in the sequence of BLF N-lobe) (Figure 2), it contains many rare

315

codons such as TTC, TCC, CCC etc for B. subitilis

316

optimization could benefit BLF N-lobe expression in B. subtilis. Based on

317

codon usage bias in Bacillus sp. OY1-2 (http://www.kazusa.or.jp/codon) 25, the

25.

31.

By

Thus, the codon

15



Page 16 of 41

318

variant mBLF N-lobe was designed to improve the protein production. The

319

mutations were designed in its coding genes as follows: in mBLF N-lobe, 16

320

types of rare codons were substituted with the synonymous ones used at the

321

highest frequency according to the international DNA sequence databases

322

In details (as shown in Figure 2), TTC in BLF-N were substituted by TTA, TCC

323

and TCA by TCG, TGC and TGG by TGT, CCC and CCG by CCA, CAG by

324

CAA, ATC and ATA by ATT, ACC by ACA, AAG by AAA, GTC by GTT, GCC

325

by GCA, GAC by GAT, GGC by GGT respectively. The mutated genes were

326

chemically synthesized by Takara Co. (Shanghai, China) then constructed on

327

pMA0911 with the promoter Pveg in B. subtilis 168.

328

25.

Insert Figure 2

329

330

Improving BLF N-lobe Expression by Promoter and

331

Codon Optimization

332

The construction of expression plasmid pMA0911-Pveg-mBLF-N was

333

shown in Figure 3. The recombinant pMA0911-Pveg-mBLF-N was transformed

334

into the competent cells of B. subtilis 168. Then, the positive clone B. subtilis

335

/pMA0911-Pveg-mBLF-N was achieved after verified by DNA sequencing. The

336

changes in mRNA and protein expression of BLF N-lobe were determined by

337

RT-PCR technique under the promoter Pveg after codon optimization. The

338

mRNA and protein expression of BLF N-lobe are shown in Figure 4. The codon

16


Page 17 of 41


339

optimization resulted in the increases in mRNA of 164.3%, and protein

340

expression of 52.7%, respectively, compared with the sample before

341

optimization as the control (P < 0.05). The protein BLF N-lobe was produced at

342

29.6 mg per liter culture after the optimization of promoter and codon.

343

Therefore, the codon optimization improved the BLF N-lobe production in B.

344

subtilis, which facilitate the further its large-scale culture, protein purification

345

and antibacterial activity measurement. As shown in Figure 5, the sequence of

346

this BLF N-lobe protein was identical to those of lactoferrin from four different

347

species: 96.52% with buffalo lactoferrin (PDB ID:1CE2 and 1BIY) 32, 32 92.45%

348

with goat lactoferrin (PDB ID: 1JW1) 34, and 74.46% with camel lactoferrin

349

(PDB ID: 1DTZ) 35. The protein BLF N-lobe is a monomeric one, with the ability

350

to bind two Fe3+ irons, together with two CO32-; the synergistic relationship

351

between metal ion and anion binding is a unique feature of transferin chemistry.

352

The 3-D structure demonstrated the presence of lactoferricin, the antibacterial

353

core, within Phe18 to Phe42 and the existence of 21 α-helices and 26

354

β-strands (Figure 6). Our experimental data is probably the first documentation

355

of expression of BLF N-lobe, in viral promoter pMA0911 in a B. subtilis system

356

using electotransformation.

357

Insert Figure 3

358

Insert Figure 4

359

Insert Figure 5

360

Insert Figure 6

17



Page 18 of 41

361

362

Large-scale Production of BLF-N and mBLF-N and

363

their Purification

364

After promoter optimization and codon engineering, the recombinant

365

strain B. subtilis /pMA0911-Pveg-mBLF-N was cultured on a larger scale of 5-L

366

fermentor with B. subtilis /pMA0911-Pveg-BLF-N as a control. Because the

367

recombinant proteins were fused with N-terminal His6-tag, they were purified to

368

homogeneity in three steps including the precipitation with 27% of ammonia

369

sulfate, then Ni-NTA affinity chromatography and a SuperdexTM 200 (10/300

370

GL) chromatography. The proteins were purified to apparent homogeneity by

371

SDS-PAGE. SDS-PAGE analysis showed that the purified BLF and mBLF was

372

around 80 kDa with a purity of over 93.6%. Lipopolysaccharide (LPS)

373

contamination, checked by Limulus Amebocyte assay (LAL Pyrochrome kit,

374

PBI International), was found to be 0.5 ± 0.05 ng/mL. Calculations of the

375

corresponding A280 value and SDS-PAGE analysis revealed that the

376

recombinant B. subtilis /pMA0911-Pveg-mBLF-N increased protein production

377

with 16.5 mg per liter of culture, while B. subtilis /pMA0911-Pveg-mBLF-N

378

produced about 10 mg per liter of culture. The production of recombinant BLF

379

in B. subtilis was much higher than that of E. coli 15.and C. vulgaris13, but lower

380

than that of P. pastoris14.

381

18


Page 19 of 41

382


Antibacterial Activity

383

Antimicrobial activity was envaluated by the filter-disc plate assay way

384

using three bacterial strains (E. coli JM109, P. aeruginosa and S. aureus)

385

according to the methods described by Kim et al. 12. The purified mBLF N-lobe

386

exhibited significant antibacterial activity against the three bacterial strains.

387

The mBLF N-lobe at a concentration of 100 ng/mL exhibited antibacterial

388

activity towards E. coli growth inhibition (Figure 7A). And 200 ng/ml mBLF-N

389

inhibited all the above strains with a large inhibition halo (Figure 7B and

390

7C).The inhibition halo size of E. coli was obviously larger than those of P.

391

aeruginosa and S. aureus under the same treated condition . And the inhibition

392

size of S. aureus was the smallest among the three ones, which might be S.

393

aureus was negative-bacterium with its stronger cell walls. In a control

394

experiment of E. coli JM109 without the mBLF-N, no zones of antibacterial

395

activity were observed. The results suggested that mBLF-N presented the

396

bactericidal effects against E. coli JM109, P. aeruginosa and S. aureus. But

397

the detailed data of antibacterial activity was required to be determined.

398

The antibacterial activity of mBLF N-lobe was measured according to the 27.

399

method described by Flores-Villaseñor et al

After incubation in a 96-well

400

microplate, the capacity of mBLF N-lobe and BLF N-lobe to inhibit the

401

cell-growth of E. coli JM109, P. aeruginosa and S. aureus was tested in

402

medium. The results suggested that the same strains showed the same

403

susceptibility to mBLF N-lobe and BLF N-lobe (Figure 8), suggesting the 19



Page 20 of 41

404

promoter and codon optimization did not alter antibacterial activity. However,

405

the three strains showed different susceptibility to mBLF N-lobe (Figure 8). E.

406

coli JM109 (DE3) was the most susceptible to mBLF N-lobe with a growth

407

inhibition of 69.8 %, compared with 66.7% and 32.2% for P. aeruginosas and S.

408

aureus under the treatment of 100 ng/ml mBLF N-lobe. mBLF N-lobe of 200

409

ng/mL completely inhibited the cell-growth of E. coli. And it showed increased

410

inhibition rates of bacterial growth, which were 70.3% and 41.5% for P.

411

aeruginosas and S. aureus respectively. Kanamycin of 60 ng/mL showed

412

complete inhibition of bacterial growth of E. coli and P. aeruginosas, while

413

inhibited 53.7% S. aureus cell-growth. Antimicrobial activity is one of the various biological functions of BLF, and

414 415

its mechanism of action and immunomodulatory interactions are of particular

416

interest. BLF can inhibit bacterial growth by its iron binding activity and

417

restriction of iron metabolism 27. It was reported that lactoferricin B derived

418

from the N-lobe of bovine lactoferrin had bactericidal activity 36, since it is

419

attributed to disruption of cell membranes of Gram-negative or -positive bateria

420

by the basic residues arrayed along the outside of the lactoferricin B molecule

421

37.

422

bacteria.

423

So mBLF N-lobe showed the inhibition for the Gram-negative and –positive

The mBLF N-lobe expressed in B. subtilis showed the lower antibacterial

424

activity than that in P. pastoris, which might result from the lower degree of

425

glycosylation of mBLF N-lobe in B. subtilis than that in P. pastoris 14. However,

20


Page 21 of 41


426

it exhibited significantly higher than BLF C-lobe in E. coli, which might be due

427

to the very important role of BLF N-lobe in B. subtilis than its C-lobe in E. coli15.

428

So the B. subtilis expression system realized high-level expression of the BLF

429

N-lobe, and it demonstrated significant antibacterial activity against selected

430

strains of E. coli, P. aeruginosas and S. aureus. The successful expression

431

and charaterization of functional mBLF N-lobe expressed in B. subtilis opens a

432

prospect for the production of natural antimicrobial agents and facilitates its

433

structure–function relationship research. Food-grade production of the

434

iron-containing BLF would be favorable to the practical applications in food and

435

pharmaceutical industries.

436

Insert Figure 7

437

Insert Figure 8

438

439

440 441

ABBREVIATIONS AND NOMENCLATURE BLF, bovine lactoferrin; GRAS, generally recognized as safe; HLF, human lactoferrin; LF, Lactoferrin; Kan, Kanamycin

442 443

21



445

Page 22 of 41

Figure Captions:

446

Figure 1. Relative mRNA expression and BLF N-lobe production under

447

the different promoters. Relative mRNA expression level of BLF N-lobe under

448

Pcat promoter was used as the control normalized to a value of 1. Different

449

treatments within relative mRNA expression or protein production represent

450

significant differences (P < 0.05). Error bars indicate SD.

451 452

Figure 2. Codon usage analysis of BLF N-lobe gene sequence. Signal

453

peptide sequence was excluded. Coding sequences are shown for each of the

454

three codon variants that were expressed. The rare codons underlined with red

455

lines are replaced the preferred codons in blue based on the codon bias

456

according to the B. subtilis database (http://www.kazusa.or.jp/codon).

457 458

Figure 3. The construciton of the recombinant plasmid

459

pMA0911-Pveg-mBLF-N. The vector contains a synthetic mBLF N-lobe gene

460

for expression in B. subtilis 168. The Pveg was included as a promoter.

461 462

Figure 4. Relative mRNA expression and BLF N-lobe production by

463

codon optimization under the Pveg promoter. The relative mRNA expression

464

before optimization was used as the control normalized to a value of 1.

465

Different treatments within relative mRNA expression or protein production

22


Page 23 of 41

466


represent significant differences (P < 0.05). Error bars indicate SD.

467 468

Figure 5. The structure and sequence alignment of BLF N-lobe with

469

several selected members of transferring family. Left columns contain the

470

Protein Data Bank accession codes of the structures. 1CE2, buffalo lactoferrin;

471

1BIY, buffalo lactoferrin 2; 1JW1, goat lactoferrin; 1DTZ, camel lactoferrin.

472

Conserved residues are boxed with blue lines. Selected residue numbers of

473

the lactoferrin are labeled above the sequence. Secondary structure elements

474

of BLF are marked on the top of the alignment. This figure was prepared with

475

the program Espript (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi/).

476 477 478

Figure 6. The 3-D structure of recombinant BLF N-lobe protein. The existence of 21 α-helices and 26 β-sheets structure was confirmed.

479 480

Figure 7. Filter-Disc plate assay for detection of antibanterial activity

481

against E. coli JM109 (A), P. aeruginosa CGMCC 1.6740 (B) and S. aureus

482

CGMCC 1.282 (C).

483

The solid medium was cooled to about 45 oC and mixed with 1 mL of the

484

precultured target cells, and then overlaid on a plate. Sterile filter discs were

485

placed on the plate and the mBLF N-lobe, BLF N-lobe and kanamycin were

486

prepared in different concentrations, and then 20 μL of these solutions were

487

spotted on the filter disc. All plates were incubated at 37 oC for 18 h.

23



Page 24 of 41

488

Antibacterial activity was evaluated by the size of the zones of cell-growth

489

inhibition. A, C, 200 ng/mL and 100 ng/mL mBLF N-lobe; B, D, 200 ng/mL and

490

100 ng/mL BLF N-lobe; E, 60 ng/mL kanamycin.

491 492

Figure 8. Antibacterial activity of BLF N-lobe. Bacteria were incubated in

493

LB medium treated with 100 ng/mL and 200 ng/mL BLF N-lobe, or 60 ng/mL

494

kanamycin. Percentage of growth was calculated relative to commercial BLF.

495

Experiments were performed in triplicate. The viable cells were calculated

496

relative to untreated bacteria in medium. Different treatments represent

497

significant differences (P < 0.05). Error bars indicate SD.

498 499 500

24


Page 25 of 41


502

Tables:

503

Table 1. Bacterial strains and plasmids used in this work. Strains/plasmids/primers

Characteristics

Sources

E. coli JM109

recA1, endA1, gyrA96, thi-1, hsd R17(rk- mk+)supE44

Invitrogen

E. coli / pMA0911-Pcat-BLF-N

E. coli JM109 containing pMA0911-Pcat-BLF-N (KmR)

This study

E. coli / pMA0911-PsacB-BLF-N

E. coli JM109 containing pMA0911-PsacB-BLF-N (KmR)

This study

E. coli / pMA0911-P43-BLF-N

E. coli JM109 containing pMA0911-P43-BLF-N (KmR)

This study

E. coli / pMA0911-Pveg-BLF-N

E. coli JM109 containing pMA0911-Pveg-BLF-N (KmR)

This study

E. coli / pMA0911-PserA-BLF-N

E. coli JM109 containing pMA0911-PserA-BLF-N (KmR)

This study

E. coli / pMA0911-Pveg-mBLF-N

E. coli JM109 containing pMA0911-Pveg-mBLF-N (KmR)

This study

B. subtilis 168

trpC2

Lab stock

B. subtilis/ pMA0911-Pcat-BLF-N

B. subtilis 168 containing pMA0911-Pcat-BLF-N (KmR)

This study

B.

B. subtilis 168 containing pMA0911-PsacB-BLF-N (KmR)

This study

B. subtilis/ pMA0911-P43-BLF-N

B. subtilis 168 containing pMA0911-P43-BLF-N (KmR)

This study

B. subtilis/ pMA0911-Pveg-BLF-N

B. subtilis 168 containing pMA0911-Pveg-BLF-N (KmR)

This study

B.

subtilis/

B. subtilis 168 containing pMA0911-PserA-BLF-N (KmR)

This study

subtilis/

B. subtilis 168 containing pMA0911-Pveg-mBLF-N (KmR)

This study

pMA0911

E. coli / B. subtilis shuttle vector

Lab stock

pMA0911-Pcat-BLF-N

pMA0911 containing BLF N-lobe with promoter Pcat

This study

pMA0911-PsacB-BLF-N

pMA0911 containing BLF N-lobe with promoter PsacB

This study

pMA0911-P43-BLF-N

pMA0911 containing BLF N-lobe with promoter P43

This study

pMA0911-Pveg-BLF-N

pMA0911 containing BLF N-lobe with promoter Pveg

This study

pMA0911-PserA-BLF-N

pMA0911 containing BLF N-lobe with promoter PserA

This study

pMA0911-Pveg-mBLF-N

pMA0911 containing mBLF N-lobe with promoter Pveg

This study

Strains

subtilis/pMA0911-PsacB-BLF-N

pMA0911-PserA-BLF-N B. pMA0911-Pveg-mBLF-N

Plasmids

504

KmR kanamycin-resistant.

505 25



Page 26 of 41

506

AUTHOR INFORMATION

507

Corresponding Authors

508

*Tel.: +86 510 85197760. Fax: +86 501 85918201.

509

E-mail: [email protected] (R.Z.Z.).

510

ORCID

511

Rongzhen Zhang: 0000-0002-5745-1190

512

Author Contributions

513

L.J. conducted the investigation; devised the methodology; did experiments;

514

and wrote the original draft. Y.X. were involved in formal analysis, and revising

515

the manuscript. R.Z.Z. and J.M.L supervised the work; reviewed and revised

516

the manuscript. L.H.L. and L.X.Z. performed formal data analysis.

517

Notes

518

The authors declare no competing financial interest.

519

FUNDING SOURCES

520

This project was supported by the National Key research and

521

Development Program of China (2018YFA0900302), the Program for

522

Advanced Talents within Six Industries of Jiangsu Province (2015-SWYY-010),

523

the National First-class Discipline Program of Light Industry Technology and

524

Engineering (LITE2018-12), and the Program of Introducing Talents of

525

Discipline to Universities (111-2-06).

526

26


Page 27 of 41


527

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TOC graphic

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33



Figure 1. Relative mRNA expression and BLF N-lobe production under the different promoters. Relative mRNA expression level of BLF N-lobe under Pcat promoter was used as the control normalized to a value of 1. Different treatments within relative mRNA expression or protein production represent significant differences (P < 0.05). Error bars indicate SD. 113x79mm (300 x 300 DPI)


Page 34 of 41

Page 35 of 41


Figure 2. Codon usage analysis of BLF N-lobe gene sequence. 160x113mm (300 x 300 DPI)



Figure 3. The construciton of the recombinant plasmid pMA0911-Pveg-mBLF-N. The vector contains a synthetic mBLF N-lobe gene for expression in B. subtilis 168. The Pveg was included as a promoter. 101x12mm (300 x 300 DPI)


Page 36 of 41

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Figure 4. Relative mRNA expression and BLF N-lobe production by codon optimization under the Pveg promoter. The relative mRNA expression before optimization was used as the control normalized to a value of 1. Different treatments within relative mRNA expression or protein production represent significant differences (P < 0.05). Error bars indicate SD. 106x79mm (300 x 300 DPI)



Figure 5. The structure and sequence alignment of BLF N-lobe with several selected members of transferring family. Left columns contain the Protein Data Bank accession codes of the structures. 1CE2, buffalo lactoferrin; 1BIY, buffalo lactoferrin 2; 1JW1, goat lactoferrin; 1DTZ, camel lactoferrin. Conserved residues are boxed with blue lines. Selected residue numbers of the lactoferrin are labeled above the sequence. Secondary structure elements of BLF are marked on the top of the alignment. This figure was prepared with the program Espript (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi/). 186x123mm (300 x 300 DPI)


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Figure 6. The 3-D structure of recombinant BLF N-lobe protein. 265x179mm (72 x 72 DPI)



Figure 7. Filter-Disc plate assay for detection of antibanterial activity against E. coli JM109 (A), P. aeruginosa CGMCC 1.6740 (B) and S. aureus CGMCC 1.282 (C). 299x103mm (300 x 300 DPI)


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Figure 8. Antibacterial activity of BLF N-lobe. 279x124mm (300 x 300 DPI)


Improving Expression of Bovine Lactoferrin N-Lobe by Promoter

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