High-Efficiency Secretion of Î²-Mannanase in Bacillus subtilis through

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High-efficiency secretion of #-mannanase in Bacillus subtilis through protein synthesis and secretion optimization Yafeng Song, Gang Fu, Huina Dong, Jianjun Li, Yuguang Du, and Dawei Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05528 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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Journal of Agricultural and Food Chemistry

High-efficiency secretion of β-mannanase in Bacillus subtilis through protein synthesis and secretion optimization Yafeng Song1, 2, Gang Fu1, 2, Huina Dong1, 2, Jianjun Li3, 4,*, Yuguang Du3, 4 Dawei Zhang1, 2,*

1 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China 2 Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China 3 National Key Laboratory of Biochemical Engineering, National Engineering Research Center for Biotechnology (Beijing), 100190, China 4 Key Laboratory of Biopharmaceutical Production & Formulation Engineering, PLA, Institute of Process Engineering, Chinese Academy of Sciences, 100190, China

*Corresponding author. [email protected] (J.Li) +86-10-82545039; [email protected] (D. Zhang) +86-022-24828749

ACS Paragon Plus Environment


1

Abstract

2

The manno endo-1, 4-mannosidase (β-mannanase, EC. 3.2.1.78) catalyzes the random

3

hydrolysis of internal (1→4)-β-mannosidic linkages in the mannan polymers. A codon

4

optimized β-mannanase gene from Bacillus licheniformis DSM13 was expressed in

5

Bacillus subtilis. When four Sec-dependent and two Tat-dependent signal peptide

6

sequences cloned from B. subtilis were placed upstream of the target gene, the highest

7

activity of β-mannanase was observed using SPlipA as a signal peptide. Then, a

8

1.25-fold activity of β-mannanase was obtained when another copy of groESL operon

9

was inserted into the genome of host strain. Finally, five different promoters were

10

separately used to enhance the synthesis of the target protein. The results showed that

11

promoter Pmglv, a modified maltose-inducible promoter, significantly elevated the

12

production of β-mannanase. After 72 h of flask fermentation, the enzyme activity of

13

β-mannanase in the supernatant when using locust bean gum as substrate reached

14

2207 U/mL. This work provided a promising β-mannanase production strain in

15

industrial application.

16

Keywords: β-mannanase, Bacillus subtilis, signal peptides, promoters, chaperones,

17

signal peptidase

18


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19

Introduction

20

β-mannanase (endo-1,4-β-D-mannan mannosidase, EC. 3.2.1.78), an extracellular

21

enzyme, is responsible for randomly cleaving the backbone of mannan-based

22

polysaccharides, releasing short β-1,4-mannooligomers as well as new chain ends1. It

23

plays an important role in the hydrolysis of hemicellulose, which is estimated to be

24

the second most abundant polysaccharide in nature because mannans constitute the

25

predominant component of hemicellulose fractions in various plant tissues2.

26

Furthermore, it displayed synergistic interactions with β-mannosidase and

27

α-galactosidase 3, due to the elaborate chemically distinctness and interlocking of

28

polysaccharides within the plant cell walls. Being one of the most significant

29

mannan-degrading enzymes, β-mannanases are widely distributed among plants,

30

marine mollusks, fungi and some species of bacteria, and many have been purified

31

and characterized4-8. The broad substrate specificities and resistance to harsh

32

conditions of some β-mannanases cloned from different species permit their industrial

33

applications, ranging from food, feed, paper and pulp biobleaching, bioactive

34

oligosaccharides production in addition to second generation biofuels9. Owing to the

35

importance and demand for mannooligosaccharides, β-mannanases have been

36

extensively researched. These efforts comprise the enzyme cloning, purification and

37

identification of the X-ray crystal structures to facilitate the elucidation of catalytic

38

mechanisms and engineering of improved enzymes with regard to product specificity

39

and catalytic efficiency.

40

β-mannanase (ManB) from Bacillus licheniformis DSM13 (ATCC14580) which



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10

41

belongs to glycoside hydrolase family 26

42

alkali-stability under a wide range of conditions, especially when substrates were

43

high-molecular-weight mannans consisting of more than six mannose monomers11.

44

The half-life time of activity (τ1/2) of ManB remained as high as approximately 80

45

hours, when incubating temperature increased up to 50oC. In addition, it also kept

46

stable within pH ranging from 5-12 after incubation for 30 min at 50oC. In many cases,

47

genes encoding β-mannanases originated from different bacteria and fungi species

48

were

49

respectively12-14. Similarly, β-mannanase (ManB) has once been expressed in E. coli

50

by applying OmpA signal peptide directing its secretion11. However, only half of the

51

enzymes secreted into the medium and half stayed within the periplasmic space. This

52

elevated costs for downstream purification process and hampered its commercial

53

implementation.

heterologously

expressed

in

, performed well in thermo-stability and

Escherichia

coli

and

Pichia

pastoris,

54

B. subtilis, a Gram-positive bacterium, is widely used in expressing a variety of

55

enzymes, therapeutic proteins and metabolites15-20 due to the Generally Recognized

56

As Safe (GRAS) status, which gives it a priority in food, beverage, feed additive,

57

pharmaceuticals applications. The most significant advantage of this bacterium is to

58

secrete proteins directly into the culture medium at high concentrations19,

59

Furthermore, genetic engineering strategies and high-efficiency transformation

60

methods22-25 make the genetic manipulation more accessible, which prompt the

61

optimizations of this host strain. Although B. subtilis has been used to express

62

mannanase, neither the regulation of transcription and translation nor the secretion


21-23

.

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63

pathway has been optimized18,

26-29

64

protease-deficient strain, and Brevibacillus brevis have been employed to express

65

mannanase Man23, but with only two vectors being investigated to improve its

66

production30. In another example, lacking systematic optimizations, a shuttle vector

67

carrying an endo-1,4-beta-mannosidase from Bacillus pumilus was expressed in B.

68

subtilis 1A751, leading to a low level of enzyme production (8.65U/mL)31. Therefore,

69

a combination of above-mentioned approaches was conducted to verify whether it

70

could reduce specific bottlenecks encountered by β-mannanase during the secretion

71

process, to further increase the final production.

. For instance, B. subtilis WB600, a

72

In the present study, we attempted to increase the synthesis and secretion of

73

β-mannanase in B. subtilis. The β-mannanase gene from B. licheniformis DSM13 was

74

codon optimized after the removal of the native signal peptide, followed by the

75

expression in pMA5. In the following optimization processes, we firstly selected the

76

most suitable signal peptide among 6 candidates, which could direct the secretion of

77

most β-mannanases into the medium. The second step was the identification of the

78

bottlenecks that hindered the secretion and release of high concentration proteins.

79

This was achieved by expressing our target protein in host strains that overexpressed

80

genes encoding bottleneck candidates, such as secretion components, chaperones or

81

signal peptidase. The final step was intended to increase the flux of the protein

82

amounts by efficient synthesis. In this phase, several constitutive promoters and

83

inducible promoters were evaluated and compared. As a result, the combination of

84

these strategies (Figure 1) considerably increased the final production of β-mannanase.



85

Furthermore, this improvement not only prompted the potential industrial application

86

of β-mannanase, but also implied the feasibility of this strategy being applied to

87

increase the production of many other heterologous proteins in B. subtilis.

88

Materials and Methods

89

Strains, plasmids, and cultivation conditions

90

Plasmids and bacteria strains used in this study were listed in Table 1 and Table 2.

91

E. coli strains were cultured at 37°C with 220 rpm orbital shaking in lysogeny broth

92

(LB) medium [1% tryptone, 0.5% yeast extract, 0.5% NaCl]. B. subtilis cells

93

harboring appropriate plasmids were cultivated at 37°C in 2×SR medium [3%

94

tryptone, 5% yeast extract and 0.6% K2HPO4, pH 7.2] in 250-mL triangular flasks.

95

Appropriate antibiotics were supplemented to the medium when required: 100 µg/mL

96

ampicillin for E. coli, 40 µg/mL kanamycin and 10 µg/mL chloramphenicol for B.

97

subtilis. Only B. subtilis 1A751 derivative strains with genes encoding Sec-secretion

98

components, signal peptidases, and molecular chaperones integrated into the genome

99

(Table 2) were cultured with media adding 10 µg/mL chloramphenicol as well as 2%

100

(w/v) of xylose. Strains containing inducible promoters were separately cultivated in

101

medium with the presence of inducers under different concentrations: 0, 0.001, 0.01,

102

and 0.1 mM of IPTG, and 0, 0.5, 1.0, 2.0% (w/v) of maltose. Cells of B. subtilis were

103

pre-cultured in LB medium overnight and inoculated to OD600 of 0.01, followed by 4

104

hours of incubation in 2×SR medium. Different final concentrations of IPTG were

105

then added to the medium with strains containing Pgrac, respectively. For the strains

106

carrying Pmglv, maltose was added to the medium immediately after the inoculation.


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107

Cell growth was monitored by measurement of the optical density at 600 nm (OD600).

108

Standard deviations are based on a minimum of three statistically independent

109

experiments.

110

General manipulation

111

The native signal peptide of β-mannanase (NCBI accession number AAU39670)

112

from B. licheniformis DSM13 was removed and the rest sequences were codon

113

optimized for B. subtilis. The sequence of the native signal peptide being removed

114

was: MKKNIVCSIFALLLAFAVSQPSYA. Plasmids were constructed by applying

115

prolonged overlap extension (POE)-PCR32 method. β-mannanase and linearized

116

vector fragment were prepared by PCR amplification using PrimerSTAR Max DNA

117

Polymerase mix containing dNTP and DNA polymerase (TaKaRa, Japan), and

118

primers (ms/ma and vs/va, respectively), according to the instructions of the

119

manufacturer. POE-PCR amplification mixture comprises Q5 DNA polymerase,

120

dNTP mix, amplified β-mannanase and linearized vector fragments. The PCR

121

protocol was as follows: denaturation at 98oC for 30 s, followed by 30 cycles of

122

denaturation at 98oC for 5 s and then annealing at 55oC for 10 s and extension at 72°C

123

for 4 min. The POE-PCR products were directly transformed into competent E. coli

124

DH5α, and positive colony samples were selected to be further validated by

125

sequencing the fragments (Genwiz, China).

126

Other plasmids were constructed following the same method. Primers used to

127

amplify the signal peptide fragments and promoter fragments, as well as other

128

oligonucleotides used in this study were listed in Table S1. The genome of B. subtilis



27

129

168 served as the template to amplify signal peptide

fragments including SPamyL,

130

SPlipA, SPnprB, SPnprE, SPphoD, SPywbN, and promoter fragments including PaprE, and P43.

131

The fragment lengths of PaprE and P43 were 200 bp and 275 bp, respectively. Pgrac

132

containing the promoter, lacO sequences and the regulator protein lacI was 1548 bp,

133

and originated from the plasmid pHT43 plasmid33. Pmglv was a modified maltose

134

inducible promoter34, 35, which was constructed by researchers in our lab (unpublished

135

work). Constructed plasmids were transformed into B. subtilis under the standard

136

competent transformation method.

137

SDS-PAGE analysis

138

Supernatant samples were harvested and separated by centrifugation (12000g, 10

139

minutes, 4oC) after fermentation in flasks for different hours to perform sodium

140

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (NuPAGE 10 %

141

Bis-Tris Gel, Commassie Brilliant Blue, Invitrogen Life Technologies, USA). Cell

142

precipitate was resuspended with 50 µL lysis buffer (50 mM Tris/HCl, 150 mM NaCl,

143

pH 8.0) per OD600. Prestained Protein Ladder, purchased from Invitrogen Life

144

Technologies, was applied to determine the molecular weights of the proteins with

145

prominent bands.

146

Enzyme assays

147

Dinitrosalicylic acid (DNS) method was performed to determine the

148

β-mannanase activity by measuring the amount of reducing sugar released from locust

149

bean gum (LBG). The substrate, 0.5% LBG (Sigma G0753) was dissolved in acetic

150

acid-sodium acetate buffer, pH 5.5. 40 µL of appropriate dilutions of culture


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151

supernatants (i.e. the secreted β-mannanase) and 40 µL of the substrate were mixed

152

and incubated at 37oC for 30 minutes. 100 µL of DNS was added to the enzyme

153

reaction mixture and boiled for 10 minutes to stop the reaction. The mixture was then

154

cooled on ice for 5 minutes and diluted with 320 µL of double distilled water before

155

measuring the absorbance at 540 nm. One unit (U) of β-mannanase activity is defined

156

as the amount of enzyme that liberates 1 µmol of reducing sugar (using D-mannose as

157

a standard) per minute under the given experimental conditions.

158

Results and Discussion

159

Host and vector selection

160

Compared to E. coli and yeast, two extensively used protein expression hosts, B.

161

subtilis possesses its own advantages in producing proteins such as its strong capacity

162

of

163

well-characterized secretion pathways25, 36. In some cases, when E. coli serves as the

164

host, large fraction of proteins might be remained within cytoplasmic or periplasmic

165

space of the cells, making the downstream separation and purification much more

166

complex. When comparable secretion capacity occurs between the two generally

167

recognized as safe species, i. e. yeast and B. subtilis, the much higher growth rate of

168

the latter gives it a priority in industrial application. Therefore, we used B. subtilis

169

1A751, a protease-deficient strain, as the host to express β-mannanase.

secreting

high

concentration

proteins

into

the

culture

medium

and

170

The multicopy plasmid pMA5 was employed as the parent plasmid to express

171

β-mannanase37. The fragment of the target protein was successfully placed

172

downstream of promoter PhpaII, and upstream of T4 terminator, resulting in



173

pMA5-Man. Thereafter, a series of pMA5-Man vectors with different signal peptide

174

fragments were constructed, to facilitate the target protein secretion.

175 176

Increased secretion of β-mannanase by selecting the most efficient signal peptide

177

Typically, to secrete β-mannanase directly into the culture medium, screening

178

one efficient signal peptide that well functioned in B. subtilis was necessary. There are

179

several well-characterized secretion pathways elucidated in B. subtilis, especially the

180

general secretory (Sec) pathway, and the twin-arginine (Tat) translocation pathway.

181

We selected four signal peptides (SPamyL, SPlipA, SPnprB, and SPnprE) from Sec

182

pathway38-40 and two (SPphoD and SPywbN) from Tat pathway41, 42, which were reported

183

to highly direct the secretion of different enzymes.

184

Six different signal peptide fragments were inserted between PhpaII and

185

β-mannanase gene of pMA5-Man, respectively. Then they were transformed into the

186

B. subtilis 1A751. After 72 h of fermentation, most of the enzymes could secrete into

187

the medium. We could easily recognize that all of the signal peptides could drive the

188

protein secrete into the medium since the prominent bands corresponding to the size

189

of 38kDa were observed on the SDS-PAGE gel (Figure 2A), which were in

190

accordance with the theoretical molecular weight of the mature protein. Among the

191

four Sec pathway signal peptides, SPlipA (1A7512) was the most efficient one (enzyme

192

activities listed in Table S2), with almost a twofold activity of SPnprB (1A7514), which

193

turned out to be the least efficient. SDS-PAGE result of the supernatant samples

194

(Figure 2A) also showed that both the two Tat-dependent signal peptides (SPphoD,


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195

SPywbN) could direct the secretion of β-mannanase, but at relatively low levels.

196

Substantial fractions of protein precursors remained within the cytoplasmic of the

197

cells harboring Tat-dependent signal peptides. This was observed from further

198

analysis of the cell samples by SDS-PAGE (Figure 2B). In addition, those remained

199

proteins had a larger size than the mature proteins, implying that most of the Tat

200

signal peptides could not be successfully removed by the signal peptidases. However,

201

when Sec-dependent signal peptides performed, only small fractions of proteins

202

remained in the cytoplasm (Figure 2B), with signal peptides being removed. Thus, we

203

concluded that β-mannanase tended to be highly secreted under the direction of

204

Sec-pathway signal peptide SPlipA. Therefore, pMA5-2Man (SPlipA) was used for the

205

following experiments.

206 207

Increase the secretion of β-mannanase by systematic gene overexpression

208

Since both the enzyme activity detection of the cytoplasmic extracts and the

209

SDS-PAGE result of the cell fraction samples (Figure 2B) indicated that some

210

β-mannanases retained in the cytoplasm, we concluded that only the most suitable

211

signal peptide itself could not guarantee their maximum production in B. subtilis, but

212

some other bottlenecks within the secretion pathway needed to be addressed.

213

Insufficiency of each component of the secretion machinery might decrease the final

214

secretion amounts of β-mannanases. It was reported that the secretion level of

215

α-amylase was remarkably improved by systematic overexpression of Sec-pathway

216

related genes to screen the main bottleneck candidates43. Thus it was suspected this



217

process to be a necessary step to distinguish the components that limited β-mannanase

218

secretion, considering the different sequences between α-amylase and β-mannanase.

219

Therefore, we transformed pMA5-2Man (SPlipA) into B. subtilis 1A751 derivative

220

strains containing the overexpression genes encoding elements of Sec secretion

221

machinery, respectively. Each single copy of these overexpressed genes integrated

222

into the B. subtilis chromosome at amyE locus via double crossing-over43, and under

223

the strict control of promoter Pxyl. Candidate recombinant strains were listed in Table

224

2. B. subtilis 1A751S1 (secY), 1A751S2 (secE), 1A751S3 (secG), 1A751S4 (secYEG),

225

1A751S5 (secDF), 1A751S6 (secA), 1A751S7 (ffh), 1A751S8 (scr), 1A751S9 (hbs),

226

1A751S10 (SRP), and 1A751S11 (ftsY) were strains which overexpressed genes of the

227

Sec-translocon components and B. subtilis 1A751P1(sipS), 1A751P 2(sipT), 1A751P3

228

(sipU), 1A751P4 (sipV), 1A751P5 (sipW), 1A751P6 (partial dnaK operon), 1A751P7

229

(groESL operon) and 1A751P8 (prsA) were strains that overexpressed different signal

230

peptidases and molecular chaperones.

231

These candidates were selected because of their importance in secretion process.

232

Among the Sec translocase complex17, SecYEG constituted the secretion channel, and

233

SecDF served as the accessory protein. SecA provided energy for crossing membrane

234

by catalyzing the hydrolysis of ATP. The signal recognition particle (SRP) consisted

235

of a GTPase named Ffh, two histone-like proteins (Hbs) and scRNA (scr), and FtsY

236

was the SRP receptor. To some extent, well cooperation or not between these elements

237

and β-mannanase considerably influenced the secretion process, as well as the

238

ATP-dependent molecular chaperones, such as DnaK and GroESL. Among the seven


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239

type I signal peptidases, five (sipS, sipT, sipU, sipV, and sipW) were identified to

240

locate on the chromosome. Either the presence of SipS or SipT was sufficient for

241

processing of precursor proteins and cell viability44.

242

After bacteria being inoculated into the 2×SR fermentation medium and cultured

243

at 37oC for 4 hours in 250-mL triangular flasks, xylose was added to a final

244

concentration of 2% (m/v) to induce the overexpression of the relevant genes in Sec

245

secretion pathway. Fermentation cultures were harvested to obtain supernatant

246

samples to detect the β-mannanase activities and perform the SDS-PAGE after 72 h of

247

incubation. Compared to B. subtilis 1A751 (pMA5-2Man), no strains that

248

overexpressed genes had increased the enzyme activities for several times. No

249

dramatic effects on the target protein secretion led to the enzyme yields almost

250

keeping equivalent to levels of the control strain B. subtilis 1A7512 (pMA5-2Man)

251

(Figure 3A and Table S2). These strains were B. subtilis 1A751S1 (secY), 1A751S2

252

(secE), 1A751S6 (secA), 1A751S8 (scr), 1A751P3 (sipU), 1A751P4 (sipV), 1A751P5

253

(sipW), 1A751P6 (partial dnaK operon), 1A751S7 (ffh), 1A751S10 (SRP), and

254

1A751P8 (prsA).

255

When separately overexpressed genes (secY, secE, and secG) encoding the

256

channel of Sec translocase complex45, there were no apparent changes in the

257

β-mannanase secretion levels. However, combinatorial overexpression of the channel

258

genes (secYEG) enhanced the secretion to 116% compared to B. subtilis 1A7512

259

(pMA5-2Man). This implied that balanced amounts of the translocase components

260

were critical for the increased number of the functional SecYEG channels, thus



261

enhancing the target protein secretion. In addition, overexpression of the translocase

262

accessory protein SecDF (1A751S5), the SRP receptor FtsY (1A751S11), had positive

263

effects on the secretion, but the improvements were not very obvious (Figure 3B).

264

Despite the fact that Ffh could assist the forming of stable SRP complex, no elevated

265

level of production was observed.

266

Not only the translocase complex plays an important role in its secretion, but also

267

many other factors affect this process. For example, the signal peptidases (SPPases)

268

were responsible for the cleavage of the N-terminal signal peptides, so that mature

269

proteins could secrete into the medium. The SipS (1A751P1) and SipT (1A751P2)

270

overexpression indeed enhanced the secretion of β-mannanase, as could be seen from

271

Figure 3A and 3B. This was consistent with the previous conclusion that SipS or SipT

272

served as the dominant SPPases 46. We supposed that overexpression of these SPPases

273

could intensify the cleavage of the signal peptides, accelerate the transportation, thus

274

reducing the accumulation of these cytoplasmic proteins. Even though increased

275

amounts of secretory β-mannanases were obtained, a small portion of mature proteins

276

still existed within the cells (Figure 4). Therefore, other secretion bottlenecks still

277

have to be discovered.

278

The overexpression of groESL operon, intracellular chaperones which were in charge

279

of mediating protein folding, decreasing aggregation, and maintaining pre-proteins in

280

translocation-competent conformations, highly increased (125% activity of the control)

281

the secretion amount of β-mannanase (Figure 3). This was similar with the result that

282

GroESL actively contributed to the Sec-dependent export process, which has been


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283

demonstrated in E. coli47. Partial dnaK operon also encoded intracellular chaperones,

284

but their overexpression almost had no positive effects on the secretion. This might

285

attribute to its preference for binding to certain polypeptide sequences48, and the

286

major function of DnaK was in response to stress affecting the Sec translocon. An

287

extracytoplasmic chaperone, PrsA, which could increase the α-amylase secretion by

288

several times43, 49, failed to assist β-mannanase with the secretion. This could be

289

probably due to the tremendously different primary sequences and folding states

290

between the two target proteins. Until now, even though we know the fact that these

291

chaperones participate in protein translocation, more knowledge is needed to explain

292

their abilities to interact with substrates and assist the folding of certain target proteins.

293

Based on that, it would be much easier to further facilitate the target protein secretion.

294

Increase the secretion of β-mannanase by using strong promoter

295

Overexpression of genes including translocon components (secYEG, secDF, ftsY),

296

SPPases (sipS, sipT), and chaperones (groESL operon), could increase the secretory

297

production of β-mannanases to different levels, with the highest (groESL operon)

298

being 125% of the control strain (Figure 3A). However, a small fraction of mature

299

enzymes still remained within the cells after strengthening the secretion pathway

300

(Figure 4). It seemed that these remained cytoplasmic proteins could not be avoided.

301

Therefore, we shifted to enhance the synthesis of enzymes by optimizing the level of

302

transcription and translation so that more proteins could be secreted. Using strong

303

promoters was one of the most efficient methods to increase the production, when no

304

obvious secretion bottlenecks hindering the transportation.



305

We selected three constitutive promoters and two inducible promoters to

306

determine the most suitable one. Promoter PhpaII was the original promoter in pMA5,

307

detected to be a relatively strong promoter50. Promoter PaprE was reported to highly

308

drive the expression of the downstream gene of α-Amylase when bacteria were

309

under stationary phase20. Promoter P43 was a well-characterized constitutive strong

310

promoter, and widely used in many protein expressions in B. subtilis30, 51, 52. The

311

inducible promoters included IPTG inducible promoter Pgrac (Figure 5A), and a

312

modified maltose inducible promoter Pmglv (Figure 5B). With the concentration of the

313

inducers increasing, the production of secreted β-mannanase increased. According to

314

the results of the SDS-PAGE, when the concentration of IPTG and maltose were 0.1

315

mM and 2% (m/v), the promoters performed better (Figure 5A and 5B) than either

316

higher or lower concentration.

317

To determine the most efficient promoter, these strains containing different

318

promoters (1A751P7, 1A751SPP1, 1A751SPP2, 1A751SPP4, 1A751SPP5) were

319

cultured in the 2×SR medium at 37oC for 72 h, with the optimized concentrations of

320

inducers. The enzyme activity produced by 1A751SPP5 (Pmglv) was the highest 2207

321

U/mL (Figure 6A and Table 3 and Table S2), with almost a 3.1-fold activity of the

322

control strain 1A751P7 (groESL operon, PhpaII). This was probably because maltose

323

served as both the inducer and carbon sources, which promoted bacteria growth, as

324

could be inferred from the higher OD600 (Figure 6B) of this strain than the control one

325

(1A751P7). Compared to other strains, the period of stationary phase of 1A751SPP5

326

(Pmglv) was much longer, which kept the cells continuously producing, accumulating


Page 16 of 39

Page 17 of 39


327

and subsequently secreting β-mannanase into the medium. The function of maltose

328

was evaluated by incubating the control strain 1A751P7 in the medium with 2% of

329

maltose (1A751P7-a) and 0.1 mM IPTG (1A751P7-b). The enzyme activity of

330

β-mannanase secreted by 1A751P7-a increased to 124% of the control strain (Table 3),

331

whereas adding IPTG did not increase the secretion of β-mannanase. 1A751SPP2

332

(P43) secreted the second highest amount of β-mannanase (Table S2), which was

333

about 1.8-fold activity of the control one. The constitutive promoter P43 was much

334

stronger than PhpaII and PaprE, even though PaprE could keep at high strength during the

335

stationary phase. 1A751SPP4 (Pgrac) could only reach 90% enzyme activity of the

336

control strain, which was still lower than the strains with promoter P43.

337

All of the strains carrying constitutive promoters could drive the transcription

338

and translation of the β-mannanase, but to extremely different levels. Promoter Pmglv

339

could prodigiously improve the secretion yields of β-mannanase. In this research,

340

selection of the suitable signal peptide, overexpression of secretion components or

341

chaperones to reduce the secretion bottlenecks, selection of the strong promoters to

342

enhance the transcription and translation, were combined to optimize and improve the

343

final production of β-mannanase to 2207 U/mL. Considering the potential application

344

of this enzyme in the feed industry, enzyme assay was conducted at 37oC, which was

345

not the optimal temperature. If the temperature was set between 50oC and 60oC, we

346

supposed that higher enzyme activity would be obtained. Nevertheless, the enzyme

347

activity produced by flask fermentation was already much higher than other reported

348

B. subtilis strains30,

31

. It is quite promising that a much higher production of



Page 18 of 39

349

β-mannanase would be obtained once this strain cultivated in fed-batch and further

350

optimization of fermentation process was conducted. In conclusion, not only the

351

valuable promoter Pmglv could be used for the production of β-mannanase, but also

352

these strategies could be adapted to improve other protein expression systems in B.

353

subtilis.

354 355

Abbreviations Used

356

SP:

357

Dinitrosalicylic acid;

358

Acknowledgement

Signal

peptide;

IPTG:

Isopropyl

β-D-1-thiogalactopyranoside;

DNS:

359

This work was supported by the National Nature Science Foundation of China

360

(31370089，21506244), State Key Development 973 Program for Basic Research of

361

China (2013CB733600), National High Technology Research and Development

362

Program of China (863 Program) (No. 2014AA093511), Nature Science Foundation

363

of Tianjin City (CN) (16JCYBJC23500, 15JCQNJC09500) and the Key Projects in

364

the Tianjin Science & Technology Pillar Program 11ZCZDSY08600.

365

Supporting Information

366

Table S1. Oligonucleotides used in this study.

367

Table S2. Enzyme activities of β-mannanases secreted by different strains of Bacillus

368

subtilis.

369

Notes

370

This work has been included in a patent application by Tianjin Institute of Industrial


Page 19 of 39


371

Biotechnology, Chinese Academy of Sciences.

372 373



374

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(51). Wang, P. Z.; Doi, R. H., Overlapping promoters transcribed by bacillus subtilis sigma 55 and

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509 510


Page 26 of 39

Page 27 of 39


511

Figure captions

512

Figure 1. Scheme of combined strategies to improve the secretion of β-mannanase.

513

Approaches include improvements of protein synthesis and secretion capacity. The

514

former was performed by application of strong promoters and codon optimization of

515

the target gene. The latter was intended to strengthen the transportation of mature

516

proteins, with signal peptides selection performed on plasmids and overexpression of

517

secretion machinery components on the chromosome.

518

Figure 2. Comparison of efficiency of different signal peptides. The molecular weight

519

of mature β-mannanase is about 38 kDa. Column Ct: plasmids without the gene of

520

β-mannanase. A: SDS-PAGE analysis of β-mannanase in the supernatant secreted by

521

B. subtilis. Four Sec-dependent signal peptides (SPamyL, SPlipA, SPnprB, and SPnprE) and

522

two Tat-dependent (SPphoD, SPywbN) signal peptides were used, and SPlipA was the most

523

efficient. B: SDS-PAGE analysis of β-mannanase of the cell samples. The two

524

Tat-dependent signal peptides could not be removed successfully, leading to the size

525

of proproteins larger than the mature ones.

526

Figure 3. Effects of overexpression genes on β-mannanase secretion. A: Relative

527

activities of β-mannanase that secreted by different B. subtilis strains (1A751S1,

528

1A751S2, 1A751S3, 1A751S4, 1A751S5, 1A751S6, 1A751S7, 1A751S8, 1A751S9,

529

1A751S10, 1A751S11, 1A751P1, 1A751P2, 1A751P3, 1A751P4, 1A751P5,

530

1A751P6, 1A751P7, 1A751P8 overexpressed secY, secE, secG, secYEG, secDF, secA,

531

ffh, scr, hbs, SRP, ftsY, sipS, sipT, sipU, sipV, sipW, partial dnaK operon, groESL

532

operon, prsA, respectively.) Column control was set as 100% activity, and the strains



533

were B. subtilis 1A7512, carrying SPlipA. Error bars represent standard deviations of

534

biological triplicates. B: SDS-PAGE analysis of β-mannanase in the supernatant

535

secreted by B. subtilis strains that overexpressed different genes.

536

Figure 4. SDS-PAGE analysis of β-mannanase cell samples of B. subtilis strains that

537

overexpressed different genes. Strains B. subtilis 1A751S1, 1A751S2, 1A751S3,

538

1A751S4,

539

1A751S11, 1A751P1, 1A751P2, 1A751P3, 1A751P4, 1A751P5, 1A751P6, 1A751P7,

540

1A751P8, overexpressed gene of secY, secE, secG, secYEG, secDF, secA, ffh, scr, hbs,

541

SRP, ftsY, sipS, sipT, sipU, sipV, sipW, partial dnaK operon, groESL operon, prsA,

542

respectively.

543

Figure 5. Optimization of the concentration of inducers to improve the β-mannanase

544

production. A: SDS-PAGE analysis of β-mannanase in the supernatant secreted by B.

545

subtilis 1A751SPP4 (Pgrac), with the concentration of IPTG increased from 0 to

546

0.1mM. B: SDS-PAGE analysis of β-mannanase in the supernatant secreted by B.

547

subtilis 1A751SPP5 (Pmglv), with the concentration of maltose increased from 0 to 2%

548

(m/v).

549

Figure 6. Comparison of β-mannanase amounts produced by strains with different

550

promoters. A: SDS-PAGE analysis of β-mannanase in the supernatant secreted by

551

strains with constitutive promoters (PhpaII, PaprE, P43) and inducible promoters (Pgrac,

552

Pmglv). B: Growth curve of B. subtilis strains with promoter PhpaII (Control) and Pmglv.

1A751S5, 1A751S6,

1A751S7, 1A751S8,


1A751S9, 1A751S10,

Page 28 of 39

Page 29 of 39


Table 1 Plasmids used in this study Plasmids

Genotype and/or relevant characteristics

pMA5

E. coli/B. subtilis shuttle vector, PhpaII, Apr, Kmr

BGSC

pMA5-Man

pMA5 derivative, β-mannanase, T4 terminator

This work

pMA5-1Man

pMA5-Man derivative, SPamyL,

This work

pMA5-2Man

pMA5-Man derivative, SPlipA,

This work

pMA5-3Man

pMA5-Man derivative, SPnprB,

This work

pMA5-4Man

pMA5-Man derivative, SPnprE,

This work

pMA5-5Man

pMA5-Man derivative, SPphoD,

This work

pMA5-6Man

pMA5-Man derivative, SPywbN,

This work

pMA5-2Man1

pMA5 derivative, PaprE, β-mannanase

This work

pMA5-2Man2

pMA5 derivative, P43, β-mannanase

This work

pMA5-2Man4

pMA5 derivative, Pgrac, β-mannanase

This work

pMA5-2Man5

pMA5 derivative, Pmglv, β-mannanase

This work



Page 30 of 39

Table 2 Strains used in this study Strains


Sources

E. coli DH5α

F−∆lacU169(-80d lacZ∆M15) supE44 hsdR17 recA1 gyrA96 endA1

Invitrogen

B. subtilis 1A751

eglS∆102 bglT/bglS∆EV aprE nprE his

BGSC

B. subtilis 1A7511

1A751 derivative, pMA5-1Man, Kmr

This work

B. subtilis 1A7512


This work

B. subtilis 1A7513


This work

B. subtilis 1A7514


This work

B. subtilis 1A7515


This work

B. subtilis 1A7516


This work

B. subtilis 1A751S1

1A751 derivative, amyE::Pxyl-secY, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751S2

1A751 derivative, amyE::Pxyl-secE, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751S3

1A751 derivative, amyE::Pxyl-secG, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751S4

1A751 derivative, amyE::Pxyl-secYEG, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751S5

1A751 derivative, amyE::Pxyl-secDF, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751S6

1A751 derivative, amyE::Pxyl-secA, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751S7

1A751 derivative, amyE::Pxyl-ffh, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751S8

1A751 derivative, amyE::Pxyl-scr, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751P1

1A751 derivative, amyE::Pxyl-sipS, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751P2

1A751 derivative, amyE::Pxyl-sipT, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751P3

1A751 derivative, amyE::Pxyl-sipU, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751P4

1A751 derivative, amyE::Pxyl-sipV, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751P5

1A751 derivative, amyE::Pxyl-sipW, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751P6

1A751 derivative, amyE::Pxyl-dnaK, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751P7

1A751 derivative, amyE::Pxyl-groESL, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751S9

1A751 derivative, amyE::Pxyl-hbs, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751S10

1A751 derivative, amyE::Pxyl-SRP, Cmr, pMA5-2Man, Kmr

This work


Page 31 of 39


Strains


Sources

B. subtilis 1A751S11

1A751 derivative, amyE::Pxyl-ftsY, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751P8

1A751 derivative, amyE::Pxyl-prsA, Cmr, pMA5-2Man, Kmr

This work

B. subtilis 1A751SPP1

1A751 derivative, amyE::Pxyl-groESL, Cmr, pMA5-2Man1(PaprE) , Kmr

This work


1A751 derivative, amyE::Pxyl-groESL, Cmr, pMA5-2Man2 (P43) , Kmr

This work


1A751 derivative, amyE::Pxyl-groESL, Cmr, pMA5-2Man4 (Pgrac) , Kmr

This work


1A751 derivative, amyE::Pxyl-groESL, Cmr, pMA5-2Man5 (Pmglv) , Kmr

This work



Page 32 of 39

Table 3 Relative activity of β-mannanase secreted by different recombinant strains Strains

Promoters

Relative

1A751P7

PhpaII

100

1A751P7-a

PhpaII

124

1A751P7-b

PhpaII

102

1A751SPP1

PaprE

32

1A751SPP2

P43

179

1A751SPP4

Pgrac

87

1A751SPP5

Pmglv

310

activity

of

B. subtilis 1A751P7-a and B. subtilis 1A751P7-b represent that the same strain B. subtilis 1A751P7 cultured in medium adding 2% (m/v) of maltose and 0.1mM IPTG, respectively.


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Figure graphics

Figure 1.



Figure 2.


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Figure 3.



Figure 4.


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Figure 5.



Figure 6


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1


TOC Graphic

2 3 4


High-Efficiency Secretion of Î²-Mannanase in Bacillus subtilis through

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