N-Glycosylation Engineering to Improve the Constitutive Expression of

Jul 6, 2017 - of Rhizopus oryzae Lipase in Komagataella phaffii ... In order to improve the secretion of Rhizopus oryzae lipase (ROL) under the contro...
0 downloads 0 Views 2MB Size
Subscriber access provided by UNIVERSITY OF CONNECTICUT

Article

N-Glycosylation engineering to improve the constitutive expression of Rhizopus oryzae lipase in Komagataella phaffii Xiaowei Yu, Min Yang, Chuanhuan Jiang, Xiaofeng Zhang, and Yan Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01884 • Publication Date (Web): 06 Jul 2017 Downloaded from http://pubs.acs.org on July 7, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

Journal of Agricultural and Food Chemistry

1

N-Glycosylation

2

constitutive expression of Rhizopus oryzae lipase in

3

Komagataella phaffii

4

Xiao-Wei Yu a,b,*, Min Yanga, Chuanhuan Jianga, Xiaofeng Zhanga, Yan Xu a,b,*

5

a

6 7 8

9

engineering

to

improve

the

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P. R. China

b

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi

214122, P. R. China *

Corresponding

authors:

Xiao-Wei

(X.-W.

Yu),

Yu,

10

[email protected]

11

+86-510-85918201, Fax: +86-510-85918201

Yan

Xu,

[email protected]

E-mail (Y.

addresses: Xu),

Tel.:

12 13 14 15 16 17 18 19 20 21 22

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 31

23

Abstract

24

Our previous studies demonstrated that the N-glycans in Rhizopus chinensis lipase

25

(RCL) was important for its secretion. In order to improve the secretion of Rhizopus

26

oryzae lipase (ROL) under the control of the GAP promoter in K. phaffii, two extra

27

N-glycosylation sites were introduced in ROL according to the position of the

28

N-glycosylation sites of RCL by sequence alignment. The results indicated that the

29

secretion level of ROL was strongly improved by N-glycosylation engineering, and

30

the highest value of extracellular enzyme activity was increased from 0.4±0.2 U/mL

31

to 207±6 U/mL in shake flask. In 7-L fermenter, the extracellular enzyme activity of

32

the mutant (2600±43 U/mL) and the total protein concentration (2.5±0.2 g/L) were

33

218- and 6.25-fold higher than these of the parent, respectively. This study presents a

34

strategy for constitutive recombinant expression of ROL using the GAP promoter

35

combined with N-glycosylation engineering, providing a potential enzyme for the

36

application in food industry.

37

Keywords: Rhizopus oryzae; lipase; N-glycosylation; Komagataella phaffii; GAP

38

promoter; Rational design;

2

ACS Paragon Plus Environment

Page 3 of 31

Journal of Agricultural and Food Chemistry

39

Introduction

40

Komagataella phaffii (formerly called Pichia pastoris) is a widely used platform for

41

the production of many heterologous proteins of medical and industrial interest.1, 2

42

Many factors can potentially affect heterologous protein production in the K. phaffii

43

expression system, such as promoter, gene sequence, gene copy number and

44

post-translational modification of proteins.3 The promoter is very important for

45

transcription initiation. The AOX1 promoter is the most widely used promoter, which

46

has been successfully used to express many different kinds of foreign genes. The

47

tightly regulated AOX1 promoter can be strongly induced by methanol and is

48

repressed by glucose, glycerol and ethanol. However, methanol is toxic and a

49

potential fire hazard.4 One of the alternative promoters is the constitutive promoter

50

(PGAP) of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme. PGAP,

51

providing a continuous transcription of the heterologous gene, has the advantages of

52

omitting the use of hazardous methanol and ease of process handling.4, 5

53

Lipases are very attractive enzymes for use in various industrial applications, such as

54

in the food processing industry for brewing and wine making, dairy processing, fruit,

55

meat, and vegetable processing,6-8 and in the energy industry for biodiesel

56

production.9, 10 Rhizopus lipases, are highly important resources of industrial lipases,

57

which are the most attractive enzymes in the application of edible oil and fat

58

industry.11 In order to enhance the enzyme production to meet industry demand,

59

several Rhizopus lipases have been expressed in Escherichia coli,

60

Saccharomyces cerevisiae13, 14 and in K. phaffii.15-20 The best productivity was gained 3

ACS Paragon Plus Environment

12

in

Journal of Agricultural and Food Chemistry

61

in the host of K. phaffii. The extracellular activity of the mature lipase from R. oryzae

62

expressed in K. phaffii reached 500 U/mL (60 mg active lipase per liter).17 And the

63

extracellular activity of this lipase was further improved to 1334 U/mL by a methanol

64

feeding strategy.15 The highest extracellular enzyme activity of ROL reported in K.

65

phaffii was 21000 U/mL with a specific activity of the crude enzyme of 2210 U/mg,

66

which was obtained with methanol induction in a 50-L bioreactor.19 However, all

67

above mentioned reports on the expression of Rhizopus lipases in K. phaffii were

68

under the AOX1 promoter induced by methanol, which is unfavorable for the usage in

69

food industry because of its toxicity.

70

N-glycosylation is important for protein maturation along the ER and Golgi traffic

71

pathway.21 The processing steps in the ER and Golgi of some enzymes and the

72

respective genes of K. phaffii are described by Delic et al..22 The discovery of the

73

quality control system in the ER has further elucidated the relationship between

74

protein N-glycosylation and its secretion,21, 23-25 which provides a theoretical basis for

75

N-glycosylation engineering. The introduction of N-glycosylation sites has been

76

successfully applied to improve pharmacokinetic and pharmacodynamic properties of

77

therapeutic proteins.26,

78

modified by N-glycosylation engineering. Sagt et al.28 demonstrated that introducing

79

an N-glycosylation site, preferably at the N terminus of cutinase, improved the protein

80

secretion levels by five-fold. Han et al.29 improved the hydrolytic efficiency and

81

specific activity of an elastase by introduction of N-glycosylation sites.

27

Enzymatic properties and production also have been

4

ACS Paragon Plus Environment

Page 4 of 31

Page 5 of 31

Journal of Agricultural and Food Chemistry

82

In our previous study, we demonstrated that the R. chinensis lipase (RCL, GenBank

83

accession no. EF405962) expressed in K. phaffii was N-glycosylated at three sites

84

(N-14, N-48 and N-60) in the prosequence and N-glycosylation had a great impact on

85

its secretion.30 RCL is homologous to the lipase from R. oryzae (ROL, 75.6% identity,

86

GenBank accession no. AF229435), while ROL contained only one potential

87

N-glycosylation site in the prosequence. In this study, we constructed the constitutive

88

expression of Rhizopus oryzae lipase (ROL) in K. phaffii under the GAP promoter to

89

avoid methanol induction, and then improved the secretion level of ROL by rational

90

design of the N-glycosylation site in the prosequence of ROL according to the

91

N-glycosylation sites in RCL.

92 93

Materials and Methods

94

Strains and plasmids

95

Escherichia coli JM109 was used as the cloning host. K. phaffii GS115 (Novagen,

96

USA) was used as the heterologous expression host. The plasmid pGAPZɑ (Novagen,

97

USA) was

98

pPIC9K-proROL were constructed and stored in our lab.31

used as gene expression

vector.

The

recombinant

plasmids

99 100

Enzymes and reagents

101

p-nitrophenyl palmitate (pNPP) was from Sigma (USA). Restriction enzymes, Taq

102

DNA polymerase, polymerase chain reaction (PCR) reagent, T4 DNA ligase,

103

PrimeSTAR(mix) polymerase, PCR reagent were obtained from Takara (China). 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 31

104

SDS-PAGE Protein Marker was provided by Beyotime Institue Biotechnology

105

(China). Primers were synthesized at Sangon Biotech (China). Gel Extraction Kit,

106

PCR Purification Kit were from Bioflux (Malaysia). Plasmid Mini Kit I was from

107

OMEGA BioTek (USA). Fluorescence quantification tubes were purchased from

108

Bio-Rad (USA). AceQTM qPCR SYBR® Green Master Mix was from Vazyme (China).

109

All other chemicals used were purchased from Sinopharm Chemical Reagent (China).

110 111

Introduction of the N-glycosylation sites in ROL

112

The proROL gene without its own signal sequence was amplified from the

113

pPIC9K-proROL vector. After treatment with EcoR I and Not I the proROL gene was

114

ligated into the pGAPZɑ vector using T4 DNA ligase, fused in frame with the

115

sequence

116

pGAPZɑ-proROL. The introduction of N-glycosylation sites in the prosequence of

117

proROL were made by point mutation using pGAPZɑ-proROL as a template. The

118

point mutations at positions A (mutate S15A16S17 to N15G16T17) and B (mutate N50T51

119

to N50L51T52) were carried out using PrimeSTAR (mix) polymerase with the primers

120

showed in Table 1 and yielded vectors pGAPZɑ-proROLA and pGAPZɑ-proROLB,

121

respectively. Then, the pGAPZɑ-proROLAB recombinant plasmid was constructed by

122

a point mutation at position B using the pGAPZɑ-proROLA plasmid as a template.

123

Subsequently, the transformants were selected on low salt LB agar plates with 25

124

µg/mL Zeocin. K. phaffii GS115 competent cells were transformed with the Avr II

125

linearized recombinant plasmids by electroporation with 750V/mm using a Gene

encoding

the

alpha-factor

signal peptide

6

ACS Paragon Plus Environment

gene,

and

yielded

Page 7 of 31

Journal of Agricultural and Food Chemistry

126

Pulser TM (Bio-Rad). The transformed cells were grown on YPD plates with 100

127

µg/mL Zeocin and cultured for confirmation of the recombinant lipases. Gene

128

manipulation and media are prepared by means of “Pichia expression Kit” from

129

Invitrogen Corporation.32

130 131

Gene copy number determination

132

The gene copy number was determined by real-time quantitative PCR with slight

133

modification.33 The designed primers were annealed to the complementary regions of

134

the GAP promoter sequence. The parent strain GS115 only contains one GAP

135

promoter. Thus, the copy number of PGAP minus one equals the copy number of the

136

lipase gene controlled under the GAP promoter. qPCR data were normalized using

137

GAPDH gene as the endogenous control (reference gene). All qPCR reactions were

138

run in triplicate on MJ chromo4 (MJ, America) using the following program: 98 °C 2

139

min, 40 cycles of 98 °C for 5 s, and 50 °C for 5 s.

140 141

Lipase Fermentation

142

The K. phaffii transformants were cultured in 100 mL of YPD medium shaken at

143

30 °C and 200 rpm in 500 mL shake flasks. The culture supernatant was collected

144

every 12 h or 24 h during culture to assay the cell growth, protein concentration, and

145

lipase activity of different mutants.

146

Feed batch fermentation experiments were performed in a 7-L bioreactor (New

147

Brunswick, BioFlo 110, USA). Two hundred milliliter of inoculum was added into 7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

148

2.6 L of a Fermentation Basal Salts Medium (40 g/L glycerol, 22.7 g/L H3PO4, 0.93

149

g/L CaSO4, 18.2 g/L K2SO4, 14.9 g/L MgSO4 ·7H2O, 4.13 g/L KOH, 7.0 g/L K2HPO4)

150

and 12 mL of trace solution. Trace solution consisted of 6 g/L CuSO4·5H2O, 0.08 g/L

151

NaI, 3.0 g/L MnSO4 · H2O, 0.2 g/L Na2MoO4 ·2H2O, 0.02 g/L H3BO3, 0.5 g/L CoCl2,

152

20 g/L ZnCl2, 65 g/L FeSO4·7H2O, 0.2 g/L biotin, and concentrated sulfuric acid, 0.5 %

153

(v/v). The fermentation conditions were maintained at 30 °C and the pH of the

154

medium was adjusted and controlled at 5.0 with the addition of 28 % (v/v) ammonium

155

hydroxide. During the fermentation, 50 % (v/v) glycerol containing 1.2 % (v/v) trace

156

solution was feed at the average rate of 12.4 g/L/h and adjusted to control DO at

157

20~40 % saturation.

158 159

Lipase activity assay

160

Lipase activity was measured on emulsified p-nitrophenyl palmitate (pNPP) as the

161

substrate according to Kordel et al..34 One volume of a 1.08 mM solution of pNPP in

162

2-propanol was mixed prior to use with nine volumes of 50 mM phosphate buffer pH

163

8.0 containing 4 g/L Triton X-100 and 1 g/L arabic gum. The standard reaction was

164

started by pre-equilibration of 2.4 mL of above mixture at 40 °C and then addition of

165

0.1 mL of enzyme solution at an appropriate dilution in 50 mM pH 8.0

166

phosphate buffer. The absorbance of the reactant against a blank without enzyme was

167

monitored at 410 nm using a UV-vis spectrophotometer (UNICO UV-3102 PC,

168

China). One enzyme unit was defined as the amount of enzyme releasing 1 µmol of

169

p-nitrophenol per minute under assay conditions (pH 8.0, 40 °C). 8

ACS Paragon Plus Environment

Page 8 of 31

Page 9 of 31

Journal of Agricultural and Food Chemistry

170 171

SDS-PAGE analysis

172

SDS-PAGE was conducted in accordance with the method of Laemmli.35 Samples of

173

extracellular proteins (the culture supernatant) of the mutants were subjected to 12%

174

SDS–PAGE using a Mini-Protein II Cell (Bio-Rad Laboratories, Hercules, CA).

175

Protein was stained with Coomassie Brilliant Blue R-250 (Amresco, Solon, OH, USA)

176

and quantified by a Molecular Imaging System Cell (Bio-Rad Laboratories, Hercules,

177

CA) using protein ladder as the standard. All samples were normalised for the

178

same cell density prior to loading on gel.

179 180

Results

181

Rational design of the N-glycosylation site in R. oryzae lipase

182

In our previous study, we demonstrated that the lipase from R. chinensis (RCL) was

183

N-glycosylated when expressed in K. phaffii and the N-glycans played a key role in

184

the lipase secretion.30 Both ROL and RCL sequences are composed of signal sequence,

185

prosequence and mature sequence (Fig. 1). As shown in Figure 1A, RCL has three

186

N-linked glycosylation sites in the prosequence (N-14, N-48, N-60), while ROL only

187

contains one potential site. Therefore, according to the N-glycosylation sites in RCL,

188

the amino acids S15A16S17 and N50T51 of ROL were mutated into the N-glycosylation

189

sites (N15G16T17 and N50L51T52) by rational design (Fig. 1B) and were named

190

r28ROLA and r28ROLB, respectively. The amino acids S15A16S17 and N50T51 of ROL

191

were mutated into N15G16T17 and N50L51T52 at the same time and were named 9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

192

Page 10 of 31

r28ROLAB.

193 194

Gene copy number determination of R. oryzae lipase

195

qPCR has been developed into an important and widely used analytical tool

196

lipase gene copy number was subsequently determined by measuring the copy

197

number ratio of the target gene to GAPDH. The results showed that the copy number

198

of R. oryzae lipase gene in all used strains in this study is one.

36

. The

199 200

Cell growth of the recombinant strains producing N-glycosylation engineered lipase

201

Lipase fermentation was carried out in shake flask to determine the difference

202

between r28ROL and variants containing additional N-glycosylation sites. As shown

203

in Figure 2, the cell growth rate of the N-glycosylation mutants were consistent with

204

that of the parent r28ROL. All strains grew quickly within 48 h and later grew slowly

205

and steadily after 72 h, indicating that the introduction of the N-glycosylation sites in

206

R. oryzae lipase did not influence biomass accumulation.

207 208

Effect of the extra N-glycosylation sites on the secretion level of R. oryzae lipase

209

In Figure 3, the total extracellular protein concentration of r28ROL and the mutants

210

introduced with the extra N-glycosylation sites were compared. The total protein

211

concentration of r28ROL (Fig. 3, Fig. 4) was apparently lower than those of the

212

mutants. And no obvious difference was observed among the mutants during

213

cultivation. The bands of r28ROL and the mutants from different sampling times were 10

ACS Paragon Plus Environment

Page 11 of 31

Journal of Agricultural and Food Chemistry

214

detected by SDS-PAGE (Fig. 4). Due to the existence of the Kex2 protease site (KR)

215

in the prosequence of ROL (Fig. 1), the potential N-glycosylation sites in the

216

prosequence of ROL were removed after the cleavage by Kex2 endoprotease. The

217

resulting products of ROL, named r28ROL, were the mature lipases attached with 28

218

amino acids of the carboxy-terminal part of the prosequence.37,

219

detected molecular weight of r28ROLA, r28ROLB and r28ROLAB were the same as

220

r28ROL. As shown in Figure 4, the masses of the mutants (r28ROLA, r28ROLB and

221

r28ROLAB) were approximately 35.0 kDa, consistent with the reported value of

222

r28ROL.13 The secretion levels of the mutants reached the highest point at 84 h and

223

later decreased. The secretion levels of r28ROLA and r28ROLAB were slightly

224

higher than that of r28ROLB at 84 h. However, the bands of r28ROL without

225

introduction of the N-glycosylation sites in the prosequence were not detected when

226

culturing from 24 h to 96 h.

227

Under GAP promoter the extracellular activity of the parent r28ROL could not be

228

detected, in agreement with no band being detected by SDS-PAGE in the supernatant.

229

We speculated that r28ROL was retained in the cell. Thus, the intracellular activity

230

was measured and the intracellular proteins were analyzed using SDS-PAGE.

231

However, neither the intracellular lipase activity nor the intracellular lipase of

232

r28ROL could be detected, which was the same case with the mutants (r28ROLA,

233

r28ROLB, r28ROLAB) (data not shown).

38

Therefore, the

234 235

Extracellular lipase activity of the recombinant strains producing N-glycosylation 11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

236

engineered lipase

237

As shown in Figure 5, the extracellular enzyme activity of r28ROL and the mutants

238

were compared during cultivation. In agreement with no band being detected by

239

SDS-PAGE, the activity of r28ROL in the supernatant was very low (0.4±0.2 U/mL),

240

while those of the mutants reached a much higher level. The highest activities of

241

r28ROLA, r28ROLB and r28ROLAB were 175±2 U/mL, 150±2 U/mL and 207±6

242

U/mL, respectively, after culturing for 84 h. The activities of the r28ROLA and

243

r28ROLAB mutants were slightly higher than that of r28ROLB.

244 245

Lipase fermentation in 7-L fermenter

246

The production of the N-glycosylation engineered lipase r28ROLAB in K. phaffii was

247

further investigated in 7-L fermenter, and compared with that of the parent r28ROL.

248

As shown in Figure 6, the extracellular enzyme activity of the mutant r28ROLAB

249

reached the maximum of 2600±43 U/mL by pNPP assay after induction of 84 h,

250

which was 218 folds higher than that of the parent r28ROL (11±1 U/mL). The total

251

extracellular protein concentration of r28ROLAB (2.5±0.2 g/L) showed an increase of

252

6.25 times over that of the parent at 84 h and the specific activity of the crude enzyme

253

r28ROLAB was 1040 U/mg. Nevertheless, the cell grow rates of both strains were

254

very similar to each other.

255 256

Discussion

12

ACS Paragon Plus Environment

Page 12 of 31

Page 13 of 31

Journal of Agricultural and Food Chemistry

257

It was unexpected that the first attempt to express ROL under the GAP promoter in K.

258

phaffii was unsuccessful, which showed nearly no lipase activity (r28ROL) in the

259

supernatant (Fig. 5.), since ROL has been successfully expressed under the AOX1

260

promoter.15-20 Compared with the GAP and AOX1 promoters, researches showed that

261

the overall efficiency of protein expression under the regulation of AOX1 or GAP

262

promoter in K. phaffii depends on individual protein.39 In several cases, the AOX1

263

promoter was found to be superior to the GAP promoter, whereas in some cases the

264

GAP promoter gained better yield.40 Researchers even combined use of these two

265

promoters in K. phaffii for enhancing the protein expression level.5 In our case, the

266

GAP promoter might be too weak to make ROL expressed. However, the reasons

267

underlying the differences between protein expression under the control of different

268

promoters is still unclear.

269

Glycosylation in K. phaffii is a common post-translational modification of proteins.

270

The roles of N-glycosylation in protein secretion have been widely studied, and

271

abundant literature has shown that a lack of N-glycosylation may cause defects in

272

particular protein secretion pathways.41, 42 It has been reported that N-glycosylation

273

can be engineered to increase protein secretion based on the amino acid sequence of

274

the protein.43 In our previous study, we demonstrated that R. chinensis lipase

275

containing the intact prosequence (proRCLCNQ) was N-glycosylated at the N-14,

276

N-48 and N-60 sites when expressed in K. phaffii, and the N-glycans on N-60 played

277

a key role in the secretion of lipase.30 The alignment of the amino acids of RCL and

278

ROL have a high degree of similarity (Fig. 1A). However, ROL has only one 13

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

279

potential N-glycosylation site in the prosequence, while there are three

280

N-glycosylation sites in the prosequence of RCL (Fig. 1A). Because the expression

281

level of ROL in K. phaffii under the GAP promoter is quite lower than that of RCL,

282

we hypothesized that the introduction of the N-glycosylation consensus sequence in

283

the prosequence of ROL designed according to the N-glycosylation sites in RCL

284

would enhance its secretion efficiency.

285

The alignment of the prosequence of RCL and ROL indicated that ROL also contains

286

the key potential N-60 site, despite of no secretion of r28ROL under the constitutive

287

promoter in this study. We speculated that the N-glycans on N-14, N-48 and N-60 in

288

r27RCLC probably function collaboratively for the efficient secretion of the lipase.

289

Based on this hypothesis, we designed three mutants to introduce the N-glycosylation

290

site (A, B and AB) in r28ROL at the conserved location to N-14 and N-48 in

291

r27RCLC, respectively (Fig.1B). Heterologous gene dosage has a great influence on

292

protein expression level in K. phaffii.33 To rule out the influence of gene copy number

293

on lipase secretion level and enzymatic activity, we determined the copy numbers of

294

proROL, proROLA, proROLB and proROLAB in K. phaffii GS115 and employed

295

strains with only one copy number of lipase gene. Therefore, the differences in

296

secretion level between r28ROL and the N-glycosylation mutants should not be

297

influenced by gene copy number. After introducing the extra N-glycosylation sites in

298

ROL, the secretion levels (Fig. 3) and extracellular enzyme activities (Fig. 5) of

299

r28ROLA, r28ROLB and r28ROLAB greatly improved compared to r28ROL.

300

Additionally, the determined extracellular activities (Fig. 5) of r28ROLAB and 14

ACS Paragon Plus Environment

Page 14 of 31

Page 15 of 31

Journal of Agricultural and Food Chemistry

301

r28ROLA were slightly higher than those of r28ROLB. These results suggested that

302

the introduced mutations in ROL has a great impact on the protein secretion in K.

303

phaffii.

304

In 7-L fermenter the extracellular enzyme activity of the mutant r28ROLAB

305

(2600±43 U/mL) and the total protein concentration (2.5±0.2 g/L) were 218- and

306

6.25-fold higher than those of the parent r28ROL, respectively (Fig. 6). Although this

307

value was still lower than the highest activity of ROL (21000U/mL) ever reported

308

under the AOX1 promoter performed in 50-L fermenter

309

r28ROLAB under the GAP promoter can be further improved by optimization of gene

310

copy number, scale-up fermentation, co-expression of chaperons and so on.

311

In conclusion, in order to simplify enzyme production compared to methanol

312

inducible expression systems this study constructed recombinant strains for

313

constitutive expression of ROL under the control of GAP promoter in K. phaffii, and

314

successfully enhanced the extracellular activity of ROL by N-glycosylation

315

engineering, providing a potential enzyme for the industry applications. The strategy

316

of introducing extra N-glycosylation sites by rational design proved to be an efficient

317

way to enhance secretion of certain enzymes produced in K. phaffii.

19

, the production of

318 319

Acknowledgments

320

Financial support from NSFC (31671799), Six Talent Peaks Project in Jiangsu

321

Province (NY-010), 333 Project in Jiangsu Province (BRA2015316), and the National

15

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

322

High Technology Research and Development Program of China (863 Program)

323

(2012AA022207) are greatly appreciated.

324

16

ACS Paragon Plus Environment

Page 16 of 31

Page 17 of 31

Journal of Agricultural and Food Chemistry

325

References

326

1.

327

for the usage in food and feed industry with Pichia pastoris. J. Biotechnol. 2015, 202,

328

118-134.

329

2.

330

pastoris: recent achievements and perspectives for heterologous protein production.

331

Appl. Microbiol. Biot. 2014, 98, 5301-5317.

332

3.

333

Pichia pastoris by enhancing the disulfide bond formation pathway in the

334

endoplasmic reticulum. J. Ind. Microbiol. Biot 2013, 40, 1241-1249.

335

4.

336

methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 2000, 24, 45-66.

337

5.

338

AOX1 promoters and optimization of culture conditions to enhance expression of

339

Rhizomucor miehei lipase. J. Ind. Microbiol. Biot 2015, 42, 1175-1182.

340

6.

341

interesterification reactions for human milk fat substitutes production: a review. Eur. J.

342

Lipid. Sci. Tech 2013, 115, 270-285.

343

7.

344

novel alkaline-stable lipase from a metagenomic library and its specific application

345

for milkfat flavor production. Microb. Cell Fact. 2014, 13, 1.

346

8.

Spohner, S. C.; Muller, H.; Quitmann, H.; Czermak, P., Expression of enzymes

Ahmad, M.; Hirz, M.; Pichler, H.; Schwab, H., Protein expression in Pichia

Sha, C.; Yu, X. W.; Zhang, M.; Xu, Y., Efficient secretion of lipase r27RCL in

Cereghino, J. L.; Cregg, J. M., Heterologous protein expression in the

He, D.; Luo, W.; Wang, Z. Y.; Lv, P. M.; Yuan, Z. H., Combined use of GAP and

Soumanou, M. M.; Pérignon, M.; Villeneuve, P., Lipase-catalyzed

Peng, Q.; Wang, X.; Shang, M.; Huang, J.; Guan, G.; Li, Y.; Shi, B., Isolation of a

Sharma, R.; Chisti, Y.; Banerjee, U. C., Production, purification, characterization, 17

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

347

and applications of lipases. Biotechnol. Adv. 2001, 19, 627-662.

348

9.

349

feedstocks and process development. Bioresource Technol. 2013, 135, 386-395.

350

10. Mounguengui, R. W. M.; Brunschwig, C.; Baréa, B.; Villeneuve, P.; Blin, J., Are

351

plant lipases a promising alternative to catalyze transesterification for biodiesel

352

production? Prog. Energ. Combust. 2013, 39, 441-456.

353

11. Yu, X.-W.; Xu, Y.; Xiao, R., Lipases from the genus Rhizopus: Characteristics,

354

expression, protein engineering and application. Prog. Lipid. Res. 2016, 64, 57-68.

355

12. Di Lorenzo, M.; Hidalgo, A.; Haas, M.; Bornscheuer, U. T., Heterologous

356

production of functional forms of Rhizopus oryzae lipase in Escherichia coli. Appl.

357

Environ. Microb. 2005, 71, 8974-8977.

358

13. Ueda, M.; Takahashi, S.; Washida, M.; Shiraga, S.; Tanaka, A., Expression of

359

Rhizopus oryzae lipase gene in Saccharomyces cerevisiae. J. Mol. Catal. B-Enzym.

360

2002, 17, 113-124.

361

14. Takahashi, S.; Ueda, M.; Atomi, H.; Beer, H. D.; Bornscheuer, U. T.; Schmid, R.

362

D.; Tanaka, A., Extracellular production of active Rhizopus oryzae lipase by

363

Saccharomyces cerevisiae. J. Ferment. Bioeng. 1998, 86, 164-168.

364

15. Minning, S.; Serrano, A.; Ferrer, P.; Sola, C.; Schmid, R. D.; Valero, F.,

365

Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris.

366

J. Biotechnol. 2001, 86, 59-70.

367

16. Resina, D.; Serrano, A.; Valero, F.; Ferrer, P., Expression of a Rhizopus oryzae

368

lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde

Hama, S.; Kondo, A., Enzymatic biodiesel production: an overview of potential

18

ACS Paragon Plus Environment

Page 18 of 31

Page 19 of 31

Journal of Agricultural and Food Chemistry

369

dehydrogenase promoter. J. Biotechnol. 2004, 109, 103-113.

370

17. Minning, S.; Schmidt-Dannert, C.; Schmid, R. D., Functional expression of

371

Rhizopus oryzae lipase in Pichia pastoris: High-level production and some properties.

372

J. Biotechnol. 1998, 66, 147-156.

373

18. Li, Z.; Li, X.; Wang, Y.; Wang, Y.; Wang, F.; Jiang, J., Expression and

374

characterization of recombinant Rhizopus oryzae lipase for enzymatic biodiesel

375

production. Bioresource Technol. 2011, 102, 9810-9813.

376

19. Wang, J.-R.; Li, Y.-Y.; Xu, S.-D.; Li, P.; Liu, J.-S.; Liu, D.-N., High-level

377

expression of pro-form lipase from Rhizopus oryzae in Pichia pastoris and its

378

purification and characterization. Int. J. Mol. Sci. 2014, 15, 203-217.

379

20. Arnau, C.; Ramon, R.; Casas, C.; Valero, F., Optimization of the heterologous

380

production of a Rhizopus oryzae lipase in Pichia pastoris system using mixed

381

substrates on controlled fed-batch bioprocess. Enzyme. Microb. Tech. 2010, 46,

382

494-500.

383

21. Kukuruzinska, M.; Lennon, K., Protein N-glycosylation: molecular genetics and

384

functional significance. Crit. Rev. Oral Biol. M 1998, 9, 415-448.

385

22. Delic, M.; Valli, M.; Graf, A. B.; Pfeffer, M.; Mattanovich, D.; Gasser, B., The

386

secretory pathway: exploring yeast diversity. FEMS Microbiol. Rev. 2013, 37,

387

872-914.

388

23. Helenius, A.; Aebi, M., Intracellular functions of N-linked glycans. Science 2001,

389

291, 2364-2369.

390

24. Jones, J.; Krag, S. S.; Betenbaugh, M. J., Controlling N-linked glycan site 19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

391

occupancy. Biochim. Biophys. Acta. 2005, 1726, 121-137.

392

25. Mitra, N.; Sinha, S.; Ramya, T. N.; Surolia, A., N-linked oligosaccharides as

393

outfitters for glycoprotein folding, form and function. Trends Biochem. Sci. 2006, 31,

394

156-163.

395

26. Hudak, J. E.; Bertozzi, C. R., Glycotherapy: new advances inspire a reemergence

396

of glycans in medicine. Chem. Biol. 2014, 21, 16-37.

397

27. Sola, R. J.; Griebenow, K., Glycosylation of therapeutic proteins an effective

398

strategy to optimize efficacy. Biodrugs 2010, 24, 9-21.

399

28. Sagt, C. M. J.; Kleizen, B.; Verwaal, R.; de Jong, M. D. M.; Muller, W. H.; Smits,

400

A.; Visser, C.; Boonstra, J.; Verkleij, A. J.; Verrips, C. T., Introduction of an

401

N-glycosylation site increases secretion of heterologous proteins in yeasts. Appl.

402

Environ. Microb. 2000, 66, 4940-4944.

403

29. Han, M.; Wang, X.; Yan, G.; Wang, W.; Tao, Y.; Liu, X.; Cao, H.; Yu, X.,

404

Modification of recombinant elastase expressed in Pichia pastoris by introduction of

405

N-glycosylation sites. J. Biotechnol. 2014, 171, 3-7.

406

30. Yang, M.; Yu, X.-W.; Zheng, H.; Sha, C.; Zhao, C.; Qian, M.; Xu, Y., Role of

407

N-linked glycosylation in the secretion and enzymatic properties of Rhizopus

408

chinensis lipase expressed in Pichia pastoris. Microb. Cell Fact. 2015, 14, 40.

409

31. Yu, X. W.; Sha, C.; Guo, Y. L.; Xiao, R.; Xu, Y., High-level expression and

410

characterization of a chimeric lipase from Rhizopus oryzae for biodiesel production.

411

Biotechnol. Biofuels 2013, 6, 29.

412

32. Invitrogen Corporation, U., Pichia expression vectors for constitutive expression 20

ACS Paragon Plus Environment

Page 20 of 31

Page 21 of 31

Journal of Agricultural and Food Chemistry

413

and purification of recombinant proteins. Catalog nos. V200-20 and V205-20. 2002.

414

33. Sha, C.; Yu, X.-W.; Li, F.; Xu, Y., Impact of gene dosage on the production of

415

lipase from Rhizopus chinensis CCTCC M201021 in Pichia pastoris. Appl. Biochem.

416

Biotech. 2013, 169, 1160-1172.

417

34. Kordel, M.; Hofmann, B.; Schomburg, D.; Schmid, R. D., Extracellular Lipase of

418

Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization,

419

and preliminary X-Ray diffraction data. J. Bacteriol. 1991, 173, 4836-4841.

420

35. Laemmli, U. K., Cleavage of structural proteins during the assembly of the head

421

of bacteriophage T4. Nature 1970, 227, 680-685.

422

36. D’haene, B.; Vandesompele, J.; Hellemans, J., Accurate and objective copy

423

number profiling using real-time quantitative PCR. Methods 2010, 50, 262-270.

424

37. Hama, S.; Tamalampudi, S.; Fukumizu, T.; Miura, K.; Yamaji, H.; Kondo, A.;

425

Fukuda, H., Lipase localization in Rhizopus oryzae cells immobilized within biomass

426

support particles for use as whole-cell biocatalysts in biodiesel-fuel production. J.

427

Biosci. Bioeng. 2006, 101, 328-333.

428

38. Yu, X. W.; Wang, L. L.; Yan, X., Rhizopus chinensis lipase: gene cloning,

429

expression in Pichia pastoris and properties. J. Mol. Catal. B-Enzym. 2009, 57,

430

304-311.

431

39. Varnai, A.; Tang, C.; Bengtsson, O.; Atterton, A.; Mathiesen, G.; Eijsink, V. G. H.,

432

Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.

433

Microb. Cell Fact. 2014, 13, 10.

434

40. Zhang, A. L.; Luo, J. X.; Zhang, T. Y.; Pan, Y. W.; Tan, Y. H.; Fu, C. Y.; Tu, F. Z., 21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

435

Recent advances on the GAP promoter derived expression system of Pichia pastoris.

436

Mol. Biol. Rep. 2009, 36, 1611-1619.

437

41. Liu, Y.; Nguyen, A.; Wolfert, R. L.; Zhuo, S., Enhancing the secretion of

438

recombinant proteins by engineering N-glycosylation sites. Biotechnol. Progr. 2009,

439

25, 1468-1475.

440

42. Kazenwadel, C.; Klebensberger, J.; Richter, S.; Pfannstiel, J.; Gerken, U.; Pickel,

441

B.; Schaller, A.; Hauer, B., Optimized expression of the dirigent protein AtDIR6 in

442

Pichia pastoris and impact of glycosylation on protein structure and function. Appl.

443

Microbiol. Biot. 2013, 97, 7215-7227.

444

43. Hou, J.; Tyo, K. E.; Liu, Z.; Petranovic, D.; Nielsen, J., Metabolic engineering of

445

recombinant protein secretion by Saccharomyces cerevisiae. FEMS Yeast Res. 2012,

446

12, 491-510.

447 448 449

22

ACS Paragon Plus Environment

Page 22 of 31

Page 23 of 31

Journal of Agricultural and Food Chemistry

450

Table 1

451

Primers used in the introduction of the N-glycosylation sites in ROL. Name

Sequence (5’-3’)

Length (bp)

GATTCTCCACTACCGCCGTCAACGGAACCGAC

46

NGT-A F1 AATTCTGCCCTCCC GGGAGGAATTGTCGGTTCCGTTGACGGCGGTA

42

NGT-A R1 GTGGAAGATC NLT-BF2

CTACATGCAAAAGAATCTTACAGAATGGTATGA

33

NLT-BR2

TCATACCATTCTGTAAGATTCTTTTGCATGTAG

33

452 453 454

23

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

455

Figure legends

456

Fig. 1 (A) Alignment of the amino acids of R. oryzae lipase and R. chinensis lipase;

457

(B) Rational design of the N-glycosylation sites in the prosequence of ROL.

458

N-glycosylation sites in RCL and the corresponding sites in ROL are indicated by

459

arrows; the Kex2 protease site “KR” is indicated by brackets; the mutation sites A and

460

B are indicated by arrows in the mutated ROL.

461

Fig. 2 Growth of the strains expressing R. oryzae lipases.

462

Fig. 3 Total protein concentration in the supernatant of the strains expressing R.

463

oryzae lipases.

464

Fig. 4 SDS-PAGE analyses of the supernatant of the strains expressing R. oryzae

465

lipases.

466

Fig. 5 Extracellular lipase activity of the strains expressing R. oryzae lipases.

467

Fig. 6 Extracellular lipase activity, protein concentration, cell concentration profiles

468

of the strains expressing R. oryzae lipases.

469

24

ACS Paragon Plus Environment

Page 24 of 31

Page 25 of 31

Journal of Agricultural and Food Chemistry

Fig. 1.

25

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Fig. 2.

26

ACS Paragon Plus Environment

Page 26 of 31

Page 27 of 31

Journal of Agricultural and Food Chemistry

Fig. 3.

27

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Fig. 4.

28

ACS Paragon Plus Environment

Page 28 of 31

Page 29 of 31

Journal of Agricultural and Food Chemistry

Fig. 5.

29

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 30 of 31

DCW/r28ROLAB Enzyme activity/r28ROLAB Protein concentration/r28ROLAB DCW/r28ROL Enzyme activity/r28ROL Protein concentration/r28ROL

280 260 240 220

DCW(g/l)

200

3000

6.0

2750

5.5

2500

5.0

2250

4.5

2000

180

1750

160

1500

140 1250

120

Enzyme activity(U/ml)

300

4.0 3.5 3.0 2.5

100

1000

80

750

1.5

500

1.0

250

0.5

0

0.0

60 40 20 0 0

10

20

30

40

50

60

70

80

90

Time (h)

30

ACS Paragon Plus Environment

100

2.0

Protein concentration (g/l)

Fig. 6.

Page 31 of 31

Journal of Agricultural and Food Chemistry

TOC Graphic

31

ACS Paragon Plus Environment