Improved Secretory Production of the Sweet-Tasting Protein, Brazzein

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Improved secretory production of the sweettasting protein, brazzein, in Kluyveromyces lactis Chorong Yun, Ji Na Kong, Juhee Chung, Myung-Chul Kim, and Kwang-hoon Kong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02446 • Publication Date (Web): 28 Jul 2016 Downloaded from http://pubs.acs.org on July 31, 2016

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Journal of Agricultural and Food Chemistry

Improved secretory production of the sweet-tasting protein, brazzein, in Kluyveromyces lactis

Cho-Rong Yun,† Ji-Na Kong,‡ Ju-Hee Chung†, Myung-Chul Kim†and Kwang-Hoon Kong† *

†

Laboratory of Biomolecular Chemistry, Department of Chemistry, College of Natural

Sciences, Chung-Ang University, 221, Huksuk-Dong, Dongjak-Ku, Seoul 156-756, Korea. ‡

Department of Neuroscience and Regenerative Medicine, Medical College of George,

Augusta University, Augusta, GA 30912, United States of America.

*

To whom correspondence should be addressed. Tel: +82-2-820-5205; Fax: +82-2-825-4736;

E-mail: [email protected]

1

ACS Paragon Plus Environment


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Abstract

2

Brazzein is an intensely sweet protein with high stability over a wide range of pH and

3

temperatures, due to its four disulfide bridges. Recombinant brazzein production through

4

secretory expression in Kluyveromyces lactis is reported, but is inefficient due to incorrect

5

disulfide formation, which is crucial for achieving the final protein structure and stability.

6

Protein disulfide bond formation requires protein disulfide isomerase (PDI) and Ero1p. Here,

7

we overexpressed KlPDI in K. lactis or treated the cells with dithiothreitol to overexpress

8

KlERO1 and improve brazzein secretion. KlPDI and KlERO1 overexpression independently

9

increased brazzein secretion in K. lactis by 1.7-2.2 fold and 1.3-1.6 fold, respectively.

10

Simultaneous overexpression of KlPDI and KlERO1 accelerated des-pE1M-brazzein

11

secretion by approximately 2.6-fold compared to the previous system. Moreover, intracellular

12

misfolded/unfolded recombinant des-pE1M-brazzein was significantly decreased. In

13

conclusion, increased KlPDI and KlERO1 expression favors brazzein secretion, suggesting

14

that correct protein folding may be crucial to brazzein secretion in K. lactis.

15 16

Keywords: Brazzein, Kluyveromyces lactis, Disulfide bond, KlERO1, KlPDI, Secretory

17

expression, Sweet protein.

18

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Introduction

20 21

In recent years, a low-calorie sugar substitute that can be healthful and naturally available

22

is in high demand since over-consumption of sugar and artificial sweeteners has various side

23

effects such as hypertension, hyperlipidemia, diabetes, and obesity.1 To meet this demand,

24

studies on sweet-tasting proteins have been pursued actively. To date, only eight sweet-tasting

25

proteins are known to elicit sweetness, and these include brazzein, curculin/neoculin, egg

26

white lysozyme, mabinlin, miraculin, monellin, pentadin, and thaumatin.2 Among these,

27

brazzein is derived from the fruit of the West African Pentadiplandra brazzeana Baillon plant

28

and has attracted much attention as a candidate sweetener, because of its high sweetness

29

intensity, sugar-like taste, and good stability at high temperature and wide range of pH.

30

Numerous attempts to produce brazzein in microorganisms and transgenic plants have

31

been performed because of the difficulty and the limitations in obtaining the natural source of

32

brazzein.3-7 These heterologous brazzein expression systems were often complicated and

33

difficult, owing to their specific N-terminal pyroglutamate and four disulfide bonds. Previous

34

studies of brazzein expression in Escherichia coli reported that recombinant brazzein was

35

insoluble, and required several purification steps resulting in a low overall yield.3-5 Brazzein

36

expression was attempted in several lactic acid bacteria; however, recombinant brazzein

37

obtained had low intensity of sweetness and low productivity.6,8 Brazzein production in

38

transgenic maize resulted in low purification yield of the recombinant protein and cross-

39

contamination.7 The expression of brazzein was attempted in Pichia pastoris and the purified

40

recombinant brazzein was obtained in a soluble and active form at approximately 30-90

41

mg/L.9 Recently, we reported the secretory expression of recombinant brazzein in the yeast

42

Kluyveromyces lactis, which is “generally regarded as safe” (GRAS).10 The recombinant

43

brazzein expressed was purified by CM-Sepharose column chromatography and 3



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approximately 104 mg/L was obtained. This yeast expression system is regarded as suitable

45

for commercialization of brazzein in the food industry because of its food grade status and

46

excellent fermentation characteristics on large scale.11

47

Potential mass production could provide a valuable opportunity for the commercial use of

48

brazzein. Brazzein secretion levels in the yeast expression system could be elevated by

49

optimizing the expression conditions, the gene copy number, overexpressing molecular

50

chaperones, foldases, genes associated with the secretory pathway, and so on.12 The native

51

structure of brazzein with four disulfide bridges suggests that the correct formation of these

52

disulfide bonds during its translocation may constitute a critical step in its secretory process

53

in yeast. Formation of protein disulfide bonds in the endoplasmic reticulum (ER) of yeast

54

requires two essential proteins, protein disulfide isomerase (PDI) and ER membrane-

55

associated protein Ero1p. PDI catalyzes the formation, isomerization, reduction of disulfide

56

bonds in substrate proteins, and can facilitate the shuffling incorrect disulfides into their

57

correct pairings.13 Ero1p oxidizes PDI through a flavin-dependent mechanism in the pathway

58

for disulfide bond formation.14 Therefore, secretion level of heterologous proteins containing

59

disulfide bonds can be elevated by increasing PDI and Ero1p activities in various expression

60

systems.

61

Here, we describe the secretory expression of brazzein in high PDI (KlPDI) and Ero1p

62

(KlERO1) gene expressing yeast K. lactis. To overexpress KlPDI, KlPDI was introduced in

63

the yeast K. lactis whereas the expression of KlERO1 was induced by treating the cells with

64

dithiothreitol (DTT).15 Using this strategy, the secretion level of recombinant brazzein was

65

elevated in K. lactis.

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Materials and methods

68 69

Strains, growth media, and chemicals

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The K. lactis strain GG799 and the expression vector pKLAC2 for heterologous protein

71

expression were obtained from a commercially available K. lactis protein expression kit

72

(New England BioLabs, USA). Brazzein production from this strain has been described in

73

detail by Jo et al. (2013).10 The transformed K. lactis strain GG799 was grown in 10 mL of

74

YPGlu medium (10 g yeast extract, 20 g peptone and 40 g glucose per liter) for 20 h at 30°C

75

and was transferred into 500 mL of YPGal medium (10 g yeast extract, 20 g peptone and 40 g

76

galactose per liter) as per the optimum expression conditions. The E. coli strain DH5α

77

(Promega, USA) was used for plasmid propagation and maintenance. This strain was grown

78

in lysogeny broth (LB) medium (Difco, USA) at 37°C. Synthesis of the brazzein encoding

79

sequence and DNA primers for gene cloning was performed by GenScript (USA) and

80

COSMO Genetech (Korea), respectively. Restriction enzymes and DNA-modifying enzymes

81

were obtained from Takara Shuzo (Japan). Acetamide and dithiothreitol (DTT) were

82

purchased from Sigma-Aldrich (USA). All the chemicals and reagents used were

83

commercially available and were of the highest reagent grade.

84 85

Construction of expression vectors and transformation

86

The brazzein gene (des-pE1M-brazzein, which lacks an N-terminal methionine in pE1M-

87

brazzein form) that we used was designed with optimized codon usage for expression in K.

88

lactis and the synthetic gene was cloned into the expression vector pKLAC2 as described in a

89

previous study.10 The resulting pKLAC2-des-pE1M-brazzein was transformed into the E. coli

90

strain DH5α for vector propagation. KlPDI (NCBI Accession No. CAB51612.1; GenBank

91

Accession No. AJ243958.1) was amplified from K. lactis genomic DNA by polymerase chain 5



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92

reaction (PCR). The oligonucleotide primers used for PCR were as follows: primer-1, 5′-

93

CCCAAGCTTATGTTGTTCAAGAATACCGTTAG-3′;

94

CGCGGATCCGCGTTACAATTCATCTTG-3′. For cloning into pKLAC2, the recognition

95

sites for Hind III and BamH I restriction enzymes were added to the 5′ and 3′ ends of the

96

primers, respectively. The amplified KlPDI and pKLAC2 were digested by Hind III and

97

BamH I and ligated to generate the pKLAC2-KlPDI vector. The resulting pKLAC2-KlPDI

98

was transformed into E. coli DH5α strain for vector propagation. pKLAC2-des-pE1M-

99

brazzein and the pKLAC2-KlPDI were linearized by digestion with Sac II and were

100

introduced into competent cells of the K. lactis GG1799 strain by chemical transformation

101

using the reagents supplied with the yeast transformation kit (New England BioLabs, USA)

102

as described previously.10 Single or multiple-copy integrants were identified by PCR.

primer-2,

5′-

103 104

KlERO1 induction by DTT treatment and northern blot analysis

105

KlERO1 expression was strongly induced in K. lactis by treatment of the cells with DTT

106

as previously reported by Lodi et al. (2005).15 To induce KlERO1 expression in K. lactis, we

107

used a stimulation method by addition of DTT in the culture medium, instead of duplicating

108

the Ero1p gene. To increase the KlERO1 expression levels, the culture conditions and DTT

109

concentration were optimized. 1 mM DTT was added to culture medium when the OD600

110

value was 0.2.

111

Total RNA was prepared by extraction with hot acidic phenol and ethanol precipitation.

112

Northern blot analysis was carried out as described by Sim et al. (2014).16 The KlERO1 probe

113

was

114

CCAGAGTATTGGCAGCCTG-3′) and ERO1-R (5′-GTCTTGCCACATCGTCATCG-3′) and

115

genomic DNA from the strain GG799 as the template.

generated

by

PCR

amplification

with

the

primers

ERO1-F

(5′-

116 6


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Analysis of secreted brazzein

118

We produced recombinant brazzein using optimized culture conditions.10 The transformed

119

K. lactis was pre-cultured in 10 mL YPGlu medium for 20 h at 30°C and was inoculated into

120

500 mL YPGal medium. The induced cells were grown for 96 h at 30°C and were separated

121

by centrifugation at 9,000 g for 20 min at 4°C. The supernatant containing secreted proteins

122

including recombinant brazzein was dialyzed using 3,500 Da MWCO membrane (Membrane

123

Filtration Products, Texas, USA) against distilled water, freeze-dried, and then subjected to

124

SDS-PAGE. The separated cells were collected for lysis to compare the remaining brazzein in

125

the cells. To compare the expression level of the recombinant brazzein, denaturing SDS-

126

PAGE was performed with 16.5% Tris-tricine gels as previously described.4 The molecular-

127

mass markers used were Polypeptide SDS-PAGE Molecular Weight Standards (Bio-Rad,

128

Hercules, CA, USA) containing triosephosphate isomerase (26.6 kDa), myoglobin (17.0

129

kDa), α-lactalbumin (14.4 kDa), aprotinin (6.5 kDa), and the insulin B chain, oxidized (3.4

130

kDa). Coomassie Blue R-250 was used for protein staining. After staining, the amount of

131

secreted brazzein was evaluated by densitometry. Densitometric analysis was performed

132

using an image analyzer (Phoretix 1D; Non Linear Dynamics Ltd., New Castle upon Tyne,

133

United Kingdom) and normalized against the HSA (Sigma) standard.

134

7



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Results and discussion

136 137

Duplication of des-pE1M-brazzein and KlPDI at chromosomal sites

138

The secretion levels of brazzein can be elevated by increasing PDI and Ero1p activities in

139

the yeast expression system. There are two ways to increase gene dosage of KlPDI in

140

brazzein producing yeast: (1) the direct introduction of KlPDI into the multi-copy vector that

141

carries the brazzein expression cassette; (2) introducing a duplication of KlPDI on the host

142

chromosome. However, the former constructs were found to severely affect yeast growth and

143

plasmid stability, indicating that KlPDI on a multi-copy vector was detrimental to the growth

144

of K. lactis cells.17 Therefore, we used the latter strategy of introducing a single duplication

145

of KlPDI on the host chromosome.

146

To introduce the duplication of KlPDI on the chromosome of brazzein producing yeast,

147

both

148

transformed into K. lactis GG799. For transformation into yeast, the plasmids pKLAC2-des-

149

pE1M-brazzein and pKLAC2-KlPDI were digested with Sac II to obtain linearized fragments

150

containing the pE1M-brazzein gene and KlPDI. The fragments were co-integrated into the

151

chromosome of GG799 and the structures of these integrants were confirmed by PCR (data

152

not shown). We selected the co-integrated strains which showed a single duplication of

153

KlPDI and multi-copy duplications of the brazzein gene, since it is reported that high gene

154

dosage of KlPDI is toxic to K. lactis.16

expression

vectors,

pKLAC2-des-pE1M-brazzein

and

pKLAC2-KlPDI,

were

155 156

Effect of KlPDI duplication on brazzein secretion

157

To elucidate the effect of KlPDI single duplication, the transcriptional level of KlPDI was

158

compared among the K. lactis strain GG799 transformant with only the brazzein gene was

159

integrated into the chromosome and the transformant with both brazzein gene and KlPDI 8


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integrated. KlPDI mRNA level in GG799 that had integrated both brazzein and PDI gene was

161

more than ~2-fold higher than that in the transformant that had integrated the brazzein gene

162

alone (Fig. 1A).

163

Our previous study has shown that secretion of des-pE1M-brazzein continued after even

164

the culture entered the stationary phase and the level of the secreted protein was observed to

165

increase even in a prolonged stationary phase.10 Therefore, the des-pE1M-brazzein level in

166

the culture supernatant was determined after 96 hours of culture. Figure 2A shows that a

167

single duplication of KlPDI also resulted in significantly increased secretion of pE1M-

168

brazzein. On the other hand, multi-copy duplications of KlPDI resulted in approximately 9-

169

17% decrease in secreted amounts of recombinant des-pE1M-brazzein (Fig. 2A, compare

170

lanes 1 and 2). Densitometric evaluation of secreted amounts indicated that GG799 that had

171

integrated both brazzein genes and a KlPDI secreted approximately 1.7-2.2 fold higher level

172

of recombinant des-pE1M-brazzein compared to the parental strain that had integrated only

173

the brazzein gene (Fig. 2A, compare lanes 1 and 3).

174 175

Induction of KlERO1 in K. lactis by DTT treatment and effect on brazzein secretion

176

In the yeast expression system, production of recombinant soluble des-pE1M-brazzein by

177

extracellular secretion could also be elevated by increasing KlERO1 expression, whose

178

product directly interacts with PDI. We established a strategy using DTT treatment to

179

overexpress KlERO1 since it has been reported that KlERO1 expression was strongly induced

180

by DTT in K. lactis cells.15

181

To identify KlERO1 induction upon DTT treatment, KlERO1 mRNA level was

182

determined by northern blotting. Figure 1B shows that addition of 1 mM DTT in the culture

183

medium resulted in significantly increased transcriptional level of KlERO1. As observed from

184

the densitometric evaluation, KlERO1 mRNA levels in DTT treated cells resulted in 9



185

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approximately 1.5-1.7 fold higher mRNA level compared to the DTT untreated cells.

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The secreted amount of recombinant des-pE1M-brazzein in culture supernatant was

187

determined under similar conditions, except for DTT addition. The secreted amount of

188

recombinant des-pE1M-brazzein in DTT treated cells resulted in approximately 1.3-1.6 fold

189

increase compared to that in DTT untreated cells (Fig. 2B). The result shows that induction of

190

KlERO1 by DTT also resulted in increased secreted levels of des-pE1M-brazzein.

191 192

Effect of simultaneous KlPDI and KlERO1 overexpression on brazzein secretion

193

In order to elucidate the effect of simultaneous KlPDI and KlERO1 overexpression on

194

brazzein secretion, des-pE1M-brazzein level in the culture supernatant was determined after

195

96 hours of culture. The secreted amount of des-pE1M-brazzein upon overexpression of

196

KlPDI and KlERO1 resulted in an arithmetic increase of secreted brazzein amounts by KlPDI

197

and KlERO1 overexpression, respectively (Fig. 3). Densitometric evaluation showed that the

198

secreted amount of des-pE1M-brazzein upon KlPDI overexpression was approximately 2-

199

fold higher than that observed in the previous expression system (Fig. 3, compare lanes 1 and

200

2). Moreover, the secreted amount of des-pE1M-brazzein upon simultaneous overexpression

201

of KlPDI and KlERO1 was approximately 2.6-fold higher than that from the previous

202

expression system (Fig. 3, compare lanes 1 and 3). The secreted recombinant des-pE1M-

203

brazzein was purified by ultrafiltration using 2,000 Da MWCO membrane (Sartorious,

204

Goettingen, Germany) and CM-Sepharose column chromatography. The purity and

205

conformational state of recombinant des-pE1M-brazzein were confirmed by SDS-PAGE,

206

HPLC, and circular dichroism as described in our previous paper (results not shown).10 The

207

sweetness of purified recombinant des-pE1M-brazzein was also evaluated by a human taste

208

panel and was found to have approximately 2130 times more sweetness than sucrose on a

209

weight basis as previously described.10 10


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We also determined the level of misfolded and unfolded des-pE1M-brazzein remaining

211

inside the cell by SDS-PAGE. As expected, simultaneous overexpression of KlPDI and

212

KlERO1 resulted in a large decrease in intracellular des-pE1M-brazzein levels remaining

213

inside the cell (Fig. 4). By densitometric evaluation, the amount of des-pE1M-brazzein

214

remaining inside the cell by simultaneous overexpression of KlPDI and KlERO1 was

215

approximately 75% lower than that of the previous expression system, demonstrating that

216

simultaneous overexpression of KlPDI and KlERO1 influences the correct folding of des-

217

pE1M-brazzein with four disulfide bonds in the ER.

218

Until now, many heterologous production systems for brazzein have been attempted in

219

foreign hosts to study its structure-sweetness relationship as well as its potential mass

220

production for commercial use. In bacterial expression systems, the yields of brazzein with

221

four disulfide-bonded structure were frequently low, because it was difficult to achieve proper

222

folding of protein in the cytosol of the cell.3-5 For production of the protein containing

223

multiple disulfide bridges, a good approach is to produce the target protein in yeast in the

224

secreted form. In yeast, proteins secreted by the cell form 0.05% of all the proteins expressed.

225

The secretory production of the recombinant protein in the culture supernatant allows a better

226

chance of proper folding due to increased oxidizing conditions in the extracellular

227

compartment and has advantages with respect to purification of the target protein. Recently,

228

we have shown the heterologous expression of a minor form of brazzein, des-pE1M-brazzein,

229

in the yeast K. lactis.10 Poirier et al. (2012)9 also reported the expression of two natural forms

230

of brazzein and a mutant, called Q1-bra in the yeast P. pastoris. From both yeast expression

231

systems, recombinant brazzein was obtained approximately in the range of 30-100 mg/L.

232

However, intracellular levels of misfolded and unfolded brazzein were still high (Fig. 3).

233

To decrease the levels of intracellular misfolded and unfolded brazzein, one strategy is to

234

facilitate the secretion of highly S-S bonded proteins by increasing PDI-Ero1p activities. 11



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235

Overexpression of KlPDI in S. cerevisiae led to increased secretion of human platelet-derived

236

growth factor,18 human lysozyme,19 antistasin,20 single chain antibody fragments (ScFVs),21

237

Pyrococcus furiosus β-glucosidase,21 and human serum albumin.14 In contrast, several

238

authors reported that secretion of other proteins was not significantly influenced by any of

239

these modifications,18, 23, 24 although many negative results often remain unpublished. In the

240

present study, we examined the secretion of a sweet-tasting protein, brazzein having four

241

disulfide bonds, in K. lactis. The result of KlPDI overexpression was consistent with the

242

above mentioned findings with the S. cerevisiae systems (Fig. 2A). Induction of KlERO1 also

243

led to increased secretion of brazzein in K. lactis (Fig. 2B). The observation that KlPDI and

244

KlERO1 overexpression led to apparently similar results might suggest that these proteins

245

acting at a common step. The observed increase in brazzein production also suggests that

246

neither PDI nor Ero1p is a limiting factor when either KlPDI or KlERO1 is overexpressed,

247

because oxidizing equivalents are known to flow directly from Ero1p to secretory proteins

248

via PDI. Moreover, simultaneous overexpression of both proteins accelerated brazzein

249

secretion, resulting in an arithmetic increase in secreted brazzein amounts by KlPDI and

250

KlERO1 overexpression (Fig. 3). This result suggests the effects of PDI and Ero1p may be

251

additive. Similar results were also observed with studies regarding the secretion of highly

252

disulfide-bonded human serum albumin.15,24 The secretion of human serum albumin that has

253

17 disulfide bonds, was increased by overexpression of KlPDI and KlERO1. Thus, to

254

facilitate proper folding and subsequent high secretion of brazzein, PDI is the important

255

factor that engages thiol-disulfide exchange with Ero1p. These results also show that this

256

modified expression system could be applied to mass production of recombinant brazzein.

257

The steadily increasing requirement for low-calorie sugar substitutes in order to reduce the

258

intake of sugar and artificial sweeteners for treating the growing number of over-weight

259

people and diabetic patients have encouraged the development of efficient biotechnological 12


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systems for the production of a sweet-tasting protein, brazzein. Overexpression of KlPDI and

261

KlERO1 in the yeast K. lactis through a secretory expression system, as described here,

262

allows for maximum brazzein secretion levels and it allows cost reduction. Moreover, it

263

allows us to produce brazzein at a large-scale, which will prove useful in the food industry.

264 265

Acknowledgements

266

This work was supported by the National Research Foundation of Korea Grant funded by the

267

Korean Government (NRF-2015R1A2A1A15053693 and 2015R1D1A1A01058326) and by

268

the Chung-Ang University Excellent Student Scholarship. We thank Prof. Kangseok Lee for

269

northern blot analysis.

270

13



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(13) Wang, L.; Wang, X.; Wang, C. C. Protein disulfide-isomerase, a folding catalyst and a redox-regulated chaperone. Free Radic. Biol. Med. 2015, 83, 305-313.

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oxidative activity of Ero1p in protein disulfide bond formation in the endoplasmic

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Kluyveromyces lactis overexpressing KlPDI1 and KlERO1. Appl. Environ. Microbiol.

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duplication of polyubiquitin and protein disulfide isomerase genes in Kluyveromyces

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lactis. Gene. 2001, 272, 103-110.

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Figure Legends

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Figure 1. (A) Transcriptional level of KlPDI. Lane M, Molecular weight marker (1kb ladder);

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lane 1, mRNA level of KlPDI in GG799 wherein only the brazzein gene was integrated into

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the chromosome; lane 2, mRNA level of KlPDI in GG799 wherein both brazzein and PDI

352

genes were integrated into the chromosome. (B) Effect of DTT on KlERO1 mRNA levels.

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Total RNA was prepared from GG799 cells growing in YPGal medium without DTT (lane 1)

354

or with 1 mM DTT (lane 2). RNA was hybridized with labeled probes against KlERO1.

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Figure 2. SDS polyacrylamide gel electrophoresis. (A) Comparison of brazzein expression

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levels by duplication of KlPDI. Lane M, Molecular weight marker (polypeptide marker); lane

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1, brazzein expressed in the host without the integrated KlPDI; lane 2, brazzein expressed in

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the host with multi-copy duplications of KlPDI; lane 3, brazzein expressed in the host with a

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single duplication of the KlPDI. (B) Comparison of brazzein expression levels upon addition

361

of DTT in K. lactis. Lane M, Molecular weight marker (polypeptide marker); lane 1, brazzein

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expressed in the host without DTT addition; lane 2, brazzein expressed in the host with DTT

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addition.

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Figure 3. SDS polyacrylamide gel electrophoresis for comparison of brazzein expression

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levels. Lane M, Molecular weight marker (polypeptide marker); lane 1, brazzein expressed

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without overexpression of proteins related to protein folding in the ER; lane 2, brazzein co-

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expressed with KlPDI; lane 3, brazzein co-expressed with KlPDI and KlERO1.

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Figure 4. SDS polyacrylamide gel electrophoresis for comparison of intracellular brazzein in

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K. lactis. Lane M, Molecular weight marker (polypeptide marker); lane 1, cell lysate of K.

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lactis expressing brazzein; lane 2: cell lysate of K. lactis co-expressing brazzein with KlPDI

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and KlERO1.

19



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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Table of Contents Graphic

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Improved Secretory Production of the Sweet-Tasting Protein, Brazzein

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