Optimization of production conditions of rice α-galactosidase II displayed on yeast cell surface

Journal Pre-proof Optimization of production conditions of rice α-galactosidase II displayed on yeast cell surface Mosi Dong, Yun Gong, Jia Guo, Jing Ma, Suhong Li, Tuoping Li PII:

S1046-5928(19)30686-2

DOI:

https://doi.org/10.1016/j.pep.2020.105611

Reference:

YPREP 105611

To appear in:

Protein Expression and Purification

Received Date: 27 December 2019 Revised Date:

2 February 2020

Accepted Date: 20 February 2020

Please cite this article as: M. Dong, Y. Gong, J. Guo, J. Ma, S. Li, T. Li, Optimization of production conditions of rice α-galactosidase II displayed on yeast cell surface, Protein Expression and Purification (2020), doi: https://doi.org/10.1016/j.pep.2020.105611. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

Author statement Mosi Dong: Writing- Original draft preparation, Validation, Yun Gong: Software, Jia Guo: Data curation, Jing Ma: Formal analysis, Suhong Li: Conceptualization, Methodology, Funding acquisition, Tuoping Li: Writing- Reviewing and Editing

1

Optimization of production conditions of rice α-Galactosidase II displayed on

2

yeast cell surface

3

Mosi Dong1, Yun Gong1, Jia Guo1, Jing Ma2, Suhong Li1*, Tuoping Li1*

4

1

5

China.

6

2

College of Food Science, Shenyang Agricultural University, Shenyang 110866,

Xingcheng Village Rehabilitation Service Centre, Xingcheng, 125100, China.

7 8

Corresponding author:

9

Suhong Li, E-mail: [email protected]; Tel.: 86-24-88487161; fax: 862488487161;

10 11

Tuoping Li, E-mail: [email protected]; Tel.: 86-24-88487161; fax: 862488487161.

12 13

Abstract

14

The yeast surface displayed rice α-galactosidase II (YSD rice α-Gal II) was

15

generated with the pYD1 vector. The expression and cultural conditions for the

16

improvement of production of YSD rice α-Gal II were optimized. The results showed

17

that several induction factors, which were the initial cell density, inoculation ratio,

18

galactose (inducer) concentration, induction time and temperature, determined the

19

activity and expression efficiency of YSD rice α-Gal II. Meanwhile, the medium

20

composition also affected its activity and production. Moreover, the production of

21

YSD rice α-Gal II was further improved by continuous feeding of galactose in the

22

fermenter level. The highest production was obtained at an initial cell density of

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OD600 = 2.9, 2 % inoculation ratio, and 2 % galactose, with 0.6 g/L compound

24

nitrogen source ((NH4)2SO4/urea=2/1, w/w) and 5 g/L sucrose, followed by

25

continuous feeding of galactose (20 g/L with flow rate of 1.5 mL/h). At such

26

conditions, the enzyme activity and productivity reached to 676.2 U/g (DCW) and

27

1548.5 U/L, respectively, 26.4- and 63.7-fold to that before optimization. The results

28

provided a basic and effective strategy for the industrial production of YSD rice α-Gal

29

II.

30 31 32

Keywords: α-Galactosidase, Yeast cell surface display, Cell weight, Fed-batch fermentation

33 34

1 Introduction

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α-Galactosidase (α-Gal, EC 3.2.1.22) catalyzes the hydrolysis of the glycosidic

36

bonds of terminal non-reducing α-galactosyl residues of galacto-oligosaccharides,

37

galacto-polysaccharides, galacto-protein and galacto-lipid [1, 2, 3]. Given its

38

properties, α-Gals are of great interest for various applications in food, feed and

39

medicine industry. In our previous study, rice α-Gal II gene (BAC84411) was cloned

40

and constructed into E. coli Origami B. The expressed rice α-Gal II showed broad

41

substrate specificity and good hydrolytic activity on polymeric galactomannans [4].

42

As such, rice α-Gal II might be used to modify galactomannan with the

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degalactosylation, to obtain the enhanced or new properties. Likes many eukaryote

44

proteins，the expression level of rice α-Gal II in E. coli is relatively lower, and the

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enzyme purification cost also is a long-standing limited factor restricting its industrial

46

application.

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For its commercial success, it is necessary to minimize the cost of enzyme

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production. This depends on the purification costs, expression level as well as the

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activity and stability of the rice α-Gal II produced. Recently, cell-surface display has

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been proved to be a promising cost-effective tool for industrial applications. It is a

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novel technique that can auto-immobilize target proteins on the cell surface of

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microorganisms by fusing an appropriate protein as an anchoring motif [5]. As such,

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cell-surface display is a comprehensive biotechnology combines gene expression,

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protein purification and enzyme immobilization [6]. Saccharomyces cerevisiae (S.

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cerevisiae) is one of the most suitable host strains for enzyme cell-surface display,

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since it allows the folding and glycosylation of expressed heterologous eukaryotic

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proteins. Its other features include generally regarded as safe status (GRAS), clear

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genetic background, easy cultivation, and cheap production [7]. Effects of cell wall

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proteins of S. cerevisiae as anchors on efficiency of the cell surface expression for an

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α-Gal were reported [8]. The yeast surface display system has also been used for

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expressing several enzymes, such as lipase [6], β-lactamase [9], glucose oxidase [10]

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pyranose dehydrogenase [11]. The nutrients and carbon source in the culture media

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were found to affect the display of a Flo1 fusion lipase on the sake yeast cell-surface

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[12]. However, expression levels of these displayed enzymes are not desirable.

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In this experiment, rice α-Gal II gene was constructed into yeast surface display

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vector pYD1 and subsequently transformed into the host of S. cerevisiae EBY100.

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The effects of several conditions, including induction and fermentation parameters

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were investigated to maximize the enzyme activity and production level for potential

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commercial application of YSD rice α-Gal II.

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2 Materials and Methods

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2.1 Materials

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S. cerevisiae EBY100 and pYD1 (Invitrogen, Carlsbad, CA, USA) were used as

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the host and vector for expression, respectively. The pET32a+-α-Gal II plasmid [4]

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used

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p-Nitrophenyl-α-D-galactose (pNPG) was purchased from Sigma Chemical Company

76

(St. Louis, MO, USA). All other chemical reagents are of analytical grade.

77

2.2 Protein expression

as

the

template

was

previously

generated

in

our

laboratory.

78

Rice α-Gal II gene (BAC84411 from O. sativa L. subsp. japonica var.

79

Nipponbare) fragment was amplified by high-fidelity PCR from pET32a+-α-Gal II

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plasmid

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5’-TAAGGTACCAGGATCCATGCTCGACAACGGGCTCGGGCG-3’

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BamH

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(reverse, EcoR I). The restricted PCR-product was ligated into the display vector

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pYD1. The resultant recombinant plasmid was confirmed by sequencing and finally

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transferred into S. cerevisiae EBY100 to obtain YSD rice α-Gal II. Minimal Dextrose

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Plate was used to screen for positive clones.

with

I)

and

the

specific

primers

of (forward,

5’-GATATCTGCAGAATTCGCTCCGCTCCTCGCTGGCCC-3’

87

Protein expression was carried out according to the manual of the pYD1 Yeast

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Display Vector Kit. A single colony of YSD rice α-Gal II was inoculated into

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YNB-CAA medium containing 2 % glucose and incubated with shaking (185 rpm) at

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30 °C overnight until the OD600 reached over 2.0. The yeast cells were harvested via

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centrifugation (5000 × g for 10 min at 4 °C), then subsequently resuspended in 200

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mL YNB-CAA medium containing galactose (2 %) using 500 mL shake flask, and

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cultured at 20 °C for 48 h. Galactose was used as inducer for the expression of YSD

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rice α-Gal II. The yeast cells were recovered by centrifugation (5000 × g for 10 min at

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4 °C), washed with McIlvaine buffer (pH 5), then freeze-dried and weighted.

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2.3 Cell immunofluorescence analysis

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The induced yeast cells were collected and washed with phosphate buffer saline

98

(PBS), resuspended in PBS with 1 mg/mL fetal bovine serum (FBS) and subsequently

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incubated on ice for 20 min. After washing with PBS, the yeast cells were labeled

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with V5 tag-FITC Mouse Monoclonal Antibody (Invitrogen, Carlsbad, CA, USA;

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excitationmax/emissionmax=494/518 nm) conjugated with FITC on ice for 1 h in dark

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with PBS containing 1 mg/mL FBS. After washing with PBS, the yeast cells were

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incubated to combine with the fluorescent antibody Anti-V5-FITC on ice for 1 h in

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dark with PBS containing 1 mg/mL FBS. The binding of Anti-V5-FITC to the yeast

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cell surface was visualized using a fluorescence microscope.

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2.4 SDS-PAGE for YSD rice α-Gal II

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The expression profile of YSD rice α-Gal II was assessed with SDS-PAGE using

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12 % w/v polyacrylamide gel as described by Laemmli [13]. The strap were stained

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with 0.25 % Coomassie Brilliant Blue R-250. Molecular masses of the protein marker

110

(TAKARA, Japan) were used ranging from 14.4 to 97.4 kDa.

111

2.5 YSD rice α-Gal II activity assay

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The activity was assayed with dry cell of YSD rice α-Gal II in McIlvaine buffer

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(pH 5) containing 2.5 mM pNPG at 45 °C for 10 min. An equal volume of 0.5 M

114

Na2CO3 was added to terminate the reaction. Cells were removed via centrifugation

115

(5000 × g for 10 min at 4 °C) and the absorbance of supernatant was measured at 405

116

nm. One unit of YSD rice α-Gal II activity was defined as the amount of enzyme that

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releases 1 µmol of p-nitrophenol per minute [4]. Total production was expressed as

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enzyme activity multiply by dry cell weight.

119

2.6 Selection of induction and culture conditions for the production of YSD rice

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α-Gal II

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The variation of induction conditions were initial cell density (OD600 = 2.3-3.7),

122

inoculation ratio (1-7 %), galactose concentrations (0.5-6 %), induction time (0-84 h)

123

and temperature (20-26 oC), respectively. Several nitrogen (urea, (NH4)2SO4, and

124

L-arginine) and carbon (glucose, glycerol, sucrose, and maltose) sources with

125

different concentration (0.1-1 g/L) and were used to select the optimal culture

126

conditions for the production of YSD rice α-Gal II. Nitrogen compounds of

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(NH4)2SO4 and urea, with different composite ratio (2/1–1/1–1/2, w/w) and

128

concentration (0.3-0.6 g/L) were also designed for selecting the optimal conditions of

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YSD rice α-Gal II. Yeast cells were incubated in a 2 L shake flask containing 1 L

130

medium at 185 rpm. The incubation temperature and time were 24 oC and 36 h, unless

131

otherwise specifically indicated. The enzyme activity, dry cell weight and total

132

production were used as the indexes to evaluate the expression efficiency of YSD rice

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α-Gal II.

134

2.7 Fed-batch fermentation of YSD rice α-Gal II

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The fermentation was carried out in 2 L fermenter (Bailun Bio, China) equipped

136

with auto-controlling system for pH, temperature, aeration, agitation and feeding. The

137

operating parameters for the fermentation process were 24 °C, 0.8 vvm aeration rate

138

and 120 rpm agitator speed for 36 h. The cultivation was carried out in 1 L above

139

optimized culture medium, continuously fed with compound of (NH4)2SO4 and urea

140

(2/1–1/1, v/v; 0.1 - 0.4 g/L), sucrose (5-30 g/L), or galactose (5-30 g/L), respectively,

141

at constant flow rate of 1.5 mL/h for 36 h.

142

2.8 Statistical Analysis

143

All data were shown as the mean ± SD of triplicates. Significant differences

144

among the treatments were determined using SPSS software (version 19.0 SPSS,

145

Chicago, IL, USA). Statistical significance was set at p < 0.05.

146

3 Results and Discussion

147

3.1 Expression of YSD rice α-Gal II

148

Full-length rice α-Gal II gene comprised of 417 amino acids was cloned into the

149

pYD1 vector, and was confirmed using PCR and DNA sequencing. Fluorescent

150

staining with antibody Anti-V5-FITC was used to detect the Aga2p fusion. As shown

151

in Fig.1a, the green fluorescence of the EBY100/pYD1 and YSD rice α-Gal II

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demonstrated the presence of Aga2p on the cell surface. The expression pattern of

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YSD rice α-Gal II induced by galactose was detected in SDS-PAGE (lane 4 of Fig.1b),

154

and a major protein band which was in agreement with predicted molecular weight

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was detected at position of 45.5 kDa (42.5 kDa of target protein and 3 kDa of Aga2

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peptide). The result indicated that rice α-Gal II protein was fused to the Aga2p of

157

pYD1 and was displayed on the yeast cell surface. Furthermore, in the initial

158

expression condition mentioned in section 2.2, YSD rice α-Gal II activity was

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confirmed as 25.6 U/g (DCW), dry cell weight and total production were 0.19 g and

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4.86 U in 200 mL culture medium, respectively.

161

3.2 Effect of induction conditions on the production of YSD rice α-Gal II

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The effects of initial cell density (represented as OD600 of cell suspension),

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inoculum ratio, inducer (galactose) concentration, incubating temperature and time on

164

the expression production of YSD rice α-Gal II were exhibited in Fig. 2. In general,

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the display of fusion proteins was relatively lower below OD600 2.0 and over 5.0 [14].

166

As shown in Fig. 2a, the enzyme activity was gradually enhanced with the increase of

167

initial cell density and the peak of enzyme activity (109.9 U/g) was observed at OD600

168

= 2.9. Proper inoculation ratio that was favorable for the expression of proteins was to

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ensure that the cells continuously grew in log-phase when S. cerevisiae was

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transferred to medium containing galactose [15]. The expression pattern of YSD rice

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α-Gal II at different inoculation ratios were shown in Fig. 2b, the highest enzyme

172

activity was 204.5 U/g (DCW) at the inoculum ratio of 2 %. It was said that, galactose

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was an effective inducer for the expression of foreign genes in the yeast surface

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display systems [16]. The galactose-regulated promoter (GAL1) gene of S. cerevisiae

175

was rapidly and efficiently activated when target protein was switched to medium

176

containing galactose [17]. Suitable galactose concentration improved glucose oxidase

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displayed on the surface of S. cerevisiae [10]. In the present experiment, enzyme

178

activity was enhanced along with the increase of galactose concentration, and reached

179

its highest activity (208.3 U/g) at galactose concentration of 2-3 % (Fig. 2c).

180

On the other hand, researches have reported that, in the surface display system,

181

more fusion proteins are displayed on the cell surface at 20-25 oC [15, 16]. The

182

expression pattern of YSD rice α-Gal II at different incubation temperature and time

183

were shown in Fig. 2d. It showed that YSD rice α-Gal II was sensitive to the

184

expression temperature. The suitable expression temperature was at 24 oC, in which,

185

the enzyme activity of YSD rice α-Gal II was obviously higher than that at 20 oC, 22

186

o

C and 26 oC. These results might due to that the proper temperature was more

187

suitable to the correct protein folding, making more protein transfer from the inside to

188

the cell surface, and improving the expression of protein [18]. Meanwhile, the longer

189

expression time period decreased the expression of YSD rice α-Gal II, since the

190

expression plasmid in S. cerevisiae would be unstable over the proper expression time,

191

in result, the expression efficiency was reduced [19]. On the other hand, the activity

192

might be lost during a long expression, due to the expression temperature was not

193

suitable for stabilizing YSD rice α-Gal II. The highest enzyme activity (240.6 U/g)

194

was obtained at 24oC incubation for 36 h.

195

3.3 Effect of nitrogen sources on the production of YSD rice α-Gal II

196

Based on the above selected expression conditions (initial cell density of OD600

197

2.9, inoculum ratio 2 %, galactose 2 %, incubation temperature 24 oC, and incubation

198

time 36 h), effects of nitrogen sources on the production of YSD rice α-Gal II were

199

investigated. As showing in Fig. 3a, L-arginine exhibited a negative effect on the

200

production of YSD rice α-Gal II. Meanwhile, addition of both (NH4)2SO4 and urea

201

significantly (p < 0.05) increased the production of YSD rice α-Gal II, compared to

202

that of control. The result was consistent with the report that adding (NH4)2SO4

203

promoted the growth of S. cerevisiae [20, 21]. However, the difference of highest

204

production between (NH4)2SO4 and urea was not statistically significant. It was

205

interesting that urea mainly increased the enzyme activity of YSD rice α-Gal II at the

206

lower concentration (0.1-0.3 g/L), while, (NH4)2SO4 mainly enhanced the growth of

207

yeast cell, represented as the significant (p < 0.05) increase of dry cell weight of YSD

208

rice α-Gal II. Therefore, to combine these two positive functionalities, effects of the

209

mixture of (NH4)2SO4 and urea on the production of YSD rice α-Gal II were further

210

investigated. Fig. 3b showed that enzyme activity, dry cell weight, and total

211

production were all significantly (p < 0.05) increased by adding of compounded

212

nitrogen source, compared to those of control. The highest production were obtained

213

at 0.6 g/L compounded with the proportion of (NH4)2SO4/urea = 2/1 (w/w).

214

3.4 Effect of carbon source on the production of YSD rice α-Gal II

215

Carbon source provide energy for microbial growth and metabolism, and also

216

affect the synthesis and accumulation of target metabolites in microorganisms [20]. As

217

for the case of YSD rice α-Gal II, Fig. 4a showed that glucose, glycerol and maltose

218

were not beneficial carbon sources either for enzyme activity or for the enzyme

219

production. In contrast, the enzyme activity and the production of YSD rice α-Gal II

220

were significantly (p < 0.05) increased by adding sucrose. This result was similar to

221

other literatures in which the lipase expression in S. cerevisiae with GAL as the

222

promoter was promoted using sucrose as one of optimal carbon source [12] and

223

production of oxidoreductase from S. cerevisiae was improved by sucrose [22]. The

224

further investigation was carried out for understanding the effect of sucrose

225

concentrations on the production of YSD rice α-Gal II. The results revealed that the

226

activity and production of YSD rice α-Gal II were significantly (p < 0.05) increased in

227

the lower concentration range (2.5-10 g/L) of sucrose, but was decreased in the

228

relatively higher concentration range (12.5-15 g/L) of sucrose, compared to those of

229

control (Fig. 4b). The highest enzyme activity and production were obtained at 5 g/L

230

of sucrose. However, there were no significant differences in dry cell weight among

231

all treatments (Fig. 4b).

232

3.5 Effect of continuous feeding on YSD rice α-Gal II in the fermenter

233

Based on the optimal induction and cultural conditions obtained in shake flask

234

experiments mentioned above, the effects of continuous feeding on the expression and

235

production of YSD rice α-Gal II in the fermenter were discussed. No significant

236

positive impacts in continuous feeding of nitrogen and carbon source were observed

237

(Fig. 5a, b). Although no significant effects on the activity and dry cell weight were

238

observed, galactose used as both substrate and inducer played an important role in the

239

expression and production of YSD rice α-Gal II, since its production were

240

significantly (p < 0.05) enhanced by continuous feeding of 10-20 g/L galactose (Fig.

241

5c). This may be related to the supplementation of galactose by continuous feeding to

242

resupply its loss. This may have aided in maintaining the optimal inducer

243

concentration during the induction process, and subsequently yielded a significant

244

higher expression of recombinant genes driven by galactose-inducible promoters [23].

245

Moreover, galactose in cultural medium was consumed rapidly in the fermentation

246

process, and reduced to half of the initial concentration after 12 h-fermentation (data

247

not shown). The highest production (1548.5 U) was obtained with continuous feeding

248

of 20 g/L galactose.

249

4 Conclusions

250

Yeast surface display is a novel expression system for the eukaryotic foreign

251

proteins. This study showed that several induction factors involving the initial cell

252

density, inoculum ratio, inducer concentration, induction time and temperature,

253

significantly affected the expression of YSD rice α-Gal II. (NH4)2SO4, urea, and

254

sucrose were also important substrates for the growth and/or expression of YSD rice

255

α-Gal II. Furthermore, the continuous feeding of galactose in the fermentation process

256

was an effective strategy to improve the production of YSD rice α-Gal II. The

257

expression and production conditions of YSD rice α-Gal II were optimized using our

258

experimental procedure which ultimately increased its production from 24.3U (the

259

initial total production measured in the condition of section 2.2 in 1 L culture) up to

260

1548.5 U, 63.7-fold increase over the initial condition. The results show potential for

261

commercial production and utilization of YSD rice α-Gal II.

262

Acknowledgements

263

This work was supported by research grants from the National Natural Science

264

Foundation of China (Grant No.31271819); Key Laboratory Program of Shenyang,

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China (Grant No.17-158-1-00).

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Conflict of Interest

267 268

The authors declare that they have no conflict of interest.

269 270

Reference

271

[1] Y.J. Hu, G.T. Tian, L.Y. Wang, H.X. Wang, T.B. Ng. A protease-resistant

272

α-galactosidase from Pleurotus djamor with broad pH stability and good

273

hydrolytic activity toward raffinose family oligosaccharides. Int. J. Biol.

274

Macromol. 94 (2017) 122-130. http://doi.org/10.1016/j.ijbiomac.2016010.005.

275

[2] P. Katrolia, H.Y. Jia, Q.J. Yan, S. Song, Z.Q. Jiang, H.B. Xu. Characterization of a

276

protease-resistant α-galactosidase from the thermophilic fungus Rhizomucor

277

miehei and its application in removal of raffinose family oligosaccharides.

278

Bioresource

279

http://doi.org/10.1016/j.biortech.2012.01.144.

Technol.

110

(2012)

578-586.

280

[3] Z.G. Han, J.W. Zhang, X.F. Jiang. J.K. Yang. Gene dosage and coexpression with

281

endoplasmic reticulum secretion-associated factors improved the secretory

282

expression of α-galactosidase. Protein Expr. Purif. 153 (2019) 83-91.

283

http://doi.org/10.1016/j.pep.2018.08.004.

284

[4] S.H. Li, T.P. Li, W.D. Kim, M. Kitaoka, S. Yoshida, M. Nakajima, H. Kobayashi.

285

Characterization of raffinose synthase from rice (Oryzasativa L. var.

286

Nipponbare).

287

http://doi.org/10.1007/s10529-006-9268-3.

288

Biotechnol.

Lett.

(2007)

635-640.

[5] A. Kondo, M. Ueda. Yeast cell-surface display-applications of molecular display.

289

Appl.

290

http://doi/org/10.007/s00253-003-1492-3.

291

29

Microbiol.

Biotechnol.

64

(2004)

28-40.

[6] Y. Liu, R. Zhang, Z.S. Lian, S.H. Wang, A.T. Wright. Yeast cell surface display for

292

lipase whole cell catalyst and its applications. J. Mol. Catal. B: Enzym. 106

293

(2014) 17-25. http://doi.org/10.1016/j.molcatb.2014.04.011.

294

[7] Z. Liu, S.H. Ho, T. Hasunuma, J.S. Chang, N.Q. Ren, A. Kondo. Recent advances

295

in yeast cell-surface display technologies for waste biorefineries. Bioresource

296

Technol. 215 (2016) 324-333. http://doi.org/10.1016/j.biotech.2016.03.132.

297

[8] J.M. Van der Vaart, R. Biesebeke, J.W. Chapman, H.Y. Toschka, F.M. Klis, C.T.

298

Verrips. Comparison of cell wall proteins of Saccharomyces cerevisiae as

299

anchors for cell surface expression of heterologous proteins. Appl. Environ.

300

Microb. 63(1997) 615-620. http://doi.org/10.0000/AEM.asm/63/.

301

[9] A. Strauss, F. Gotz. In vivo immobilization of enzymatically active polypeptides

302

on the cell surface of Staphylococcus carnosus. Mol. Microbiol. 21 (1996)

303

491-500. http://doi.org/10.1111/j.1365-2958.1996.tb02558.x.

304

[10] M. Blazic, G. Kovacevic, O. Prodanovic, R. Ostafe, M. Gavrovic-Jankulovic, R.

305

Fischer, R. Prodanovic. Yeast surface display for the expression, purification and

306

characterization of wild-type and B11 mutant glucose oxidases. Protein Expr.

307

Purif. 89 (2013) 175-180. http://doi.org/10.1016/j.pep.2013.03.014.

308

[11] I. Gal, O. Schlesinger, L. Amir, L. Alfonta. Yeast surface display of

309

dehydrogenases in microbial fuel-cells. Bioelectrochemistry. 11 (2016) 53-60.

310

http://doi.org/10.1016/j.bioelechem.2016.07.006.

311

[12] T. Tanino, E. Noguchi, S. Kimura, H. Sahara, Y. Hata, H. Fukuda, A. Kondo.

312

Effect of cultivation conditions on cell-surface display of Flo1 fusion protein

313

using

sake

yeast.

33

(2007)

232-237.

Biochem.

Eng.

J.

314

fglhttp://doi.org/10.1016/j.bej.2006.11.001.

315

[13] U.K. Laemmli. Cleavage of structural proteins during the assembly of the head of

316

bacteriophage T4. Nature. 227 (1970) 680-685. http://doi.org/10.1038/227680a0.

317

[14] T. Tanaka, A. Kondo. Cell-surface display of enzymes by the yeast

318

Saccharomyces cerevisiae for synthetic biology. FEMS Yeast Research. (2014)

319

http://doi.org/10.1111/1567-1364.12212.

320

[15] pYD1 Yeast Display Vector Kit. Catalog no. V835-01. 10 (2002) 25-0259.

321

[16] E.T. Boder, K.D. Wittrup. Yeast surface display for directed evolution of protein

322

expression, affinity, and stability. Methods Enzymol. 328 (2000) 430–444. http://

323

www.ncbi.nlm.nih.gov/pubmed/11075358.

324

[17] P.J. Bhat, T.V. Murthy. Transcriptional control of the GAL/MEL regulon of yeast

325

Saccharomyces cerevisiae: mechanism of galactose-mediated signal transduction.

326

Mol.

327

http://doi:10.1046/j.1365–2958.2001.02421.x.

Microbiol.

40

(2001)

1059–1066.

328

[18] K. Takayama, S. Suye, K. Kuroda, M. Ueda, T. Kitagucihi, K. Tsuchiyama, T.

329

Fukuda, W. Chen, A. Mulchandani. Surface display of organophosphorus

330

hydrolase on Saccharomyces cerevisiae. Biotechnol. Prog. 22 (2006) 939-943.

331

http://doi.org/10.1021/bp060107b.

332

[19] C. Andreu, M.O. Del. Yeast arming by the Aga2p system: effect of growth

333

conditions in galactose on the efficiency of the display and influence of

334

expressing leucine-containing peptides. Appl. Microbiol. (2013) 97 9055-9069.

335

[20] W.W. Yu, W. Chen, Y. Teng, Y.J. Chen, M.D. Xu, S.L. Yang. Optimization of

336

fermentation process of engineered yeast for production of artemisnic acid. Fine

337

Chemicals. 36 (2019) 856-864.

338

[21] W.P. Zhang, X.R. Zhao, G.C. Du, H.J. Zou, J.W. Fu, J.W. Zhou, J. Chen.

339

Nitrogen catabolite repression in Saccharomyces cerevisiae and its effect on

340

safety of fermented foods. J. Appl. Environ. Biol. 18 (2012) 862-872.

341

http://doi.org/10.3724/SP.J.1145.2012.00862.

342

[22] Q. Guo, H. Zabed, H.H. Zhang, X. Wang, J.H. Yun, G.Y. Zhang, M.M. Yao, W.J.

343

Sun, X.H. Qi. Optimization of fermentation medium for a newly isolated yeast

344

strain (Zygosaccharomyces rouxii JM-C46) and evaluation of factors affecting

345

biosynthesis of D-arabitol. LWT-Food Sci. Technol. 99 (2019) 319-327.

346

http://doi.org/10.1016/j.lwt.2018.09.086.

347

[23] M.D. Kim, T.H. Lee, H.K. Lim, J.H. Seo. Production of antithrombotic hirudin in

348

GAL1-disrupted Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 65

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(2004) 259–262. http://doi.10.1007/s00253.004.1598.2.

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Figure captions

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Fig. 1 Expression and display of YSD rice α-Gal II on the yeast surface based on

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pYD1 system. (a) Green immunofluorescence staining of yeast cells decorating the

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YSD rice α-Gal II, (b) Expression pattern of the YSD rice α-Gal II detected by

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SDS-PAGE. Lane1, molecular maker; Lane 2, EBY100; Lane 3, EBY100/pYD1;

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Lane 4, YSD rice α-Gal II.

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Fig. 2 Effects of induction factors on YSD rice α-Gal II expression. (a) OD600, (b)

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inoculum ratio, (c) galactose concentration, (d) temperature and time.

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Fig. 3 Effect of nitrogen sources on YSD rice α-Gal II expression. (a) different

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nitrogen sources, (b) the proportion of compound nitrogen source ((NH4)2SO4 /urea,

362

w/w). Non-adding of (NH4)2SO4, urea, or L-arginine on optimal conditions selected in

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Fig.2 was used as a control. Values not sharing a common letter are significantly

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different between treatments at p < 0.05, where letters in the lower case, upper case

365

and lower case apostrophe indicate significant differences for enzyme activity, total

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production, and dry cell weight, respectively.

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Fig.4 Effect of carbon sources on YSD rice α-Gal II expression. (a) different carbon

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sources, (b) different concentration of sucrose. Non-adding of carbon sources was

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used as a control. Values not sharing a common letter are significantly different

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between treatments at p < 0.05, where letters in the lower case, upper case and lower

371

case apostrophe indicate significant differences for enzyme activity, total production,

372

and dry cell weight, respectively.

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Fig 5 Effect of continuous feeding of nitrogen source (a), sucrose (b), and galactose (c)

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on the production of YSD rice α-Gal II. Treatments with no-feedings were used as a

375

control. Values not sharing a common letter are significantly different between

376

treatments at p < 0.05, where letters in the lower case, upper case and lower case

377

apostrophe indicate significant differences for enzyme activity, total production, and

378

dry cell weight, respectively.

a

EBY100/pYD1

YSD rice α-Gal II

Immunofluoresce

Visible light

EBY100

11

10µm

b

1

2

3

4

75 kDa 60 kDa YSD α-Gal II 45.5 kDa

45 kDa 35 kDa 25 kDa

Fig.1

Fig.2

Fig.3

Fig.4

Fig.5

Highlights Yeast surface display (YSD) rice α- Gal II was constructed. Culture conditions increased the activity and expression efficiency of YSD rice α-Gal II. Fed-batch fermentation remarkably increased total production of YSD rice α-Gal II.