Improved polysaccharide production by homologous co

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Food and Beverage Chemistry/Biochemistry

Improved polysaccharide production by homologous cooverexpression of phosphoglucomutase and UDP glucose pyrophosphorylase genes in the mushroom Coprinopsis cinerea Jiangsheng Zhou, Yang Bai, Rujuan Dai, Xiaoli Guo, Zhonghua Liu, and Sheng Yuan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01343 • Publication Date (Web): 25 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Journal of Agricultural and Food Chemistry

Improved

polysaccharide

production

by

homologous

co-overexpression

of

phosphoglucomutase and UDP glucose pyrophosphorylase genes in the mushroom Coprinopsis cinerea

Jiangsheng Zhou§, Yang Bai§, Rujuan Dai, Xiaoli Guo, Zhong-Hua Liu*, Sheng Yuan* Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing, PR China 210023

Running Title: Polysaccharide from Coprinopsis cinerea

§ Co-first authors * To whom correspondence should be addressed: Dr Sheng Yuan, College of Life Science, Nanjing Normal University, 1 Wenyuan Rd, Xianlin University Park, Nanjing, 210023, PR China. Tel: 86-25-85891067 (O), Fax: 86-25-85891067 (O), E-mail: [email protected] Dr Zhong-hua Liu, College of Life Science, Nanjing Normal University, 1 Wenyuan Rd, Xianlin University Park, Nanjing, 210023, PR China. Tel: 86-13584093709, E-mail: [email protected],

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ACS Paragon Plus Environment


1

ABSTRACT

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Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this

3

report, increases in polysaccharide production by homologous co-overexpression or

4

individual homologous overexpression of phosphoglucomutase and UDP glucose

5

pyrophosphorylase gene in Coprinopsis cinerea, which participate in polysaccharide

6

biosynthesis. The transcription levels of the target genes were upregulated

7

significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP

8

strain. The maximum intracellular polysaccharide content obtained in the

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oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight

10

improvement in polysaccharide production was obtained in the oePGM and oeUGP

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strains. Extracellular polysaccharide production was enhanced by 75% in the

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oePGM-UGP strain when compared with that of the WT strain, whereas

13

improvements of 30% and 16% were observed for the oePGM and oeUGP strains,

14

respectively. These results show that multiple interventions in polysaccharide

15

biosynthesis pathways of Basidiomycetes might improve polysaccharide yields when

16

compared with that of single interventions.

17 18

KEYWORDS: Coprinopsis cinerea, polysaccharides, homologous overexpression,

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phosphoglucomutase, UDP glucose pyrophosphorylase

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INTRODUCTION

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In China, mushrooms are traditionally used to enhance flavor and texture of foods

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as well as offering nutritional and pharmaceutical characteristics.1-3 Extensive

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investigations have confirmed that many mushrooms, including Ganoderma lucidum

24

(G. lucidum), Lentinula edodes (L. edodes) and Grifola frondosa (G. frondosa)

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species, possess antioxidative, antitumor, antinociceptive, anti-inflammatory,

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hepatoprotective and hypoglycemic effects.4 The polysaccharides present in

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mushrooms are postulated to be the main biologically active components. Because of

28

their potential health-promoting properties, numerous studies on the biological and

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pharmacological activities of polysaccharides derived from mushrooms have been

30

performed. Studies show that polysaccharides extracted from various mushroom

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species often exhibit different biological activities. This difference in activity is due

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to the large diversity of the chemical structures and chain conformations of these

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polysaccharides.2,

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pharmacological

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immunomodulatory and anti-inflammatory activities.5 Polysaccharides from

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Cordyceps sinensis have significant biological activities, e.g., antioxidant, antitumor,

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immunomodulatory activity and a hypoglycemic effect.4, 6 Polysaccharides from G.

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frondosa possess antioxidant activity, antitumor activity and significant free radical

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scavenging activity.7 The polysaccharide lentinan has been shown to exhibit strong

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antitumor, anticoagulatory and antiviral activity.4

4

Polysaccharides from Ganoderma have a wide spectrum of activity,

such

as

antitumor,

antimicrobial,

antioxidant,

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The biosynthetic pathways of mushroom polysaccharides including intracellular

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polysaccharides (IPS) and extracellular polysaccharides (EPS) involve the sugar

43

nucleotide synthesis pathway, and pathways involved in the assembly of the 3



44

repeating monosaccharide units and synthesis of the polymer.8, 9 In the pathway of

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polysaccharide biosynthesis, phosphoglucomutase (PGM) and UDP glucose

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pyrophosphorylase (UGP) are two key enzymes of sugar nucleotide biosynthesis.

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PGM occupies a branching point between the catabolic and anabolic pathways,

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which catalyzes the reversible interconversion of glucose 6-phosphate (G6P) to

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glucose 1-phosphate (G1P).10 Then G1P is catalyzed by UGP to form uridine

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diphosphate (UDP-D-Glc),11 which is used as a substrate to synthesize the linear

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β-1,3-glucan chains by a plasma membrane-bound glucan synthase complex and

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then extruded into the periplasmic space.12

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Recently, mushroom polysaccharides produced by submerged fermentation have

54

received significant interest as a promising alternative because of the potential

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advantages of high productivity and ease of controlling product quality.13 Several

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approaches including optimization of the fermentation conditions, addition of

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inducers and use of stimulatory agents have been used to obtain high yields of

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mushroom polysaccharides.14-17 However, greater quantity and better quality of

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mushroom polysaccharides are needed to meet the growing market demands.

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Because of the potential bottleneck of low levels of nucleotide sugar precursors in

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polysaccharides production, up-regulating the expression levels of particular

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biosynthetic genes by metabolic engineering represents a promising approach.18-20

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Numerous efforts have shown a positive correlation between the activities of

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enzymes involved in nucleotide sugar precursors biosynthesis with polysaccharides

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production.21 Moreover, genetic manipulation to increase expression of genes

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involved in sugar nucleotide biosynthesis to improve polysaccharide production has

67

been successfully performed in Streptococcus thermophilus, Lactobacillus helveticus 4


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68

and Saccharomyces cerevisiae.18,

22, 23

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shown that multiple interventions in metabolic pathways are more likely to be

70

successful, whereas single interventions are not sufficient to enhance the yields of

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desired products because of competition in different reactions for substrates and

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cofactors.18 Currently, there are no reports about enhancing polysaccharide

73

production in Basidiomycota by multiple interventions in polysaccharide

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biosynthesis metabolic pathways. Only a minor improvement in polysaccharide

75

production was observed by overexpression of the pgm or ugp gene in G. lucidum.24,

76

25

77

multiple interventions in selected metabolic pathways remains to be elucidated.

Recent metabolic engineering efforts have

Thus, a possible improvement of polysaccharide production in Basidiomycetes by

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In this study, Coprinopsis cinerea (C. cinerea) was used as the model organism to

79

examine mushroom polysaccharides production because C. cinerea widely used as

80

edible mushroom throughout the subtropical and tropiacl region of the world due to

81

its nutritional and medicinal benefits,26,

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hypoglycemic, antioxidant and acetylcholinesterase inhibitory pharmacological

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effects.28-30 In addition, C. cinerea has a relatively short life cycle, is accessible to

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genetic manipulation and has a well characterized genetic background.31-34 We first

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constructed the pCcpab1-EXP and pCccbx-EXP plasmids for genetic transformation

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in C. cinerea. We then cloned the pgm and ugp genes from C. cinerea, and

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constructed the oePGM, oeUGP and oePGM-UGP strains. Cell growth, IPS content,

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EPS production and the transcript levels of the two genes were investigated in

89

submerged cultures of C. cinerea. The results showed clearly that multiple

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interventions in polysaccharide biosynthesis metabolic pathways

91

for improving the yield of polysaccharides from mushrooms when compared with

27

and Coprinopsis polysaccharides has

5


is more effective


92

single interventions in Basidiomycetes. This research should aid development of

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effective measures through regulation of multiple genes in polysaccharide

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biosynthetic pathways to overproduce mushroom polysaccharides, especially for

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edible and medicinal mushrooms from G. lucidum, Cordyceps militaris, G. frondosa

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and L. edodes.

97 98

MATERIALS AND METHODS

99

Strains and Culture Conditions. C. cinerea strain AmutBmut (A43mut, B43mut,

100

pab-1) was purchased from the Japan Collection of Microorganisms (JCM) and

101

maintained on potato dextrose yeast agar (PDYA) medium (300 g diced potatoes, 20

102

g glucose, 5 g yeast extract, 15 g agar and 1 L distilled water) in a Petri dish at

103

28 °C.35 To prepare the seed culture, 7-day-old mycelia in PDYA medium were

104

transformed to 50 mL seed culture medium (35 g glucose, 5 g peptone, 2.5 g yeast

105

extract, 1 g KH2PO4·H2O, 0.5 g MgSO4·7H2O, 0.05 g vitamin B1 and 1 L distilled

106

water) in a 250 mL conical flask by scraping with a sterile blunt spatula and

107

incubating at 28 °C in a rotary at 120 rpm for 2 d. For the shake-flask culture

108

experiments, 5 mL of the seed medium was incubated in a 250 mL conical flask

109

containing 45 mL of fermentation medium (35 g lactose, 5 g peptone, 5 g yeast

110

extract, 1 g KH2PO4·H2O, 0.5 g MgSO4·7H2O, 0.05 g vitamin B1 1 L distilled

111

water).36 For plasmid construction and amplification, the Escherichia coli DH5α

112

strain was used and grown in Luria-Bertani medium containing 100 µg/mL

113

ampicillin.

114

Vector Construction. For constructing the pCcpab1-EXP plasmid, the pab1

115

fragment containing native promoter and terminator was cloned from the pCcpab1 6


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116

plasmid (in our lab) using primers pUC19-pab1 F and pab1 R listed in Table S1. The

117

A. bisporus gpdII promoter and A. nidulans trpC terminator were amplified from the

118

pCcEXP plasmid (in our lab) using primers pab1-AbgpdII F and pab1-AbgpdII R

119

(Table S1), and pab1-AntrpC F and pab1-AntrpC R (Table S1), respectively.37 The

120

three amplified fragments were fused to the pUC19 vector digested with EcoRI and

121

HindIII using the ClonExpress MultiS One Step Cloning Kit (C112, Vazyme) and

122

according to the manufacturer’s instructions. DNA sequencing confirmed the

123

plasmid was constructed successfully.

124

In order to develop the vector using carboxin resistance in C. cinerea, a mutated

125

iron-sulfur subunit of the dehydrogenase (sdi1) gene was obtained from the genomic

126

DNA of C. cinerea using primers β-tub-Pro F, β-tub-Pro R, β-tub-Ter F and

127

β-tub-Ter R listed in Table S1, as described by Kilaru et al.37 The β tubulin promoter

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and the β tubulin terminator were then amplified from the genomic DNA of C.

129

cinerea using the β-tub-Pro F and β-tub-Pro R primers (Table S1), and the β-tub-Ter

130

F and β-tub-Ter R primers (Table S1), respectively.38 The three fragments were

131

ligated into the pUC19 plasmid digested by SmaI using the ClonExpress MultiS One

132

Step Cloning Kit (C112, Vazyme) to yield the carboxin resistance vector.

133

Subsequently, the carboxin resistance vector was digested by XbaI and inserted into

134

the A. bisporus gpdII promoter and A. nidulans trpC terminator cloned from the

135

plasmid pCcEXP using the cbx-AbgpdII F and cbx-AbgpdII R primers (Table S1),

136

and the cbx-AntrpC F and cbx-AntrpC R primers (Table S1), respectively. The

137

generated pCccbx-EXP vector was used to transform into C. cinerea with a selection

138

marker of carboxin resistance.

139

The pgm gene was amplified from genomic DNA of C. cinerea using primers 7



Page 8 of 33

140

pab1-PGM F and pab1-PGM R listed in Table S1, and inserted into pCccbx-EXP

141

digested by XhoI and KpnI using the ClonExpress II One Step Cloning Kit (C112,

142

Vazyme) to generate the plasmid pCcpab1-EXP-PGM. Construction of the

143

pCcpab1-EXP-UGP plasmid was similar to that of pCcpab1-EXP-PGM, except the

144

gene cloning primers pab1-UGP F and pab1-UGP R listed in Table S1 were used.

145

The pCccbx-EXP-PGM plasmid was generated by digesting pCccbx-EXP with NcoI

146

and EcoRV, and subsequent insertion of the pgm gene fragment amplified by the

147

cbx-PGM F and cbx-PGM R primers listed in Table S1.

148

Construction of the Overexpression Strains. Strain C. cinerea AmutBmut was

149

used as the recipient strain. The protoplasts were prepared by enzymatic digestion of

150

the fungal cell walls of spores obtained from the yeast malt glucose (YMG) (4 g of

151

yeast extract, 10 g of malt extract, 4 g of glucose, and 1 L of distilled water) agar

152

medium using a cellulase/chitinase solution, as described by Dörnte et al.34 The

153

overexpression

154

transformed into the protoplasts by PEG-mediated transformation to give the

155

overexpression PGM (oePGM) transformants and overexpression UGP (oeUGP)

156

transformants. For genomic PCR analysis, the stable transformants were identified

157

by detecting the fusion fragment of A. bisporus gpdII and the pgm or ugp gene by

158

PCR using primers check-PGM F and check-PGM R , and check-UGP F and

159

check-UGP R listed in Table S1, respectively, which should give a 1.72 kb fragment

160

in oePGM transformants or a 1.87 kb fragment in oeUGP transformants, further

161

confirmed by DNA sequencing (GenScript, Nanjing). The co-overexpression

162

PGM-UGP (oePGM-UGP) transformants were obtained by transforming the plasmid

163

pCccbx-EXP-PGM into the protoplasts prepared from oeUGP transformants, and the

vectors

pCcpab1-EXP-PGM

and

pCcpab1-EXP-UGP

8


were

Page 9 of 33


164

transformants were selected on minimal medium containing 5 µg/mL carboxin.33

165

Integration of the fusion fragment A. bisporus gpdII and pgm or ugp gene in

166

oePGM-UGP transformants was checked by PCR and confirmed by DNA

167

sequencing, as described above for the oePGM or oeUGP transformants.

168

Measurements of Cell Dry Weight, Residual Medium Sugar, and Intracellular

169

and Extracellular Polysaccharides. Cell dry weight (DW) was obtained by vacuum

170

filtering the sample through a Whatman no. 4 filter paper, washing the filtrate three

171

times with distilled water and drying at 50 °C to a constant weight.17,

172

fermentation supernatant was collected and stored for analysis of residual sugars by

173

the 3,5-dinitrosalicylic acid (DNS) method. 39

36

The

174

For analysis of IPS content, the dried mycelia were extracted with 1 M NaOH at

175

60 °C for 1 h. The supernatant was then used to measure residual sugars using the

176

phenol-sulfuric acid method according to Tang et al.17

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The crude EPS was precipitated by addition of four volumes of 95% (v/v) ethanol

178

and stored at 4 °C overnight. After centrifugation at 13,000 g for 15 min, the

179

insoluble components were separated and suspended in 1 M NaOH at 60 °C for 1 h.

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The supernatant was then assayed by the phenol-sulfuric acid method.17

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Nucleic Acid Extraction and qRT-PCR Measurement of Gene Expression.

182

The mycelia were harvested and then ground to a fine powder in a mortar with liquid

183

nitrogen. The genomic DNA was extracted using the UNIQ-10 Column Fungal

184

Genomic DNA Isolation Kit (B511375, Sagon Biotech) and the RNA was extracted

185

using the Spin Column Fungal Total RNA Purification Kit (B518659, Sagon

186

Biotech), as described by the manufacturers. cDNA synthesis and quantitative PCR

187

were performed according to a method described previously.40 The β-tubulin gene 9



188

was used as the internal control gene to standardize the mRNA levels.40 Relative

189

gene expression was calculated according to the 2−∆∆CT method. The primers used

190

for qPCR are listed in Table S1.

191

Statistical analysis. Data used in this study are reported as the mean of three

192

independent sample measurements. The error bars indicate the standard deviations

193

from the averages of the three independent samples. Data were analyzed with the

194

two-tailed Student t-test. The difference between contrasting treatments when P

Improved polysaccharide production by homologous co

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