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Efficient co-production of mannanase and cellulase by transformation of a codon-optimized endo-mannanase gene from Aspergillus niger into Trichoderma reesei Xianhua Sun, Xianli Xue, Mengzhu Li, Fei Gao, Zhenzhen Hao, Huoqing Huang, Huiying luo, Lina Qin, Bin Yao, and Xiaoyun Su J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05114 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 5, 2017

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Journal of Agricultural and Food Chemistry

1

Efficient co-production of mannanase and cellulase by

2

transformation of a codon-optimized endo-mannanase gene

3

from Aspergillus niger into Trichoderma reesei

4

Xianhua Sun1, Xianli Xue1, Mengzhu Li1, Fei Gao1, Zhenzhen Hao1, Huoqing Huang1,

5

Huiying Luo1, Lina Qin2*, Bin Yao1*, Xiaoyun Su1* 1

6

Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed

7

Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China

8

2

National Engineering Research Center of Industrial Microbiology and Fermentation

9

Technology; College of Life Sciences, Fujian Normal University, Fuzhou, Fujian

10

350108, China

11 12

* Address correspondence to Lina Qin: National Engineering Research Center of

13

Industrial Microbiology and Fermentation Technology; College of Life Sciences,

14

Fujian Normal University, Fuzhou, Fujian 350108, China. Tel: +86-591-22868612,

15

E-mail: [email protected]; Bin Yao and Xiaoyun Su: Key Laboratory for Feed

16

Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese

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Academy of Agricultural Sciences, No. 12 South Zhongguancun Street, Beijing

18

100081,

19

[email protected].

China.

Tel.:

+86-10-82106094.

E-mail:

20 21 22

1

ACS Paragon Plus Environment

[email protected];


23

Abstract

24

Cellulase and mannase are both important enzyme additives of animal feeds.

25

Expressing the two enzymes simultaneously within one microbial host could

26

potentially lead to cost saving in animal feeding. For this purpose, we codon

27

optimized the Aspergillus niger Man5A gene to the codon usage bias of Trichoderma

28

reesei. By comparing the free energy and the local structure of the nucleotide

29

sequences, one optimized sequence was finally selected and transformed into the T.

30

reesei pyridine auxotrophic strain TU-6. The codon optimized gene was expressed to

31

a higher level than the original one. Further expressing the codon optimized gene in a

32

mutated T. reesei strain in fed-batch cultivation resulted in co-production of the

33

cellulase and mannanase up to 1,376 U·mL-1 and 1,204 U·mL-1, respectively.

34 35

Key words: mannanase; cellulase; Trichoderma reesei; feed additive

2


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Introduction

37

Glycoside hydrolases serve as routine additives to animal feeds in the current

38

husbandry industry

39

cellulases are of huge demand since many kinds of animal feeds are plant-derived and

40

they contain plant cell wall polysaccharides. Decomposition of these polysaccharides,

41

mainly constituted by cellulose and hemicellulose by an in vivo enzymatic manner,

42

has been shown to be beneficial for growth performance of livestock and poultry 3.

43

The feeds derived from certain kinds of plants, such as palm kernel cake and copra

44

meal, are rich in cellulose and mannan 4. Adding cellulase and mannanase into these

45

feed stuffs will facilitate release of the nutritional components in the gastrointestinal

46

tract.

1, 2

. Among the glycoside hydrolases used as feed additives,

47

Trichoderma reesei is a saprophytic filamentous fungus with prominent ability to

48

produce producing cellulase 5. It can also be used as a host to produce heterologous

49

genes 6. Proteins such as xylanase 7, lipase 8, and β-glucosidase 9 of bacterial, fungal,

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and even mammalian origin 10 have been successfully expressed in this organism.

51

Traditionally, enzymes used for animal feeds are produced individually by

52

different organisms. However, production of two or even more enzymes in one

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microorganism, instead of multiple hosts, may save fermentation cost and therefore be

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economically more beneficial. Endo-mannanase is an enzyme that can randomly

55

degrade the internal chain of homogenous or heterogeneous mannans

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useful in enzymatic treatment of mannan-rich animal feeds. The Aspergillus niger

57

GH5 endo-mannanase AnMan5A is an excellent endo-mannanase for potential usage 3


11, 12

. It is very


58

in animal feed: it is highly active on different kinds of mannans (locust bean gum,

59

konjac glucomannan, and guar gum), thermostable by retaining 38.2% residual

60

activity at 75°C for 4 h, and active at 40°C (near body temperature)13. In this study,

61

we carried out the expression of the A. niger Man5A in T. reesei by using both the

62

original and one codon-optimized gene. The properties of the recombinant AnMan5A

63

were measured. Co-expression of AnMan5A gene with cellulase in a mutated strain

64

with improved cellulase production performance was carried out in fed-batch

65

fermentation.

66 67

4


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68

Materials and Methods

69

Microbial strains and growth conditions

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The Escherichia coli Trans1 strain (Transgen, Beijing, China) was used as a host for

71

plasmid construction and maintenance in this work. The S. cerevisiae AH109

72

auxotrophic strain was used as the host for assembly of the expressing cassettes by

73

DNA assembler

74

peptone dextrose medium supplemented with adenine (YPDA). The T. reesei strains

75

used to overexpress the mannanase gene were the uridine auxotrophic TU-6 (ATCC

76

MYA-256) strain and the T. reesei SUS1 strain, which is a mutant of QM9414

77

developed in our lab with enhanced cellulase producing ability and lower viscosity

78

after multiple rounds of mutagenesis. The T. reesei strains were maintained on potato

79

dextrose agar (PDA) at 28 °C for sporulation.

80

Codon optimization

81

The AnMan5A gene from A. niger LW-113 was synthesized by GenScript Inc.

82

(Nanjing, China). The codon of AnMan5A was optimized by using the Gene Designer

83

software15

84

(http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=51453).

85

optimized AnMan5A nucleotide sequences were uploaded into the RNA structure

86

software version 5.8.1 16 for analyses of the secondary structure and the free energy.

87

Plasmid construction

88

We used DNA assembler, a method allowing quick assembly of several DNA

89

fragments

14

. Then the yeast AH109 strain was cultivated at 30 °C in yeast

according

to

the

codon

usage

14

bias

of

T.

reesei Ten

, to construct the plasmid for expressing AnMan5A in TU-6. The 5



90

EcoRI-restriction digested pRS424 plasmid (New England Biolabs, Beverly, MA) was

91

mixed with the cbh1 promoter (amplified using Trcbh1pF/Trcbh1pR primer pairs

92

from the genomic DNA of TU-6, Table S1), cbh1 terminator (amplified using

93

Trcbh1tF/Trcbh1tR primer pairs from the genomic DNA of TU-6, Table S1), and the

94

AnMan5A gene (amplified using Trman5AF/Trman5AR primer pairs from

95

synthesized gene, Table S1) and these DNA fragments were co-transformed into

96

AH109. The transformants were selected on yeast synthetic dropout medium without

97

tryptophan. Assembly of the fragments in the pRS424 were verified by PCR from the

98

transformant colonies with the gene specific primers of Yz-pcbh1pF/Yz-man5AR

99

(Table S1) spanning the joint region of cbh1 promoter and AnMan5A. For the

100

codon-optimized AnMan5A, our aim is to first evaluate its expression in the model

101

strain TU-6 and then test its co-production with cellulase in the mutant strain SUS1.

102

Unlike TU-6, the latter strain is resistant to co-transformation of two plasmids

103

containing the gene of interest and the selection marker, respectively, which is

104

commonly used in gene transformation of T. reesei

105

manipulation, its expressing cassette was amplified using the primers of

106

Trman5A(opt)F/Trman5A(opt)R and inserted into the BamHI and EcoRI sites of a

107

plasmid pAPA, which was constructed in our lab and contained a pyr4 marker gene

108

flanked by two tandem repeated DNA fragments (ampicillin-resistance genes). The

109

repeated DNA fragments were designed for potential use in looping out the pyr4

110

selection marker gene in the T. reesei transformants.

111

Transformation of T. reesei 6


17

. Therefore, for ease of

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The transformation of T. reesei was basically the same as that described in 17. Briefly,

113

young mycelia cultured in MM-glucose (2 %) for 20 h were harvested and 10 mg/mL

114

of Lysing Enzymes from Trichoderma harzianum (L1412, Sigma-Aldrich, St. Louis,

115

MO) were added to release protoplasts from the mycelia. A pSKpyr4 plasmid

116

harboring the pyr4-expressing cassette

117

AnMan5A-bearing expression plasmid into TU-6 and selected on MM-glucose plates

118

without addition of uridine. For the codon-optimized AnMan5A, the expressing

119

plasmid was transformed into TU-6 and SUS1, respectively. The transformants were

120

also selected on MM-glucose plates without uridine. After 5-7 days of cultivation at

121

28ºC, transformants could be clearly visualized and they were screened for integration

122

of the AnMan5A-expressing cassette in the chromosome by PCR using the primers

123

Yz-pcbh1pF/Yz-man5AR (Table S1).

124

Induction of AnMan5A expression in T. reesei

125

For induction of recombinant AnMan5A (both the wild-type and codon-optimized)

126

expression in T. reesei in shake flask fermentation, 107 fresh spores were inoculated

127

into 50 mL of liquid MM supplemented with 2 % glucose and shaken at 28 ºC for 48

128

h. The mycelia were filtered through a 200-mesh sifter and washed with sterile water

129

to remove residual glucose. Then equal amounts of mycelia based on wet weight from

130

the

131

codon-optimized one) were transferred to 100 mL MM-Avicel (2%) and the culture

132

was continued at 28ºC for induction of mannase expression. From day 2 to 7 post

133

induction, 2 mL of the culture supernatants were periodically collected for assay of

parent

strain

TU-6

and

its

8

was co-transformed with the original

AnMan5A transformants

7


(wild-type

and


134

enzymatic activities and protein concentrations. The fed-batch cultivation of T. reesei

135

was essentially the same as that carried out in

136

Avicel cellulose The operating volume was 10 L. Ammonium hydroxide was used to

137

provide nitrogen and maintain the pH at 5.5. The fermentation temperature was 28°C.

138

Assay of mannanase and cellulase activities and protein concentration

139

For assay of the endo-mannase activity, 100 µL of appropriately diluted crude enzyme

140

(fermentation broth) were mixed with 5 mg/mL of locust bean gum in 0.2 M

141

Na2HPO4-0.1 M citric acid buffer (pH 5.0) with a total volume of 1 mL. The reaction

142

was incubated at 50 ºC for 15 min. The reducing sugars released were determined

143

using the 3,5-dinitrosalicylic acid (DNS) method 19. One unit of mannanase activity

144

was defined as the amount of enzyme liberating 1 µmol of reducing sugar in one

145

minute. The cellulase activity of T. reesei can be presented as the overall cellulase

146

activity or divided into endo-glucanase, exo-glucanase, and β-glucosidase activities 20.

147

For assay of the overall cellulase activity, the reaction included one strip of Whatman

148

No.1 filter paper (6 × 1 cm) and 100 µL of culture supernatant in a 50 mM acetate

149

buffer (pH 4.8) and was incubated at 50 ºC for 1 h.21 One unit of the overall cellulase

150

activity was defined as the amount of enzyme that released 1 µmol of reducing sugar

151

per hour under the assay conditions. For endo-glucanase, sodium carboxymethyl

152

cellulose (CMC-Na, 10 mg/mL, purchased from Sigma-Aldrich, St. Louis, MO) was

153

used as the substrate and the reaction was carried out in the McIlvaine buffer (pH 5.0)

154

and incubated at 50 ºC for 30 min. One unit of the endo-glucanase activity was

155

defined as the amount of enzyme that released 1 µmol of reducing sugar per min

18

with the change of Solka Floc to

8


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156

under the assay conditions. The assay of cellobiohydrolase I (CBHI) activity was

157

essentially

158

4-methylumbelifery-β-D-lactopyranoside (MUL) as the substrate and incubated at 50

159

ºC for 20 min 22. One unit of CBHI activity was defined as the amount of enzyme that

160

released 1 µmol of 4-Methylumbelliferyl (4-MU) per min under the assay condition.

161

The

162

(p-Nitrophenol-β-D-glucopyranoside) as the substrate and incubated at 50 ºC for 10

163

min. One unit of β-glucosidase activity was defined as the amount of enzyme that

164

released 1 µmol of pNP in one hour. The protein concentration was determined with a

165

BCA-200 Protein Assay Kit (Pierce, Rockford, IL) by using bovine serum albumin as

166

the standard.

167

Assay of specific enzyme production rates

168

The specific mannanase production rate from day 1 to 6 was calculated. Because the

169

inducing culture contains insouble cellulose Avicel prohibiting us from directly

170

measuring the mycelia weight, we used the mycelia protein as a representative of the

171

fungal biomass. The mycelia of the strains expressing AnMan5A and codon optimized

172

AnMan5A were collected in different time periods from day 1 to 6 and the method

173

described by Jayaraman 23 was adopted to assay the mycelia proteins.

174

Effect of pH and temperature on the biochemical properties of AnMan5A

175

To determine the optimal pH of recombinant AnMan5A, the fermentation broth of the

176

codon-optimized AnMan5A transformant on 5 days post induction was incubated with

177

5 mg/mL of locust bean gum in 0.2 M Na2HPO4-0.1M citric acid buffer with the pH

the

same

β-glucosidase

as

that

activity

described

was

by

Beiley

determined

9


et

al.

using

using

pNPG


178

ranging from 2.5 to 7.5. The optimal temperature of recombinant AnMan5A was

179

determined at the optimal pH with the temperatures changing from 30 to 90 °C. To

180

determine the pH-stability, the crude enzyme was first pretreated in a buffer with

181

varying pHs (pH 1.0–2.0: 100 mM Glycine-HCl; pH 3.0–8.0: 100 mM McIlvaine

182

buffer; pH 9.0–12.0: 100 mM Glycine-NaOH) at 37°C for 1 h. To determine the

183

thermostability, the crude enzyme was pre-incubated at 75°C and 80°C for different

184

periods of time (10 min, 20 min, 30 min, 40 min, 50 min, and 1 h). For both

185

pH-stability and thermostability assays, the residual activity after pretreatment was

186

determined under its optimal conditions (pH 4.5 and 80 °C). The released reducing

187

sugars were measured using the DNS method.

188

Mass spectrometry analysis

189

The fermentation broth for TU-6 expressing the wild-type AnMan5A was first

190

resolved on the SDS–PAGE gel and the protein bands at ~43 kDa were excised and

191

used for in-gel trypsin digestion. The digestion solution was first incubated for 60 min

192

at 4 °C and then the sample was incubated for 14 h at 37 °C. To extract the peptide

193

fragments from the tryptic digests, 20 µl of 5% (v/v) TFA were added and incubated

194

for 60 min at 37 °C. Thereafter, 20 µl of 50% (v/v) acetonitrile (containing 2.5% (v/v)

195

TFA acid) was added to gel pieces and incubated for 60 min at 30 °C. The extracted

196

peptides were dissolved in 0.1% formic acid in distilled water. LC−MS/MS analysis

197

was run on Easy-nLC 1000 (Thermo Fisher Scientific, Bremen, Germany) coupled

198

Q-Exactive HF (Thermo Fisher Scientific) mass spectrometer. Samples were loaded

199

onto a reversed-phase trap column at a flow rate of 5 µL/min. Buffer A (0.1% formic 10


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200

acid in water) and buffer B (0.1% formic acid in acetonitrile) were used as mobile

201

phase buffer. Peptides were separated on an analytical column at a flow rate of 350

202

µL/min using the following gradients: from 3 to 8% buffer B in 1 min, from 8 to 28%

203

buffer B in 10 min, from 28 to 90% buffer B in 2 min, and remaining at 90% buffer B

204

for 7 min. The peptides were eluted from the analytical column and directly injected

205

into the mass spectrometer via nano-ESI source. Ion signals were collected in a

206

data-dependent mode. The RAW MS/MS data were searched against a composite

207

database for protein identification using in-house PEAKS software (version 8.0).

208

Quantitative reverse transcription PCR analysis

209

For quantitative reverse transcription PCR (qRT-PCR), the mycelia of TU-6 and of the

210

strains expressing wild-type AnMan5A and codon-optimized AnMan5A, respectively,

211

in shake flask fermentation were collected at 24 h post Avicel induction. The mycelia

212

were pulverized in liquid nitrogen using a pestle and mortar. Total RNA was extracted

213

from frozen lyophilized mycelia using the TRIzol reagent (Thermo Fisher Scientific,

214

Waltham, MA). One µg of RNA was treated with DNAse I and reverse-transcribed to

215

cDNA using the First Strand cDNA Maxima Synthesis kit (TOYOBO, Shanghai,

216

China). qRT-PCR was performed in a QuantStudio 6 Flex Real-Time PCR System

217

(Applied Biosystems, San Diego, CA) using a TransScript Green One-Step qRT-PCR

218

SuperMix (TransGen, Beijing, China). The actin(act) gene was used as an

219

endogenous reference gene. The primers used for qRT-PCR were actF/R, Man5AF/R1,

220

Pcbh1F/R, Pcbh2F/R, Pegl1F/R, and Pbgl1/F/R and their sequences were given in

221

Table S1. The following amplification conditions were used：95 ℃for 10 min, then 40 11



222

cycles including 94℃ for 10s, 60 ℃ for 20 s, and 72 ℃ for 30 s.

223

Deglycosylation

224

Endo-β-N-acetylglucosaminidase H (Endo H) from New England Biolabs (Ipswich,

225

MA) was used to remove N-glycosylation. The reaction system containing 9 µL

226

protein (fermentation broth of TU-6 expressing original and codon-optimized

227

AnMan5A and of Pichia pastoris expressing AnMan5A) and 1 µL Denaturing Buffer

228

(0.5% SDS, 40 mM DTT) was boiled at 100 ºC for 10 min. Then, 2 µL GlycoBuffer 3

229

(50 mM sodium acetate, pH 6) and 1 µL Endo H were added and the mixture was

230

incubated at 37 ºC for 2 h. The Endo H-treated enzymes were analyzed by SDS-PAGE.

231

For expression of AnMan5A in P. pastoris, the synthesized gene was cloned into the

232

EcoRI and NotI restriction sites of pPIC9 (Invitrogen, Carlsbad, CA) and the

233

recombinant plasmid was transformed into GS115.

12


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234

Results

235

Codon optimization of AnMan5A

236

To improve expression of a heterologous gene, codon optimization is commonly

237

employed to avoid rare codons that may appear in the gene of interest. For this

238

purpose, the AnMan5A gene was codon optimized using the Gene Designer software

239

15

240

more parameters were considered in addition to the algorithms of the software. These

241

included i) the free energy of the mRNAs, which is indicative of their stability and ii)

242

the local secondary structure near the start codon (ATG) of the transcripts. For this

243

consideration, we generated 10 codon-optimized AnMan5A gene sequences by

244

repeated running Gene Designer. Even with the same seed AnMan5A amino acid

245

sequence, codon usage table, and algorithm, the software calculated quite different

246

nucleotide sequences in each run. Since the cbh1 promoter and terminator were used

247

to drive expression of AnMan5A gene, these ten nucleotide sequences were

248

computationally placed between the 5’ untranslated region (UTR)-the cbh1 signal

249

sequence and the 3’ UTR. These combinatorial mRNA sequences were calculated for

250

their secondary structure and free energy using the RNA structure software. From the

251

calculation, the optimized gene AnMan5A-7 was selected for further expression

252

analysis, which had a relatively low free energy and loose local structure at the start

253

codon (Fig. 1 and Fig. S1).

254

Construction of plasmids for AnMan5A expression in T. reesei

255

Both the original and the codon-optimized AnMan5A gene were synthesized by the

according to the codon bias of T. reesei. In the codon optimization process, two

13



256

GenScript company (Nanjing, China). The original AnMan5A gene was mixed with

257

the T. reesei cbh1 promoter and terminator, and transformed together with EcoRI

258

linearized pRS424 into the S. cerevisiae AH109 strain. With this DNA assembly

259

method via yeast in vivo homologous recombination, the cbh1 promoter, AnMan5A

260

gene, and cbh1 terminator were one-step assembled in the pRS424 plasmid (Fig. 2A).

261

The codon-optimized AnMan5A gene was also assembled into pRS424, and then the

262

expressing cassette for AnMan5A was further amplified and inserted into the BamHI

263

and EcoRI sites of a plasmid pAPA for transformation of a mutant T. reesei strain

264

using the Gibson assembly method (Fig. 2B). The plasmid pAPA harbors a

265

pyr4-expressing cassette conferring uridine autotroph, which is flanked by two

266

ampicillin-resistance encoding genes. The repeating ampicillin-resistance DNA

267

fragments can be used for future potential looping out the pyr4 gene in the T. reesei

268

transformants.

269

Expression of AnMan5A in T. reesei

270

The whole expression plasmids bearing the original and codon-optimized AnMan5A

271

genes were individually transformed into TU-6, a uridine auxotroph mutant of T.

272

reesei QM9414

273

AnMan5A-expressing cassettes and then cultivated in shake-flasks for initial screen of

274

the mannanase-producing strains. For both the original and codon-optimized

275

AnMan5A genes, one representative transformant was selected, which had the highest

276

mannanase activity in the initial screen. These two transformants, with the TU-6 host

277

strain, were cultivated for detailed analysis of the mannanase and cellulase activities

24

. The transformants were first detected for integration of the

14


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278

and protein production.

279

The original AnMan5A gene was successfully expressed in T. reesei. On

280

SDS-PAGE gel, it appeared as a smear centering at around 43 kDa (Fig. 2C). Mass

281

spectrometry analysis unequivocally supported this conclusion (Fig. S2). Interestingly,

282

two clear bands, but not a smear, were observed on the SDS-PAGE gel for the

283

codon-optimized mannanase (Fig. 2C). From the SDS-PAGE analysis, codon

284

optimization apparently enhanced expression of this gene. In accordance with this

285

analysis, while only low mannanase activity was observed for TU-6, the transformant

286

bearing the original AnMan5A had much higher mannanase activity, peaking at day 4

287

with activity of 2.9 U·mL-1. Codon optimization largely improved the mannanase

288

activity, with highest activity appearing at day 6 with 9.3 U·mL-1 (Fig. 3A). The

289

specific mannanase production rate of codon-optimized AnMan5A from day 1 to day

290

6 was 133.3 U·g mycelia protein·h-1, higher than 32.7 U·g mycelia protein·h-1 for the

291

original AnMan5A. The extracellular protein concentrations of the three strains had

292

the same pattern with that of the mannanase activity (Fig. 3B). The difference in the

293

CBH1 (Fig. 3C) and endo-glucanase (Fig. 3D) activities of TU-6 and the two strains

294

were not large. However, it appeared that the more AnMan5A was expressed, the less

295

β-glucosidase activity was observed (Fig. 3E). As the overall cellulase activity is the

296

synergy among the exo-glucanase, endo-glucanase, and β-glucosidase, the overall

297

cellulase activity of TU-6 expressing codon-optimized AnMan5A was the lowest

298

among the three strains (Fig. 3F).

299

Transcript levels of AnMan5A and main cellulase genes 15



300

Total RNA was extracted from the mycelia collected at 24 h post Avicel cellulose

301

induction and the transcript levels of AnMan5A and main cellulase genes were

302

quantified by qRT-PCR. As shown in Fig. 4, the transcript level of AnMan5A(opt)

303

was nearly the same as that of wild-type AnMan5A. Therefore, the transcription could

304

not account for the large elevation in the mannase activity. The observed higher

305

mannanase activity for AnMan5A(opt)-expressing strain was most likely from

306

enhanced translation efficiency caused by codon optimization. Further analysis

307

indicated that two cellobiohydrolases (cbh1 and cbh2) and one main endo-glucanase

308

(egl1) did not change much in TU-6 and the two strains expressing AnMan5A, in

309

accordance to the comparable cellobiohydrolase and endo-glucanase activity in the

310

three strains (Fig. 3C, D). Interestingly, we noted that, while the extracellular

311

β-glucosidase activity decreased significantly in AnMan5A-expressing strains, the

312

mRNA level of the major extracellular β-glucosidas gene bgl1 was very similar in

313

these three strains.

314

Biochemical properties of recombinant AnMan5A

315

Since the parent strain TU-6 displayed very low endo-mannanase activity (Fig. 3A),

316

the biochemical properties of the recombinant AnMan5A could be estimated using the

317

crude enzyme, i.e. the culture supernatant. The codon-optimized AnMan5A had a

318

maximal activity at pH4.5 and exhibited over 50% activity between pH 3.5 to 6.0 (Fig.

319

5A). It had an optimal temperature of 80 °C and retained 27% activity at 40°C, a

320

temperature close to animal body temperature (Fig. 5B). The recombinant enzyme

321

was quite stable: over 60% residual activity was retained after pretreatment of 16


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AnMan5A between pH 2.0 to 12.0 for 1 h (Fig. 5C). Moreover, the residual activity of

323

AnMan5A only slightly reduced to 78% after treatment at 75 °C for 1 h (Fig. 5D).

324

Co-production of cellulase and mannanase by recombinant T. reesei in fed-batch

325

cultivation

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The codon-optimized AnMan5A gene was further transformed and expressed in SUS1,

327

a mutant strain of QM9414 with improved cellulase activity. In the fed-batch

328

cultivation, the maximal endo-glucanase activity reached 1,376 U·mL-1 (Fig. 6A) and

329

the endo-mannanase activity maximized to 1,204 U·mL-1 (Fig. 6B), respectively.

330

17



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331

Discussion

332

We proposed that co-production of two or more feed enzymes could potentially save

333

costs of animal husbandry. Among the commonly used microbes, T. reesei is a good

334

candidate for co-expression of cellulase with another feed enzyme for the following

335

two reasons: i) it is by itself a prominent cellulase producer

336

could be over 100 g·L-1 5. Indeed, by simply transforming an endo-mannanase gene

337

into T. reesei, we were able to express cellulase and mannanase simultaneously within

338

one microbe with enzyme activities up to 1,376 U·mL-1 and 1,204 U·mL-1,

339

respectively. Both enzymes have great potential as feed enzymes and have been at

340

least tested or commercialized for their usage in the feed industry. Therefore, to the

341

best of the authors’ knowledge, this is the first report of co-expression of two feed

342

enzymes, specifically cellulase and mannanase, in one microbe. However, we noticed

343

that expression of β-glucosidase was negatively affected by the over-expression of

344

AnMan5A. Higher expression of AnMan5A led to lower β-glucosidase activity

345

(compare Fig. 3A and Fig. 3E). Given that the transcription of bgl1 was similar in all

346

three strains studied (Fig. 4), one reason could be that the recombinant AnMan5A

347

competed for certain secretory pathway components which were also used by the

348

β-glucosidase for secretion. However, more evidence is needed to support this

349

hypothesis in the future.

20

and ii) its productivity

350

The recombinant AnMan5A produced in T. reesei had an optimal pH of 4.5 and

351

temperature of 70°C, while these parameters for AnMan5A expressed in P. pastoris

352

were pH3.5 and 70°C, respectively13. The AnMan5A expressed in P. pastoris has 18


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353

38.2% residual activity after 1 h of incubation at 75°C. The AnMan5A expressed in T.

354

reesei is also thermostable by keeping 78% residual activity after pretreating at 75 °C

355

for 1 h, a feature favorable for application in feed industry since the feed pelleting

356

involves processing at high temperatures

357

result of different glycosylation patterns, which may have profound effects on

358

enzyme’s thermostability

359

broth of recombinant TU-6 expressing wild-type and codon-optimized AnMan5A and

360

of the AnMan5A expressed in Pichia pastoris indicated that the mannanase was

361

differently glycosylated (Fig. S3). It is not known why the electrophoresis patterns of

362

AnMan5A expressed from the original and codon-optimized genes were slightly

363

different.

25

. The different thermostability may be a

26

. Indeed, deglycosylation analysis of the fermentation

364

Codon optimization successfully improved expression of the Man5A gene from A.

365

niger in T. reesei. Many factors could affect heterologous gene expression in a

366

microbial host 6. For example, scrutinizing the local structure at the start codon and

367

mutating the corresponding coding nucleotides have been successfully used for

368

improving expression of a 1,3-1,4-β-D-glucanase from Fibrobacter succinogens in P.

369

pastoris 27. Since there is no such study reported for manipulating heterologous gene

370

expression in T. reesei, we employed this strategy and combined it with considering

371

the free energy of the transcript. We found that repeated running of Gene Designer

372

produced optimized genes with much differing nucleotide sequences. These

373

sequences were calculated to have varying free energies and secondary structures near

374

the start codon. From these calculations, we selected one optimized gene with 19



375

apparently more favorable parameters and this simple method proved to be useful for

376

codon optimization of AnMan5A. This strategy could be also used for testing

377

expression of other heterologous genes in T. reesei in the future.

378

In conclusion, an endo-mannanase gene from A. niger was transformed in T.

379

reesei TU-6. Its expression was successfully improved by codon optimization which

380

was carried out by comparing the free energies and local secondary structures near the

381

start codon of the computationally designed transcripts. Introduction of the

382

codon-optimized AnMan5A expression cassette in a T. reesei mutant resulted in

383

co-production of cellulase and mannanase at high titers of 1,376 U·mL-1 and 1,204

384

U·mL-1, respectively.

385

20


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386

Supporting Information Available

387

Primers used in this study (Table S1), Predicted secondary structures of the transcripts

388

of codon-optimized AnMan5A gene (Figure S1), Mass spectrometry analysis of the

389

expressed recombinant AnMan5A (Figure S2), and Deglycosylation analysis of

390

AnMan5A (Fig. S3).

391 392

Funding sources

393

This research was supported by a grant from the National Key R&D Program of

394

China (2016YFD0501409-02), the National Natural Science Foundation of China

395

(31400067), the China Modern Agriculture Research System (CARS-42), and the

396

Elite Youth Program of Chinese Academy of Agricultural Sciences.

397 398

CONFLICTS OF INTEREST

399

The authors declare that they have no conflicts of interest with the contents of this

400

article.

401 402

21



403

Figure legends

404

Figure 1. Codon optimization of AnMan5A gene for expression in T. reesei. The free

405

energy and local secondary structure as analyzed by the RNAstructure software were

406

shown in (A) and (B), respectively. The arrow indicated the start codon, where the

407

translation initiates.

408

Figure 2. Expression of AnMan5A in T. reesei. A and B: Schematic diagrams for the

409

expression cassette of the wild-type AnMan5A (A) and the codon-optimized

410

AnMan5A gene (B). C: SDS-PAGE analysis of the fermentation broth of T.

411

reesei-expressing AnMan5A. M: protein molecular mass marker; lane 1: TU-6; lane 2:

412

T. reesei expressing the wild-type AnMan5A; lane 3: T. reesei expressing the

413

codon-optimized AnMan5A. The arrows indicate the recombinant AnMan5A.

414

Figure 3. Endo-mannanase and cellulase activities of the fermentation broth in the

415

shake flask culture of the recombinant T. reesei strains harboring the wild-type or

416

codon-optimized AnMan5A. A: The endo-mannanase activity using locust bean gum

417

as the substrate. B: The overall extracellular protein concentration. C: The CBH1

418

activity. D: The endo-glucanase activity. E: The β-glucosidase activity. F: The overall

419

cellulase activity measured using filter paper as the substrate. All enzymatic activities

420

were measured at 50°C and pH 5.0. TU-6, AnMan5A, and AnMan5Aopt: the crude

421

enzymes from the parent strain TU-6, the TU-6 expressing the wild-type AnMan5A,

422

and that expressing the codon-optimized AnMan5A, respectively.

423

Figure 4. Relative transcript level of AnMan5A and main cellulase genes in TU-6 and

424

the two T. reesei strains expressing the wild-type AnMan5A and codon-optimized 22


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425

AnMan5A, respectively, as analyzed by qRT-PCR. The mycelia collected at 24 h post

426

Avicel induction were used for RNA extraction and reverse transcription quantitative

427

PCR. For AnMan5A, its relative expression level in the T. reesei strain expressing the

428

wild-type AnMan5A was set as 1.0. For cbh1, cbh2, egl1, and bgl1, the relative

429

expression level of the genes in TU-6 were set as 1.0.

430

Figure 5. Biochemical properties of the AnMan5A crude enzyme in the fermentation

431

broth of TU-6 expressing the codon-optimized gene. A: The optimal pH of the

432

recombinant AnMan5A. B: The optimal temperature. C: The stability of AnMan5A at

433

different pHs. D: Thermostability. For determination of optimal pH and temperature,

434

the maximal activities were set as 100%. For pH and thermostability assays, the

435

activity of the enzyme prior to pretreatment was set as 100%.

436

Figure 6. Co-production of cellulase and endo-mannanase by T. reesei SUS-1

437

harboring the codon-optimized AnMan5A gene in fed-batch cultivation. A: The

438

endo-glucanase activity. B: The endo-mannanase activity.

439

23



440

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Efficient Coproduction of Mannanase and ... - ACS Publications

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