Efficient Xylitol Production from Cornstalk Hydrolysate Using

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Efficient xylitol production from cornstalk hydrolysate using engineered Escherichia coli whole cells Ziyue Chang, Dong Liu, Zhengjiao Yang, Jinglan Wu, Wei Zhuang, Huanqing Niu, and Hanjie Ying J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04666 • Publication Date (Web): 22 Nov 2018 Downloaded from http://pubs.acs.org on November 24, 2018

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Journal of Agricultural and Food Chemistry

Efficient xylitol production from cornstalk hydrolysate using engineered Escherichia coli whole cells

Ziyue Chang,† Dong Liu,†,* Zhengjiao Yang, Jinglan Wu, Wei Zhuang, Huanqing Niu, Hanjie Ying. State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing 211816, China.

†These

authors contributed equally to this work

*Corresponding

author:

Fax: +86 25 86990001; Tel: +86 25 86990666. E-mail: [email protected]

1

ACS Paragon Plus Environment


1 2

Abstract: Economic transformation of lignocellulose hydrolysate into valued-added products

3

is of particular importance for energy and environmental issues. In this study, xylose

4

reductase and glucose dehydrogenase were cloned into plasmid pETDuet-1 and then

5

simultaneously expressed in Escherichia coli BL21(DE3), which was used as

6

whole-cell catalyst for the first time to convert xylose into xylitol coupled with

7

gluconate production. When tested with reconstituted xylose and glucose solution, 0.1

8

g/mL cells could convert 1 M xylose and 1 M glucose completely and produced 145.81

9

g/L xylitol with a yield of 0.97 (g/g) and 184.85 g/L gluconic acid with a yield of 1.03

10

(g/g) in 24 hours. Subsequently, the engineered cells were applied in real cornstalk

11

hydrolysate, which generated 30.88 g/L xylitol and 50.89 g/L gluconic acid. The cells

12

were used without penetration treatment and CaCO3 was used to effectively regulate the

13

pH during the production, which further saved costs.

14 15

Keywords: whole cell catalysis, cornstalk hydrolysate, xylose reductase, glucose

16

dehydrogenase, xylitol

17 18 19 20 21

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1.

Introduction

23 24

Xylitol is used as an alternative to sucrose, fructose, and various artificial

25

sweeteners in food because of its high sweetening power and its usefulness as a sucrose

26

substitute for diabetics.1, 2 Xylitol can also serve as a valuable synthetic building block

27

in many industries. It is identified as one of the top 12 value-added chemicals to be

28

produced from biomass by the US department of energy (DOE).3

29

In recent years, lignocellulosic wastes have been recognized as the most promising

30

alternative low-cost raw materials that can be exploited to produce xylitol. 4-6 Thus,

31

economical xylitol production methods that can be put into these potential sources are

32

particularly important. Nowadays, most xylitol productions are based on chemical

33

process from pure xylose. Biotechnological production of xylitol is an attractive

34

alternative to chemical production because it can be operated under much milder

35

conditions directly from the lignocellulose-derived sugar mixtures.7 Biological methods

36

include fermentation and enzymatic production. Compared to fermentation, enzymatic

37

production could achieve a 100% theoretical yield.8 This high conversion ratio is due to

38

the direct transformation of xylose into xylitol which is less likely achieved through

39

fermentation because of deviation of xylose to cell maintenance.4 So far, enzymatic

40

production of xylitol has been reported with purified enzymes as well as exogenous

41

cofactors NAD(P)H, but use of purified enzymes and cofactors would increase the cost

42

despite of the 100% conversion of xylose.4, 9 To address this problem, whole-cell

3



43

catalysis can be employed with no need for enzyme purification. Meanwhile, because

44

enzymes and native cofactors are encapsulated inside cells, no exogenous cofactors are

45

required and the efficiency of cofactor recycling could also be improved.10-12 Whole-cell

46

catalysis has already been reported for xylitol production from arabitol.13 However, it

47

has so far not been reported for xylitol production from lignocellulose hydrolysate. Here,

48

we engineered E. coli cells as whole-cell catalyst for xylitol production and

49

demonstrated the viability of this simple, practical process with real straw hydrolysate

50

for the first time. The scheme of xylitol production through whole-cell catalysis is

51

shown in Fig. 1.

52 53

2.

Materials and methods

54 55

2.1. Materials

56 57

E. coli BL21(DE3) (Stratagene, USA) was used as the host. Plasmid pET28a(+)

58

(Novagen, USA) and pETDuet-1 (Novagen, USA) were used as the over-expression

59

vectors. Glucose dehydrogenase from Bacillus cereus was a commercial product

60

obtained from Amano (Milton Keynes, UK). Cornstalks was provided by a local factory

61

(Lianyungang, China). The chemical composition of the raw cornstalk (on a dry weight

62

basis) was 20.0% hemicellulose, 38.7% cellulose, 18.1% lignin, 3.9% ash and 19.3%

63

unknown components. Cellulase (245 FPU/mL) was obtained from Tianguan Co.

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(Nanyang, China). All other chemicals were of analytical grade and purchased from

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local suppliers.

66 67

2.2. Cloning of XR and GDH gene

68 69

Xylose reductase (XR) gene of Candida tenuis CBS 4435 (teXR) (GenBank

70

Accession number AF074484.1) and Candida tropicalis IFO 0618 (trXR) (GenBank

71

Accession number AB002106.1) were synthesized by Genewiz Biotech Co. (Suzhou,

72

China) after codon optimization for E. coli, and then they were amplified by PCR using

73

primers (teXR-up and teXR-dn were used to clone teXR; trXR -up and trXR -dn were

74

used to clone trXR), followed by ligation to EcoRI/HindIII-linearized plasmid

75

pET28a(+) based on homologous recombination using the infusion one-step clone kit

76

(Vazyme Biotech Co., Nanjing, China), yielding pET-28a-teXR and pET-28a-trXR,

77

respectively.

78

Plasmid pETDuet-1 has two expression cassettes and can express two genes in

79

E. coli BL21(DE3) simultaneously.14 Glucose dehydrogenase (GDH) gene of Bacillus

80

cereus ATCC 14579 (GenBank Accession number BC4715) was also synthesized by

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Genewiz Biotech Co. after codon optimization for E. coli and then trXR and GDH were

82

amplified by PCR using primers (XR-up and XR-dn were used to clone trXR; GDH-up

83

and GDH-dn were used to clone GDH). Next, PCR product of XR was ligated with

84

NcoI/SalI-linearized plasmid pETDuet-1, yielding pETDuet-XR. Then PCR product of

5



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GDH was ligated with BglII/XhoI-linearized plasmid pETDuet-XR, yielding

86

pETDuet-XR-GDH. All these methods were also based on homologous recombination

87

using the infusion one-step clone kit. Then three recombinant plasmids were

88

transformed into E. coli DH5a for general cloning and cells were plated on LB agar

89

supplemented with the appropriate antibiotics (50 μg/mL kanamycin for E. coli DH5a

90

with pET-28a-teXR and pET-28a-trXR; 100 μg/mL ampicillin for E. coli DH5a with

91

pETDuet-XR-GDH) and incubated at 37oC for about 12 h to select positive

92

transformants that were all confirmed by ApaI and XhoI digestion and sequencing. All

93

primers are listed in Table 1.

94 95

2.3. Expression of xylose reductase and glucose dehydrogenase gene

96 97

The plasmids pET-28a-teXR, pET-28a-trXR and pETDuet-XR-GDH were

98

transformed into the competent cells of E. coli BL21(DE3) for expression respectively,

99

and the recombinants were named as E. coli (pET-28a-teXR), E. coli (pET-28a-trXR)

100

and E. coli (pETDuet-XR-GDH). Similarly, the empty vectors pET28a(+) and

101

pETDuet-1 were also transformed into E. coli BL21(DE3), yielding corresponding

102

plasmid-control strains. The strains were transferred into LB and incubated at 37oC, 200

103

rpm for 12 h, and then 2% cultured medium was incubated into fresh LB medium for

104

further cultivation. When the cell OD600 nm was about 0.6, IPTG was added to the broth

105

to a final concentration of 1 mM. Then cultivation was continued for a further 12 h at

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corresponding temperature (Fig. 3; Fig. 4). The cells were then harvested by

107

centrifugation and washed twice with 50 mM Tris-HCl buffer (pH 7.0). Then they were

108

resuspended in appropriate volume of the same buffer and disrupted by ultrasonic

109

treatment. Cell-free extract was obtained after the debris was removed by centrifugation

110

at 8,000 g for 10 min at 4oC. The expression of enzymes was determined by SDS-PAGE.

111

The culture of E. coli (pET-28a-teXR), E. coli (pET-28a-trXR) was added with

112

kanamycin to a final concentration of 50 μg/mL and culture of E. coli

113

(pETDuet-XR-GDH) was added ampicillin to a final concentration of 100 μg/mL when

114

needed.

115 116

2.4. Enzymatic assays

117 118

Enzyme activities of teXR, trXR and GDH were determined by spectrophotometric

119

analysis at 25oC and 340 nm, using NAD(P)H and NAD(P)+ , respectively. One XR or

120

GDH unit (U) was defined as the amount of enzyme required for formation or

121

consumption of 1 mM NAD(P)H per min. The protein concentration of the protein

122

solution extract was determined according to the Bradford method with bovine serum

123

albumin (BSA) as the protein standard.15

124 125

2.5. Xylitol production through enzymatic process from pure xylose and glucose

126

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127

The optimum temperature of XR (25oC) was applied in this reaction9 and then

128

catalytic conditions were explored with pure xylose and glucose. The pH of reaction

129

was reported to be controlled by addition of Tris-hydroxymethyl-aminomethane in

130

previous research.4, 9 In this study, to lower production costs, CaCO3 (0.1 g/mL) was

131

used to stabilize the pH at around 6.2 by neutralizing gluconic acid produced in the

132

reaction. For the optimization of bioconversion conditions, substrate and cofactor

133

concentrations were investigated using single factor experiments. The liquid samples

134

were analyzed by HPLC, equipped with refractive index detector. The concentrations of

135

glucose, xylose and xylitol were analyzed using Aminex HPX-87C column at 80oC with

136

ultrapure water as mobile phase at 0.6 mL/min. After glucose was detected to be

137

completely consumed, the concentration of gluconic acid was analyzed using Aminex

138

HPX-87H column at 45oC with 5 mM H2SO4 as mobile phase at 0.4 mL/min.

139 140

2.6. Xylitol production through whole-cell catalysis from pure xylose and glucose

141 142

Cells induced at 30oC were harvested by centrifugation and washed twice with 50

143

mM Tris-HCl buffer (pH 7.0) for whole-cell catalysis. The reactions were carried out at

144

25oC /200 rpm using 1 M xylose, 1 M glucose and 1 M CaCO3. Pure water was added

145

to the required volume of the reaction. Cell addition and reaction time were further

146

explored. The products were detected by HPLC described above.

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2.7. Cornstalk pretreatment and enzymatic hydrolysis

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Cornstalk was knife milled, screened to 30-50 mesh and dried to constant weight.

150 151

The pretreatment processes were carried out in a high-pressure reactor equipped with a

152

temperature controller and mechanical agitator by Yanzheng Co. in China. The reactor

153

was made of 316 stainless steel with the total volume of 250 mL. Reactions were

154

performed using 10.0 g cornstalk (dry basis) with 60 vt% aqueous ethanol, and 4 wt%

155

NaOH as a catalyst. The pretreatment was conducted at 110oC and for 90 min. After

156

completion, the reactor was cooled using a water bath, and the obtained solid and liquid

157

fractions were separated by filtration. The solids were washed with water (2 × 25 mL)

158

and dried at 105oC to constant weight and then were crushed by mill.16 Finally, the

159

pretreated cornstalk was hydrolyzed at 50oC /150 rpm for 24 h in 50 mM sodium citrate

160

buffer (pH 4.8) and cellulase was added at 15 FPU per gram of substrate. The pretreated

161

mixture was filtered to obtain straw hydrolysate and the hydrolysate composition of

162

sugars was determined by HPLC, obtaining 32.04 g/L of xylose, 51.57 g/L of glucose

163

and 3.01g/L of arabinose. The scheme is shown in Fig. 2. Straw hydrolysate was

164

adjusted to pH 7 with NaOH and then was used for xylitol production through either

165

enzymatic catalysis or whole-cell catalysis described above.

166 167

3.

Results and discussion

168

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169

3.1. Screen of xylose reductase genes and characterization of the enzyme activities

170 171

Two XR genes were cloned into the pET28a(+) plasmid and confirmed by

172

restriction enzyme reaction and sequencing respectively. Their expressions in E. coli

173

BL21(DE3) were analyzed by SDS–PAGE (Fig. 3). The apparent sizes of teXR and

174

trXR as migrated on the SDS-PAGE were in good agreement with the calculated

175

molecular weight (teXR, trXR and GDH are approximately 43 kD, 36.6 kD, and 31 kD,

176

respectively). The results showed that expression of both teXR and trXR was successful.

177

They were highly expressed and well soluble in E. coli as indicated by the SDS-PAGE.

178

Enzyme activity assay showed that for both teXR and trXR, NADPH was much

179

more efficient than NADH as a cofactor (Table 2). Compared to teXR, trXR showed a

180

higher specific activity when using NADPH as a cofactor. Then, their catalytic

181

performances were preliminarily investigated using 0.1 M xylose as substrate, coupled

182

to a reaction catalyzed by GDH (Table 3). Consistent with the enzyme activity assay,

183

the trXR gave the highest xylitol yield with NADPH as a cofactor, while it had no

184

productivity with NADH as a cofactor. In contrast, teXR could apparently use NADH

185

as a cofactor, but the xylitol yield was lower than that of trXR. This was more apparent

186

on a high titer of xylose. It was found that when xylose titer was increased to 1 M,

187

xylitol yield of teXR with NADH was only 0.1 g/g and the reaction ended in 3 h (data

188

not shown). With NADPH, teXR could obtain a result comparable to that of trXR in

189

enzyme catalysis. However, further experiments showed that it was not efficient in

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whole-cell catalysis (Supporting Information, Experiment S1 and S2). Therefore, this

191

study decided to adopt only the trXR with NADPH as a cofactor in subsequent

192

experiments.

193 194

3.2. Integration of glucose dehydrogenase in Escherichia coli

195 196

Since the NADPH-dependent trXR was demonstrated to be the most efficient XR

197

for xylitol production, an NADP+ dependent GDH from B. subtilis was selected to

198

couple it instead of the commercial GDH which was active on both NADH and

199

NADPH (Table 2). SDS-PAGE analysis of E. coli (BL21-XR-GDH) showed that two

200

clear bands of trXR and GDH were observed (Fig. 4), implying that they were

201

successfully co-expressed in recombinant strains. Three different temperatures (20oC,

202

25oC, 30oC) were investigated for expression of these proteins. While XR was most

203

highly expressed at 20oC, the GDH was expressed most highly at 25oC. Finally, the

204

engineered cells were cultivated at 25oC considering 25oC gave adequate expression for

205

both proteins as well as relatively higher cell concentrations.

206 207

3.3. Xylitol production through enzymatic catalysis from pure xylose and glucose

208 209

Effects of substrate and NADPH concentrations were systematically investigated

210

by using trXR and GDH. Substrate concentration was a critical factor in this reaction.

11



211

Low substrate concentrations resulted in low productivities, whereas high substrate

212

concentrations might inhibit the enzymes. The effect of xylose and glucose

213

concentration on substrate consumption and product formation was tested in the range

214

of 0.8-1.5 M (Fig. 5a). The results indicated that xylitol concentration was significantly

215

increased by increasing the initial substrates concentration from 0.8 M to 1.0 M.

216

Thereafter, increasing substrate concentration would decrease xylitol production

217

dramatically, implying that 1 M substrate was optimum for xylitol production. Under

218

this condition, 1 M CaCO3 could help to maintain an appropriate reaction pH (6.2). The

219

effect on substrate consumption and product formation from NADPH addition was also

220

studied in a range of 0.1-1.0 mM. Fig. 5b indicated that maximum rate and

221

concentration were achieved at 0.5 mM NADPH. The final reaction mixture was

222

determined as 1 M xylose, 1 M glucose, 5 U/mL trXR, 4 U/mL GDH, 0.5 mM NADPH

223

and 1 M CaCO3, carried out in pure water. It produced 146.23 g/L xylitol with a yield of

224

0.97 (g/g) and 185.05 g/L gluconic acid with a yield of 1.03 (g/g) in a total period of 24

225

h.

226 227

3.4. Xylitol production through whole-cell catalysis from pure xylose and glucose

228 229

Whole-cell biocatalysis with engineered E. coli (BL21-XR-GDH) in the range of

230

0.01-0.2 g/mL of wet cells was conducted for 24 h. The results showed that the yield

231

and reaction rate were gradually increased with the increase in cell amount (Fig. 5c). A

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xylitol concentration of 145.81 g/L was obtained with 0.1 g/mL of wet cells without

233

exogenous supplementation of cofactors. Use of CaCO3 maintained the reaction pH at

234

around 6.2, otherwise the pH would be decreased rapidly and reaction would be

235

inhibited (Supporting Information, Experiment S3). Different substrate concentrations

236

were also investigated and results showed that 1 M substrate was optimal (Supporting

237

information, Experiment S4). Thus, 1 M xylose, 1 M glucose, 0.1 g/mL wet cells and 1

238

M CaCO3 were used as a final system for the following experiments. It should be noted

239

here that, both the yield and concentration of the products were comparable to those

240

obtained with the crude enzymes described above. This was because the host cells used

241

in whole-cell catalysis were no longer in a normal state of life and could not catabolize

242

the sugars (Supporting Information, Experiment S5). Thus, the host cells were carriers

243

of the enzymes and only the overexpressed XR and GDH were predominantly involved

244

in the catalytic reaction. Consistent with this observation, glucose and xylose were

245

utilized simultaneously during the whole-cell catalysis (Fig. 5c), indicating cellular

246

integrity and phosphotransferase system (PTS) of the whole-cells might be impaired.

247 248

3.5. Xylitol production from cornstalk hydrolysate

249 250

The results showed that xylitol production by whole-cell catalysis from cornstalk

251

hydrolysate was over within 4 h with 0.1 g/mL of wet cells. Xylose and glucose were

252

completely consumed, and 30.88 g/L of xylitol and 50.89 g/L of gluconic acid were

13



253

produced. Decreasing the cell amount by 5-fold to 0.02 g/mL, the reaction would cease

254

in 20 h with the product concentration and conversion ratio remaining unchanged (Table

255

4). Xylitol yield with crude enzymes was almost the same as that of whole-cell catalysis,

256

but the concentration of substrates and products were decreased to (80±3)％ compared

257

to whole-cell catalysis due to the dilution of hydrolysate by the enzyme solution. The

258

cornstalk hydrolysate also contained about 3.01 g/L arabinose which was converted to

259

2.9 g/L arabitol by XR. Although arabitol and xylitol are both pentosyl alcohols, it was

260

reported that suitable calcium resin could be used to separate the two alcohols

261

efficiently.21

262 263

3.6. Comparison with other studies

264 265

Production of xylitol from xylose requires NAD(P)H. For fermentation, the

266

NAD(P)H is provided by catabolizing glucose to end products such as acetate and CO2.

267

For enzymatic process, the NAD(P)H is provided by dehydrogenation of the

268

co-substrate glucose to gluconate. In some cases, other electron donors such as glycerol

269

were tried but were not as efficient as glucose.17 By fermentation, one molecule of

270

glucose could be metabolized to acetyl-CoA through glycolysis while four molecules of

271

NADH equivalents are typically generated. NADH could be transformed into NADPH

272

to be used for NADPH-dependent XR in E. coli.3 In contrast, only one molecule of

273

NADPH is produced when glucose is converted into gluconic acid by enzyme catalysis.

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So theoretically, catabolism of glucose in fermentation could provide much more

275

NAD(P)H per glucose.3,18 However, fermentation has been complicated by well-known

276

problems such as the preference of glucose over xylose by cells, un-matched cofactor

277

specificities, as well as diversion of glucose to cell growth and competing products. As

278

a result, production of xylitol from xylose by fermentation was still at the cost of

279

considerable amounts of glucose.3, 18 Attempts were also made to directly produce

280

xylitol solely from glucose by engineered strains, but the yields were too low.19, 20

281

Compared to fermentation, enzymatic production of xylitol would also consume glucose

282

but can gain equimolar gluconate as a value-added co-product. Enzymatic catalysis is

283

also particularly suitable for producing xylitol from lignocellulosic hydrolysate wherein

284

xylose and glucose concurrently exist. In this study, highly efficient XR and GDH were

285

screened and used to engineer E. coli as a whole-cell biocatalyst. The whole cells'

286

ability to actively take up and catabolize sugars appeared to be impaired under the

287

catalysis conditions. As a result, glucose and xylose entered the host cells

288

simultaneously probably in a manner of diffusion and became available for xylitol

289

production, with little sugar being diverted to the metabolism of the host. The

290

whole-cell catalysis required no enzyme preparation and could make use of intracellular

291

cofactors to avoid exogenous NADPH addition. The pH regulation was also simplified.

292

Considering downstream purification, xylitol and gluconic acid can be separated using

293

strongly basic anion exchange resins (e.g., Cl- and OH- form).23 They can also be

294

separated by electrodialysis. The charged molecules of gluconic acid can be removed

15



295

under applied electrical potential whereas uncharged xylitol molecules remain in the

296

process stream.24 Table 4 lists the representative best results of xylitol production with

297

different strategies at present. Compared to these results, the present study obtained the

298

highest product yield of 331 g/L of xylitol and gluconate, with the highest xylitol

299

productivity of 6.1 g/L/h yet reported. When the whole-cells were applied to the

300

cornstalk hydrolysate, the sugars in the cornstalk hydrolysate were completely

301

converted with product yields much higher than those from other studies.4 Moreover,

302

the substrate concentration suitable for the whole-cell catalysis was found to be up to 1

303

M, indicating that the method developed here can be applied to cornstalk hydrolysate of

304

higher concentrations, thus would have a great potential in cost-effective production

305

xylitol from lignocellulosic feedstock.

306 307

4.

Declarations of interest

308 309

None.

310 311 312 313

Acknowledgments We thank the National Engineering Research Center for Biotechnology for providing straw and assisting us in hydrolysis of lignocellulose.

314 315

Supporting Information

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316 317


Experimental results concerning teXR performance, substrate concentrations, CaCO3 addition and sugar utilization in whole-cell catalysis.

318 319 320

Funding Sources This work was supported by the Jiangsu Provincial Natural Science Foundation of

321

China (Grant No.: BK20150938); the National Nature Science Foundation of China

322

(Grant No.: 21706123); the Major Research Plan of the National Natural Science

323

Foundation of China (21390204); the key program of the National Natural Science

324

Foundation of China (21636003); the Program for Changjiang Scholars and Innovative

325

Research Team in University (IRT_14R28); the Priority Academic Program

326

Development of Jiangsu Higher Education Institutions (PAPD), and the Jiangsu

327

Synergetic Innovation Center for Advanced Bio-Manufacture.

328 329 330

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Figure captions:

Fig. 1 The scheme of xylitol production through whole-cell catalysis. Xylose was directly reduced to xylitol by xylose reductase (XR), wherein NADPH was required. The NADPH could be regenerated in a reaction catalyzed by glucose dehydrogenase (GDH) to produce gluconate from glucose that was concurrently present in straw hydrolysate. Substrates and products were able to enter and leave the cells with no particular penetration. Fig. 2 The scheme of straw pretreatment and hydrolysis Fig. 3 The SDS-PAGE analysis of crude enzyme from E. coli (pET-28a-teXR) and E. coli (pET-28a-trXR) induced at 20oC. Lane M: marker; Lane 1: soluble fraction of E. coli (pET-28a) (control); Lane 2: insoluble fraction of E. coli (pET-28a) (control); Lane 3: soluble fraction of E. coli (pET-28a-teXR); Lane 4: insoluble fraction of E. coli (pET-28a-teXR); Lane 5: soluble fraction of E. coli (pET-28a-trXR); Lane 6: insoluble fraction of E. coli (pET-28a-trXR). Fig. 4 The SDS-PAGE analysis of crude enzyme from E. coli (BL21-XR-GDH) Lane M: marker; Lane 1: soluble fraction of E. coli (pETDuet-1) (control) induced at 20oC; Lane 2: insoluble fraction of E. coli (pETDuet-1) (control) induced at 20oC; Lane

22


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3, 5, 7: soluble fraction of E. coli (BL21-XR-GDH) induced at 20oC, 25oC, 30oC; Lane 4, 6, 8: insoluble fraction of E. coli (BL21-XR-GDH) induced at 20oC, 25oC, 30oC. Fig. 5a The effect of different concentrations of xylose and glucose on substrate consumption and product formation. The reactions were carried out at 25oC /200 rpm using 5 U/mL trXR, 4 U/mL GDH, 1 M CaCO3 and 0.5 mM NADPH in pure water. The pH was maintained at 6.2. Fig. 5b The effect of different concentrations of NADPH on substrate consumption and product formation. The reactions were carried out at 25oC / 200 rpm using 1 M xylose, 1 M glucose, 5 U/mL trXR, 4 U/mL GDH and 1 M CaCO3 in pure water. The pH was maintained at 6.2. Fig. 5c The effect of wet cell amount on substrate consumption and product formation. The reactions were carried out at 25oC /200 rpm using 1 M xylose, 1 M glucose and 1 M CaCO3 in pure water. The pH was maintained at 6.2.

Table 1 Oligonucleotides used in this study

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Page 24 of 35

Oligos

Sequence (5’→3’)

teXR-up

ATGGGTCGCGGATCCGAATTCATGAGCGCAAGTATCCCAGACA

teXR-dn

CTCGAGTGCGGCCGCAAGCTTTTAAACGAAGATTGGAATGTTGTCC

trXR-up

ATGGGTCGCGGATCCGAATTCATGAGCACCACCCCGACAA

trXR-dn

CTCGAGTGCGGCCGCAAGCTTTTAAACAAAAATCGGGATGTTGTC

XR-up

TAAGAAGGAGATATACCATGGGCATGAGTACCACCCCTACCAT

XR-dn

TGCGGCCGCAAGCTTGTCGACTTAAACAAAAATCGGAATGTTATCC

GDH-up

AGATATACATATGGCAGATCTCATGTATAGCGATTTAGCC GC

GDH-dn

GTTTCTTTACCAGACTCGAGTTAGCCACGACCAGCTTGAA

Underlined are restriction sites: GAATTC, EcoRI; AAGCTT, HindIII; CCATGG, NcoI; GTCGAC, SalI; AGATCT, BglII; CTCGAG, XhoI.

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Table 2 Enzyme activity assay for XRs Enzyme

Cofactor

Specific activity (U/mg)

teXR

NADH

1.39

teXR

NADPH

5.96

trXR

NADH

Extremely low

trXR

NADPH

7.00

GDH (purchased)

NAD+

59.60

GDH (purchased)

NADP+

73.75

25



Page 26 of 35

Table 3 Characterization of the catalytic performances of XRs Xylose

Glucose

XR

GDH

Cofactor

(M)

(M)

(U/mL)

(U/mL)

(mM)

Reaction time Xylitol yield (h)

(g/g)

Xylitol titer (M)

0.1

0.1

4 (teXR)

5

4 (NADH)

2

0.902

0.0890

0.1

0.1

4 (teXR)

5

4 (NADPH)

4

0.912

0.0907

0.1

0.1

4 (trXR)

5

4 (NADH)

4

0

0

0.1

0.1

4 (trXR)

5

4 (NADPH)

4

0.915

0.0913

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Page 27 of 35


Table 4 Representative best results of xylitol production with different strategies Substrates

Strategy

Products

Fermentation on glucose and xylose xylose 37.5 g/L and batch fermentation by glucose 27 g/L recombinant E. coli xylose ~189 g/L and glucose ~94.5 g/L

fed-batch fermentation by recombinant E. coli

Engineered production solely from glucose glucose ~200 g/L fermentation by recombinant Pichia pastoris directly on glucose glucose 100 g/L fermentation by recombinant B. subtilis directly on glucose Enzymatic catalysis with a co-substrate xylose 12 g/L and XR 0.05 g/g; GlyDH 0.02 g/g; glycerol 7.4 g/L NAD+ 0.1 mM. sugarcane bagasse XR 0.2 U/mL; GDH 0.2 U/mL; hydrolysate (xylose 18.6 NADPH 0.2 mM. g/L and glucose 5.6 g/L) xylose 150 g/L and 0.1 g/mL wet cells glucose 180 g/L corn stalk hydrolysate (xylose 32 g/L and glucose 51.6 g/L) corn stalk hydrolysate (xylose 32 g/L and glucose 51.6 g/L)

0.1 g/mL wet cells

0.02 g/mL wet cells

Xylitol productivity (g/L/h)

Reference

xylitol 38 g/L and glycolysis products xylitol 172.4 g/L and glycolysis products

0.79

(3)

1.57

(18)

xylitol 15.2 g/L and D-arabitol ~9.0 g/L xylitol 23 g/L, D-ribulose 4.5 g/L, and ribitol 2.0 g/L

0.29

(19)

0.08

(20)

xylitol 1.2 g/L and DHA 0.8 g/L xylitol ~3.9 g/L and gluconate 5.0 g/L xylitol 146 g/L and gluconate 185 g/L xylitol 30.88 g/L and gluconate 50.89 g/L xylitol 30.92 g/L and gluconate 50.94 g/L

0.025

(17)

0.33

(4)

6.1

This study

7.72

This study

1.54

This study

27



Fig. 1

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Fig. 2

29



Fig. 3

30


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Fig. 4

31



Fig. 5a

32


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Fig. 5b

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Fig. 5c

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TOC graphic

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Efficient Xylitol Production from Cornstalk Hydrolysate Using

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