Improved Xylitol Production from d

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Improved xylitol production from D-arabitol by enhancing the coenzyme regeneration efficiency of the pentose phosphate pathway in Gluconobacter oxydans Sha Li, Jinliang Zhang, Hong Xu, and Xiaohai Feng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05509 • Publication Date (Web): 04 Jan 2016 Downloaded from http://pubs.acs.org on January 19, 2016

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Journal of Agricultural and Food Chemistry

1 2

Improved xylitol production from D-arabitol by enhancing the coenzyme

3

regeneration efficiency of the pentose phosphate pathway in Gluconobacter

4

oxydans

5

Sha Li a,b, Jinliang Zhang b, Hong Xu a,b,*, Xiaohai Feng a,b

6 7 8

a

9

University of Technology, Nanjing 210009, PR China

State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing

10

b

11

Nanjing 211816, PR China

College of Food Science and Light Industry, Nanjing University of Technology,

12 13 14 15 16 17

Corresponding author

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Hong Xu; Nanjing University of Technology;

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Tel/Fax: +86-25-58139433;

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E-mail address: [email protected] (H. Xu)

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1

ACS Paragon Plus Environment


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Abstract: Gluconobacter oxydans is used to produce xylitol from D-arabitol. This study aims to

24

improve xylitol production by increasing the coenzyme regeneration efficiency of the pentose

25

phosphate pathway in G. oxydans. Glucose-6-phosphate dehydrogenase (G6PDH) and

26

6-phosphogluconate dehydrogenase (6PGDH) were overexpressed in G. oxydans. Real-time PCR

27

and enzyme activity assays revealed that G6PDH/6PGDH activity and coenzyme regeneration

28

efficiency increased in the recombinant G. oxydans strains. Approximately 29.3 g/L xylitol was

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obtained, with a yield of 73.2%, from 40 g/L D-arabitol in the batch biotransformation with the G.

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oxydans PZ strain. Moreover, the xylitol productivity (0.62 g/L/h) was 3.26-fold of the wild type

31

strain (0.19 g/L/h). In repetitive batch biotransformation, the G. oxydans PZ cells were used for

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five cycles without incurring a significant loss in productivity. These results indicate that the

33

recombinant G. oxydans PZ strain is economically feasible for xylitol production in industrial

34

bioconversion.

35 36

Keywords:

37

regeneration.

Xylitol;

Gluconobacter

oxydans;

Pentose phosphate pathway;

38 39 40 41 42 43 44

2


Coenzyme

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Introduction

46

The genus Gluconobacter performs rapid but incomplete oxidation of a wide range of

47

substrates 1. Gluconobacter oxydans is used in several biotechnological applications 2, including

48

production of 2-keto-L-gulonic acid, 5-keto-D-gluconic acid, and 6-amino-L-sorbose

49

applications involve the production of xylitol 5, a pentahydroxy sugar alcohol. Xylitol exhibits

50

sweetness similar to sucrose and thus can be used as an alternative natural sweetener. Xylitol also

51

prevents dental cavities and is suitable for diabetic patients because the metabolism of this sugar is

52

insulin independent 6-8. Therefore, xylitol is widely used in food and chemical industries.

3,4

. These

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Xylitol is mainly produced chemically by reducing D-xylose 9. Recently, xylitol production

54

through fermentation has attracted much attention. Researchers have also gained much progress in

55

xylitol bioproduction with xylose as feedstock. Several yeast strains, including Debaryomyces

56

hansenii , Candida tropicalis and Candida parapsilosis, have been used for xylitol production, to

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obtain high xylitol productivity (Table. S1). However, chemical and fermentation processes rely

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on the hydrolysis and purification of D-xylose from hemicellulose–xylan hydrolysates, thereby

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causing serious contamination10. A two-step process, with D-glucose as the initial substrate, has

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been developed as an alternative technique for xylitol production because of the technological and

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economic advantages of D-glucose compared with D-xylose 11. D-glucose is initially converted into

62

D-arabitol

63

membrane-bound

64

dehydrogenase (XDH) 5. D-arabitol is almost completely converted into D-xylulose by m-AraDH,

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but only ~25% of D-xylulose is converted into xylitol by XDH

66

restrictive enzyme that controls the overall bioconversion process probably because of its confined

by yeasts and then transformed to xylitol by G. oxydans D-arabitol

dehydrogenase

(m-ArDH)

and

5,15,16

3


12-14

, which contains

NAD-dependent

xylitol

. Therefore, XDH is a key


67

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activity or coenzyme support.

68

Studies have been conducted to increase xylitol yield. Overexpressing the xdh gene in G.

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oxydans, resulted in 11-fold enhancement in XDH activity compared with that in the wild-type

70

strain. Nevertheless, the xylitol yield was still less than 30% 17. Given that XDH is highly specific

71

for the cofactor NADH

72

NADH is sufficient.

73

18

, researchers must ensure that the supply of reducing potential from

In other bacterial strains, NADH can be regenerated via the Embden Meyerhof pathway and 19, 20

74

the TCA cycle during the metabolism of glucose or other carbohydrates

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Gluconobacter lacks essential enzymes, including phosphofructokinase, and succinate

76

dehydrogenase 21. As such, the oxidative pentose phosphate pathway (PPP) is an important route

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in the phosphorylative breakdown of sugars in Gluconobacter. Moreover, PPP is mainly used to

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generate ATP and regenerate coenzyme in Gluconobacter

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activities of glucose-6-phosphate dehydrogenase (G6PDH, zwf) and 6-phosphogluconate

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dehydrogenase (6PGDH, gnd) in the soluble fraction of G. oxydans cells, as well as in the cloned

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and expressed Escherichia coli strains. The results showed that G6PDH and 6PGDH exhibit

82

NAD+/NADP+ dual coenzyme specificity. Thus, PPP could be the main factor responsible for

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NADH regeneration in G. oxydans 23.

22

. However,

. Previous studies examined the

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This study aims to increase the coenzyme regeneration efficiency of PPP for xylitol

85

production. The effects of the constitutive expression of G6PDH/6PGDH in G. oxydans on

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coenzyme regeneration and xylitol conversion were investigated. Basing on the results, this study

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established an efficient and concise route for xylitol production in G. oxydans.

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Methods

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Strains, plasmids, and chemicals

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The xylitol producing strain G. oxydans NH-10 was isolated from soil and deposited at the

92

China General Microbiological Culture Collection Center with accession number CGMCC 2709.

93

Escherichia coli JM109 was used as a host for plasmid construction and maintenance. E. coli

94

HB101 harboring helper plasmid pRK2013 was used for transformation experiments. G. oxydans

95

PZ or G. oxydans PG (this study) were used for zwf and gnd expression and xylitol production,

96

respectively. The broad host range vector pBBR1MCS-5 was used for expression of zwf or gnd in

97

G. oxydans NH-10 24. Restriction enzymes, Bacterial Genomic DNA Extraction Kit, T4 DNA

98

ligase, exTaq polymerase, and DNA and protein markers were obtained from TaKaRa (Dalian,

99

China). The strains and plasmids used in this study are listed in Table 1.

100 101

Medium and culture conditions

102

All E. coli strains were cultivated at 37 oC in lysogeny broth (LB) medium (yeast extract, 5 g/L;

103

tryptone, 10 g/L; NaCl, 10 g/L; pH 7.0) with the addition of 10 µg/mL gentamicin or 50 µg/mL

104

kanamycin to maintain the plasmids. Recombinant G. oxydans strains were cultivated in YPG

105

medium (glucose, 30 g/L; sorbitol, 10 g/L; yeast extract, 20 g/L; (NH4)2 SO4, 0.5 g/L; KH2PO4 ,

106

1.5 g/L; MgSO4·7H2O, 0.5 g/L) containing 50 µg/mL gentamicin, and 20 g/L CaCO3 to control pH.

107

G. oxydans was cultivated in 1000 mL shake flasks containing 200 mL of YPG medium at 30 oC

108

and 200 rpm on a rotary shaker, or in a 7.5 L fermentor (New Brunswick Scientific, USA)

109

containing 4.0 L of YPG medium with pH and temperature automatically maintained at 6.0 and 30

110

o

C, respectively. Agitation and aeration were controlled at 600 rpm and 1 vvm, respectively. Cell

5



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111

cultivations were initiated by inoculating 2% (v/v) of 16 h precultures of G. oxydans strains. After

112

24 h, the cells reached early stationary phase and were harvested by centrifugation at 8000 g and 4

113

o

114

in D-arabitol solution for xylitol bioproduction.

C for 10 min, washed twice with 100 mM potassium phosphate buffer (pH 6.0), and resuspended

115 116

Vector construction and transfer into G. oxydans NH-10

117

In order to express the G6PDH/6PGDH genes in G. oxydans NH-10, two plasmids containing

118

either zwf or gnd driven by the tufB promoter were constructed, respectively. Based on the

119

available sequence information for G. oxydans (Accession number CP000009), oligonucleotide

120

primers were designed to amplify the zwf, gnd and promoter tufB genes from genomic DNA of G.

121

oxydans

122

5′-CGGGGTACCTATCAGGTTCCGGTTGAAG -3′ (the Kpn I site is underlined); PtufB-R,

123

5′-CCGCTCGAGACCTGGAACGGGAGTAAG-3′ (the Xho I site is underlined). zwf-F,

124

5′-CACTCGAGGAGGTCAGAGAGTCTT-3′

125

5′-GAGGATCCTATCGTCGCTCAAAT-3′

126

5′-CATCTCGAGACAAAGCACTGGCAG-3′ (the Xho I site is underlined); gnd-R, 5′-

127

GTAGGATCCGACGCAGGCTCATTT-3′ (the BamH I site is underlined).

NH-10

by

PCR.

The

primers

(the

(the

were

as

follows:

PtufB-F,

Xho

I

site

is

underlined);

zwf-R,

BamH

I

site

is

underlined).

gnd-F,

128

The PtufB PCR product and plasmid pBBR1MCS-5 were digested with restriction enzymes

129

Kpn I and Xho I, and ligated with T4 DNA ligase (MBI Fermentas) to generate plasmid

130

pBBR-PtufB. Then the zwf and gnd PCR products were digested and cloned into the Xho I- BamH I

131

restriction sites of pBBR-PtufB to generate the recombinant plasmids pBBR-PtufB-zwf and

132

pBBR-PtufB-gnd. Transformants were selected on LB agar plates containing 10 µg/mL gentamicin,

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133

and confirmed by colony PCR and restriction enzyme digestion. The expression plasmids

134

pBBR-PtufB-zwf and pBBR-PtufB-gnd were transferred into G. oxydans NH-10 by triparental mating

135

as described previously 25.

136

For determination of plasmid stability, recombinant strains were cultured in glycerol

137

medium without antibiotics for 3 days. Then the cultures were diluted and spread onto

138

antibiotics-free agar plates. After cultivation, 100–200 colonies were randomly transferred with

139

sterile toothpicks from each of these plates to agar plates containing 50 µg/mL gentamicin.

140

Plasmid stability was calculated by measuring the ratio of the numbers of colonies on the

141

gentamicin-containing agar plates to that on nonselective agar plates.

142 143

Quantitative real-time PCR

144

For real-time reverse transcription PCR (RT-PCR) experiments, 0.5 mL preculture of the G.

145

oxydans strains were inoculated into 250-mL shake flasks containing 50 mL YPG medium and

146

grown to late exponential phase (20 h) at 30 oC and 200 rpm on a rotary shaker. Then the cells

147

were harvested for total RNA isolation by centrifugation at 10,000 g and 4 oC for 1 min. Total

148

RNA was extracted using RNAiso Plus reagent (TaKaRa, Dalian) and treated with DNase I

149

(TaKaRa,

150

pyrocarbonate-treated water. RNA concentrations and purity were calculated by measuring

151

absorbance at 260 and 280 nm using a NanoDrop 1000 Spectrophotometer (Thermo Scientific)

152

and by analysis on a 1.2% (w/v) agarose gel. For transcriptional analysis of each gene, RT-PCR

153

was carried out with a first-strand cDNA synthesis kit (Promega, USA) using total RNA as

154

template. Gene expression analysis was performed by quantitative real-time PCR performed with

Dalian).

Purified

RNA was

resuspended

in

50

7


µl

of

0.1%

dimethyl


Page 8 of 35

155

the StepOnePlus™ Real-Time PCR System (Applied Biosystems, USA). The primers were as

156

follows:

157

5′-CGTTCTGGACCGTCTCTTTC-3′. gnd-F, 5′-CGGTTACTGCATGATGTTCG-3′; gnd-R,

158

5′-CTCGTTAAGCGTCTCCGAAC-3′. xdh-F,

159

5′-TTGGCGGTCCCTTCACA-3′. The 16S rRNA gene was used as an internal standard, using the

160

primers 5′- GCGGTTGTTACAGTCAGATG -3′ and 5′- GCCTCAGCGTCAGTATCG -3′. Data

161

were collected using Opticon2 software. Data represent the mean of three independent

162

experiments. Gene expression patterns were calculated using the the 2-

163

changes in gene expression were calculated by measuring relative abundance of PCR products in

164

samples, normalized to the internal standard.

zwf-F,

5′-GAGAACCTGAACCGCTACGA-3′;

zwf-R,

5′-GTGCCTTCCACGTCCTCAA-3′; xdh-R,

△△Ct

method

26

. Relative

165 166

Biotransformation of D-arabitol into xylitol by using the recombinant G. oxydans PZ/PG resting

167

cell system

168

Initial small-scale biotransformation was performed in shake flasks. Recombinant G. oxydans

169

cells were dispersed at a concentration of 10% (cell wet weight, w/v) in a reaction solution

170

containing 30 g/L D-arabitol, and then incubated at 30 °C on a rotary shaker. The first reaction,

171

oxidation of D-arabitol to D-xylulose, occurred under 220 rpm for 7 h. The second reaction,

172

reduction of D-xylulose to xylitol, was performed after adding 5% glucose (w/v) to the mixture

173

under 50 rpm.

174

Scaled up production of xylitol was performed in a 7.5 L bioreactor (NBS, USA) with an

175

effective working volume of 4.5 L. The pH and temperature of the reactor was maintained at 6.0

176

and 30 °C, respectively. Agitation and aeration rates were regulated at 600 rpm and 1.0 vvm for 7

8


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177

h. The agitation rate was then adjusted to 150 rpm after adding 5% glucose (w/v) as co-substrate

178

for another 40 h. When the reaction reached the equilibrium, the cells were recovered by

179

centrifugation and resuspended in fresh reaction mixture for biotransformation. The composition

180

of the final media after cell collection was determined by HPLC.

181 182

Analytic methods

183

Enzymatic activity assay

184

Cell-free extracts of G. oxydans NH-10 and the recombinant G. oxydans strains were prepared

185

by ultrasonication, followed by centrifugation at 8000 rpm for 15 min at 4 °C. The supernatants

186

were used for the enzymatic activity assays. Dehydrogenases were assayed by a routine method

187

used for NAD(P)H-linked enzymes by recording the rate of increase or decrease of NAD(P)/H at

188

340 nm with respective substrates. The activities of G6PDH and 6PGDH were measured

189

spectrophotometrically at 30 °C. The reactions were initiated by adding the enzyme solution to a

190

final volume of 2 mL as a standard condition and monitored for 0.5-1.0 min by recording the rate

191

of NAD(P)H depletion at 340 nm. The reaction mixture for G6PDH activity assay contained

192

10 mM glycine–NaOH (pH 10.0), 1 mM NADP, and 10 mM glucose-6-phosphate. Meanwhile, the

193

mixture for 6PGDH activity assay contained 10 mM potassium phosphate buffer (pH 6.0) 1 mM

194

NAD, and 50 mM 6-phosphogluconate. One enzyme unit was defined as the amount of enzyme

195

that reduces 1 µmole of pyridine dinucleotide per minute. The protein concentration of the

196

cell-free extract supernatant was determined via Bradford method 27. The XDH activity was

197

determined as described previously 16.

198

NAD+ and the TTN (total turnover number) assay 9



199

Total turnover number (TTN) was used to indicate the coenzyme regeneration efficiency in this

200

process. TTN is defined as the total number of moles of xylitol formed per mole of NAD+. The

201

NAD+ was extracted by collecting 1 mL sample in a tube. The sample was centrifuged at 12,000

202

rpm for 1 min. The supernatant was removed and 300 µL of 0.2 M HCl was added to extract

203

the NAD+. The samples was placed in a 50 °C water bath for 10 min and then cooled on ice. The

204

extract was neutralized by adding 300 µL of 0.1 M NaOH dropwise. The cellular debris was

205

removed by centrifuging at 12,000 rpm for 5 min. Supernatant was transferred to new tube and

206

stored at -20 °C. A very sensitive cycling assay was used to determine the intracellular

207

concentration of NAD+. The assay was performed using a reagent mixture consisting of equal

208

volumes of 1.0 M Bicine buffer (pH 8.0), absolute ethanol, 40 mM EDTA (pH 8.0), 4.2 mM

209

MTT and twice as much of 16.6 mM PES (phenazine ethosulfate), previously incubated at 30 °C.

210

The reaction mixture including : 50 µL neutralized extract , 0.3 mL water , 0.6 mL reagent

211

mixture and 50 µL of yeast ADH. The absorbance at 570 nm was recorded for 10 min at 30 °C.

212

Standard solution of NAD+ was calibrated 28.

213

Quantification of D-arabitol and xylitol

214

D-arabitol and xylitol concentrations in the mixture were determined by HPLC equipped with

215

a refractive index detector (RI-101, Shodex, Japan). The reaction products were filtered through

216

0.22 µm membrane filters prior to HPLC analysis. The samples (20 µL) were injected onto a

217

Rezex RCM-Monosaccharide Ca2+ column（Phenomenex，USA）. The mobile phase was water

218

with a flow rate of 0.4 mL/min at 70 °C. D-arabitol and xylitol were used as standards.

219

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Results

221

Construction and expression of recombinant vectors in G. oxydans NH-10

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PPP is vital for NADH production; in this study, PPP was activated by overexpressing the

223

G6PDH (zwf)/6PGDH (gnd) genes in G. oxydans NH-10 to increase intracellular NADH

224

concentration. The zwf, gnd, and tufB promoter genes were amplified from G. oxydans NH-10

225

genomic DNA by PCR reaction (Fig. 1).

226

Plasmids used to overexpress G6PDH/6PGDH were constructed and transformed into G.

227

oxydans NH-10 through tri-parental conjugation. The strain harboring the pBBR-PtufB-zwf plasmid

228

was named G. oxydans PZ, and the other strain harboring the pBBR-PtufB-gnd was designated as G.

229

oxydans PG. Successful transformation was verified by PCR and restriction enzyme digestion of

230

the recombinant plasmids (Fig.1). The biochemical performance of the three strains was assessed

231

and compared. The recombinant strains grew at approximately the same rate as the wild-type

232

strain, indicating that the expression vectors did not affect the physiological status of G. oxydans.

233

Figure 2 shows the activities of G6PDH (Fig. 2a) and 6PGDH (Fig. 2b) in the strains during

234

fermentation. For example, the activity of G6PDH was 4.25-fold higher in G. oxydans PZ (1.26 ±

235

0.02 U/mg) than that in G. oxydans NH-10 (0.24±0.01 U/mg) but remained at the same level as

236

that in G. oxydans PG (0.24±0.02 U/mg). By contrast, the activity of 6PGDH was two fold higher

237

in G. oxydans PG (0.143±0.006 U/mg), and 29.7% more active in G. oxydans PZ (0.061±0.007

238

U/mg) than that in the wild type (0.047±0.005 U/mg). Nevertheless, no significant change in XDH

239

activity (0.05±0.01 U/mg ) was observed among the strains.

240 241

RT-PCR analysis of the transcription of G6PDH and 6PGDH genes in different G. oxydans strains

11



242

Cellular mRNA was detected by real-time RT-PCR to demonstrate the overexpression of

243

G6PDH and 6PGDH genes in different G. oxydans strains. Overexpression of G6PDH and

244

6PGDH showed no effects on the cell growth of G. oxydans. All cells used to isolate RNA were

245

cultured at a constant period to minimize the effect on RT-PCR analysis because gene expression

246

varied significantly in different growth phases 29. The results showed that the transcription level of

247

G6PDH in G. oxydans PZ was 12-fold higher than that in G. oxydans NH-10 (Fig. 3).

248

Overexpression of the zwf gene significantly promoted G6PDH gene transcription. In G. oxydans

249

PG, the transcript of the 6PGDH gene was approximately five times higher than that in G. oxydans

250

NH-10. This observation could be attributed to the overexpression of the gnd gene. The

251

transcription levels of the XDH gene were stabilized not only in the wild type strain but also in the

252

recombinant stains.

253 254

Biotransformation of D-arabitol to xylitol via the recombinant systems in shake flasks

255

D-arabitol was converted into xylitol by using recombinant G. oxydans strains. Differences in

256

xylitol production among the three strains are depicted in Fig. 4. Xylitol production almost

257

reached the maximum yield after 48 h. The G. oxydans PZ strain produced 18.8 g/L xylitol, with a

258

yield of 62.7%, whereas the G. oxydans PG strain produced 14.6 g/L xylitol, with a yield of 48.7%.

259

Only 7.5 g/L xylitol was produced using the G. oxydans NH-10 strain (yield of 25%). Xylitol yield

260

increased in both recombinant systems compared with that in the wild type. TTN, defined as the

261

number of moles of xylitol formed per mole of NADP+/NAD+, was (1.15 ± 0.20) × 104 mol/mol in

262

G. oxydans PZ and (7.70 ± 0.16) × 103 mol/mol in G. oxydans PG. Based on the TTN number,

263

the recombinant systems were more efficient than G. oxydans NH-10 (1180 mol/mol). These

12


Page 12 of 35

Page 13 of 35


264

results also indicated that the amounts of xylitol accumulated by the tested strains were consistent

265

with those obtained from the activity of control enzymes in the PPP measurements.

266

The recombinant G. oxydans PZ strain was selected as the xylitol-producing strain. Several

267

key factors of biotransformation were optimized. The effects of substrate concentration on xylitol

268

production are shown in Table 2. In particular, xylitol yield decreased and conversion was

269

prolonged with increasing D-arabitol concentration. The maximum xylitol yield (73%) was

270

achieved at 20 g/L D-arabitol. Xylitol productivity (0.56 g/L/h) was more efficient when using

271

40 g/L D-arabitol as the substrate. Based on xylitol productivity, 40 g/L D-arabitol was selected as

272

the optimum concentration. The amount of added recombinant cells also influenced xylitol

273

production. As shown in Fig. 5, xylitol yield was improved by 10% by increasing cell

274

concentration from 5% to 15% (m/v). However, only a slightly higher xylitol yield was observed

275

when 12.5% or 15% recombinant cells were used. As such, the recombinant system was the most

276

efficient when the amount of added cells was 12.5% and generated a maximum xylitol yield of

277

70%.

278 279

Biotransformation of D-arabitol into xylitol in a 7.5 L fermentor

280

Scaled-up biotransformation in pH-controlled conversion was performed to assess the

281

potential use of the PZ strain in industrial xylitol production. As shown in Fig. 6, D-arabitol was

282

immediately oxidized at high rates within the initial 7 h after the cells were added to the bioreactor.

283

For both strains, less than 5 g/L D-arabitol was retained, and only a small amount of xylitol was

284

produced in the reaction mixture. D-xylulose was reduced to xylitol under low agitation speed and

285

DO concentration. After another 40 h of biotransformation, all the supplied D-arabitol (40 g/L) in

13



286

the reaction mixture was consumed, and about 29.3 g/L xylitol was obtained using the G. oxydans

287

PZ strain. In contrast to the wild G. oxydans NH-10 strain (9.2 g/L ), G. oxydans PZ showed

288

improved xylitol titer upon incubation in the same reaction solution. The xylitol productivity of G.

289

oxydans PZ was significantly increased to 0.62 g/L/h, in contrast to 0.19 g/L/h for G. oxydans

290

NH-10. Thus, overexpression of G6PDH in G. oxydans increased xylitol productivity by

291

approximately 226% compared with that in G. oxydans NH-10.

292 293

Repeated-batch biotransformation of D-arabitol to xylitol by G. oxydans PZ

294

In repetitive batch experiments with the G. oxydans PZ strain, six runs were repeated in a 7.5

295

L bioreactor under defined conditions. Fig. 7 shows that the cells could be recycled four times

296

without an evident decrease in xylitol production. Only a small decrease in the fifth cycle was

297

observed. However, a remarkable decrease in D-xylitol yield (drop to 45%) was observed in the

298

sixth cycle. During the repeated-batch experiments, approximately 128 ± 5 g of xylitol was

299

accumulated in each round of biotransformation with a slight increase in the reaction time from

300

the first cycle to the fifth cycle. A total of 640 g of xylitol was formed within 245 h in the five runs

301

of the reaction; hence, the resulting average xylitol productivity is 0.54 g/L/h.

302

Discussion

303

Xylitol is an important compound widely used in food and chemical industries. Microbial

304

production from D-glucose is technically feasible and harmless to the environment, and can be

305

performed using a two-step process. G. oxydans can produce xylitol from D-arabitol via

306

D-xylulose.

307

studies implied that improvements in XDH activity alone are insufficient to increase xylitol yields

This bioprocess is mainly affected by XDH activity and coenzyme supply. Previous

14


Page 14 of 35

Page 15 of 35


308

17

309

from D-xylulose 11. When 0.5 mmol/L NAD+ was added for NADH regeneration, a yield close to

310

100% was achieved. However, the high cost and limited availability of pyridine cofactors hindered

311

the economic viability of industrial-scale biotransformation using an XDH in situ cofactor

312

regeneration step.

. Mayer et al. employed purified XDH and formate dehydrogenase to catalyze xylitol production

313

Introduction of dehydrogenases by adding co-substrates, such as ethanol or D-glucose, is a

314

convenient and useful method for regenerating pyridine cofactors; this process can also promote

315

the recycling of nicotinamide coenzymes

316

transformed co-expression plasmids encoding XDH and glucose dehydrogenase/alcohol

317

dehydrogenase into Escherichia coli Rosetta (DE3). As a result, the xylitol yield was significantly

318

enhanced to 92%/85.2% using 30 g/L D-arabitol. Therefore, the engineered G. oxydans PXPG

319

strain was constructed to co-express the XDH gene and glucose dehydrogenase gene in the wild G.

320

oxydans strain. The xylitol yield was increased to 40.8% 25. Although xylitol yield was increased

321

by these co-expression systems, the yield remains insufficient for large-scale production of xylitol.

322

Considering that the overall xylitol production is limited by coenzyme deficiency, this study

323

was conducted to enhance coenzyme production in G. oxydans strains. PPP is an important route

324

in the phosphorylative breakdown of sugars, which in turn generates ATP and regenerates

325

coenzyme in Gluconobacter sp. 22. G6PDH and 6PGDH, which are key enzymes in oxidative PPP,

326

can reduce NADP+ and NAD+; therefore, NADPH and NADH can be generated in PPP 23. In the

327

present study, G6PDH or 6PGDH expression was enhanced to produce xylitol from D-arabitol in

328

G. oxydans. Physiological status of the resulting recombinant strains confirmed that the expression

329

vector with individual genes did not significantly affect the growth of the host strain. Several

30

. For example, Zhou et al.

15


16

constructed and


Page 16 of 35

330

previous reports concluded that a TTN of 103–105 may be sufficient to obtain an economically

331

viable process 31. In these two recombinant systems, the TTN of xylitol forming NAD+ was (1.15

332

± 0.20) × 104 in G. oxydans PZ and (7.70 ±0.16) × 103 in G. oxydans PG. These values were

333

higher than that in G. oxydans NH-10, thereby suggesting that the two processes are economically

334

feasible. G. oxydans PZ was more efficient than G. oxydans PG not only in terms of cofactor

335

regeneration but also in xylitol production. This observation could be due to the higher activities

336

of G6PDH and 6PGDH in G. oxydans PZ than those in the wild strain. However, only 6PGDH

337

activity was increased by 38.3% in the G. oxydans PG strain. In addition, the transcription level of

338

the G6PDH gene in G. oxydans PZ was higher than that of the 6PGDH gene in G. oxydans PG.

339

G6PDH is the first enzyme in the PPP and specific for NADP as a coenzyme

32

. 6PGDH is

340

the second enzyme in the PPP and specific for NADH as coenzyme, which catalyzes the oxidative

341

decarboxylation of 6-phosphogluconate to D-ribulose-5-phosphate. Recombinant G. oxydans

342

strains with enhanced PPP and high enzyme activities could possibly produce high NADH and

343

NADPH contents during intermediary metabolism because G6PDH produces NADPH and

344

6PGDH produces NADH under physiological conditions. Moreover, nicotinamide dinucleotide

345

transhydrogenase (NDTH，Gox310-312) can convert NADPH into NADH 22. This process may

346

maintain the balance of oxidation states between pyridine nucleotides. In xylitol production, this

347

process more efficiently occurred by enhancing the expression of the 6PGDH gene, which was

348

loaded in the upstream of the PPP.

349

The maximum xylitol yield (73.2%) was obtained by using 40 g/L D-arabitol as substrate and

350

12.5% (w/v) recombinant G. oxydans PZ cells. This result was different from that reported by

351

Zhou et al., in their study, the optimal D-arabitol concentration was 30 g/L and the amount of cells

16


Page 17 of 35


352

added was 10% to produce xylitol by G. oxydans NH-10 16. In addition, the xylitol yield achieved

353

by the G. oxydans PZ strain was higher than that by the G. oxydans PXPG strain (40.8%)

354

constructed by Zhang et al.

355

promote xylitol production by enhancing the coenzyme regeneration efficiency of PPP in G.

356

oxydans strain.

25

. The present study is the first investigation that attempted to

357

The stability of the free G. oxydans PZ cells was higher than that of E. coli

358

Rosetta/Duet-xdh-gdh cells, which can recover 40% of the initial activity after four cycles of

359

biotransformation during xylitol production

360

biotransformation from D-arabitol to xylitol using G. oxydans NH-10 and E. coli

361

Rosetta/Duet-xdh-gdh as biocatalyst, the recombinant G. oxydans PZ strain can produce xylitol

362

from D-arabitol directly. Therefore, the later process is simpler.

16

. Furthermore, compared with the two-step

363

In conclusion, xylitol production from D-arabitol was improved by overexpressing either

364

G6PDH or 6GPDH gene in the wild G. oxydans strain. Increased coenzyme regeneration

365

efficiency of PPP was evaluated. The recombinant G. oxydans PZ strain produced xylitol at 40 g/L

366

D-arabitol and displayed a substantially increased xylitol productivity in a resting cell system. In

367

repetitive batch biotransformation, the cells can be used for five cycles without a significant loss

368

in xylitol production. From an economic viewpoint, the results are of great interest for xylitol

369

production using the G. oxydans PZ cells on an industrial scale.

370 371

Acknowledgements

372

This work was supported by National Basic Research Program of China (2013CB733600), the

373

National High Technology Research and Development Program of China (2012AA021503). The

17



374

authors thank Dr. Matthew I Pena (Rice University, USA) for read-through.

375 376

Supporting Information available

377

Table. S1 Production of xylitol by microorganisms using different feedstock

378

This material is available free of charge via the Internet at http://pubs.acs.org.

379 380 381 382 383 384 385 386 387

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388

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389

1. Arun, G.; Vinay, K.; Singh, G. N.; Qazi, A. K. Gluconobacter oxydans: Its Biotechnological

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Applications. J. Mol. Microbiol. Biotechnol. 2001, 3, 445-456. 2. Deppenmeier, U.; Hoffmeister, M.; Prust, C. Biochemistry and biotechnological applications of Gluconobacter strains. Appl. Microbiol. Biotechnol. 2002, 60, 233-242.

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3. Muynck, C. D.; Pereira, C. S. S.; Naessens, M.; Parmentier, S.; Soetaert, W.; Vandamme, E. J.

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The genus Gluconobacter oxydans: comprehensive overview of biochemistry and

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biotechnological applications. Crit. Rev. Biotechnol. 2007, 27, 147-171.

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4. Matsushita, K.; Fujii, Y.; Ano, Y.; Toyama, H.; Shinjoh, M.; Tomiyama, N.; Miyazaki,

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T.; Sugisawa, T.; Hoshino, T.; Adachi, O. 5-Keto-D-gluconate production is catalyzed by a

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quinoprotein glycerol dehydrogenase, major polyol dehydrogenase, in Gluconobacter species.

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5. Suzuki, S.; Sugiyama, M.; Mihara, Y.; Hashi, K.; Yokozeki, K. Novel enzymatic method for

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the production of xylitol from D-arabitol by Gluconobacter oxydans. Biosci. Biotechnol.

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Biochem. 2002, 66, 2614-2620.

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6. Emodi, A. Xylitol, its properties and food applications. Food Technol. 1978,32, 20-32.

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7. Amaechi, B. T.; Higham, S. M; Edgar, W. M. The influence of xylitol and fluoride on dental

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erosion in vitro. Arch. Oral. Biol. 1998, 43, 157-161. 8. Makinen, K. K. The rocky road of xylitol to its clinical application. J. Dent. Res. 2000,79, 1352-1355. 9. Winkelhausen, E.; Kuzmanova, S. Microbial conversion of D-xylose to xylitol. J. Ferment. Bioeng. 1998, 86, 1-14.

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10. Parajó, J. C.; Domı́nguez, H.; Domı́nguez, J. M. Biotechnological production of xylitol. Part 3:

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Operation in culture media made from lignocellulose hydrolysates. Bioresour. Technol. 1998,

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66, 25-40.

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11. Mayer, G.; Kulbe, K. D.; Nidetzky, B. Utilization of xylitol dehydrogenase in a combined

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microbial/enzymatic process for production of xylitol from D-glucose. Appl. Biochem.

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Biotechnol. 2000, 100, 577-589.

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12. Escalanate, J.; Caminal, G.; Figueredo, M.; Mas, C. Production of arabitol from glucose by Hansenula polymorpha. J. Ferment. Bioeng. 1990, 70, 228-231. 13. Morgan, J. W.; Witter, L. D. Effect of sugars on D-arabitol production and glucose metabolism in Saccharomyces rouxii. J. Bacteriol. 1979, 138, 823-831. 14. Blakley, E. R.; Spencer, J. F.T. Studies on the formation of D-arabitol by osmophilic yeasts. Can. J. Biochem. Physiol. 1962, 40, 1737-1748.

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15. Adachi, O.; Fujii, Y.; Ghaly, M. F.; Toyama, H.; Shinagawa, E.; Matsushita, K.

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Membrane-bound quinoprotein D-arabitol dehydrogenase of Gluconobacter suboxydans IFO

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3257: a versatile enzyme for the oxidative fermentation of various ketoses. Biosci. Biotechnol.

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Biochem. 2001, 65, 2755-2762.

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16. Zhou, P.; Li, S.; Xu, H.; Feng, X. H.; Ouyang, P. K. Construction and co-expression of a

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xylitol dehydrogenase and a cofactor regeneration enzyme in Escherichia coli for the

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production of xylitol from D-Arabitol. Enzyme Microb. Technol. 2012, 51, 119-124.

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17. Sugiyama, M.; Suzuki, S.; Tonouchi, N.; Yokozeki, K. Cloning of the xylitol dehydrogenase

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gene from Gluconobacter oxydans and improved production of xylitol from D-arabitol. Biosci.

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Biotechnol. Biochem. 2003, 67, 584-591.

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18. Ehrensberger, A. H.; Elling, R. A.; Wilson, D. K. Structure-guided engineering of xylitol dehydrogenase cosubstrate specificity. Struct. 2006, 14, 567-575. 19. Donk, W. A.; Zhao, H. M. Recent developments in pyridine nucleotide regeneration. Curr. Opin. Biotechnol. 2003, 14, 421-426. 20. Liu, L. M.; Li, Y.; Shi, Z. P. Enhancement of pyruvate productivity in Torulopsis glabrata： Increase of NAD(+) availability. J. Biotechnol. 2006, 126, 173-185.

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21. Rauch, B.; Pahlke, J.; Schweiger, P.; Deppenmeier, U. Characterization of enzymes involved

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in the central metabolism of Gluconobacter oxydans. Appl. Microbiol. Biotechnol. 2010, 88,

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711–718.

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22. Prust, C.; Hoffmeister, M.; Liesegang, H.; Wiezer, A.; Fricke, W. F.; Ehrenreich, A.;

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Gottschalk, G.; Deppenmeier, U. Complete genome sequence of the acetic acid bacterium

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Gluconobacter oxydans. Nat. Biotechnol. 2005, 23, 195-200.

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23. Tonouchi, N.; Sugiyama, M.; Yokozeki, K. Coenzyme specificity of enzymes in the oxidative

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pentose phosphate pathway of Gluconobacter oxydans. Biosci. Biotechnol. Biochem. 2003, 67,

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2648-2651.

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24. Li, M.; Wu, J.; Li, X.; Lin, J.; Wei, D. Enhanced production of dihydroxyacetone from

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glycerol by overexpuression of glycerol dehydrogenase in an alcohol dehydrogenase-deficient

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mutant of Gluconobacter oxydans. Bioresour. Technol. 2010, 101, 8294-8299.

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25. Zhang, J. L.; Li, S.; Xu, H.; Zhou, P.; Zhang, L. J.; Ouyang, P. K. Purification of xylitol

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dehydrogenase and improved production of xylitol by increasing XDH activity and NADH

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supply in Gluconobacter oxydans. J. Agric. Food. Chem. 2013, 61, 2361-2367.

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26. Rajeevan, M. S.; Ranamukhaarachchi, D. G.; Vernon, S. D.; Unger, E. R. Use of real-time

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quantitative PCR to validate the results of cDNA array and differential display PCR

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technologies. Methods, 2001, 25, 443-51.

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27. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. 28. Berrios R. Metabolic engineering of cofactors (NADH/NAD+) in Escherichia coli. Degree paper. Houston: Rice University, 2002, 24-25.

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29. Quintero, Y.; Poblet, M.; Guillamón, J. M.; Mas, A. Quantification of the expression of

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reference and alcohol dehydrogenase genes of some acetic acid bacteria in different growth

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conditions. J. Appl. Microbiol. 2009,106, 666-674.

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30. Weckbecker, A.; GrÖger, H.; Hummel, W. Regeneration of nicotinamide coenzymes:

464

principles and applications for the synthesis of chiral compounds. Adv. Biochem. Eng. Bio.

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2010, 120, 195-242.

466 467

31. Zhao, H. M.; Donk, W. A. Regeneration of cofactors for use in biocatalysis. Curr. Opin. Biotechnol. 2003,14, 583-589.

468

32. Adachi, O.; Matsushita, K.; Shinagawa, E.; Ameyama, M. Occurrence of old yellow enzyme

469

in Gluconobacter suboxydans, and the cyclic regeneration of NADP. J. Biochem. 1979, 86,

470

699-709.

471 472 473 474 475

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476

Figures and Tables

477

Figures Caption:

478

Fig. 1. Identification of plasmids pBBR-PtufB -zwf/gnd extracted from the recombinant strain by

479

PCR and enzyme digestion. Lane M2k: DNA marker 2000bp; Lane M15k: DNA Marker 15000bp;

480

1: PCR PtufB; 2: PCR zwf; 3: PCR PtufB +zwf; 4: pBBR-PtufB-zwf digested with BamH I/Kpn I; 5:

481

pBBR-PtufB- gnd digested with BamH I/Kpn I; 6: PCR PtufB +gnd; 7: PCR gnd; 8:PCR PtufB.

482 483

Fig. 2. G6PDH (a) and 6PGDH (b) activities of different G. oxydans stains. Error bars represent

484

standard deviation from mean for three batches.

485 486

Fig. 3. The transcription level analysis of zwf, gnd and xdh genes at different strains. Error bars

487

represent standard deviation of three samples taken from the same RNA sample.

488

Relative quantity (RQ) =2

489

(HGC)]; TGS: Target gene of sample; HGS: House-keeping gene of sample; TGC: Target gene of

490

control; HGC: House-keeping gene of control.

(－△△CT)

; △△CT= [△CT (TGS)－△CT (HGS)]－[△CT (TGC)－△CT

491 492

Fig. 4. Comparison of xylitol production among different G. oxydans strains in a resting cell

493

system in shake flasks. Error bars represent standard deviation from mean for three batches.

494 495

Fig. 5. Effect of the addition of recombinant G. oxydans PZ cells on xylitol production. Error bars

496

represent standard deviation from mean for three batches.

497

23



498

Fig. 6 Comparison of xylitol production between G. oxydans PZ (open) and G. oxydans NH-10

499

(filled) in a 7.5 L bioreactor. Error bars represent standard deviation from mean for three batches.

500 501

Fig. 7. Xylitol production from D-arabitol by G. oxydans PZ in repeated biotransformation. Each

502

point represents the average value of two independent experiments.

503 504

24


Page 24 of 35

Page 25 of 35


Tables Caption:

Table 1

Bacterial strains and plasmids used in this study Source

Strain and Plasmid

or

Relevant characteristics

reference G. oxydans NH-10

Wide type strain

This lab

recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, E. coli JM109

Stratagene +

q

△(lac-proAB)/F[traD36, proab , lac1 , lacZ△M15] F–, thi-1, hsdS20 (rB–, mB–), supE44, recA13, ara-14, This lab

E. coli HB101 leuB6, proA2, lacY1, galK2, rpsL20 (strr), xyl-5, mtl-1. pBBR1MCS-5

Gmr ;Broad-host-range cloning vector

This lab

pMD18-T vector

Ampr

Takara

pRK2013

Kmr; helper plasmid for triparental mating

This lab

Gmr; a derivarive of pBBR1MCS-5, harboring tufB This study

pBBR-PtufB promoter Gmr; a derivarive of pBBR1MCS-5, harboring pBBR-PtufB-zwf

This study G6PDH gene under tufB promoter Gmr;a derivarive of pBBR1MCS-5, harboring 6PGDH

pBBR1-PtufB-gnd

This study gene under tufB promoter

Abbreviations:

Ampr,

ampicillin-resistant;

Kmr,

kanamycin-resistant;

chloramphenicol-resistant.

25


Gmr,


Page 26 of 35

Table 2 Effect of D-arabitol concentration on xylitol production by G. oxydans PZ strain Xylitol D- arabitol

Conversion time

Xylitol titer

Xylitol yield

concentration (g/L)

(h)

(g/L)

(%)

productivity (g/L/h) 20

40

14.6

73

0.37

30

48

19.5

65

0.41

40

48

26.8

67

0.56

50

54

27.5

55

0.51

60

66

25.2

42

0.38

26


Page 27 of 35


Fig.1 303x196mm (96 x 96 DPI)



Fig.2a 192x143mm (96 x 96 DPI)


Page 28 of 35

Page 29 of 35


Fig.2b 193x144mm (96 x 96 DPI)



Fig.3 265x192mm (96 x 96 DPI)


Page 30 of 35

Page 31 of 35


Fig. 4 296x210mm (300 x 300 DPI)



Fig. 5 289x202mm (150 x 150 DPI)


Page 32 of 35

Page 33 of 35


Fig. 6 296x210mm (300 x 300 DPI)



Fig. 7 289x204mm (150 x 150 DPI)


Page 34 of 35

Page 35 of 35


Graphical Abstract 259x167mm (96 x 96 DPI)


Improved Xylitol Production from d

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