Improved xylitol production from d-arabitol by enhancing the coenzyme regeneration efficiency of the pentose phosphate pathway in Gluconobacter oxydans

Article abstract of DOI:10.1021/acs.jafc.5b05509

Gluconobacter oxydans is used to produce xylitol from d-arabitol. This study aims to improve xylitol production by increasing the coenzyme regeneration efficiency of the pentose phosphate pathway in G. oxydans. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were overexpressed in G. oxydans. Real-time PCR and enzyme activity assays revealed that G6PDH/6PGDH activity and coenzyme regeneration efficiency increased in the recombinant G. oxydans strains. Approximately 29.3 g/L xylitol was obtained, with a yield of 73.2%, from 40 g/L d-arabitol in the batch biotransformation with the G. oxydans PZ strain. Moreover, the xylitol productivity (0.62 g/L/h) was 3.26-fold of the wild type strain (0.19 g/L/h). In repetitive batch biotransformation, the G. oxydans PZ cells were used for five cycles without incurring a significant loss in productivity. These results indicate that the recombinant G. oxydans PZ strain is economically feasible for xylitol production in industrial bioconversion.

Full text of DOI:10.1021/acs.jafc.5b05509

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Article
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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Improved xylitol production from D-arabitol by enhancing the coenzyme
regeneration efficiency of the pentose phosphate pathway in Gluconobacter
oxydans
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Sha Li a,b, Jinliang Zhang b, Hong Xu a,b,*, Xiaohai Feng a,b
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a
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State Key Laboratory of MaterialsꢀOriented Chemical Engineering, Nanjing
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University of Technology, Nanjing 210009, PR China
b
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College of Food Science and Light Industry, Nanjing University of Technology,
Nanjing 211816, PR China
Corresponding author
Hong Xu; Nanjing University of Technology;
Tel/Fax: +86ꢀ25ꢀ58139433;
Eꢀmail address: xuh@njtech.edu.cn (H. Xu)
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Abstract: Gluconobacter oxydans is used to produce xylitol from Dꢀarabitol. This study aims to
improve xylitol production by increasing the coenzyme regeneration efficiency of the pentose
phosphate pathway in G. oxydans. Glucoseꢀ6ꢀphosphate dehydrogenase (G6PDH) and
6ꢀphosphogluconate dehydrogenase (6PGDH) were overexpressed in G. oxydans. Realꢀtime PCR
and enzyme activity assays revealed that G6PDH/6PGDH activity and coenzyme regeneration
efficiency increased in the recombinant G. oxydans strains. Approximately 29.3 g/L xylitol was
obtained, with a yield of 73.2%, from 40 g/L Dꢀarabitol in the batch biotransformation with the G.
oxydans PZ strain. Moreover, the xylitol productivity (0.62 g/L/h) was 3.26ꢀfold of the wild type
strain (0.19 g/L/h). In repetitive batch biotransformation, the G. oxydans PZ cells were used for
five cycles without incurring a significant loss in productivity. These results indicate that the
recombinant G. oxydans PZ strain is economically feasible for xylitol production in industrial
bioconversion.
Keywords: Xylitol; Gluconobacter oxydans; Pentose phosphate pathway; Coenzyme
regeneration.
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Introduction
The genus Gluconobacter performs rapid but incomplete oxidation of a wide range of
1
2
substrates . Gluconobacter oxydans is used in several biotechnological applications , including
production of 2ꢀketoꢀLꢀgulonic acid, 5ꢀketoꢀDꢀgluconic acid, and 6ꢀaminoꢀLꢀsorbose 3,4. These
5
applications involve the production of xylitol , a pentahydroxy sugar alcohol. Xylitol exhibits
sweetness similar to sucrose and thus can be used as an alternative natural sweetener. Xylitol also
prevents dental cavities and is suitable for diabetic patients because the metabolism of this sugar is
insulin independent 6ꢀ8. Therefore, xylitol is widely used in food and chemical industries.
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Xylitol is mainly produced chemically by reducing
D
ꢀxylose . Recently, xylitol production
through fermentation has attracted much attention. Researchers have also gained much progress in
xylitol bioproduction with xylose as feedstock. Several yeast strains, including Debaryomyces
hansenii , Candida tropicalis and Candida parapsilosis, have been used for xylitol production, to
obtain high xylitol productivity (Table. S1). However, chemical and fermentation processes rely
on the hydrolysis and purification of
causing serious contamination10. A twoꢀstep process, with
been developed as an alternative technique for xylitol production because of the technological and
economic advantages of ꢀglucose compared with ꢀglucose is initially converted into
ꢀxylose 11.
ꢀarabitol by yeasts and then transformed to xylitol by G. oxydans 12ꢀ14, which contains
Dꢀxylose from hemicellulose–xylan hydrolysates, thereby
Dꢀglucose as the initial substrate, has
D
D
D
D
membraneꢀbound
D
ꢀarabitol dehydrogenase (mꢀArDH) and NADꢀdependent xylitol
ꢀarabitol is almost completely converted into ꢀxylulose by mꢀAraDH,
D
ꢀxylulose is converted into xylitol by XDH 5,15,16. Therefore, XDH is a key
dehydrogenase (XDH) 5.
D
D
but only ~25% of
restrictive enzyme that controls the overall bioconversion process probably because of its confined
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activity or coenzyme support.
Studies have been conducted to increase xylitol yield. Overexpressing the xdh gene in G.
oxydans, resulted in 11ꢀfold enhancement in XDH activity compared with that in the wildꢀtype
strain. Nevertheless, the xylitol yield was still less than 30% 17. Given that XDH is highly specific
for the cofactor NADH 18, researchers must ensure that the supply of reducing potential from
NADH is sufficient.
In other bacterial strains, NADH can be regenerated via the Embden Meyerhof pathway and
19,
the TCA cycle during the metabolism of glucose or other carbohydrates
20. However,
Gluconobacter lacks essential enzymes, including phosphofructokinase, and succinate
dehydrogenase 21. As such, the oxidative pentose phosphate pathway (PPP) is an important route
in the phosphorylative breakdown of sugars in Gluconobacter. Moreover, PPP is mainly used to
generate ATP and regenerate coenzyme in Gluconobacter 22. Previous studies examined the
activities of glucoseꢀ6ꢀphosphate dehydrogenase (G6PDH, zwf) and 6ꢀphosphogluconate
dehydrogenase (6PGDH, gnd) in the soluble fraction of G. oxydans cells, as well as in the cloned
and expressed Escherichia coli strains. The results showed that G6PDH and 6PGDH exhibit
NAD+/NADP+ dual coenzyme specificity. Thus, PPP could be the main factor responsible for
NADH regeneration in G. oxydans 23.
This study aims to increase the coenzyme regeneration efficiency of PPP for xylitol
production. The effects of the constitutive expression of G6PDH/6PGDH in G. oxydans on
coenzyme regeneration and xylitol conversion were investigated. Basing on the results, this study
established an efficient and concise route for xylitol production in G. oxydans.
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Methods
Strains, plasmids, and chemicals
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The xylitol producing strain G. oxydans NHꢀ10 was isolated from soil and deposited at the
China General Microbiological Culture Collection Center with accession number CGMCC 2709.
Escherichia coli JM109 was used as a host for plasmid construction and maintenance. E. coli
HB101 harboring helper plasmid pRK2013 was used for transformation experiments. G. oxydans
PZ or G. oxydans PG (this study) were used for zwf and gnd expression and xylitol production,
respectively. The broad host range vector pBBR1MCSꢀ5 was used for expression of zwf or gnd in
G. oxydans NHꢀ10 24. Restriction enzymes, Bacterial Genomic DNA Extraction Kit, T4 DNA
ligase, exTaq polymerase, and DNA and protein markers were obtained from TaKaRa (Dalian,
China). The strains and plasmids used in this study are listed in Table 1.
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Medium and culture conditions
All E. coli strains were cultivated at 37 oC in lysogeny broth (LB) medium (yeast extract, 5 g/L;
tryptone, 10 g/L; NaCl, 10 g/L; pH 7.0) with the addition of 10 ꢁg/mL gentamicin or 50 ꢁg/mL
kanamycin to maintain the plasmids. Recombinant G. oxydans strains were cultivated in YPG
medium (glucose, 30 g/L; sorbitol, 10 g/L; yeast extract, 20 g/L; (NH4)2 SO4, 0.5 g/L; KH2PO4 ,
1.5 g/L; MgSO4·7H2O, 0.5 g/L) containing 50 ꢁg/mL gentamicin, and 20 g/L CaCO3 to control pH.
o
G. oxydans was cultivated in 1000 mL shake flasks containing 200 mL of YPG medium at 30 C
and 200 rpm on a rotary shaker, or in a 7.5 L fermentor (New Brunswick Scientific, USA)
containing 4.0 L of YPG medium with pH and temperature automatically maintained at 6.0 and 30
oC, respectively. Agitation and aeration were controlled at 600 rpm and 1 vvm, respectively. Cell
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cultivations were initiated by inoculating 2% (v/v) of 16 h precultures of G. oxydans strains. After
24 h, the cells reached early stationary phase and were harvested by centrifugation at 8000 g and 4
oC for 10 min, washed twice with 100 mM potassium phosphate buffer (pH 6.0), and resuspended
in Dꢀarabitol solution for xylitol bioproduction.
Vector construction and transfer into G. oxydans NHꢀ10
In order to express the G6PDH/6PGDH genes in G. oxydans NHꢀ10, two plasmids containing
either zwf or gnd driven by the tufB promoter were constructed, respectively. Based on the
available sequence information for G. oxydans (Accession number CP000009), oligonucleotide
primers were designed to amplify the zwf, gnd and promoter tufB genes from genomic DNA of G.
oxydans
NHꢀ10
by
PCR.
The
primers
were
as
follows:
PtufBꢀF,
5′ꢀCGGGGTACCTATCAGGTTCCGGTTGAAG ꢀ3′ (the Kpn I site is underlined); PtufBꢀR,
5′ꢀCCGCTCGAGACCTGGAACGGGAGTAAGꢀ3′ (the Xho I site is underlined). zwfꢀF,
5′ꢀCACTCGAGGAGGTCAGAGAGTCTTꢀ3′ (the Xho
5′ꢀGAGGATCCTATCGTCGCTCAAATꢀ3′ (the BamH
I
site is underlined); zwfꢀR,
site is underlined). gndꢀF,
I
5′ꢀCATCTCGAGACAAAGCACTGGCAGꢀ3′ (the Xho I site is underlined); gndꢀR, 5′ꢀ
GTAGGATCCGACGCAGGCTCATTTꢀ3′ (the BamH I site is underlined).
The PtufB PCR product and plasmid pBBR1MCSꢀ5 were digested with restriction enzymes
Kpn I and Xho I, and ligated with T4 DNA ligase (MBI Fermentas) to generate plasmid
pBBRꢀPtufB. Then the zwf and gnd PCR products were digested and cloned into the Xho Iꢀ BamH I
restriction sites of pBBRꢀPtufB to generate the recombinant plasmids pBBRꢀPtufBꢀzwf and
pBBRꢀPtufBꢀgnd. Transformants were selected on LB agar plates containing 10 ꢁg/mL gentamicin,
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and confirmed by colony PCR and restriction enzyme digestion. The expression plasmids
pBBRꢀPtufBꢀzwf and pBBRꢀPtufBꢀgnd were transferred into G. oxydans NHꢀ10 by triparental mating
as described previously 25.
For determination of plasmid stability, recombinant strains were cultured in glycerol
medium without antibiotics for 3 days. Then the cultures were diluted and spread onto
antibioticsꢀfree agar plates. After cultivation, 100–200 colonies were randomly transferred with
sterile toothpicks from each of these plates to agar plates containing 50 ꢁg/mL gentamicin.
Plasmid stability was calculated by measuring the ratio of the numbers of colonies on the
gentamicinꢀcontaining agar plates to that on nonselective agar plates.
Quantitative realꢀtime PCR
For realꢀtime reverse transcription PCR (RTꢀPCR) experiments, 0.5 mL preculture of the G.
oxydans strains were inoculated into 250ꢀmL shake flasks containing 50 mL YPG medium and
o
grown to late exponential phase (20 h) at 30 C and 200 rpm on a rotary shaker. Then the cells
o
were harvested for total RNA isolation by centrifugation at 10,000 g and 4 C for 1 min. Total
RNA was extracted using RNAiso Plus reagent (TaKaRa, Dalian) and treated with DNase I
(TaKaRa, Dalian). Purified RNA was resuspended in 50 ꢁl of 0.1% dimethyl
pyrocarbonateꢀtreated water. RNA concentrations and purity were calculated by measuring
absorbance at 260 and 280 nm using a NanoDrop 1000 Spectrophotometer (Thermo Scientific)
and by analysis on a 1.2% (w/v) agarose gel. For transcriptional analysis of each gene, RTꢀPCR
was carried out with a firstꢀstrand cDNA synthesis kit (Promega, USA) using total RNA as
template. Gene expression analysis was performed by quantitative realꢀtime PCR performed with
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the StepOnePlus™ RealꢀTime PCR System (Applied Biosystems, USA). The primers were as
follows:
5′ꢀCGTTCTGGACCGTCTCTTTCꢀ3′. gndꢀF, 5′ꢀCGGTTACTGCATGATGTTCGꢀ3′; gndꢀR,
5′ꢀCTCGTTAAGCGTCTCCGAACꢀ3′. xdhꢀF, 5′ꢀGTGCCTTCCACGTCCTCAAꢀ3′; xdhꢀR,
zwfꢀF,
5′ꢀGAGAACCTGAACCGCTACGAꢀ3′;
zwfꢀR,
5′ꢀTTGGCGGTCCCTTCACAꢀ3′. The 16S rRNA gene was used as an internal standard, using the
primers 5′ꢀ GCGGTTGTTACAGTCAGATG ꢀ3′ and 5′ꢀ GCCTCAGCGTCAGTATCG ꢀ3′. Data
were collected using Opticon2 software. Data represent the mean of three independent
△△
Ct
experiments. Gene expression patterns were calculated using the the 2ꢀ
method 26. Relative
changes in gene expression were calculated by measuring relative abundance of PCR products in
samples, normalized to the internal standard.
Biotransformation of Dꢀarabitol into xylitol by using the recombinant G. oxydans PZ/PG resting
cell system
Initial smallꢀscale biotransformation was performed in shake flasks. Recombinant G. oxydans
cells were dispersed at a concentration of 10% (cell wet weight, w/v) in a reaction solution
containing 30 g/L
D
ꢀarabitol, and then incubated at 30 °C on a rotary shaker. The first reaction,
ꢀarabitol to ꢀxylulose, occurred under 220 rpm for 7 h. The second reaction,
ꢀxylulose to xylitol, was performed after adding 5% glucose (w/v) to the mixture
oxidation of
reduction of
D
D
D
under 50 rpm.
Scaled up production of xylitol was performed in a 7.5 L bioreactor (NBS, USA) with an
effective working volume of 4.5 L. The pH and temperature of the reactor was maintained at 6.0
and 30 °C, respectively. Agitation and aeration rates were regulated at 600 rpm and 1.0 vvm for 7
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h. The agitation rate was then adjusted to 150 rpm after adding 5% glucose (w/v) as coꢀsubstrate
for another 40 h. When the reaction reached the equilibrium, the cells were recovered by
centrifugation and resuspended in fresh reaction mixture for biotransformation. The composition
of the final media after cell collection was determined by HPLC.
Analytic methods
Enzymatic activity assay
Cellꢀfree extracts of G. oxydans NHꢀ10 and the recombinant G. oxydans strains were prepared
by ultrasonication, followed by centrifugation at 8000 rpm for 15 min at 4 °C. The supernatants
were used for the enzymatic activity assays. Dehydrogenases were assayed by a routine method
used for NAD(P)Hꢀlinked enzymes by recording the rate of increase or decrease of NAD(P)/H at
340 nm with respective substrates. The activities of G6PDH and 6PGDH were measured
spectrophotometrically at 30 °C. The reactions were initiated by adding the enzyme solution to a
final volume of 2 mL as a standard condition and monitored for 0.5ꢀ1.0 min by recording the rate
of NAD(P)H depletion at 340 nm. The reaction mixture for G6PDH activity assay contained
10 mM glycine–NaOH (pH 10.0), 1 mM NADP, and 10 mM glucoseꢀ6ꢀphosphate. Meanwhile, the
mixture for 6PGDH activity assay contained 10 mM potassium phosphate buffer (pH 6.0) 1 mM
NAD, and 50 mM 6ꢀphosphogluconate. One enzyme unit was defined as the amount of enzyme
that reduces 1 µmole of pyridine dinucleotide per minute. The protein concentration of the
cellꢀfree extract supernatant was determined via Bradford method 27. The XDH activity was
determined as described previously 16.
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NAD+ and the TTN (total turnover number) assay
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Total turnover number (TTN) was used to indicate the coenzyme regeneration efficiency in this
process. TTN is defined as the total number of moles of xylitol formed per mole of NAD+
. The
NAD+ was extracted by collecting 1 mL sample in a tube. The sample was centrifuged at 12,000
rpm for 1 min. The supernatant was removed and 300 µL of 0.2 M HCl was added to extract
the NAD+. The samples was placed in a 50 °C water bath for 10 min and then cooled on ice. The
extract was neutralized by adding 300 µL of 0.1 M NaOH dropwise. The cellular debris was
removed by centrifuging at 12,000 rpm for 5 min. Supernatant was transferred to new tube and
stored at ꢀ20 °C. A very sensitive cycling assay was used to determine the intracellular
concentration of NAD+. The assay was performed using a reagent mixture consisting of equal
volumes of 1.0 M Bicine buffer (pH 8.0), absolute ethanol, 40 mM EDTA (pH 8.0), 4.2 mM
MTT and twice as much of 16.6 mM PES (phenazine ethosulfate), previously incubated at 30 °C.
The reaction mixture including : 50 µL neutralized extract , 0.3 mL water , 0.6 mL reagent
mixture and 50 µL of yeast ADH. The absorbance at 570 nm was recorded for 10 min at 30 °C.
Standard solution of NAD+ was calibrated 28.
Quantification of D-arabitol and xylitol
Dꢀarabitol and xylitol concentrations in the mixture were determined by HPLC equipped with
a refractive index detector (RIꢀ101, Shodex, Japan). The reaction products were filtered through
0.22 ꢁm membrane filters prior to HPLC analysis. The samples (20 ꢁL) were injected onto a
Rezex RCMꢀMonosaccharide Ca2+ column（Phenomenex，USA）. The mobile phase was water
with a flow rate of 0.4 mL/min at 70 °C. Dꢀarabitol and xylitol were used as standards.
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Results
Construction and expression of recombinant vectors in G. oxydans NHꢀ10
PPP is vital for NADH production; in this study, PPP was activated by overexpressing the
G6PDH (zwf)/6PGDH (gnd) genes in G. oxydans NHꢀ10 to increase intracellular NADH
concentration. The zwf, gnd, and tufB promoter genes were amplified from G. oxydans NHꢀ10
genomic DNA by PCR reaction (Fig. 1).
Plasmids used to overexpress G6PDH/6PGDH were constructed and transformed into G.
oxydans NHꢀ10 through triꢀparental conjugation. The strain harboring the pBBRꢀPtufBꢀzwf plasmid
was named G. oxydans PZ, and the other strain harboring the pBBRꢀPtufBꢀgnd was designated as G.
oxydans PG. Successful transformation was verified by PCR and restriction enzyme digestion of
the recombinant plasmids (Fig.1). The biochemical performance of the three strains was assessed
and compared. The recombinant strains grew at approximately the same rate as the wildꢀtype
strain, indicating that the expression vectors did not affect the physiological status of G. oxydans.
Figure 2 shows the activities of G6PDH (Fig. 2a) and 6PGDH (Fig. 2b) in the strains during
fermentation. For example, the activity of G6PDH was 4.25ꢀfold higher in G. oxydans PZ (1.26 ±
0.02 U/mg) than that in G. oxydans NHꢀ10 (0.24±0.01 U/mg) but remained at the same level as
that in G. oxydans PG (0.24±0.02 U/mg). By contrast, the activity of 6PGDH was two fold higher
in G. oxydans PG (0.143±0.006 U/mg), and 29.7% more active in G. oxydans PZ (0.061±0.007
U/mg) than that in the wild type (0.047±0.005 U/mg). Nevertheless, no significant change in XDH
activity (0.05±0.01 U/mg ) was observed among the strains.
RTꢀPCR analysis of the transcription of G6PDH and 6PGDH genes in different G. oxydans strains
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Cellular mRNA was detected by realꢀtime RTꢀPCR to demonstrate the overexpression of
G6PDH and 6PGDH genes in different G. oxydans strains. Overexpression of G6PDH and
6PGDH showed no effects on the cell growth of G. oxydans. All cells used to isolate RNA were
cultured at a constant period to minimize the effect on RTꢀPCR analysis because gene expression
varied significantly in different growth phases 29. The results showed that the transcription level of
G6PDH in G. oxydans PZ was 12ꢀfold higher than that in G. oxydans NHꢀ10 (Fig. 3).
Overexpression of the zwf gene significantly promoted G6PDH gene transcription. In G. oxydans
PG, the transcript of the 6PGDH gene was approximately five times higher than that in G. oxydans
NHꢀ10. This observation could be attributed to the overexpression of the gnd gene. The
transcription levels of the XDH gene were stabilized not only in the wild type strain but also in the
recombinant stains.
Biotransformation of Dꢀarabitol to xylitol via the recombinant systems in shake flasks
Dꢀarabitol was converted into xylitol by using recombinant G. oxydans strains. Differences in
xylitol production among the three strains are depicted in Fig. 4. Xylitol production almost
reached the maximum yield after 48 h. The G. oxydans PZ strain produced 18.8 g/L xylitol, with a
yield of 62.7%, whereas the G. oxydans PG strain produced 14.6 g/L xylitol, with a yield of 48.7%.
Only 7.5 g/L xylitol was produced using the G. oxydans NHꢀ10 strain (yield of 25%). Xylitol yield
increased in both recombinant systems compared with that in the wild type. TTN, defined as the
number of moles of xylitol formed per mole of NADP+/NAD+, was (1.15 ± 0.20) × 104 mol/mol in
G. oxydans PZ and (7.70 ± 0.16) × 103 mol/mol in G. oxydans PG. Based on the TTN number,
the recombinant systems were more efficient than G. oxydans NHꢀ10 (1180 mol/mol). These
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results also indicated that the amounts of xylitol accumulated by the tested strains were consistent
with those obtained from the activity of control enzymes in the PPP measurements.
The recombinant G. oxydans PZ strain was selected as the xylitolꢀproducing strain. Several
key factors of biotransformation were optimized. The effects of substrate concentration on xylitol
production are shown in Table 2. In particular, xylitol yield decreased and conversion was
prolonged with increasing
achieved at 20 g/L ꢀarabitol. Xylitol productivity (0.56 g/L/h) was more efficient when using
40 g/L ꢀarabitol as the substrate. Based on xylitol productivity, 40 g/L ꢀarabitol was selected as
Dꢀarabitol concentration. The maximum xylitol yield (73%) was
D
D
D
the optimum concentration. The amount of added recombinant cells also influenced xylitol
production. As shown in Fig. 5, xylitol yield was improved by 10% by increasing cell
concentration from 5% to 15% (m/v). However, only a slightly higher xylitol yield was observed
when 12.5% or 15% recombinant cells were used. As such, the recombinant system was the most
efficient when the amount of added cells was 12.5% and generated a maximum xylitol yield of
70%.
Biotransformation of Dꢀarabitol into xylitol in a 7.5 L fermentor
Scaledꢀup biotransformation in pHꢀcontrolled conversion was performed to assess the
potential use of the PZ strain in industrial xylitol production. As shown in Fig. 6, Dꢀarabitol was
immediately oxidized at high rates within the initial 7 h after the cells were added to the bioreactor.
For both strains, less than 5 g/L Dꢀarabitol was retained, and only a small amount of xylitol was
produced in the reaction mixture. Dꢀxylulose was reduced to xylitol under low agitation speed and
DO concentration. After another 40 h of biotransformation, all the supplied Dꢀarabitol (40 g/L) in
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the reaction mixture was consumed, and about 29.3 g/L xylitol was obtained using the G. oxydans
PZ strain. In contrast to the wild G. oxydans NHꢀ10 strain (9.2 g/L ), G. oxydans PZ showed
improved xylitol titer upon incubation in the same reaction solution. The xylitol productivity of G.
oxydans PZ was significantly increased to 0.62 g/L/h, in contrast to 0.19 g/L/h for G. oxydans
NHꢀ10. Thus, overexpression of G6PDH in G. oxydans increased xylitol productivity by
approximately 226% compared with that in G. oxydans NHꢀ10.
Repeatedꢀbatch biotransformation of Dꢀarabitol to xylitol by G. oxydans PZ
In repetitive batch experiments with the G. oxydans PZ strain, six runs were repeated in a 7.5
L bioreactor under defined conditions. Fig. 7 shows that the cells could be recycled four times
without an evident decrease in xylitol production. Only a small decrease in the fifth cycle was
observed. However, a remarkable decrease in Dꢀxylitol yield (drop to 45%) was observed in the
sixth cycle. During the repeatedꢀbatch experiments, approximately 128 ± 5 g of xylitol was
accumulated in each round of biotransformation with a slight increase in the reaction time from
the first cycle to the fifth cycle. A total of 640 g of xylitol was formed within 245 h in the five runs
of the reaction; hence, the resulting average xylitol productivity is 0.54 g/L/h.
Discussion
Xylitol is an important compound widely used in food and chemical industries. Microbial
production from
Dꢀglucose is technically feasible and harmless to the environment, and can be
performed using a twoꢀstep process. G. oxydans can produce xylitol from
Dꢀarabitol via
Dꢀxylulose. This bioprocess is mainly affected by XDH activity and coenzyme supply. Previous
studies implied that improvements in XDH activity alone are insufficient to increase xylitol yields
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17. Mayer et al. employed purified XDH and formate dehydrogenase to catalyze xylitol production
from D
ꢀxylulose 11. When 0.5 mmol/L NAD+ was added for NADH regeneration, a yield close to
100% was achieved. However, the high cost and limited availability of pyridine cofactors hindered
the economic viability of industrialꢀscale biotransformation using an XDH in situ cofactor
regeneration step.
Introduction of dehydrogenases by adding coꢀsubstrates, such as ethanol or Dꢀglucose, is a
convenient and useful method for regenerating pyridine cofactors; this process can also promote
16
the recycling of nicotinamide coenzymes 30. For example, Zhou et al.
constructed and
transformed coꢀexpression plasmids encoding XDH and glucose dehydrogenase/alcohol
dehydrogenase into Escherichia coli Rosetta (DE3). As a result, the xylitol yield was significantly
enhanced to 92%/85.2% using 30 g/L Dꢀarabitol. Therefore, the engineered G. oxydans PXPG
strain was constructed to coꢀexpress the XDH gene and glucose dehydrogenase gene in the wild G.
oxydans strain. The xylitol yield was increased to 40.8% 25. Although xylitol yield was increased
by these coꢀexpression systems, the yield remains insufficient for largeꢀscale production of xylitol.
Considering that the overall xylitol production is limited by coenzyme deficiency, this study
was conducted to enhance coenzyme production in G. oxydans strains. PPP is an important route
in the phosphorylative breakdown of sugars, which in turn generates ATP and regenerates
coenzyme in Gluconobacter sp. 22. G6PDH and 6PGDH, which are key enzymes in oxidative PPP,
can reduce NADP+ and NAD+; therefore, NADPH and NADH can be generated in PPP 23. In the
present study, G6PDH or 6PGDH expression was enhanced to produce xylitol from Dꢀarabitol in
G. oxydans. Physiological status of the resulting recombinant strains confirmed that the expression
vector with individual genes did not significantly affect the growth of the host strain. Several
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previous reports concluded that a TTN of 103–105 may be sufficient to obtain an economically
viable process 31. In these two recombinant systems, the TTN of xylitol forming NAD+ was (1.15
± 0.20) × 104 in G. oxydans PZ and (7.70 ±0.16) × 103 in G. oxydans PG. These values were
higher than that in G. oxydans NHꢀ10, thereby suggesting that the two processes are economically
feasible. G. oxydans PZ was more efficient than G. oxydans PG not only in terms of cofactor
regeneration but also in xylitol production. This observation could be due to the higher activities
of G6PDH and 6PGDH in G. oxydans PZ than those in the wild strain. However, only 6PGDH
activity was increased by 38.3% in the G. oxydans PG strain. In addition, the transcription level of
the G6PDH gene in G. oxydans PZ was higher than that of the 6PGDH gene in G. oxydans PG.
G6PDH is the first enzyme in the PPP and specific for NADP as a coenzyme 32. 6PGDH is
the second enzyme in the PPP and specific for NADH as coenzyme, which catalyzes the oxidative
decarboxylation of 6ꢀphosphogluconate to Dꢀribuloseꢀ5ꢀphosphate. Recombinant G. oxydans
strains with enhanced PPP and high enzyme activities could possibly produce high NADH and
NADPH contents during intermediary metabolism because G6PDH produces NADPH and
6PGDH produces NADH under physiological conditions. Moreover, nicotinamide dinucleotide
transhydrogenase (NDTH，Gox310ꢀ312) can convert NADPH into NADH 22. This process may
maintain the balance of oxidation states between pyridine nucleotides. In xylitol production, this
process more efficiently occurred by enhancing the expression of the 6PGDH gene, which was
loaded in the upstream of the PPP.
The maximum xylitol yield (73.2%) was obtained by using 40 g/L Dꢀarabitol as substrate and
12.5% (w/v) recombinant G. oxydans PZ cells. This result was different from that reported by
Zhou et al., in their study, the optimal Dꢀarabitol concentration was 30 g/L and the amount of cells
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added was 10% to produce xylitol by G. oxydans NHꢀ10 16. In addition, the xylitol yield achieved
by the G. oxydans PZ strain was higher than that by the G. oxydans PXPG strain (40.8%)
constructed by Zhang et al. 25. The present study is the first investigation that attempted to
promote xylitol production by enhancing the coenzyme regeneration efficiency of PPP in G.
oxydans strain.
The stability of the free G. oxydans PZ cells was higher than that of E. coli
Rosetta/Duetꢀxdhꢀgdh cells, which can recover 40% of the initial activity after four cycles of
biotransformation during xylitol production 16. Furthermore, compared with the twoꢀstep
biotransformation from Dꢀarabitol to xylitol using G. oxydans NHꢀ10 and E. coli
Rosetta/Duetꢀxdhꢀgdh as biocatalyst, the recombinant G. oxydans PZ strain can produce xylitol
from Dꢀarabitol directly. Therefore, the later process is simpler.
In conclusion, xylitol production from Dꢀarabitol was improved by overexpressing either
G6PDH or 6GPDH gene in the wild G. oxydans strain. Increased coenzyme regeneration
efficiency of PPP was evaluated. The recombinant G. oxydans PZ strain produced xylitol at 40 g/L
Dꢀarabitol and displayed a substantially increased xylitol productivity in a resting cell system. In
repetitive batch biotransformation, the cells can be used for five cycles without a significant loss
in xylitol production. From an economic viewpoint, the results are of great interest for xylitol
production using the G. oxydans PZ cells on an industrial scale.
Acknowledgements
This work was supported by National Basic Research Program of China (2013CB733600), the
National High Technology Research and Development Program of China (2012AA021503). The
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authors thank Dr. Matthew I Pena (Rice University, USA) for readꢀthrough.
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Supporting Information available
Table. S1 Production of xylitol by microorganisms using different feedstock
This material is available free of charge via the Internet at http://pubs.acs.org.
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Figures and Tables
Figures Caption:
Fig. 1. Identification of plasmids pBBRꢀPtufB ꢀzwf/gnd extracted from the recombinant strain by
PCR and enzyme digestion. Lane M2k: DNA marker 2000bp; Lane M15k: DNA Marker 15000bp;
1: PCR PtufB; 2: PCR zwf; 3: PCR PtufB +zwf; 4: pBBRꢀPtufBꢀzwf digested with BamH I/Kpn I; 5:
pBBRꢀPtufBꢀ gnd digested with BamH I/Kpn I; 6: PCR PtufB +gnd; 7: PCR gnd; 8:PCR PtufB.
Fig. 2. G6PDH (a) and 6PGDH (b) activities of different G. oxydans stains. Error bars represent
standard deviation from mean for three batches.
Fig. 3. The transcription level analysis of zwf, gnd and xdh genes at different strains. Error bars
represent standard deviation of three samples taken from the same RNA sample.
Relative quantity (RQ) =2(－△△CT ; △△CT= [△CT (TGS)－△CT (HGS)]－[△CT (TGC)－△CT
)
(HGC)]; TGS: Target gene of sample; HGS: Houseꢀkeeping gene of sample; TGC: Target gene of
control; HGC: Houseꢀkeeping gene of control.
Fig. 4. Comparison of xylitol production among different G. oxydans strains in a resting cell
system in shake flasks. Error bars represent standard deviation from mean for three batches.
Fig. 5. Effect of the addition of recombinant G. oxydans PZ cells on xylitol production. Error bars
represent standard deviation from mean for three batches.
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504
Fig. 6 Comparison of xylitol production between G. oxydans PZ (open) and G. oxydans NHꢀ10
(filled) in a 7.5 L bioreactor. Error bars represent standard deviation from mean for three batches.
Fig. 7. Xylitol production from Dꢀarabitol by G. oxydans PZ in repeated biotransformation. Each
point represents the average value of two independent experiments.
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Tables Caption:
Table 1 Bacterial strains and plasmids used in this study
Source
or
Strain and Plasmid
G. oxydans NHꢀ10
E. coli JM109
Relevant characteristics
reference
This lab
Wide type strain
recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1,
Stratagene
This lab
△
(lacꢀproAB)/F[traD36, proab+, lac1q, lacZ
△M15]
F–, thiꢀ1, hsdS20 (rB–, mB–), supE44, recA13, araꢀ14,
leuB6, proA2, lacY1, galK2, rpsL20 (strr), xylꢀ5, mtlꢀ1.
Gmr ;Broadꢀhostꢀrange cloning vector
Ampr
E. coli HB101
pBBR1MCSꢀ5
pMD18ꢀT vector
pRK2013
This lab
Takara
Kmr; helper plasmid for triparental mating
Gmr; a derivarive of pBBR1MCSꢀ5, harboring tufB
promoter
This lab
pBBRꢀPtufB
This study
This study
This study
Gmr;
a derivarive of pBBR1MCSꢀ5, harboring
pBBRꢀPtufBꢀzwf
G6PDH gene under tufB promoter
Gmr;a derivarive of pBBR1MCSꢀ5, harboring 6PGDH
gene under tufB promoter
pBBR1ꢀPtufBꢀgnd
Abbreviations:
Ampr,
ampicillinꢀresistant;
Kmr,
kanamycinꢀresistant;
Gmr,
chloramphenicolꢀresistant.
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Table 2 Effect of Dꢀarabitol concentration on xylitol production by G. oxydans PZ strain
Xylitol
Dꢀ arabitol
Conversion time
(h)
Xylitol titer
(g/L)
Xylitol yield
(%)
productivity
(g/L/h)
0.37
concentration (g/L)
20
30
40
50
60
40
48
48
54
66
14.6
19.5
26.8
27.5
25.2
73
65
67
55
42
0.41
0.56
0.51
0.38
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Fig.1
303x196mm (96 x 96 DPI)
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Fig.2a
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Fig.2b
193x144mm (96 x 96 DPI)
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