Engineering of xylose reductase and overexpression of xylitol dehydrogenase and xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha
© Dmytruk et al; licensee BioMed Central Ltd. 2008
Received: 11 June 2008
Accepted: 23 July 2008
Published: 23 July 2008
The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45 – 48°C). Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation. However, to be economically viable, the main characteristics of xylose fermentation of H. polymorpha have to be improved.
Site-specific mutagenesis of H. polymorpha XYL1 gene encoding xylose reductase was carried out to decrease affinity of this enzyme toward NADPH. The modified version of XYL1 gene under control of the strong constitutive HpGAP promoter was overexpressed on a Δxyl1 background. This resulted in significant increase in the KM for NADPH in the mutated xylose reductase (K341 → R N343 → D), while KM for NADH remained nearly unchanged. The recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase on Δxyl1 background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48°C. A significant increase in ethanol productivity (up to 7.3 times) was shown in this recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably to 0.9 mg × (L × h)-1 as compared to 4.2 mg × (L × h)-1 for the wild type strain.
Recombinant strains of H. polymorpha engineered for improved xylose utilization are described in the present work. These strains show a significant increase in ethanol productivity with simultaneous reduction in the production of xylitol during high-temperature xylose fermentation.
Fuel ethanol produced from lignocellulosics is an environmentally friendly alternative liquid fuel to petroleum derived transportation liquid fuels. Xylose represents a significant constituent of the hemicellulose xylan fraction of lignocellulosics. Due to this, it is necessary to ferment xylose efficiently to ethanol to improve process economics for the production of this renewable liquid fuel .
Some yeasts, filamentous fungi and bacteria are able to convert xylose to ethanol. Yeasts and most other fungi first reduce xylose to xylitol using xylose reductase, EC 184.108.40.206 (XR). This enzyme has strong preference for NADPH as a co-factor. In the following step, xylitol is oxidized to xylulose with a strictly NAD-dependent xylitol dehydrogenase, EC 220.127.116.11 (XDH) . The difference in cofactor specificity in the first steps for xylose metabolism results in cofactor imbalance with reduced ethanol production and accumulation of xylitol [3–8]. Xylitol production can be reduced by metabolic engineering approaches directed to optimize the expression levels of XR and XDH [9–12], by changing the cofactor specificity of XR from NADPH to NADH [13, 14], or by modifying the redox metabolism of the host cell [15–17]. Another approach used to bypass redox imbalance during xylose fermentation relies on the expression of fungal or bacterial xylose isomerase, EC 18.104.22.168 (XI) thereby directly converting xylose to xylulose with no requirement for NADPH or NADH as cofactors [18, 19]. In the subsequent step, xylulose conversion can be further streamlined through the overexpression of xylulokinase, EC 22.214.171.124 (XK) (the third enzyme in the xylose metabolism that converts xylulose to xylulose-5-phosphate). The C5 sugar, xylulose-5-phosphate, is the entry point to the pentose phosphate pathway and into central metabolism. The overexpression of xylulokinase has been shown to enhance both aerobic and anaerobic xylose utilization in XR-XDH – as well as XI-carrying strains [5, 20]. Overexpression of XK is necessary to overcome the naturally low expression level of this enzyme [3, 5] and has been shown to result in more efficient conversion of xylose to ethanol [5, 21].
The thermotolerant methylotrophic yeast Hansenula polymorpha is capable for alcoholic fermentation of xylose at elevated temperatures (45 – 48°C) [22–24]. Such property of the yeast defines it as a good candidate for use in efficient process of simultaneous saccharification and fermentation (SSF). This process combines enzymatic hydrolysis of lignocellulosic material with subsequent fermentation of released sugars in the same vessel. Among the major advantages for this yeast using are (i) well developed methods of molecular genetics and (ii) availability of genome sequence for the type strain CBS4732 [25, 26].
In this article, we describe the construction of a recombinant H. polymorpha strain overexpressing the modified XR (K341R N343D) together with native XDH and XK on a Δxyl1 background. Xylose consumption, ethanol and xylitol production of this strain in comparison with those of strains overexpressing the native XR, XDH and XK are presented.
Materials and methods
Strains and media
Strains and plasmids used in the present investigation.
Δxyl1, pX1N-Z (GAPp-XYL1-AOXt)
Δxyl1, pX1M-Z (GAPp-XYL1mod-AOXt)
Δxyl1, pX1N-Z-X2 (GAPp-XYL1-AOXt, GAPp-XYL2-XYL2t)
Δxyl1, pX1M-Z-X2 (GAPp-XYL1mod-AOXt, GAPp-XYL2-XYL2t)
Δxyl1, pX1N-Z-X2 (GAPp-XYL1-AOXt, GAPp-XYL2-XYL2t), pGLG61/HpXYL3 (GAPp-XYL3-AOXt)
Δxyl1, pX1M-Z-X2 (GAPp-XYL1mod-AOXt, GAPp-XYL2-XYL2t), pGLG61/HpXYL3 (GAPp-XYL3-AOXt)
The E. coli DH5α strain (Φ80d lacZ ΔM15, recA 1, endA 1, gyrA 96, thi-1, hsdR 17(rK-, mK+), supE 44, relA 1, deoR, Δ(lacZYA-argF)U169) was used as a host for propagation of plasmids. Strain DH5α was grown at 37°C in LB medium as described previously . Transformed E. coli cells were maintained on a medium containing 100 mg L-1 of ampicillin.
Standard cloning techniques were used as described . Genomic DNA of H. polymorpha was isolated using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). Restriction endonucleases and DNA ligase (Fermentas, Vilnius, Lithuania) were used according to the manufacturer specifications. Plasmid isolation from E. coli was performed with the Wizard® Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA). PCR-amplification of the fragments of interest was done with Platinum® Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA) according to the manufacturer specification. PCRs were performed in GeneAmp® PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). Transformation of the yeast H. polymorpha by electroporation was carried out as described previously .
The H. polymorpha XYL2 gene with own terminator and the HpGAP promoter were amplified from the genomic DNA of CBS4732 using the corresponding pairs of primers Ko134 (GAT AAT ATA GAA ACA AAA TGA ACA ATC CTT CTG CTG)/Ko133 (ACA GGA TCC ATC CAT GAG AAA CG) and L1 (CTC GGA TCC CAA TTA TCA TTA ATA ATC)/Ko135 (CAG CAG AAG GAT TGT TCA TTT TGT TTC TAT ATT ATC). Primers L1 and Ko133 were used to obtain the fragment containing the XYL2 gene with own terminator driven with the HpGAP promoter by the overlap PCR. This fragment was cloned into the Bam HI linearized plasmids pX1N-Z i pX1M-Z, resulting in the recombinant constructs pX1N-Z-X2 and pX1M-Z-X2, respectively (Figure 1).
The expression cassette containing prGAP-XYL3-trAOX was obtained as Sac II restriction fragment from the plasmid pKO8/GAP/HpXYL3  and cloned into the Sac II linearized plasmids pGLG61 . The resulting plasmid was designated pGLG61/HpXYL3 (Figure 1). The accuracy of constructed plasmids was verified by sequencing. Constructed plasmids are presented in Table 1.
The XR activity in cell extracts was determined spectrophotometrically at 37°C. The XR assay mixture contained: Tris-HCl buffer (pH 7.0) 100 mM, NADPH 0.15 mM and xylose 350 mM. The reaction was started with cell extract addition . To evaluate KM towards NADPH or NADH, XR activities were measured with four different concentrations of cofactors 20, 50, 100 and 150 μM in triplicate.
The XDH activity in cell extracts was determined spectrophotometrically at 37°C. The XDH assay mixture contained: Tris-HCl buffer (pH 8.8) 100 mM, MgCl2 10 mM, NAD 3 mM and xylitol 300 mM. The reaction was started with cell extract addition .
The XK activity in cell extracts was determined spectrophotometrically at 37°C as described before , with some modifications. The XK assay mixture contained: Tris-HCl buffer (pH 7.8) 50 mM, MgCl2 5 mM, NADH 0.2 mM, phosphoenolpyruvate 1 mM, D-xylulose 8.5 mM, lactate dehydrogenase (EC 126.96.36.199) (Fluka, St. Louis, MO, USA) 10 U, pyruvate kinase (EC 188.8.131.52) (Fluka, St. Louis, MO, USA) 0.05 U, and ATP 2 mM. The reaction was started with addition of cell extract. For the XK assay, a blank without pyruvate kinase and lactate dehydrogenase was used to minimize interference of XDH activity in H. polymorpha.
All assay experiments were repeated at least twice.
Cells of transformants were grown in 100 ml of YPX medium (1% yeast extract, 2% peptone, 4% xylose) in Erlenmeyer flasks (bottle size – 300 ml) for 2 days and then inoculated into the 40 ml of YNB medium with 12% xylose in 100 ml Erlenmeyer flasks. Fermentation was carried out at the temperature of 48°C with limited aeration (140 revolutions × min-1). Concentrations of ethanol in medium were determined using alcohol oxidase/peroxidase-based enzymatic kit "Alcotest" . Concentrations of xylitol in medium were determined enzymaticaly as described earlier (Enzymatic determination of D-sorbitol and xylitol, R-Biopharm GmbH, Darmstadt, Germany) with slight modifications. Nitrotetrazolium Blue (NTB) 12 mM and phenazine methosulfate 15 mM were used instead iodonitrotetrazolium chloride and diaphorase, respectively. The absorbance of the reduced NTB was measured at 570 nm. Concentrations of xylose from fermentation in mineral medium were analyzed by chemical method as described before . The biomass was determined turbidimetrically in a FEK-56M photoelectric colorimeter (cuvette, 3 mm, light filter no. 6) with gravimetric calibration.
Experiments were performed at least twice.
Engineering of XR
To generate strains with overexpression of native or modified versions of XR or strains with simultaneous overexpression of native or modified XR together with XDH, the H. polymorpha Δxyl1  strain was transformed with Sac I linearized plasmids pX1N-Z and pX1M-Z or pX1N-Z-X2 and pX1M-Z-X2, respectively. The transformants were grown on YPD medium supplemented with zeocin. The presence of expression cassettes in the transformants was examined by PCR using corresponding primers. To express the XK, the recombinant plasmid pGLG61/HpXYL3 was transformed into the recipient strain H. polymorpha overexpressing native or modified versions of XR and XDH. The transformants were grown on YPD medium in the presence of increasing concentrations of G418 addition. The highest concentration of G418, which allows the transformants to grow, was 0.4 mg × ml-1. Colonies able to grow on the selective medium appeared after 3 days of incubation with frequency of up to 20 transformants × mg-1 DNA. The transformants were stabilized by cultivation in non-selective media for 10–12 generations with further shifting to the selective media with G418. The presence of recombinant XYL3 gene driven by the HpGAP promoter in genomic DNA of stable transformants was confirmed by PCR. As pGLG61-based plasmids promote multiple integration into the genome of recipient strains , constructed strains were examined by Southern hybridisation to select recombinant strains with equal amount of XK expression cassette. The strains bearing 3 copies of XK expression cassettes were selected (data not shown). Constructed yeast strains are represented in Table 1.
Biochemical analysis of constructed strains
XR, XDH, XK activities, ethanol and xylitol productivity of H. polymorpha transformants and control strain
Activity [U (mg protein-1)]
Productivity [mg (L h)-1]/[mg (L g h)-1]
0.091 ± 0.005/9 ± 0.5
0.016 ± 0.001/100 ± 5.5
0.551 ± 0.035
6.3 ± 0.3/2.75 ± 0,11
3.6 ± 0.2/1.56 ± 0.1
0.01 ± 0.001/152 ± 8.4
0.014 ± 0.001/112 ± 6.5
0.504 ± 0.028
9.8 ± 0.4/4.45 ± 0.19
3.2 ± 0.2/1.45 ± 0.07
1.3 ± 0.07
12 ± 0.5/5.7 ± 0.3
3.1 ± 0.2/1.48 ± 0.07
1.485 ± 0.087
18.4 ± 0.9/8.76 ± 0.35
1.6 ± 0.1/0.76 ± 0.04
0.375 ± 0.021
13.8 ± 0.6/6.4 ± 0.3
2.8 ± 0.1/1.3 ± 0.06
0.366 ± 0.019
54.7 ± 2.7/21.9 ± 1.1
0.9 ± 0.04/0.36 ± 0.02
0.034 ± 0.025/7 ± 0.4
0.012 ± 0.001/85 ± 4.7
0.697 ± 0.049
0.156 ± 0.009
7.5 ± 0.4/3.3 ± 0.2
4.2 ± 0.2/1.8 ± 0.09
As described earlier , natural xylose-utilizing yeasts display alcoholic fermentation only when their native XR possessed NADH-linked activity. The XR of H. polymorpha belongs to enzymes with dual cofactor specificity, however NADPH is strongly preferred (>10-fold over NADH). In the present study, we focused our efforts on engineering an XR with increased KM for NADPH. Lysine and asparagine residues were substituted for arginine and aspartic acid, respectively, at the positions 341 and 343 in the cofactor binding site using the site-specific mutagenesis, based on sequence data of XR gene of C. tenuis . The modified version of XR gene under control of the strong constitutive HpGAP promoter was overexpressed on the Δxyl1 background. This resulted in significant increase of KM for NADPH, while KM for NADH remained nearly unchanged. The results are in good agreement with reported features of modified XR from C. tenuis . The constructed XRm strain showed a slight increase in ethanol productivity as compared to the wild type strain, while the overexpression of native XR had no positive effect. Xylitol production of these strains varied insignificantly. The cloned mutated XR gene results in significant lower specific activity with NADPH and in an increase in ethanol productivity of the host strain. To enable further improvement in ethanol production, XDH was expressed together with the modified XR. Overexpression of enzymes for initial two stages of xylose utilization pathway resulted in a 2.4-fold improvement of ethanol productivity accompanied by a 2.6-fold decrease in xylitol production.
In our previous work we developed H. polymorpha strains co-overexpressing E. coli XI and own XK. These strains were characterized by significant improvement of ethanol production during xylose fermentation . In present study, the constructed strain XRm/XDH/XK overexpressing the modified XR together with XDH and XK is characterized with significant increase in ethanol productivity (up to 7.3 times) as compared to the wild type strain. It is worth mentioning that xylitol production by this strain is reduced considerably: 0.9 mg × (L × h)-1 versus 4.2 mg × (L × h)-1 by the wild type strain. Additional overexpression of XDH and XDH together with XK led to gradual increase in ethanol productivity and simultaneously decrease in xylitol production. It could be assumed that the initial stages of xylose utilization are limiting in alcoholic fermentation of xylose in H. polymorpha. The fermentation profiles of XRm/XDH/XK and XRn/XDH/XK are represented in Figure 3. The consumption of xylose by both constructed H. polymorpha strains and the wild type strain during the fermentation was rather low (Figure 3). This may suggest that xylose uptake in H. polymorpha is quite inefficient and corresponding genes coding putative xylose transporters should be cloned and overexpressed. In addition, bottlenecks downstream of XR cannot be excluded but this has to be determined. The fermentation profile revealed reutilization of synthesized ethanol. The reason of this phenomenon is not understood. We have isolated H. polymorpha 2EtOH-mutant which is characterized by significant decrease in synthesized ethanol consumption (Ishchuk et al., unpublished). The molecular nature of the corresponding mutation, however, remains unknown.
Recombinant strains of H. polymorpha constructed in this study showed significant increase in ethanol productivity during high-temperature xylose fermentation. On the other hand, ethanol production from xylose is still very low as compared to the best current xylose fermenting strains [14, 18, 35]. Therefore, further efforts have to be applied to improve the xylose alcoholic fermentation in the thermotolerant yeast H. polymorpha.
In the present work, co-overexpression of mutated form of XR (K341 → R N343 → D) together with native XDH and XK resulted in significant increase in ethanol productivity with simultaneous reduction of xylitol production during high-temperature xylose fermentation.
We thank Dr. M. Gonchar for valuable comments and O. Ishchuk for participation in the cloning. This work was partially supported by DAAD (individual grant Ref.: 332/FSC-e).
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