R E S E A R C H A R T I C L E

Overexpressionof pyruvate decarboxylase in theyeastHansenulapolymorpha results in increased ethanol yield in high-temperaturefermentationof xyloseOlena P. Ishchuk1, Andriy Y. Voronovsky1, Oleh V. Stasyk1, Galina Z. Gayda1, Mykhailo V. Gonchar1,Charles A. Abbas2 & Andriy A. Sibirny1,3

1Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine; 2Archer Daniels Midland Co J.R. Randall Research Center, Decatur, IL, USA; and 3Department of

Biotechnology and Microbiology, Rzeszow University, Rzeszow, Poland

Correspondence: Andriy A. Sibirny, Institute

of Cell Biology, NAS of Ukraine, Drahomanov

Street 14/16, Lviv, 79005, Ukraine. Tel.: 1380

32 261 2108; fax: 1380 32 261 2148; e-mail:

sibirny@cellbiol.lviv.ua

Received 4 February 2008; revised 15 July 2008;

accepted 15 July 2008.

First published online 22 August 2008.

DOI:10.1111/j.1567-1364.2008.00429.x

Editor: Patrizia Romano

Keywords

PDC1 gene; pyruvate decarboxylase; high-

temperature fermentation; xylose; fuel ethanol;

Hansenula polymorpha .

Abstract

Improvement of xylose fermentation is of great importance to the fuel ethanol

industry. The nonconventional thermotolerant yeast Hansenula polymorpha

naturally ferments xylose to ethanol at high temperatures (48–50 1C). Introduc-

tion of a mutation that impairs ethanol reutilization in H. polymorpha led to an

increase in ethanol yield from xylose. The native and heterologous (Kluyveromyces

lactis) PDC1 genes coding for pyruvate decarboxylase were expressed at high levels

in H. polymorpha under the control of the strong constitutive promoter of the

glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This resulted in

increased pyruvate decarboxylase activity and improved ethanol production from

xylose. The introduction of multiple copies of the H. polymorpha PDC1 gene

driven by the strong constitutive promoter led to a 20-fold increase in pyruvate

decarboxylase activity and up to a threefold elevation of ethanol production.

Introduction

Several factors currently drive the need for alternatives to

fossil fuels. Global warming and an increase in petroleum

prices make renewable resources, especially plant biomass, a

desirable alternative as a source for conversion into liquid

fuel, especially ethanol which is the most popular.

The major constituent of plant biomass is lignocellulose.

Upon hydrolysis, lignocellulose yields a mixture of mono-

meric hexoses (glucose, mannose and galactose) and pen-

toses (D-xylose and L-arabinose). Among these, glucose is the

most abundant, followed by xylose and mannose with other

sugars present in much lower concentrations (Jeffries & Shi,

1999). Fermentation of both glucose and xylose is essential

for economical conversion of biomass into ethanol (Aristi-

dou & Penttila, 2000). Most microorganisms are able to

ferment glucose but few have been reported to utilize xylose

efficiently and even fewer ferment this pentose to ethanol.

However, a competitive process for fuel ethanol production

from lignocellulosic material requires the development of

microorganisms capable of active xylose fermentation.

The methylotrophic yeast Hansenula polymorpha is one of

the most important industrially applied nonconventional

yeasts (Gellissen, 2000, 2002). In addition to its use as a host

for heterologous protein production, this yeast serves as a

good model organism to study the mechanisms of perox-

isomal biogenesis and degradation, regulation of methanol

metabolism, nitrate assimilation and stress response

(Navarro et al., 2003; Dunn et al., 2005; Ubiyvovk et al.,

2006; van der Klei et al., 2006). Hansenula polymorpha is able

to ferment xylose (Ryabova et al., 2003), and is one of the

most thermotolerant yeast species (Guerra et al., 2005). Thus,

it has the potential to be used in fuel ethanol production.

However, to be commercially viable, the alcoholic fermenta-

tion in H. polymorpha needs to be improved substantially.

Pyruvate decarboxylase is a key enzyme in alcoholic

fermentation. This enzyme catalyzes the conversion of

pyruvate into acetaldehyde and carbon dioxide. In Sacchar-

omyces cerevisiae, the metabolism of acetaldehyde depends

on the oxygen supply. Pyruvate is oxidized to acetyl-CoA

by the enzymes of the pyruvate dehydrogenase complex

(Pronk et al., 1996) or is reduced to ethanol by pyruvate

FEMS Yeast Res 8 (2008) 1164–1174c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

decarboxylase and alcohol dehydrogenase. In S. cerevisiae,

fermentation occurs under aerobic conditions (De Deken,

1966) and with direct competition for pyruvate between

pyruvate decarboxylase and pyruvate dehydrogenase.

Attempts to overexpress the PDC1 gene in S. cerevisiae do

not result in higher ethanol yield from glucose (Schaaff

et al., 1989; van Hoek et al., 1998).

The methylotrophic yeast, H. polymorpha, belongs to the

respiratory Crabtree-negative yeast species (Verduyn et al.,

1992). Maximum production of ethanol in H. polymorpha

occurs under oxygen-limited conditions (Ryabova et al.,

2003; Voronovsky et al., 2005). Under these conditions,

increasing pyruvate decarboxylase activity may be impor-

tant for pyruvate distribution between pyruvate dehydro-

genase complex and the pyruvate decarboxylase. To

test this hypothesis, we decided to clone and overexpress

in H. polymorpha the structural gene for pyruvate decar-

boxylase, PDC1, and to study the effect of its overexpress-

ion on ethanol production. The H. polymorpha ORF of

the PDC1 gene was cloned under the control of the

H. polymorpha strong constitutive promoter of glyceralde-

hyde-3-phosphate dehydrogenase gene (GAPDH) and

introduced into the yeast. The PDC1 gene overexpression

resulted in increased pyruvate decarboxylase activity and

elevated ethanol production during high-temperature

xylose fermentation.

Materials and methods

Strains and growth conditions

The yeast strains used in this study are listed in Table 1. Yeast

strains H. polymorpha NCYC495 leu1-1 and 2EthOH� were

used as recipient strains for PDC1 gene overexpression.

2EthOH� is a UV-induced mutant derived from the parental

strain NCYC495 leu1-1, which is unable to utilize ethanol as

a carbon source and exhibits improved ethanolic fermenta-

tion of xylose (see the main text).

Both NCYC495 leu1-1 and 2EthOH�were maintained on

minimal medium containing 0.67% YNB (Difco, Detroit,

MI) supplemented with 2% sucrose and leucine at 40 mg L�1

at 37 1C. The 3Leu1 strain was used as a control strain for

NCYC495 leu1-1 transformants carrying the PDC1 gene

expression cassettes.

The H. polymorpha CBS4732s strain (Lahtchev et al.,

2002) was used as a source of PDC1 gene. It was kindly

provided by Dr Lahtchev (Institute of Microbiology, Bulgar-

ian Academy of Sciences, Sofia, Bulgaria). The strain was

maintained on YPD medium (0.5% yeast extract, 1%

peptone and 2% glucose) at 37 1C.

The Kluyveromyces lactis strain CBS 2359 was used as

a source of the PDC1 gene. It was maintained on YPD

at 30 1C.

Yeast transformants were selected either on YNB medium

with 2% sucrose or on YPS medium (0.5% yeast extract, 1%

peptone and 2% sucrose) supplemented with geneticin at

1 g L�1 or zeocin at 140 mg L�1.

The Escherichia coli strain DH5a [F80dlacZDM15, recA1,

endA1, gyrA96, thi-1, hsdR17 (rK�, mK

1), supE44, relA1, deoR,

D(lacZYA-argF) U169] was used in experiments that re-

quired a bacterial host. The bacterial strain was grown at

37 1C in a rich [Luria–Bertani (LB)] medium as described in

Sambrook et al. (1989). Transformed E. coli cells were

maintained on a medium containing 100 mg L�1 of ampi-

cillin or erythromycin.

Molecular biology techniques

Plasmid DNA isolations from E. coli were carried out using

the NucleoSpins Plasmid QuickPure (Macherey-Nagel,

Germany). Taq DNA polymerase and VentRs DNA polymer-

ase (both New England Biolabs) were used for analytical and

preparative PCR, respectively. T4 DNA ligase, T4 DNA

polymerase and restriction enzymes were purchased from

Fermentas, Lithuania.

Preparations of genomic DNA from yeast species were

carried out using the DNeasys Tissue Kit (Qiagen, Germany).

Transformation of H. polymorpha was performed using

electroporation as described previously (Faber et al., 1994).

Southern blotting analysis was performed using the

Amersham ECL Direct Nucleic Acid Labeling and Detection

System (GE Healthcare).

Cloning of the PDC1 gene of H. polymorpha

The complete sequence of H. polymorpha ORF of the PDC1

gene is not available in current databases. Only the 949-bp

Table 1. Yeast strains used in this study

Strain Description References

H. polymorpha

NCYC495 leu1-1 leu2 Gleeson & Sudbery

(1988)

2EthOH� leu2 This study

CBS4732s leu2 Lahtchev et al.

(2002)

3Leu1 NCYC495 leu1-1

derivative, leucine

prototroph, the strain was

obtained by

transformation of

NCYC495 leu1-1 with the

plasmid pKO8-GAPpr

(Voronovsky et al., 2005)

This study

K. lactis CBS 2359 Wild type Wesolowski-Louvel

et al. (1996), Kiers

et al. (1998)

FEMS Yeast Res 8 (2008) 1164–1174 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

1165PDC1 overexpression in Hansenula polymorpha

internal part of the gene is present in the genome database

‘Genolevures’ for Pichia angusta/H. polymorpha (http://

cbi.labri.fr/Genolevures/index.php, NCBI accession number

AL433358). Therefore, we cloned the entire ORF. For this

purpose the inverse PCR approach was used. The primer

pairs were designed to amplify the regions flanking the 949-

bp sequence of PDC1 ORF: K1 and K2; K3 and K4 (Table 2).

A range of restriction endonucleases was used to choose the

appropriate ones, which are located not far from the PDC1

ORF and present on the 949-bp sequence. Genomic DNA of

H. polymorpha CBS4732s strain was digested with each of

these restriction endonucleases and self-ligated. Recovered

DNA fragments were used as templates for PCR with the

inverse primers: K1/K2 and K3/K4 (Table 2). The c. 3.9-kb

fragment was obtained using the inverse PCR (primers K3/

K4) where the sample of H. polymorpha genomic DNA

digested with SalI was used as a template. The c. 3.4-kb

fragment was obtained using the inverse PCR (primers K1/

K2) where the sample of H. polymorpha genomic DNA

digested with SacI was used as a template. The obtained

PCR fragments were cloned into the multiple cloning site of

the plasmid pUC19 and sequenced. Using nucleotide BLAST

with available yeast sequences (http://www.ncbi.nlm.nih.

gov/blast/Blast.cgi), the entire H. polymorpha ORF of the

PDC1 gene was detected.

Construction of plasmids

The recombinant plasmid pKO81prGAP1PDC1Hp (Fig.

1a) was constructed on the basis of plasmid pKO8-GAPpr

(Voronovsky et al., 2005). The genomic DNA of H. poly-

morpha strain CBS4732s served as a template to isolate the

ORF of the PDC1 gene with primers K10 and K11 (Table 2).

The PCR fragment was treated using restriction endonu-

cleases NdeI and NotI. Restriction sites of the endonucleases

flank the PCR fragment (underlined, Table 2). The resulting

fragment was cloned into the NdeI/NotI-linearized plasmid

pKO8-GAPpr.

The plasmid pGLG611prGAP1PDC1Hp (Fig. 1b) was

constructed on the basis of vector pGLG61 (Sohn et al.,

1999). The vector was kindly provided by Dr Kang HA

(Korea Research Institute of Bioscience and Biotechnology,

Taejon, Korea). The plasmid pKO81prGAP1PDC1Hp (Fig.

1a) served as a template to isolate the fragment carrying

promoter GAPDH-ORF PDC1-terminator AOX with pri-

mers IS5 and IS6 (Table 2). Sites of NarI restriction

endonuclease were incorporated into the primers (under-

lined, Table 2). The PCR fragment was cut using NarI and

cloned into the plasmid pGLG61.

The plasmid ploxZeoloxPDC1Hp (Fig. 1c) was con-

structed on the basis of pGLG611prGAP1PDC1Hp (Fig.

1b). pGLG611prGAP1PDC1Hp was cut using PstI. A 7.56-

kb fragment was ligated with a 1.1-kb fragment containing

zeocin resistance gene Zeor amplified from the plasmid

pPICZ-B (Invitrogen, Carlsbad, CA) with primers Ko58

and Ko59 (Table 2) and treated using PstI.

The plasmid p19L21prGAP1PDC1Kl (Fig. 1d) was

constructed on the basis of p19L2 (Voronovsky et al.,

2002). The expression cassette containing H. polymorpha

GAPDH promoter and the AOX terminator was isolated

from the pKO8-GAPpr (Voronovsky et al., 2005) with

restriction enzymes BamHI and SacI and ligated with

BamHI/SacI-digested p19L2. The resulting plasmid was cut

with HindIII and ligated with the HindIII-digested PCR

fragment carrying K. lactis ORF of the PDC1 gene [amplified

from the genomic DNA of K. lactis CBS2359 using primer

pair: IS3 and IS4 (Table 2)].

Ethanol production assay

Ethanol fermentation was carried out in 100-mL flasks

containing 40 mL YNB medium with 12% or 8% xylose or

12% glucose. Yeast cells were inoculated in the media to a

final density of 2 mg mL�1 and cultivated at 37 or 48 1C

with restricted aeration (140 r.p.m.) for 5 days. Samples

of medium for ethanol production assay were taken each

day. The concentration of ethanol in the medium was

determined using the ‘Alcotest’ kit (Gonchar et al.,

2001). Fermentation experiments were performed at least

twice.

Table 2. Oligonucleotides used in this work

Name Sequences

ACT1F 50-TGTCGTCCCAGTTGGTAACG-30

ACT1R 50-GGCCCAATCCAAGAGAGGTAT-30

IS3 50-GCGAAGCTTATGTCTGAAATTACATTAGG-30

IS4 50-CATAAGCCTTTAGTTCTTAGCGTTGGTAG-30

IS5 50-GCGGGCGCCCCAATTATCATTAATAATCACTC-30

IS6 50-TAAGGCGCCAGCATCTTGACAATCAGCAG-3 0

IS271 50-TGGTCTTGCGGCTGCTCTGTTCACC-3 0

IS272 50-GTAAAGATCAAGGGCGTAGGTGCCCAG-3 0

IS273 50-GTCTTCTCCAAGGATTTCCATAGAGCACATC-3 0

IS274 50-GCCAATGTTCAAGTAGATGCTCTTTGACTG-3 0

IS275 50-CTACGTCTCCGACAGACTCGAGGC-30

IS276 50-ACAGCCTTGACCTGGGTGTAGCTCTC-30

IS277 50-GACACCGCCACCTACGTCTCCAAC-30

IS278 50-ACCAATTCTCACAGCCTTCCACTGGGTG-3 0

IS279 50-GCCTACCTGTTCACTCAAGACATCAATCGG-30

IS280 50-GCTGAATGCTGCCAAGCCGGCTTC-30

K1 50-TGGTCCTCGCTGAAGGCCGACTTGC-3 0

K2 50-GCGGTGTGTACATCGGAGTTCTGTCG-30

K3 50-AGTCGCCGACACCAAAGGTGGTCAC-3 0

K4 50-GCCATTGCGGGCATGATGGCCGAG-3 0

K10 50-CGCCATATGTCTGAATCCCAACTACC-30

K11 50-TTTGCGGCCGCTTAAGCTGCATTGATCTGC-30

Ko58 50-CGGGGTACCTGCAGATAACTTCGTATAGCATAC-30

Ko59 50-CGGGGTACCTGCAGTAATTCGCTTCGGATAAC-30

FEMS Yeast Res 8 (2008) 1164–1174c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

1166 O.P. Ishchuk et al.

Preparation of cell-free extracts

Cell samples of the third day of fermentation were harvested

using low-speed centrifugation (1699 g) and washed with

100 mM potassium phosphate buffer pH 7.5 containing

2 mM MgCl2 and 1 mM dithiotreitol. Cells were resus-

pended in the washing buffer and extracts were prepared

using glass beads with vigorous vortexing at 14 1C. Un-

broken cells and debris were removed using centrifugation

at 20 817 g. The supernatant was used as a cell-free extract

for enzyme assays.

Enzyme assays

The pyruvate decarboxylase and alcohol dehydrogenase

activities were measured according to the methods described

earlier (Postma et al., 1989).

Samples for the enzyme activity measurements were

taken from the cultures on the third day of fermentation.

The enzyme activity was measured directly after the pre-

paration of cell-free extracts. Experiments were performed

at least twice.

Native polyacrylamide gel electrophoresis(PAGE)

Cell-free extracts isolated from xylose-grown cells of

NCYC495 (wild type) and of the 2EthOH� mutant were

used for native protein PAGE. To visualize enzyme bands

in native PAGE, a modified mixture was used: 10 mM

NAD, 0.1 mM nitrotetrazolium blue, 0.003 mM phenazine

methosulfate in 50 mM K, Na-phosphate buffer, pH 7.5,

with ethanol (up to 500 mM) for an alcohol dehydro-

genase assay, and benzylaldehyde (up to 10 mM) with

addition KCl (up to 100 mM) for an unspecific aldehyde

dehydrogenase assay (Maidan et al., 1997; Wang et al.,

1998).

RIKSc BgNdB(a)

(b)

(c)

(d)

Nt H

pKO8+prGAP+PDC1Hp c. 8.6 kb

LEU2 ScErtrAOX_HpORI prGAP_Hp

ORF PDC1_Hp

H Nt

prGAP_HptrAOX_Hp

PXb

bla APH TEL188

RI

ORIORF PDC1_Hp

Nr NrNd

prGAPHp

pGLG61+prGAP+PDC1Hp c. 9.4 kb

PNr Nr

H Nt

Nd P

prGAP_HptrAOX_Hp

P

bla TEL188 ORIORF PDC1_Hp

P

ZeoploxZeoloxPDC1Hp c. 8.66 kb

RIKSc RI

trAOX_Hp

HH

ORIlacZ

Sl Xb BSp P

prom.lacZ bla

LEU2 Sc

prGAP_ HpORF PDC1_Kl

p19L2+prGAP+PDC1Kl c. 8.52 kb

Fig. 1. Linear schemes of the plasmids pKO81prGAP1PDC1Hp, pGLG611prGAP1PDC1Hp, ploxZeoloxPDC1Hp and p19L21prGAP1PDC1Kl. The

Hansenula polymorpha PDC1 ORF is shown as a brick box, the Kluyveromyces lactis PDC1 ORF is shown as a checked box, the promoter of

glyceraldehydes-3-phosphate dehydrogenase (GAPDH) of H. polymorpha – box with vertical hatches, the terminator of alcohol oxidase of H.

polymorpha – box with slanting hatches, the LEU2 gene of Saccharomyces cerevisiae – gray box, the geneticin resistance gene, APH – white box with

black spots, the telomeric region (TEL188) (Sohn et al., 1999) as an autonomously replicating sequence – black box, the zeocin resistance gene, Zeor –

gray box with white spots, loxP sequences – boxes with wavy lines. Restriction sites: B, BamHI; H, HindIII; Sc, SacI; Bg, BglII; K, KpnI; RI, EcoRI; Xb, XbaI; P,

PstI; SalI, Sl; SphI, Sp; NdeI, Nd; NotI, Nt; NarI, Nr.

FEMS Yeast Res 8 (2008) 1164–1174 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

1167PDC1 overexpression in Hansenula polymorpha

Protein determination

Protein was determined using the Lowry method (Lowry

et al., 1951) with bovine serum albumin as a standard.

Reverse transcription (RT)-PCR analysis

Total RNA was extracted from yeast cells using the Trizol

method (Invitrogen) following the manufacturer’s protocol.

RNA was quantified using UV spectrophotometry and diluted

in RNAse-free water. Single-stranded cDNA was synthesized

using MuLV reverse transcriptase (First Strand cDNA Synthesis

Kit, Fermentas, Vilnius, Lithuania). Quantitative RT-PCR

analysis was carried out using gene-specific primer pairs and

cDNA as a template. The following primer pairs were used

(Table 2): IS271 and IS272 for the 30 fragment of H. poly-

morpha ORF116 (Hp_contig12); IS273 and IS274 for the 30

fragment of H. polymorpha ORF168 (Hp_contig15); IS275 and

IS276 for the 30 fragment of H. polymorpha ORF226 (Hp_con-

tig01); IS277 and IS278 for the 30 fragment of H. polymorpha

ORF313 (Hp_contig08); IS279 and IS280 for the 30 fragment

of H. polymorpha ORF529 (Hp_contig47); and ACT1F and

ACT1R for the 30 fragment of H. polymorpha ORF of the ACT1

gene (orf262, Hp_contig01). Sequences of the ORFs men-

tioned above were taken from the H. polymorpha genome

database (Rhein Biotech GmbH, Dusseldorf, Germany).

Results and discussion

High-temperature xylose and glucosefermentation in H. polymorpha

Hansenula polymorpha achieves optimal growth rates at tem-

peratures around 38–45 1C and yet it grows at up to 47 1C with

no thermal death and without any decrease in biomass yield

(Cabeca-Silva & Madiera-Lopes, 1984; van Uden, 1984). A

more recent study showed that H. polymorpha is one of the

most thermotolerant yeast species, with a maximum tolerated

temperature of 50 1C (Guerra et al., 2005). These observations

make this species a promising organism for the biofuel

industry as the ability to grow at high temperatures could

considerably reduce ethanol production costs by improving

fermentation of lignocellulosic material simultaneously with

enzymatic hydrolysis, thereby significantly reducing the need

to cool fermentors [Simultaneous Saccharification and Fer-

mentation (SSF) process] (Banat et al., 1998; McMillan et al.,

1999; Hari Krishna et al., 2001).

In the current study, the glucose and xylose fermentation

profiles of H. polymorpha NCYC495 leu1-1 were compared at

the optimal growth temperature of 37 1C and at a higher

temperature of 48 1C. The latter temperature was used

because according to the published data (Guerra et al.,

2005), temperatures higher than 48 1C induce heat shock in

this yeast. Ethanol accumulation profiles were similar for

glucose and xylose at both 37 and 48 1C; however, at the high

temperature, ethanol accumulated in the first 2 days and

disappeared during further incubation (Fig. 2). The amount

of ethanol lost at 48 1C totaled up to 40% of the synthesized

product (A.A. Sibirny & B.V. Kshanovska, unpublished

data). The loss could have been due to evaporation but could

have also been due to reutilization of the accumulated

ethanol. To test whether the loss was due to reutilization of

the accumulated ethanol, a mutant of H. polymorpha unable

to utilize ethanol as the sole carbon and energy source was

isolated. For this, the parental strain NCYC495 leu1-1 was

UV mutagenized as described in Johnson et al. (1999), and

the resulting glucose-growing colonies were replica plated on

YNB medium supplemented with 1% (v/v) ethanol. Several

clones were identified from c. 10 000 tested that were unable

to grow on 1% ethanol as the sole carbon source. However,

most of them still reutilized accumulated ethanol during

xylose fermentation, although to a lesser extent compared

with the parental strain NCYC 495 leu1-1 (data not shown).

One of the isolated mutants designated as 2EthOH�

utilized the least amount of accumulated ethanol during

xylose fermentation (Fig. 2) and, therefore, was studied in

more detail. The mutant exhibited a wild-type growth rate

on media supplemented with glucose, sucrose, glycerol or

methanol. The 2EthOH� strain was further tested for the

ability to utilize ethanol catabolites, acetate and succinate, as

carbon sources, and exhibited a wild-type growth rate on

both of these substrates (data not shown). Therefore, it was

assumed that this mutant probably has a metabolic block in

one of the two enzymatic stages of ethanol conversion to

acetate: either alcohol dehydrogenase, acetaldehyde dehy-

drogenase, or both these activities.

To determine possible enzymatic defects leading to the

inability of the mutant 2EthOH� to utilize ethanol and allow

utilization of acetate, activities of alcohol and aldehyde

dehydrogenases were analyzed in cell-free extracts of strains

NCYC495 (wild type) and 2EthOH�. Cells were cultivated in

a xylose-containing medium under conditions used for

alcoholic fermentation (see Materials and methods) for 3

days. Extracts were loaded on PAGE and were used for native

electrophoresis. Both the 2EthOH� mutant and the wild-

type strains have five alcohol dehydrogenase bands of

identical molecular mass (Fig. 3). However, the 2EthOH�

(line 2, Fig. 3) has an additional highly intensive band that is

absent in the wild-type strain. In addition, the intensity of

three bands of the alcohol dehydrogenases differed between

the 2EthOH� and the wild-type strains. Aldehyde dehydro-

genase activity was substantially lower in 2EthOH�mutant

the extract relative to that of the wild-type strain (Fig. 4). It

is interesting to note that one band of aldehyde dehydro-

genase was totally absent in mutant 2EthOH� extracts (Fig.

4). In S. cerevisiae, acetate is mainly produced by the

cytosolic Ald6p and by a mitochondrial route involving

FEMS Yeast Res 8 (2008) 1164–1174c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

1168 O.P. Ishchuk et al.

Ald5p (Saint-Prix et al., 2004). The H. polymorpha BLAST

search against S. cerevisiae ALD6/ALD5 protein sequences

revealed five ORF sequences (ORF 116, 168, 226, 313 and

529) showing 61–74% homology to the query gene. The

comparison of mRNA quantity of the five aldehyde dehy-

drogenase homologs of H. polymorpha using RT-PCR

showed that the 2EthOH� strain had decreased expression

of four aldehyde dehydrogenase genes (ORF 116, 226, 313

and 529) compared with the control 3Leu1 strain (Fig. 5).

Although the underlying molecular basis of the 2EthOH�

mutation is yet to be determined, we suggest that it is

impaired in a gene involved in the regulation of enzymes of

primary ethanol metabolism along with some other en-

zymes involved in xylose and glucose fermentation.

The 2EthOH� mutant has a significantly reduced ability

to consume accumulated ethanol as the sole carbon source

(Fig. 2). The 2EthOH� strain exhibited a higher level of

ethanol accumulation from xylose than that of NCYC495

leu1-1 during fermentation at 37/48 1C. It yielded approxi-

mately a threefold higher ethanol concentration on the third

day of fermentation (Fig. 2a and b). At the same time, the

mutant 2EthOH� grew and fermented glucose more slowly

relative to the wild-type strain NCYC 495 leu1-1 (Fig. 2c and

d). The reasons for the observed phenomena are unknown.

Apparently, mutation in the 2EthOH� strain oppositely

affects glucose and xylose fluxes to ethanol.

Overexpression of the PDC1 gene in the H.polymorpha wild-type strain and the mutant2EthOH�

One of the key aims of our study was to determine the effect

of pyruvate decarboxylase overexpression, a key enzyme in

3

2.5

2

1.5

1

0.5

01 2 3 4 5

Days

Eth

anol

(g

L–1)

37 °C

1

2

3

2.5

2

1.5

1

0.5

01 2 3 4 5

Days

Eth

anol

(g

L–1)

48 °C

1

2

(a) (b)

60

50

40

30

20

10

01 2 3 4 5

Days

Eth

anol

(g

L–1)

37 °C

1

2

(c) 60

50

40

30

20

10

01 2 3 4 5

Days

Eth

anol

(g

L–1)

48 °C

1

2

(d)

Fig. 2. Ethanol production of Hansenula

polymorpha strains during fermentation.

(a) Xylose fermentation at 37 1C (YNB112%

xylose), (b) xylose fermentation at 48 1C

(YNB112% xylose), (c) glucose fermentation at

37 1C (YNB112% glucose) and (d) glucose fer-

mentation at 48 1C (YNB112% glucose). Strains:

1, NCYC495 leu1-1, 2, 2EthOH�.

1 2

Fig. 3. Alcohol dehydrogenase activity of Hansenula polymorpha strains

visualized on native protein PAGE. Protein samples were taken from cell-

free extracts of cells on the third day of xylose fermentation at 48 1C. Lane

1, NCYC495; lane 2, 2EthOH�. Protein (0.1 mg) was loaded to each lane.

Total ADH activities were 0.035 U in NCYC495 and 0.2 U in 2EthOH�.

FEMS Yeast Res 8 (2008) 1164–1174 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

1169PDC1 overexpression in Hansenula polymorpha

alcoholic fermentation, on xylose fermentation of H. poly-

morpha. The NCYC495 leu1-1 was used as a recipient strain.

The plasmid pKO81prGAP1PDC1Hp (Fig. 1a) was linear-

ized and transformed into the strain. Transformants were

selected by leucine prototrophy on the YNB medium with

2% sucrose as the sole carbon source. The transformants

were stabilized by alternative cultivation in nonselective

(YPD) and selective (YNB with 2% sucrose) media. Trans-

formants that remained prototrophs after this cultivation

(the stable ones) were checked for the presence of the

desirable constructs in their genome (GAPDH promoter

fused to PDC1 ORF with the AOX terminator) using PCR

(data not shown). The ethanol production from glucose,

D-xylose and L-arabinose and pyruvate decarboxylase activ-

ities were studied in the transformants in comparison with

the control 3Leu1 strain that was obtained after transfor-

mation by the empty vector pKO8-GAPpr (Voronovsky

et al., 2005). The overexpression of the H. polymorpha

PDC1 gene under control of the H. polymorpha GAPDH

promoter in all transformants resulted in an increased

pyruvate decarboxylase activity and showed a positive effect

on alcoholic fermentation of both glucose and xylose. In one

of the transformants, PDC1Hp-4, the pyruvate decarbox-

ylase activity was 41-fold higher relative to that of the

parental strain (Fig. 6b), and this increase was accompanied

by a 2.3-fold higher ethanol yield from xylose (Fig. 6a). On

the medium with L-arabinose, transformants were charac-

terized by better growth; however, no ethanol was accumu-

lated on this pentose, similar to the parental strain (Table 3).

Because the expression of PDC1 in NCYC495 leu1-1 was

successful, it was decided to use the same approach for

the 2EthOH� strain, a better ethanol producer from xylose

(Fig. 2). In the case of 2EthOH� transformation, the

plasmids promoting multicopy integration were used:

pGLG611prGAP1PDC1Hp and ploxZeoloxPDC1Hp (Fig.

1b and c). The plasmid pGLG611prGAP1PDC1Hp is a

derivative of pGLG61, and due to the presence of the

telomeric autonomous replication sequence and the bacter-

ial aminoglycoside-3-phosphotransferase (APH, genetecin

resistance) gene, this vector promotes copy-number-con-

trolled integration of plasmid tandem repeats into the

genome (Sohn et al., 1999). The vector ploxZeoloxPDC1Hp

contains the Zeor gene (conferring zeocin resistance) flanked

by loxP sequences. The sequences provide the efficient

excision of the marker gene after integration (loxP/Cre),

and, as a result, the possibility for repeated transformation

exists with the same marker after its rescue (Gueldener et al.,

1996, 2002; Steensma & Ter Linde, 2001).

The 2EthOH� strain was transformed with the plasmid

pGLG611prGAP1PDC1Hp and corresponding transfor-

mants were selected on YPS medium supplemented with

1 g L�1 geneticin (G418). 2EthOH� transformants with the

plasmid ploxZeoloxPDC1Hp were selected on YPS medium

supplemented with 140 mg L�1 zeocin. The stability of the

corresponding transformants was checked by alternative

1 2

Fig. 4. Aldehyde dehydrogenase activity of Hansenula polymorpha

strains visualized on native protein PAGE. Protein samples were extracted

with 0.1% Triton from disrupted cells debris of cells on the third day of

xylose fermentation at 48 1C. Lane 1, NCYC495; lane 2, 2EthOH�.

Protein (0.1 mg) was loaded to each lane.

1 2

ACT1

ORF 116

ORF 168

ORF 226

ORF 313

ORF 529

Fig. 5. RT-PCR of Hansenula polymorpha aldehyde dehydrogenase

genes. RT-PCR reaction on cDNA of H. polymorpha strains, 1 – 3Leu1,

control strain, Leu1 transformant; 2 – 2EthOH�. Primers were used for

ORF 116, 168, 226, 313, 529 selected on the basis of BLAST results against

ALD6 Saccharomyces cerevisiae. Primers for actin (ACT1) were used as a

control.

FEMS Yeast Res 8 (2008) 1164–1174c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

1170 O.P. Ishchuk et al.

cultivation in nonselective (YPS) and selective media (YPS

with geneticin/zeocin). The presence of desirable recombi-

nant constructs (GAPDH promoter fused to PDC1 ORF

with the AOX terminator) in the genome of stable transfor-

mants was confirmed by PCR (data not shown).

2EthOH� transformants carrying the PDC1-expression

cassette were shown to have improved fermentation of

xylose as compared with the recipient strain. In these

transformants ethanol synthesis and ethanol productivity

during xylose fermentation at 48 1C were c. 2.3- and 3.0-fold

higher, respectively (Figs 7 and 8). Pyruvate decarboxylase

activity was substantially higher: a 14-fold increase for

2EthOH�/pGLG611PDC1Hp-12 transformant, a 20-fold

increase for 2EthOH�/pGLG611PDC1Hp-13 and almost a

19-fold increase for 2EthOH�/ploxZeoloxPDC1Hp-10 (Fig.

9). Transformants were also characterized by an increase in

pyruvate decarboxylase activity during cultivation in glu-

cose medium and accumulated elevated amounts of ethanol

in glucose medium relative to the 2EthOH� transformant

with an empty vector (although the accumulation was lower

than that in the wild-type transformant) (Table 3, Fig. 10).

In the medium with L-arabinose, transformants with ele-

vated pyruvate decarboxylase were characterized by better

growth; however, no ethanol was accumulated (Table 3).

The pyruvate decarboxylase activity varied in 2EthOH�

transformants. This could have been due to the different

plasmid copy numbers in their genomes. The Southern

blotting experiment showed that selected transformants

(a2–a4) (Fig. 11) with improved fermentation of xylose had

different copy numbers of PDC1 expression cassettes. Trans-

formants 2EthOH�/pGLG611PDC1Hp-12 and 2EthOH�/

pGLG611PDC1Hp-13 contained approximately seven to

nine copies, whereas the transformant 2EthOH�/ploxZeo-

loxPDC1Hp-10 had just five copies of the cassette (the copy

numbers were compared with the intensity of signal of the

genomic DNA of the recipient strain (a1) that carries just the

one copy of PDC1) (Fig. 11).

Overexpression of the PDC1 gene of K. lactis inthe H. polymorpha wild-type strain

We decided to determine whether the H. polymorpha

fermentation parameters could be improved by overexpres-

sion of heterologous pyruvate decarboxylase. For this

purpose, we cloned the ORF of the K. lactis PDC1 gene

into the expression cassette for H. polymorpha and intro-

duced the resulting construct into an H. polymorpha wild-

type strain. The plasmid p19L21prGAP1PDC1Kl (Fig. 1d)

1.2

1

0.8

0.6

0.4

0.2

03Leu+ PDC1Hp-4

Ethanol (g L–1) PDC1 activity (U mg–1)

20

(b)(a)

15

10

5

03Leu+ PDC1Hp-4

Fig. 6. Ethanol production (a) and specific activ-

ity of Pdc1 (b) of Hansenula polymorpha

NCYC495 transformants. 3Leu1 – control strain,

Leu1 transformant; PDC1Hp-4 – the transfor-

mant NCYC495 leu1-1/pKO81prGAP1PDC1Hp.

The samples for ethanol and pyruvate decarbox-

ylase assays were taken from the third day of

fermentation of xylose (YNB18% xylose,

37 1C and 140 r.p.m.).

Table 3. Fermentation profiles of Hansenula polymorpha strains at 48 1C under restricted aeration (140 r.p.m.) in the YNB media supplied with

different carbon sources (12% arabinose, 12% xylose, 12% glucose)

Strains

L-Arabinose D-Xylose D-Glucose

OD

(l600 nm)

Ethanol

(g L�1)

Pdc1 activity

(U mg�1)

OD

(l600 nm)

Ethanol

(g L�1)

Pdc1 activity

(U mg�1)

OD

(l600 nm)

Ethanol

(g L�1)

Pdc1 activity

(U mg�1)

3Leu1 10.5 0.0 0.4� 0.06 8.24 0.7� 0.05 0.1� 0.02 12.4 14.4�0.9 0.21� 0.03

PDC1Hp-4 11.2 0.0 9.2� 0.5 11.0 1.2� 0.1 4.1� 0.2 13.0 27.9�1.3 3.2� 0.15

2EthOH� 10.68 0.0 0.07� 0.01 11.1 0.9� 0.2 0.21�0.06 11.6 7.8�0.4 0.26� 0.04

2EthOH�/ploxZeoloxPDC1Hp-10 11.12 0.0 0.88� 0.04 14.1 1.6� 0.1 2.2� 0.2 16.2 10.8�0.7 2.9� 0.15

2EthOH�/pGLG611PDC1Hp-12 12.16 0.0 0.79� 0.04 11.2 1.5� 0.08 3.3� 0.14 15.2 8.97�0.5 1.8� 0.1

2EthOH�/pGLG611PDC1Hp-13 12.84 0.0 1.3� 0.07 13.5 1.3� 0.08 1.32�0.07 15.4 9.0�0.5 1.94� 0.09

Samples were taken for analysis on the second day of fermentation.

FEMS Yeast Res 8 (2008) 1164–1174 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

1171PDC1 overexpression in Hansenula polymorpha

was linearized and transformed into the NCYC495 leu1-1

strain. Pyruvate decarboxylase activity and ethanol produc-

tion from xylose were studied in stable Leu1 transformants

carrying the K. lactis PDC1 expression cassette. One of the

transformants of PDC1Kl was characterized with a 13-fold

increase in pyruvate decarboxylase activity (Fig. 12a) and a

2.2-fold increase in ethanol production (Fig. 12b) as

5

6a1a2a3a4

3

4

Bio

mas

s (g

L–1

)

0

1

2

Days

2.0

2.5

1.0

1.5

Eth

anol

(g

L–1)

0.0

0.5

Days0 1 2 3 4 50 1 2 3 4 5

a1a2a3a4

Fig. 7. Ethanol production and biomass accu-

mulation during xylose fermentation at 48 1C

(YNB112% xylose). Transformants: a1, 2EthOH�;

a2, 2EthOH�/pGLG611PDC1Hp-12; a3,

1.5a1

1.0

a2a3a4

0.5

0.0

Days

Eth

anol

g g

–1 o

f bio

mas

s

0 1 2 3 4 5

Fig. 8. Ethanol productivity during xylose fermentation at 48 1C

(YNB112% xylose). Strains: a1, 2EthOH�; a2, 2EthOH�/pGLG611

PDC1Hp-12; a3, 2EthOH�/pGLG611PDC1Hp-13; a4, 2EthOH�/ploxZeo

loxPDC1Hp-10.

60

50

40

30

20

10

0a1 a2 a3 a4

U m

g–1 o

f pro

tien

Fig. 9. Specific activity of pyruvate decarboxylase of Hansenula poly-

morpha transformants during xylose fermentation at 48 1C (YNB112%

xylose). Transformants: a1, 2EthOH�; a2, 2EthOH�/pGLG611PDC

1Hp-12; a3, 2EthOH�/pGLG611PDC1Hp-13; a4, 2EthOH�/ploxZeo

loxPDC1Hp-10.

30

25

20

15

10

5

00 24 48 72

Time (h)

6

5

4

3

2

1

Eth

anol

(g

L–1)

Fig. 10. Ethanol production during glucose fermentation at 48 1C

(YNB112% glucose). Strains: 1, 3Leu1; 2, PDC1Hp-4; 3, 2EthOH�; 4,

2EthOH�/ploxZeoloxPDC1Hp-10; 5, 2EthOH�/pGLG611PDC1Hp-12; 6,

2EthOH�/pGLG611PDC1Hp-13.

0.09 0.045 0.009

Genomic DNA (µg)

a1

a2

a3

a4

Fig. 11. Dot-blot hybridization for PDC1 gene copy estimation. Geno-

mic DNA: a1, 2EthOH�; a2, 2EthOH�/pGLG611PDC1Hp-12; a3,

2EthOH�/pGLG611PDC1Hp-13; a4, 2EthOH�/ploxZeoloxPDC1Hp-10.

ECL-labeled fragment containing the Hansenula polymorpha PDC1 gene

was used as a probe.

FEMS Yeast Res 8 (2008) 1164–1174c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

1172 O.P. Ishchuk et al.

compared with the control 3Leu1 strain. These results

demonstrate that overexpression of the native PDC1 gene

as well as the gene of K. lactis in H. polymorpha improves

ethanol fermentation performance.

The results of this study, along with another work from

our laboratory (Dmytruk et al., 2008), show that the high-

temperature fermentation of xylose by H. polymorpha can be

improved using metabolic engineering. The overexpression

of a single PDC1 gene under the control of a strong GAPDH

promoter resulted in a threefold increase in ethanol produc-

tion. We plan to combine overexpression of pyruvate

decarboxylase with those of bacterial xylose isomerase and

native xylulokinase in H. polymorpha to achieve further

increases in ethanol production from xylose at high tem-

peratures. We also plan to ascertain the molecular nature of

the mutation in the 2EthOH� strain that led to a substantial

increase in xylose alcoholic fermentation.

Acknowledgements

We thank Dr H.A. Kang (Korean Research Institute of

Bioscience and Biotechnology, Taejon, Korea) for kindly

providing the plasmid vector pGLG61 and Dr K. Lahtchev

(Institute of Microbiology, Bulgarian Academy of Sciences,

Sofia, Bulgaria) for providing the H. polymorpha strain

CBS4732s. This work was supported in part by Archer

Daniels Midland Co., Decatur, IL. Access to the H. poly-

morpha genome database was kindly provided by Rhein

Biotech GmbH (Dusseldorf, Germany).

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