Overexpression of pyruvate decarboxylase in the ... - CyberLeninka

RESEARCH ARTICLE

Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results in increased ethanol yield in high-temperature fermentation of xylose Olena P. Ishchuk1, Andriy Y. Voronovsky1, Oleh V. Stasyk1, Galina Z. Gayda1, Mykhailo V. Gonchar1, Charles A. Abbas2 & Andriy A. Sibirny1,3 1

Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine; 2Archer Daniels Midland Co J.R. Randall Research Center, Decatur, IL, USA; and 3Department of ´ University, Rzeszow, ´ Poland Biotechnology and Microbiology, Rzeszow

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: [email protected] 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; hightemperature 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). Introduction 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 monomeric hexoses (glucose, mannose and galactose) and pentoses (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 (Aristidou & 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. 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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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 peroxisomal 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 fermentation 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 Saccharomyces 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) 1164–1174

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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 important for pyruvate distribution between pyruvate dehydrogenase complex and the pyruvate decarboxylase. To test this hypothesis, we decided to clone and overexpress in H. polymorpha the structural gene for pyruvate decarboxylase, PDC1, and to study the effect of its overexpression on ethanol production. The H. polymorpha ORF of the PDC1 gene was cloned under the control of the H. polymorpha strong constitutive promoter of glyceraldehyde-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 fermentation 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 L1 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, Bulgarian 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. FEMS Yeast Res 8 (2008) 1164–1174

Table 1. Yeast strains used in this study Strain

Description

References

H. polymorpha NCYC495 leu1-1

leu2

Gleeson & Sudbery (1988) This study Lahtchev et al. (2002) This study

2EthOH CBS4732s

leu2 leu2

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) K. lactis CBS 2359 Wild type

Wesolowski-Louvel et al. (1996), Kiers et al. (1998)

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 L1 or zeocin at 140 mg L1. The Escherichia coli strain DH5a [F80dlacZDM15, recA1, 1 endA1, gyrA96, thi-1, hsdR17 (r K, mK ), supE44, relA1, deoR, D(lacZYA-argF) U169] was used in experiments that required 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 L1 of ampicillin 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 Vents R DNA polymerase (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 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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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 949bp 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. polymorpha 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 endonucleases 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 primers IS5 and IS6 (Table 2). Sites of NarI restriction endonuclease were incorporated into the primers (underlined, Table 2). The PCR fragment was cut using NarI and cloned into the plasmid pGLG61. The plasmid ploxZeoloxPDC1Hp (Fig. 1c) was constructed on the basis of pGLG611prGAP1PDC1Hp (Fig. 1b). pGLG611prGAP1PDC1Hp was cut using PstI. A 7.56kb fragment was ligated with a 1.1-kb fragment containing 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Table 2. Oligonucleotides used in this work Name

Sequences

ACT1F ACT1R IS3 IS4 IS5 IS6 IS271 IS272 IS273 IS274 IS275 IS276 IS277 IS278 IS279 IS280 K1 K2 K3 K4 K10 K11 Ko58 Ko59

5 0 -TGTCGTCCCAGTTGGTAACG-3 0 5 0 -GGCCCAATCCAAGAGAGGTAT-3 0 5 0 -GCGAAGCTTATGTCTGAAATTACATTAGG-3 0 5 0 -CATAAGCCTTTAGTTCTTAGCGTTGGTAG-3 0 5 0 -GCGGGCGCCCCAATTATCATTAATAATCACTC-3 0 5 0 -TAAGGCGCCAGCATCTTGACAATCAGCAG-3 0 5 0 -TGGTCTTGCGGCTGCTCTGTTCACC-3 0 5 0 -GTAAAGATCAAGGGCGTAGGTGCCCAG-3 0 5 0 -GTCTTCTCCAAGGATTTCCATAGAGCACATC-3 0 5 0 -GCCAATGTTCAAGTAGATGCTCTTTGACTG-3 0 5 0 -CTACGTCTCCGACAGACTCGAGGC-3 0 5 0 -ACAGCCTTGACCTGGGTGTAGCTCTC-3 0 5 0 -GACACCGCCACCTACGTCTCCAAC-3 0 5 0 -ACCAATTCTCACAGCCTTCCACTGGGTG-3 0 5 0 -GCCTACCTGTTCACTCAAGACATCAATCGG-3 0 5 0 -GCTGAATGCTGCCAAGCCGGCTTC-3 0 5 0 -TGGTCCTCGCTGAAGGCCGACTTGC-3 0 5 0 -GCGGTGTGTACATCGGAGTTCTGTCG-3 0 5 0 -AGTCGCCGACACCAAAGGTGGTCAC-3 0 5 0 -GCCATTGCGGGCATGATGGCCGAG-3 0 5 0 -CGCCATATGTCTGAATCCCAACTACC-3 0 5 0 -TTTGCGGCCGCTTAAGCTGCATTGATCTGC-3 0 5 0 -CGGGGTACCTGCAGATAACTTCGTATAGCATAC-3 0 5 0 -CGGGGTACCTGCAGTAATTCGCTTCGGATAAC-3 0

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 mL1 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. FEMS Yeast Res 8 (2008) 1164–1174

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Nt H

(a)

Nd

B

prGAP_Hp

ORI

K

Sc Bg

RI

trAOX_Hp ORF PDC1_Hp

Er

LEU2 Sc

pKO8+prGAP+PDC1Hp c. 8.6 kb

(b) H Nt Nr bla

Nd

trAOX_Hp

Nr

Xb

prGAP Hp

prGAP_Hp ORF PDC1_Hp

P

RI APH

TEL188

ORI

pGLG61+prGAP+PDC1Hp c. 9.4 kb

(c) H Nt Nr

bla

Nd

trAOX_Hp

Nr

P

prGAP_Hp

P TEL188

Zeo

ORF PDC1_Hp

ORI

ploxZeoloxPDC1Hp c. 8.66 kb

(d) Sp P

Sl Xb B K

prom. lacZ

RI

Sc RI H

H trAOX_Hp

prGAP_ Hp LEU2 Sc

ORF PDC1_Kl

lacZ

bla

ORI

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.

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 resuspended in the washing buffer and extracts were prepared using glass beads with vigorous vortexing at 14 1C. Unbroken 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. FEMS Yeast Res 8 (2008) 1164–1174

The enzyme activity was measured directly after the preparation 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 dehydrogenase 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). 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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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. polymorpha ORF116 (Hp_contig12); IS273 and IS274 for the 3 0 fragment of H. polymorpha ORF168 (Hp_contig15); IS275 and IS276 for the 3 0 fragment of H. polymorpha ORF226 (Hp_contig01); 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 3 0 fragment of H. polymorpha ORF of the ACT1 gene (orf262, Hp_contig01). Sequences of the ORFs mentioned above were taken from the H. polymorpha genome database (Rhein Biotech GmbH, Düsseldorf, Germany).

Results and discussion High-temperature xylose and glucose fermentation in H. polymorpha Hansenula polymorpha achieves optimal growth rates at temperatures 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 Fermentation (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 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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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 dehydrogenase, 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 wildtype 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 dehydrogenase 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 dehydrogenase 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) 1164–1174

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3

Ethanol (g L–1)

(b)

37 °C

2.5

1

2

2

1.5 1 0.5 1

2

(c) 60

4

2

1

1.5

2 1 0.5

5

1

2

3 Days

(d) 60

4

40 30 20

1

10

5

48 °C

50 Ethanol (g L–1)

Ethanol (g L–1)

3 Days

37 °C

50

1

40

2

30 20 10

2

0

0 1

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 dehydrogenase 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 enzymes 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 approximately 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 mutant 2EthOH One of the key aims of our study was to determine the effect of pyruvate decarboxylase overexpression, a key enzyme in FEMS Yeast Res 8 (2008) 1164–1174

48 °C

0

0

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 fermentation at 48 1C (YNB112% glucose). Strains: 1, NCYC495 leu1-1, 2, 2EthOH.

3 2.5

Ethanol (g L–1)

(a)

2

3 Days

4

5

1

1

2

3 Days

4

5

2

Fig. 3. Alcohol dehydrogenase activity of Hansenula polymorpha strains visualized on native protein PAGE. Protein samples were taken from cellfree 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.

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1

2

1

2

ACT1

ORF 116 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.

alcoholic fermentation, on xylose fermentation of H. polymorpha. The NCYC495 leu1-1 was used as a recipient strain. The plasmid pKO81prGAP1PDC1Hp (Fig. 1a) was linearized 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. Transformants 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 activities were studied in the transformants in comparison with the control 3Leu1 strain that was obtained after transformation 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 decarboxylase 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 characterized by better growth; however, no ethanol was accumulated 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 bacter2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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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.

ial aminoglycoside-3-phosphotransferase (APH, genetecin resistance) gene, this vector promotes copy-number-controlled 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 transformants were selected on YPS medium supplemented with 1 g L1 geneticin (G418). 2EthOH transformants with the plasmid ploxZeoloxPDC1Hp were selected on YPS medium supplemented with 140 mg L1 zeocin. The stability of the corresponding transformants was checked by alternative FEMS Yeast Res 8 (2008) 1164–1174

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Ethanol (g L–1)

(a)

PDC1 activity (U mg–1)

(b)

1.2

20

1 15 Fig. 6. Ethanol production (a) and specific activity of Pdc1 (b) of Hansenula polymorpha NCYC495 transformants. 3Leu1 – control strain, Leu1 transformant; PDC1Hp-4 – the transformant NCYC495 leu1-1/pKO81prGAP1PDC1Hp. The samples for ethanol and pyruvate decarboxylase assays were taken from the third day of fermentation of xylose (YNB18% xylose, 37 1C and 140 r.p.m.).

0.8 0.6

10

0.4 5 0.2 0

0 3Leu+

3Leu+

PDC1Hp-4

PDC1Hp-4

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) L-Arabinose

D-Xylose

D-Glucose

Strains

OD Ethanol Pdc1 activity OD Ethanol (l600 nm) (g L1) (U mg1) (l600 nm) (g L1)

Pdc1 activity OD Ethanol (U mg1) (l600 nm) (g L1)

3Leu1 PDC1Hp-4 2EthOH 2EthOH/ploxZeoloxPDC1Hp-10 2EthOH/pGLG611PDC1Hp-12 2EthOH/pGLG611PDC1Hp-13

10.5 11.2 10.68 11.12 12.16 12.84

0.1 0.02 4.1 0.2 0.21 0.06 2.2 0.2 3.3 0.14 1.32 0.07

0.0 0.0 0.0 0.0 0.0 0.0

0.4 0.06 9.2 0.5 0.07 0.01 0.88 0.04 0.79 0.04 1.3 0.07

8.24 11.0 11.1 14.1 11.2 13.5

0.7 0.05 1.2 0.1 0.9 0.2 1.6 0.1 1.5 0.08 1.3 0.08

12.4 13.0 11.6 16.2 15.2 15.4

Pdc1 activity (U mg1)

14.4 0.9 0.21 0.03 27.9 1.3 3.2 0.15 7.8 0.4 0.26 0.04 10.8 0.7 2.9 0.15 8.97 0.5 1.8 0.1 9.0 0.5 1.94 0.09

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

cultivation in nonselective (YPS) and selective media (YPS with geneticin/zeocin). The presence of desirable recombinant constructs (GAPDH promoter fused to PDC1 ORF with the AOX terminator) in the genome of stable transformants 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 glucose 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 elevated pyruvate decarboxylase were characterized by better growth; however, no ethanol was accumulated (Table 3). FEMS Yeast Res 8 (2008) 1164–1174

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. Transformants 2EthOH/pGLG611PDC1Hp-12 and 2EthOH/ pGLG611PDC1Hp-13 contained approximately seven to nine copies, whereas the transformant 2EthOH/ploxZeoloxPDC1Hp-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 in the H. polymorpha wild-type strain We decided to determine whether the H. polymorpha fermentation parameters could be improved by overexpression 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 introduced the resulting construct into an H. polymorpha wildtype strain. The plasmid p19L21prGAP1PDC1Kl (Fig. 1d) 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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2.5 a1 a2 a3 a4

5 4

Ethanol (g L–1)

Biomass (g L–1)

6

3 2

a1 a2 a3 a4

2.0 1.5 1.0

Fig. 7. Ethanol production and biomass accumulation during xylose fermentation at 48 1C (YNB112% xylose). Transformants: a1, 2EthOH; a2, 2EthOH/pGLG611PDC1Hp-12; a3,

0.5

1

0.0

0 0

1

2

3

4

0

5

1

2

3

4

30

1.5 a1 a2 a3 a4

Ethanol (g L–1)

Ethanol g g–1 of biomass

5

Days

Days

1.0

0.5

25

1

20

2

15

3

10

4

5

5 6

0 0 0.0 0

1

2

3 Days

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.

24 48 Time (h)

72

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

Genomic DNA (µg) 0.045 0.009

60

a1

U mg–1 of protien

50 40 30 20

a2

10 0 a1

a2

a3

a4

Fig. 9. Specific activity of pyruvate decarboxylase of Hansenula polymorpha 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.

was linearized and transformed into the NCYC495 leu1-1 strain. Pyruvate decarboxylase activity and ethanol production 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 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

c

a3

a4

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

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(a)

Ethanol (g L–1)

(b)

PDC1 activity (U mg–1)

16

1.2

14

1

12 0.8

10

Fig. 12. Specific activity of pyruvate decarboxylase (a) and ethanol accumulation (b) of Hansenula polymorpha transformants during xylose fermentation at 37 1C (YNB18% xylose). Transformants: 1, 3Leu1; 2, PDC1Hp-4; 3, PDC1Kl-3. The samples for estimation of ethanol concentration and pyruvate decarboxylase assay were taken from the third day of fermentation of xylose.

8

0.6

6

0.4

4 0.2

2 0

0 1

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 hightemperature 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 production. 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 temperatures. 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. polymorpha genome database was kindly provided by Rhein Biotech GmbH (Düsseldorf, Germany).

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