ORIGINAL PAPER

The yeast Dekkera bruxellensis genome contains two orthologsof the ARO10 gene encoding for phenylpyruvate decarboxylase

Anna Theresa de Souza Liberal • Marcelo Falsarella Carazzolle •

Goncalo Amarante Pereira • Diogo Ardaillon Simoes •

Marcos Antonio de Morais Jr.

Received: 21 December 2011 / Accepted: 9 April 2012 / Published online: 22 April 2012

� Springer Science+Business Media B.V. 2012

Abstract The yeast Dekkera bruxellensis possesses

important physiological traits that enable it to grow in indus-

trial environments as either spoiling yeast of wine production

or a fermenting strain used for lambic beer, or fermenting

yeast in the bioethanol production process. In this work, in

silico analysis of the Dekkera genome database allowed the

identification of two paralogous genes encoding for phenyl-

pyruvate decarboxylase (DbARO10) that represents a unique

trait among the hemiascomycetes. The molecular analysis of

the theoretical protein confirmed its protein identity. Upon

cultivation of the cell in medium containing phenylpyruvate,

both increases in gene expression and in phenylpyruvate

decarboxylase activity were observed. Both genes were dif-

ferentially expressed depending on the culture condition and

the type of metabolism, which indicated the difference in

the biological function of their corresponding proteins.

The importance of the duplicated DbARO10 genes in the

D. bruxellensis genome was discussed and represents the first

effort to understand the production of flavor by this yeast.

Keywords Ehrlich pathway � Gene expression �RT-qPCR

Introduction

The yeast D. bruxellensis, through its anamorph Bretta-

nomyces bruxellensis, is considered to be the worst wine

contaminant. It is responsible for the formation of volatile

phenols and tetrahydropyridines that cause off-flavors

during the aging period in barrels. In contrast, its impor-

tance in the production of the traditional Belgian lambic

beer (Loureiro and Malfeito-Ferreira 2003) and fuel-

ethanol have been reported (Liberal et al. 2007; Blomqvist

et al. 2010). On the other hand, its cells can produce

4-ethyl-phenol that is regarded as an important component

of the wine flavor under certain concentrations (Chatonnet

et al. 1995). The accumulation of 4-ethyl-phenol and

4-vinylphenol at certain levels seems to be dependent on

the cell growth and on the activity of hydroxycinnamate

decarboxylase (Barata et al. 2008; Benito et al. 2009). In

addition, the assimilation by the yeast cells of the amino

acids present in wort or must promotes the production of

higher alcohols and esters with flavoring characteristics

(Dickinson et al. 2003). Aromatic amino acids (tryptophan,

tyrosine and phenylalanine), branched-chain amino acids

(leucine, isoleucine and valine) and methionine are

assimilated by the Ehrlich pathway. They are first transa-

minated by transaminases to produce glutamate from

2-oxoglutarate and the corresponding 2-oxo acid from the

assimilated amino acids. In the next step, the 2-oxo acids

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11274-012-1054-x) contains supplementarymaterial, which is available to authorized users.

A. T. de Souza Liberal � D. A. Simoes � M. A. de Morais Jr.

Interdepartmental Research Group on Metabolic Engineering,

Federal University of Pernambuco, Recife, Brazil

M. F. Carazzolle � G. A. Pereira

Department of Genetics and Evolution, Institute of Biology,

University of Campinas, Campinas, Brazil

D. A. Simoes

Department of Biochemistry, Federal University

of Pernambuco, Recife, Brazil

M. A. de Morais Jr. (&)

Department of Genetics, Federal University of Pernambuco, Av.

Moraes Rego, 1235, Cidade Universitaria, Recife,

PE 50670-901, Brazil

e-mail: marcos.morais@pesquisador.cnpq.br

123

World J Microbiol Biotechnol (2012) 28:2473–2478

DOI 10.1007/s11274-012-1054-x

are decarboxylated into the corresponding aldehydes which

are further reduced to the corresponding fusel alcohol by

the alcohol dehydrogenases, at the expense of NAD(P)H

(Dickinson et al. 2003; Hazelwood et al. 2008). The partial

genome of D. bruxellensis was recently published and a

number of orthologous and non-orthologous S. cerevisiae

genes were identified (Woolfit et al. 2007). However, no

studies have been reported on the identification and char-

acterization of decarboxylase-encoding genes. Thus, the

search for these genes in the D. bruxellensis genome may

help to better understand the contribution that this yeast to

the final sensory quality of the end product. In this study,

we provide an identification of two paralogs of phenyl-

pyruvate decarboxylase-encoding genes, which differs not

only in their nucleotides and proteins, but also in their

gene expression profile in response to the type of carbon

and nitrogen available in the medium. Since no other

hemiascomycete yeasts seem to carry two copies of this

gene, we start to question the biological and evolutionary

role of this enzyme activity in the yeast metabolism.

Materials and methods

Strains

Dekkera bruxellensis GDB 248 (Liberal et al. 2007) and

Saccharomyces cerevisiae JP1 (Silva-Filho et al. 2005)

were collected from a bioethanol production plant. The

type strain D. Bruxellensis CBS 74/CLIB 316 was provided

by the CLIB collection (INRA-Grignon, France).

Dekkera genome project

The genome of the D. bruxellensis wine strain CBS 2499

was described by Woolfit et al. (2007). The nucleotide

sequences were kindly provided by Prof. Jure Piskur (Lund

University, Sweden) and assembled in a local database at

the University of Campinas (http://www.lge.ibi.unicamp.

br/dekkera/), Brazil.

Computational analysis

Nucleotide sequences of the 2-oxo acid decarboxylases

encoding genes PDC1, PDC5, PDC6, THI3 and ARO10

were collected from the Saccharomyces Genome Data-

base—SGD (http://www.yeastgenome.org/) and used as a

means of searching for homologous genes in genetically

close ascomycetes by BLASTx analysis, using Genole-

vures (http://www.genolevures.org/), GenBank (http://

www.ncbi.nlm.nih.gov/) and Dekkera project (http://www.

lge.ibi.unicamp.br/dekkera/) databases. Sequences with

e-value lower than -20 were collected (Table S1—

supplementary data) from the yeasts D. bruxellensis (Db

genes), Kluyveromyces lactis (Kl genes), Candida glabrata

(Cg genes), Ashbya gossypii (Ag genes), Candida albicans

(Ca genes), Debaryomyces hansenii (Dh genes) and Yarr-

owia lipolytica (Yl genes), and their amino acid sequences

were obtained by theoretical translation using BioEdit

Sequence Alignment Editor Program v.7.0 (http://www.

mbio.ncsu.edu/BioEdit/bioedit.html).

The similarities and divergences among the sequences

(nucleotides and amino acids) were calculated by multiple

alignment analysis using the ClustalW tool of EMBL-EBI

database (http://www.ebi.ac.uk/) and used to construct

similarity matrices of protein sequences in the Bioedit pro-

gram (Table S2—supplementary data). Phylogenetic anal-

yses were performed in the Molecular Evolutionary Genetic

analysis—MEGA program (http://www.megasoftware.

net/). Gene clustering analysis was performed by the

Neighbor -Joining Method using p-distance. Confirmation of

nucleotide sequences were provided by DNA sequencing of

the entire genes (see primers at Table S3—supplementary

data) in ABI Prism 3100 device, using the Technological

Service Platform at the Osvaldo Cruz Foundation (Recife,

Brazil).

Culture conditions, RNA extraction and cDNA

synthesis

Yeast cells were pre-cultivated overnight at 30 �C in

synthetic defined (SD) medium (YNB at 1.7 g/L; ammo-

nium sulfate at 5 g/L; glucose at 20 g/L) with agitation

(150 rpm) and used for gene expression analysis. For long

term induction during cell growth, yeast cells were

transferred to YNB media containing different C and N

sources (Table 1) to initial concentration of 0.1 (A600nm).

The cell cultures were incubated at 30 �C in a rotator

shaker (200 rpm) and the cells were collected when they

reached a cell density of 1.0 (A600nm) at the exponential

growth phase in the same physiological state, for RNA

extraction. The growth time varied according to the car-

bon and nitrogen sources. For short-term gene induction,

cells from the pre-inoculum in synthetic defined medium

were recovered when reached concentration of 1.0

(A600nm). Five milliliters were recovered and centrifuged,

and the yeast cells were washed in water and re-sus-

pended to the same volume in YNB media with different

C and N sources (Table 1). After 1 h incubation at 30 �C

the cells were collected for RNA extraction. Each con-

dition was tested in duplicate and each sample was col-

lected in triplicate and immediately frozen in liquid

nitrogen for RNA extraction.

Yeast total RNA was isolated by using the NucleoSpin�

RNA II kit and following the manufacturer’s instructions

(Macherey–Nagel, Germany). RNA was quantified by

2474 World J Microbiol Biotechnol (2012) 28:2473–2478

123

means of the spectrophotometric method (Nanovue, GE

HealthCare) and its integrity evaluated by agarose gel 1 %

electrophoresis. For cDNA synthesis, 500 ng of total RNA

was used for each reverse transcription reaction tube

(40 ll) using ImProm-IITM Reverse Transcription System

Promega II kit and following the manufacturer’s instruc-

tions (Promega, USA).

Primer design for qPCR

The nucleotide sequence of the D. bruxellensis DbEFA1

(translation Elongation Factor, Alpha chain) and DbYNA1

(transcription factor of the nitrate assimilation regulon)

genes were recovered from the GenBank Nucleotide

database (accession numbers EF552481 and EF364427,

respectively) and the nucleotide sequences of the D. brux-

ellensis genes DbEFB1 and the ARO10 homologous were

recovered from D. bruxellensis database (http://www.lge.

ibi.unicamp.br/dekkera), as described above. Primer design

was performed by on-line Genscript primer design software

at advanced mode (http://www.genscript.com/cgi-bin/tools

) with the following parameters: primer length between 17

and 25 bases, Tm value around 59 �C and amplicon length

between 70 and 110 bp. Primer pairs were analyzed by

means of the on-line Netprimer tool (www.premierbiosoft.

com/netprimer/netprlaunch/netprlaunch.html) for self-hybrid,

duplex, hairpins and loops The primer pairs ranked over

90 % were selected for RT-qPCR experiments (Table S4—

supplementary data). Reference genes DbEFA1, DbEFB1

and DbYNA1 were used for data normalization.

Analysis of relative gene expression by RT-qPCR

Real-time PCR assays were optimized for maximum effi-

ciency via experimental design, using SYBR Green PCR

Master Mix (Applied Biosystems, USA). The chosen

parameters were SYBR Green (5 ll), primers 200 nM

(0.4 ll each), H2O (3.2 ll) and cDNA (1 ll). The tempera-

ture–time profile (95 �C for 10 min and 40 cycles at 95 �C

for 15 s and 60 �C for 1 min) was optimized for the ABI

Prism 7300 (Applied Biosystems). The amplification curves

were analyzed with the aid of SDS v.2.0 software (Applied

Biosystems). Negative PCR control (without template) and

negative RT control (RNA as template in Real Time PCR

assays, i.e. RNA not reverse-transcribed to cDNA) were run

for quality improvement and the detection of contamination

by genomic DNA. The relative gene expression values are

the average of three technical replicates, with biological

duplicates, totalizing 6 samples for each condition. The mean

Cq values for each growth medium were then plotted in

Microsoft Excel 2007 worksheets to create a suitable input

file for geNorm applet (Vandesompele et al. 2002). The first

stage in the geNorm analysis was to determine the stability of

each reference gene, which represents the gene expression

stability as the average pairwise variation for that gene with

all the other tested references (Vandesompele et al. 2002).

Enzyme assay

Dekkera bruxellensis GDB 248 was cultivated in YNB-

glucose containing either ammonia or phenylalanine to cell

concentration of 1 (A600nm) as above and harvested by cen-

trifugation. The cells were washed in 200 mM HEPES (pH

7.2) and suspended in the same buffer containing 1 mM DTT

and 1 mM PMSF. Glass beads (Ø460–600 lm) were added

to the equivalent of one volume of cell suspension and the

cells were broken with five cycles of vortexing for 1 min,

with 1 min of interval on ice to avoid protein denaturation.

Following centrifugation at 10,000g for 5 min at 4 �C, cell-

free extracts were collected for protein determination by

Lowry’s method. Enzyme specific activity for phenylpyru-

vate decarboxylase using phenylpyruvate (Sigma) as a sub-

strate was measured according to Vuralhan et al. (2003).

Results

In silico gene identification

Five 2-oxo acid decarboxylases gene of S. cerevisiae were

recovered from the SGD and its orthologous from

Table 1 Relative expression of DbARO10 genes of D. bruxellensisGDB 248 cells in different conditions

Test condition Relative gene expression (RQ)

C-sourcea N-sourceb dbARO10-1 dbARO10-2

During cell growth

Glucosec Ammoniumc 1 1

Glucose Phenylalanine 95.7 35.6

Sucrose Ammonium 4.68 2.69

Glycerol Ammonium 1.42 11.08

Ethanol Ammonium -4.96 23.59

Short-term incubation

Glucose Ammonium 1 1

Glucose Phenylalanine 923 -2.69

Glucose Leucine 5.24 20.32

Glucose Proline 2.17 2.13

Glucose Tryptophan 1 1

Glucose Valine 1 -16.66

Sucrose Ammonium 19 -4.78

Glycerol Ammonium 10.41 -5.18

Ethanol Ammonium 7 -2.02

a Carbon source: glucose, 20 g/L; sucrose, 19 g/L; ethanol, 19.5 mL/

L; glycerol, 16.3 mL/Lb Nitrogen source: (NH4)SO2, 5 g/L; phenylalanine, 3.1 g/L; trypto-

phan, 3.8 g/L; leucine, 6 g/L; valine, 5.4 g/L; proline, 5.2 g/L

World J Microbiol Biotechnol (2012) 28:2473–2478 2475

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genolevures ascomycetes database and Genbank (Table

S1—supplementary material). Afterwards, the Dekkera

genome database was browsed using keywords that resul-

ted in the identification of two contigs (e-values\-20) that

displayed complete sequences from ATG to the stop

codons. Phylogenetic analysis placed the contigs 2774 and

3332 at the ARO10 gene cluster (Fig. 1), which encodes for

phenylpyruvate decarboxylase in S. cerevisiae (Vuralhan

et al. 2003). These were designated DbARO10-1 (Gene-

Bank accession number HQ693757) and DbARO10-2

(GeneBank accession number HQ693758), respectively.

Both ARO10 paralogous genes clustered with their ortho-

logs from Debaryomyces hansenii and Candida albicans

(Fig. 1), in accordance with the previous whole genome

clustering analysis (Woolfit et al. 2007). Genetic similarity

matrices were prepared from the corresponding amino acid

sequences for all the genes that were recovered and

revealed that the two isoforms of Aro10p were only 37.7 %

similar to each other (Table S2—supplementary material).

The DbAro10-1p contained 583 aa (c.a. 66 kDa) and

included two thiamine pyrophosphate pyruvate/indolpyru-

vate binding domains at N-terminal (aa 20–125) and

C-terminal (aa 415–505). The isoform Db-Aro10-2p con-

tained 593 aa (c.a. 67 kDa) and also included two thiamine

pyrophosphate (TPP) pyruvate(PDC)/indolpyruvate(IPDC)

binding domains at N-terminal (aa 40–140) and C-terminal

(aa 425–520). Both TPP/PDC domains were overlapping

two dimer interface domains that might be involved in

protein–protein interactions. These data showed that both

contigs represented two different loci in the yeast genome.

DbARO10 gene expression

The phylogenetic analysis revealed that both D. bruxell-

ensis decarboxylase-encoding genes were orthologous to

the S. cerevisiae ARO10 gene, which is involved in phen-

ylalanine catabolism and regulated by this amino acid

(Vuralhan et al. 2003). Thus, gene expression of DbARO10

genes was determined at the exponential growth phase in

synthetic medium containing phenylalanine as the nitrogen

source. During the cell growth, both DbARO10-1 and

DbARO10-2 were induced by phenylalanine both in type

strain CBS 74 and in the industrial strain GDB 248

(Fig. 2). Overall, DbARO10-1 was more induced than

DbARO10-2 and both genes was four times higher in the

industrial strain GDB 248 than in the type strain CBS 74

and that induction of DbARO10-1 was overall 2.6 times

higher than DbARO10-2 in the exponential growth phase of

both strains (Fig. 2). Afterwards, relative gene expression

was measured only in the industrial strain since the type

Fig. 1 Phylogenetic analysis of

the phenylpyruvate

decarboxylase encoding genes

of D. bruxellensis and their

positioning within the

hemiascomycetes group.

Nucleotide sequences were used

to prepare the p-distance matrix

and clustered by the Neighbor-

Joining method

2476 World J Microbiol Biotechnol (2012) 28:2473–2478

123

strain did not grow, or grew badly, in most of the different

conditions tested. Since that strain was isolated from the

ethanol fermentation using sugar cane, gene expression

was measured upon growth on sucrose. Interestingly, both

genes were induced in that carbon source, even in the

presence of ammonia, and again DbARO10-1 was more

induced than DbARO10-2 (Table 1). Gene expression was

also measured when yeast cells were grown under fully

respiratory metabolism, using glycerol or ethanol as carbon

source. In this condition only DbARO10-2 gene was

induced, while DbARO10-1 displayed a five-fold repres-

sion in ethanol relative to glucose (Table 1). Despite that

huge increase in gene transcript, the overall specific

activity of phenylpyruvate decarboxylase in the yeast cell-

free extract increased only 2.7 times in phenylalanine

compared to ammonia, which was comparable to the

increase in S. cerevisiae (Table 2).

For short-term response, the results showed that

DbARO10-1 was highly induced by immediate exposure to

phenylalanine, leucine and proline as nitrogen source as

well as in the presence of sucrose, glycerol and ethanol as

carbon source (Table 1). On the other hand, DbARO10-2

gene was induced only in the presence of leucine and

proline, and repressed in most of incubation condition

tested in those conditions (Table 1), including in the

presence of p-cumaric acid (-12.5 times). Interestingly,

phenylalanine first repressed DBARO10-2, while trypto-

phan had no effect on short-term expression of both genes

(Table 1).

Discussion

In the present study, we identified two different genes that

encode for phenylpyruvate decarboxylases (Phe-Pdc) in the

yeast D. bruxellensis, while only one gene exists in the

genomes of S. cerevisiae (Vuralhan et al. 2003) and other

phylogenetically closed hemiascomycetes (Fig. 1). In

general terms, the phylogeny of ARO10 gene products

reflected the whole phylogenetic analysis performed by

Woolfit et al. (2007) for the same yeast species, and

corroborated the topology #2 showed in that work. In

addition, the level of protein similarity of 37.7 % for both

isoforms was close to the calculated average similarity of

35.2 % for the hemiascomycetes Aro10p orthologous

analyzed. Thus, it indicates that paralogous DbARO10

genes in D. bruxellensis genome evolved from a duplica-

tion of an ancestral gene, which attests to its importance for

the yeast metabolism. Why D. bruxellensis presents two

copies of this gene?

The Phe-Pdc activity can be attributed to the decar-

boxylation of phenylpyruvate to phenyl-acetaldehyde dur-

ing the catabolism of phenylalanine (Vuralhan et al. 2003,

2005). Phenylalanine is found in grape and barley wort and

can be converted to 2-ethyl-phenol by the yeast cells during

fermentation that is responsible for the floral bouquet of the

beverages (Hazelwood et al. 2008). The induction of

DbARO10-1 gene by phenylalanine (96-fold) was higher

than that observed for S. cerevisiae (30-fold) (Vuralhan

et al. 2005), although the two works used different

approaches. Phe-PDC activity was observed in our

S. cerevisiae industrial strain growing in ammonium, despite

its absence in CEN.PK113-7D laboratory strain (Vuralhan

et al. 2003). However, the similar enzyme activities in the

industrial (69 nmol min-1 mgPRT-1) (Table 2) and labo-

ratory (53 nmol min-1 mgPRT-1) (Vuralhan et al. 2003)

strains of S. cerevisiae grown in phenylalanine ensure the

Fig. 2 Expression of D. bruxellensis genes in the type strain CBS 74

(gray columns) and the industrial strain GDB 248 (black columns)

cultivated in synthetic medium with phenylalanine as a nitrogen

source, relative to the same genes expressed in ammonia-based

medium (reference expression level of 1.0)

Table 2 Specific enzyme activity of phenylpyruvate decarboxylase

in cell free extract of yeast cells grown with different nitrogen sources

Yeast Specific activity

(nmol min-1 mgPRT-1)

Fold increase

(Phe/Amm)

Ammonia Phenylalanine

D. bruxellensis GDB248 15.5 42.1 2.72

S. cerevisiae JP1 27.4 69.3 2.53

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reliability of the assay. The induction of both DbARO10

genes by leucine (Table 1) indicates that their enzymes

could also decarboxylase the aldehydes from branched-

chain amino acids, as was shown for S. cerevisiae Aro10p

(Vuralhan et al. 2005; Boer et al. 2007). Nevertheless, the

products of two paralogous genes seem to perform different

biological functions. DbARO10-1 gene is immediately

induced upon exposure to phenylalanine, and its expression

decrease almost 10 times as the cultivation continues while

DbARO10-2 is lately induced almost 95 times in this

N-source. Another remarkable result was observed when

calculating the ratio of gene expression between long-term

and short-term incubation in respiratory carbon sources: an

over 10-times decrease in gene transcript of DbARO10-1

and an over 50-times increase in gene transcript of

DbARO10-2. This indicates that DbARO10-2 is induced

lately during the cultivation under respiratory metabolism

and maybe in response to putative oxidative damages

produced during cell growth.

Other phenolic compounds such as p-coumaric acid are

also present in the must from red grapes and their metab-

olisms contribute to the flavor and taste of the wine (Benito

et al. 2009). The decarboxylation of p-coumaric acid

produces 4-vinyl-phenol in a reaction that is dependent on

the hydroxycinnamate (p-coumarate) decarboxylase activ-

ity (Benito et al. 2009). Moreover, other decarboxylases

should are expected to act on 3-hydroxyphenylpyruvate

(from tyrosine), 3-indole pyruvate (from tryptophan) and

2-oxo-c-methylthio-butyrate (from methionine). The gene

expression results showed that any of the DbARO10 genes

responded to two of these substrates (tryptophan and

p-cumaric acid). Thus, alternative decarboxylases should

be present in the proteome of D. bruxellensis that were not

yet identified.

In conclusion, the results showed an unequivocal exis-

tence of two paralogous genes that encode for phenyl-

pyruvate decarboxylase enzymes in the genome of

D. bruxellensis and that they are involved in different

biological mechanisms in this yeast. A detailed study on

the function of each one is necessary to answer the question

on why this yeast needs to copies of this gene.

Acknowledgments This work was supported by grants from the

Brazilian funding agencies CNPq (Conselho Nacional de Desen-

volvimento Cientıfico e Tecnologico) though the UFPE-UNICAMP

cooperation program ‘‘casadinho’’, CAPES (Coordenacao de Apoio

ao Pessoal de Ensino Superior) and FACEPE (Fundacao de Apoio a

Ciencia e tecnologia do Estado de Pernambuco).

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