EurekaBrewing.wordpress.

com September 2015

Insight into the Dekkera anomala

YV396 genome
Samuel H. Aeschlimann*
*EurekaBrewing.wordpress.com (powered by Blackwell Brewery)

Abstract
The yeast Dekkera (anamorph Brettanomyces) is associated with various industrial fermentations and
currently experiences an increase of interest in the Wild Ale beer brewing scene. The notorious spoilage
yeast is capable of forming volatile phenols with flavor descripties like wet animal, musty. Due to the high
abundance of spoilage in the wine industry, so far only genomes of Brettanomyces bruxellensis have been
published and discussed. Other species from the Dekkera taxa include Dekkera anomala mainly associated
with beers such as Belgian Lambics. The current draft Dekkera anomala YV396 genome assembly consists
of 30 contigs with an estimated genome size of 12.9 Mb. Addressing the degree of completeness of the
genome revealed the existance of a set of tRNAs capable of encoding the standard 20 amino acids including
two tRNAs encoding for selenocysteine. Furthermore the entire set of 248 CEGs could be found in the
assembly indicating a rather complete draft genome. 4,000 genes were predicted of which the main part
has homologues in Brettanomyces bruxellensis. Two phenolic acid decarboxylses as well as one vinylphenol
reductase - two enzymes possibly associated with volatile phenols - could be further identified.
The genome as well as the proteome provide a useful resource for additional analysis of pathways associates
with flavor compounds in beer production.

Figure 1: Micrograph of a Dekkera anomala strain (EYL012 Brettanomyces cantillon VI)

EurekaBrewing.wordpress.com September 2015

Contents
I

Introduction

II Results and Discussion

I
Genome assembly . . . . . . . . . . .
II
tRNA analysis . . . . . . . . . . . . .
III Gene prediction . . . . . . . . . . . .
IV Gene annotation . . . . . . . . . . . .
V
Carbohydrate metabolism . . . . . .
VI Phenolic acid metabolism . . . . . .
VI.1 Phenolic acid decarboxylase
VI.2 Vinylphenol reductase . . . .

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III Methods
I
Genome assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II
Gene prediction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III Gene annotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I.

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Introduction

Dekkera (anamorph Brettanomyces) are notorious beverage spoilage yeasts associated with a variety
of fermentation products such as wine, beer, cider, kombucha as well as sourdough [Yakobson,
2010; White and Zainasheff, 2010]. Due to its spoilage potential and impact on wine quality, a lot
of research has been conducted on Dekkera to further understand the associated pathways leading
to the unwanted off-flavors. Volatile phenols (4-ethylphenol, 4-ethylguaiacol and 4-ethylcatechol)
with sensory descriptives like animal, musty are especially in the focus of the wine industry as
these compounds exhibit low detection thresholds [Curtin et al., 2012]. Beside its importance in
the wine industry, Dekkera is also present in Belgian beers like Lambics and Gueuzes where the
volatile phenols created by Dekkera species are all part of the desired beer profile [Spitaels et al.,
2014].
Next generation sequencing enables us to look at the genomic setup of organisms to better
understand the organisms and metabolic pathways. This publication discusses the genome
assembly created by Vervoort et al. [2015], the gene prediction as well as certain pathways linked
to beer production.

EurekaBrewing.wordpress.com September 2015

II.
I.

Results and Discussion

Genome assembly

The current D. anomala genome consists of 30 contigs with an estimated genome size of about
12.9 Mb (Tab 1). The estimated genome is similar in size to the close relative D. bruxellensis or
Saccharomyces cerevisiae [Goffeau et al., 1996; Curtin et al., 2012]. Looking at the GC content of the
D. anomala draft genome in comparison to other Dekkera genomes reveales a similar distribution
pattern (Fig 2).
Table 1: D. anomala YV396 genome statistics (accession number GCA_001005505.1)

Name
Total sequence length [bp]
Number of contigs [n]
Contig N50 [bp]

12,880,761
30
1,112,461

Figure 2: GC plots for D. bruxellensis CBS2499 [Pikur et al., 2012], D. bruxellensis AWRI1499 [Curtin et al., 2012],
D. bruxellensis LAMAP 2480 [Valdes et al., 2014] and D. anomala YV396 [Vervoort et al., 2015]

EurekaBrewing.wordpress.com September 2015

II.

tRNA analysis

To assess the completeness of the genome assembly in terms of tRNAs, tRNAscan-SE (Vers 1.3.1,
default settings) established by Lowe and Eddy, 1997 was run on the genome to predict tRNAs.
tRNAscan-SE predicted 123 tRNAs in the D. anomala genome and a complete set of tRNAs
sufficient to encode the standard 20 amino acids including two tRNAs encoding for selenocysteine
(not shown). The real number of tRNAs was not addressed since the tRNAs were only used as a
parameter to address the completeness of the genome assembly.

III.

Gene prediction

The results of the Augustus gene prediction, as further explained in chapter II, are summarized in
Tab 2. 4,050 potential genes and 4,160 potential transcripts could be predicted. The average coding
sequence length is about 1,600 bp with the longest transcript length of 14,904 bp (Fig 3a). Exons
are generally shortern than 4,000 bp (Fig 3b) whereas introns are commonly shorter than 1,000 bp
(Fig 3c). A gene in the genome of D. anomala contains on average 1.3 exons and 0.3 introns.
The shortest protein (67 aa) encodes a 40S ribosomal protein s28-a (part of the small ribosomal
subunit) whereas the longest 4,967 encodes for midasin (nuclear chaperone). RPS28A, the 40S
ribosomal protein homologue in S. cerevisiae has a length of 67 aa as well (Uniprot). MDN1, the
midasin homologue in S. cerevisiae has a length of 4,910 aa (Uniprot). The average protein length
distribution is shown in Fig 3d).

IV.

Gene annotation

Blast2GO annotated about 3,000 out of the 4,160 predicted genes (Fig 8). Woolfit et al., 2007 reported
about 3,000 genes for a D. bruxellensis assembly, Curtin et al., 2012 about 4,969 and Pikur et al.,
2012 of about 5,600 genes.
To address the completeness of the assembly in terms of proteins, a set of 248 highly conserved
core proteins deposited by Parra et al., 2009 was searched for in the predicted protein sequences.
A blastp analysis revealed hits for 219 CEGs (core eukaryotic genes) with a coverage greater
than 70% and e-value threshold of 1e-10. A tblastn analsyis further revealed hits for 28 of the
remaining CEGs. The remaining CEG candidate (KOG3479 Mitochondrial import inner membrane
translocase, subunit TIM9 [Intracellular trafficking, secretion, and vesicular transport]) could be
found in the initial blastp results (hit = coverage of 69% and a sequence identity of 92%) but could
not be picked up by the blast-result parsing scripts due to the sequence coverage threshold set to
70%. The hereby presented draft genome assembly of D. anomala therefore very likely contains the
entire set of 248 CEG proteins.
All the investigations to address the completeness of the genome indicate a rather complete
genome assembly of D. anomala strain YV396. The next chapters discuss certain pathways and
enzymes of importance to beer production.

0.0008

0.0008

0.0007

0.0007

0.0006

0.0006

0.0005

0.0005

Frequency

Frequency

EurekaBrewing.wordpress.com September 2015

0.0004
0.0003

0.0004
0.0003

0.0002

0.0002

0.0001

0.0001

0.0000
0

2000

4000

6000

8000

10000 12000 14000 16000

Transcript sequence length [bp]

0.0000
0

(a) Transcript sequence length distribution

3000

Exon length [bp]

4000

5000

6000

0.0020

0.008

0.0015

0.006

Frequency

Frequency

2000

(b) Exon sequence length distribution

0.010

0.004

0.0010

0.0005

0.002
0.000
0

1000

200

400

600

Intron length [bp]

800

1000

0.0000
0

(c) Intron sequence length distribution

500

1000

1500

2000

Protein sequence length [aa]

2500

3000

(d) Protein sequence length distribution

Figure 3: Length distribution histograms for D. anomala transcripts, exons, introns as well as proteins

Table 2: D. anomala YV396 gene prediction statistics

Name
Total predicted genes
Average gene length [bp]
Total predicted transcripts
Average transcript length
Introns [n]
Average intron length [bp]
Exons [n]
Average exon length [bp]

4,050
1,715
4,160
1,742
1,277
473
5,435
1,221

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V.

Carbohydrate metabolism

Various enzymes are part of the carbohydrate metabolism in yeasts (Fig 4). The most important
ones for beer production are glucosidases cleaving sugars from polysaccharides such as maltose,
sucrose or even starch and cellulose.
At least one alpha-glucosidase (EC 3.2.1.20) could be predicted in the D. anomala genome which
was however not annotated by the Blast2GO pipeline (not colorized EC 3.2.1.20 in Fig 4). Alphaglucosidase is capable of cleaving terminal 1>4 linked sugars of higher sugars such as sucrose and
starch. The predicted D. anomala alpha-glucosidase is 949 amino acids in length and shows high
sequence identities to alpha-glucosidase found in other yeasts like Candida albicans.
One beta-glucosidase candidate protein (also called cellulase, EC 3.2.1.21) could be predicted in
the D. anomala genome. This enzyme can cleave 1>4 beta-D-glucose from polysaccharides like
cellulose. It is 841 amino acids in length and shows high sequence identity to a D. bruxellensis
beta-glucosidase (90% sequence identity, total length 841 amino acids, EIF45415.1, deposited by
Curtin et al., 2012). The sequence of the predicted D. anomala beta-glucosidase from this genome
assembly was already deposited on NCBI by Vervoort et al., 2015 with the accession number
AKS48904.1.

Figure 4: Enzymes linked to starch and sucrose metabolism - Enzymes highlighted in color could be predicted and
annotated on the D. anomala genome

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VI.

Phenolic acid metabolism

The spoilage potential of Brettanomyces is mainly associated with volatile phenols like 4-ethylphenol
(animal, leather, horse sweat) and 4-ethylguaiacol (spicy, clove-like) [Doss, 2008]. These compounds
have rather low sensory threshold levels and can easily be picked up in products like wine
[Campolongo et al., 2010]. Two key enzymes are involved in the metabolism of hydroxycinnamic
acids into volatile phenols (Fig 5). The hydroxycinnamic acids originate from cinnamic acids
which are metabolized by an esterase [Kheir et al., 2013]. Cinnamic acids are part of plant cell
walls and are associated with antimicrobial function Godoy et al., 2009.
The first enzyme, a phenolic acid decarboxylase (padc), is responsible for the conversion of
hydroxycinnamic acids into vinyl derivatives. Another enzyme (vinylphenol reductase (vpr)) then
reduces the vinyl derivatives into ethyl phenols. The following two chapters discuss PADC and
VPR in more detail.

Figure 5: Phenolic acid metabolism - based on two enzymes. First row of compounds (top-bottom) represent hydroxycinnamic acids, second row represent vinyl derivaties, third row represent ethyl derivatives

VI.1

Phenolic acid decarboxylase

Edlin et al., 1995 first identified and described the function of a D. anomala enzyme capable of
transforming p-coumaric acids into 4-vinyl derivaties: phenolic acid decarboxylase (padc). PADC
have homologues in other species such as S. cerevisiae, Lactobacillus plantarum, Pediococcus pentosaceus
or Pseudomonas fluorescens [Harris et al., 2009]. The first sequencing projects of D. bruxellensis by
Woolfit et al., 2007 revealed a protein with sequence similarities to Protoplast secreted protein 2
(PST2) found in S. cerevisiae. PST2 is however not linked to any hydroxycinnamic acid metabolism
up to today.
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(a) Phylogenetic tree

(b) PADC homologue structure

(c) PST2 homologue structure

Figure 6: (a) Phylogenetic analysis of PADC homologues from different species - analysis performed using
http://www.phylogeny.fr (b) SWISS-MODEL for PADC homologue (c) SWISS-MODEL for PST2 homologue

The D. anomala draft genome contains two different sequences that can be linked to either PST2 or
PADC (Fig 6a). The PADC homologue is 176 amino acids in length with close sequence identities
to a predicted protein from a fungi called Nectria haematococca (XP_003042417) and contains a
phenolic acid decarboxylase domain (IPR008729). The PST2 homologue is around 258 amino acids
in length with close sequence identities to B. bruxellensis protoplast secreted protein 2 precursor,
EIF46519, and contains a flavodoxin binding site. These results suggest the presence of two
proteins that might be involved in the metabolism of hydroxycinnamic acids into vinyl derivatives.
To further evaluate the possible function of the two proteins, a protein structure prediction was
performed using SWISSPROT [Biasini et al., 2014; Arnold et al., 2006]. The D. anomala PADC
homologue revealed a structre modeled on a ferulic acid decarboxylase model working as homodimers (Fig 6b). Indicating a possible function as a decarboxylase like PADC. The structure of the
PST2 homologue was modeled based on a flavoprotein wrbA and resulted in a homo-tetramer
structure (Fig 6c). Further investigations are necessary to understand and address whether these
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two phenolic acid decarboxylase protein candidates are actually linked to the phenolic acid
metabolism.
VI.2

Vinylphenol reductase

Vinylphenol reductase (VPR) is a very unique enzyme present only in yeasts like Dekkera [Tchobanov
et al., 2008]. The enzyme can turn vinyl derivatives such as 4-vinyl guaiacol, 4-vinylphenol or 4vinylcatechol into compounds like 4-ethyl guaiacol, 4-ethyl phenol or 4-ethyl catechol respectively
(Fig 5).
Tchobanov et al., 2008 were of the first to characterize VPR. The authors purified VPR from D.
bruxellensis and charazterized the enzyme in more detail. The empirically determined protein
weight was around 26 kDa (SDS-PAGE) and a length of 210 amino acids was determined by
peptide sequencing. The authors then generated an in silico DNA sequence from the peptide
sequence. Parts of the DNA sequence were later on confirmed by Campolongo et al., 2010.
The draft genome of Dekkera anomala contains one predicted protein sequence with high sequence
similarity to the in silico sequence provided by Tchobanov et al., 2008. The retreived sequence is
210 amino acids in length and shows about 77.6% sequence identity compared to the predicted
in-silico sequence (Fig 7). Although the VPR activity is reported to be NADH dependent [Kheir
et al., 2013], no NADH binding site could be predicted for both sequences.

Figure 7: Sequence alignment of predicted VPR protein from D. anomala (top) versus the predicted in-silico sequence
from Tchobanov et al., 2008 (bottom)

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III.
I.

Methods

Genome assembly

The draft genome assembly of Dekkera anomala strain YV396 (isolated from a Belgian brewery) was
retrieved from GenBank (accession number LCTY00000000.1; June 2015) deposited in May 2015
by KU Leuven Vervoort et al. [2015]. Illumina HiSeq data (100x coverage) was assembled into a
genome using SOAPdenovo v.1.05. The statistics for the obtained assembly are summarized in
Tab. 1).

II.

Gene prediction

Gene prediction on contigs was performed using the AUGUSTUS web-service (AUGUSTUS
parameter project identifier: pichia_stipitis, UTR prediction: false, report genes on both strands,
alternative transcripts few, allowed gene structure: predict any number of (possibly partial) genes,
ignore conflicts with other strand: false) [Stanke et al., 2006, 2008]. The gene prediction statistics
are summarized in Tab. 2.

III.

Gene annotation

Gene annotation was performed by Blast2GO including remote blastx on NCBI and InterProScan
for domain predictions [Conesa et al., 2005]. GO-term mapping and annotation performed by
Blast2GO pipeline. Close to 3,000 out of the predicted 4,160 sequences could be annotated by
Blast2GO (Fig 8). Another subset of about 600 sequences could be mapped to a biological function
without a GO term and about 460 sequences only resulted in BLAST hits which could not be
further associated with a protein function.
Most abundant species associated with the best blastx hits were Dekkera bruxellensis, Ogataea
polymorpha and Pichia kudriavzevi (not shown).

Figure 8: Distribution of predicted D. anomala genes after Blast2GO annotation

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