S HOR T COMMUN I CA T I ON

Genome sequence of Wickerhamomyces anomalus DSM 6766reveals genetic basis of biotechnologically important

antimicrobial activities

Jessica Schneider1, Oliver Rupp1, Eva Trost1, Sebastian Jaenicke1, Volkmar Passoth2, AlexanderGoesmann1, Andreas Tauch1 & Karina Brinkrolf1

1Center for Biotechnology, Bielefeld University, Bielefeld, Germany; and 2Department of Microbiology, Swedish University of Agriculture Science,

Uppsala, Sweden

Correspondence: Karina Brinkrolf, Center

for Biotechnology, Bielefeld University,

Universitatsstrasse 27, 33615 Bielefeld,

Germany. Tel.: +495211068763; fax:

+4952110689041; e-mail: kbrinkro@cebitec.

uni-bielefeld.de

Received 22 September 2011; revised 13

December 2011; accepted 24 January 2012.

Final version published online 23 February

2012.

DOI: 10.1111/j.1567-1364.2012.00791.x

Editor: Isak Pretorius

Keywords

next-generation sequencing; killer protein;

volatile compound.

Abstract

The ascomycetous yeast Wickerhamomyces anomalus (formerly Pichia anomala

and Hansenula anomala) exhibits antimicrobial activities and flavoring features

that are responsible for its frequent association with food, beverage and feed

products. However, limited information on the genetic background of this yeast

and its multiple capabilities are currently available. Here, we present the draft

genome sequence of the neotype strain W. anomalus DSM 6766. On the basis of

pyrosequencing, a de novo assembly of this strain resulted in a draft genome

sequence with a total size of 25.47 Mbp. An automatic annotation using RAPYD

generated 11 512 protein-coding sequences. This annotation provided the basis

to analyse metabolic capabilities, phylogenetic relationships, as well as biotechno-

logically important features and yielded novel candidate genes of W. anomalus

DSM 6766 coding for proteins participating in antimicrobial activities.

The kingdom Fungi includes a variety of biotechnologi-

cally important yeast species. One such representative is

Wickerhamomyces anomalus that was hitherto known as

Pichia anomala or Hansenula anomala and was recently

assigned to the genus Wickerhamomyces (Kurtzman &

Suzuki, 2010). Wickerhamomyces anomalus exhibits a

multitude of biotechnologically important characteristics

in flavor enhancement, food and feed processing, biopre-

servation, dairy fermentation and waste water treatment.

This versatility is encouraged by the ability to tolerate

extreme environmental conditions like oxidative, salt,

osmotic stress, as well as pH and temperature shocks

(Walker, 2011). Furthermore, a broad-range antimicro-

bial activity against a variety of species from the king-

doms Fungi (Aspergillus, Penicillium, Fusarium) and

Bacteria (Erwinia, Enterobacteriaceae and Streptococci) is

a characteristic feature of W. anomalus strains that is rel-

evant to the improvement of industrial processes (Ols-

torpe & Passoth, 2011; Walker, 2011). This antimicrobial

activity is because of the biosynthesis of volatile com-

pounds such as ethyl acetate, isoamyl acetate and ethyl

propionate (Druvefors et al., 2005; Masoud et al., 2005)

or mediated by killer proteins (Passoth et al., 2006).

Killer proteins are often assigned to glucanases that are

able to convert glucan, a structural component of cell

walls of yeasts and other fungi, to simpler glucose mole-

cules (Satyanarayana & Kunze, 2009). Owing to its anti-

microbial effect, W. anomalus is used for biopreservation

of inoculated cereal feed grain (Olstorpe et al., 2010),

the prevention of beer gushing and spoilage (Laitila

et al., 2011), and the improvement of bioethanol yields

in airtight storage of wheat (Passoth et al., 2009). More-

over, glucanases originating from W. anomalus strains

can be used to improve the biotechnological synthesis of

recombinant proteins produced with other yeasts by

causing a controlled lysis of the cell wall of the respec-

tive strain along with a release of expressed proteins

(Satyanarayana & Kunze, 2009). The toxin derivatives of

W. anomalus may be used also to develop novel vaccines

(Polonelli et al., 2011).

ª 2012 Federation of European Microbiological Societies FEMS Yeast Res 12 (2012) 382–386Published by Blackwell Publishing Ltd. All rights reserved

YEA

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The diversity of application examples for W. anomalus

emphasizes the biotechnological potential of W. anomalus

strains and reinforces the demand for basic genomic

investigations. Therefore, the draft genome sequence of

the diploid neotype strain W. anomalus DSM 6766 was

established by whole-genome-shotgun and paired-end

sequencing with the Genome Sequencer FLX system and

subsequent data assembly with the GS De Novo Assembler

version 2.5 from 454 Life Sciences. In total, 925 Mb of

genomic information were used to generate 3311 contigu-

ous sequences (� 200 bp) in 356 scaffolds that sum up

to a total sequence length of 25.47 Mbp (NCBI master

record AEGI00000000.2). A summary of the sequencing

approach and the de novo assembly is displayed in

Table 1. An automated regional and functional annota-

tion of the draft genome sequence was achieved with RA-

PYD (Schneider et al., 2011). RAPYD is an efficient

bioinformatics platform that covers eukaryotic gene pre-

diction, genome annotation, comparative genomics and

metabolic pathway reconstruction. For W. anomalus DSM

6766, 11 512 protein-coding sequences were predicted,

whereof 78.57% were classified as single-exon genes. The

predicted protein-coding regions cover 63.36% of the

genome sequence, and their average length is 1408 bp.

Detailed results of the automatic annotation process are

summarized in Table 2. Access to the draft genome

sequence and its annotation is provided via the RAPYD

platform (https://rapyd.cebitec.uni-bielefeld.de).

The annotation was used for comparative yeast genom-

ics and phylogenetic analysis. The genome sequences of

Komagataella pastoris (PRJEA62483), Candida glabrata

(PRJNA13831), Candida dubliniensis (PRJEA34697),

Scheffersomyces stipitis (PRJNA16843), Saccharomyces cere-

visiae (PRJNA128), Klyveromyces lactis (PRJNA12377),

Debaromyces hansenii (PRJNA12410), Yarrowia lipolytica

(PRJNA12414) and W. anomalus NRRL Y-366 were anal-

ysed. Among these yeasts, the genomes of S. cerevisiae

and C. dubliniensis were selected as representatives of the

Saccharomyces and Candida clade, respectively (Butler

et al., 2009).

All these yeasts share a total set of 1020 proteins, the

core genome, which was used to calculate a phylogenetic

tree (Fig. 1a). An analysis of singletons revealed 3087 spe-

cies-specific proteins for W. anomalus DSM 6766 that

have no significant homology to proteins of the other

selected yeasts, displaying a huge number of proteins spe-

cific to the newly described genus Wickerhamomyces. As

an example, S. cerevisiae share only 1799 proteins with

W. anomalus DSM (Fig. 1b). Thus, the singletons of W.

anomalus DSM 6766 can be deduced in more detail when

closely related species of the Wickerhamomyces genus are

sequenced in the future.

Furthermore, bioinformatics analyses of proteins of

W. anomalus DSM 6766 were focused on the genome-

wide identification of biocontrol agents including volatile

compounds and b-glucanases. Sequences of identified

protein candidates were deposited at the NCBI (JN701425

–JN701452).The major volatile compound produced by W. anoma-

lus is ethyl acetate. In general, ethyl acetate can be pro-

duced by two different reactions in yeast involving either

an alcohol acetyltransferase (EC 2.3.1.84) using ethanol

and acetyl coenzyme A as precursors or an esterase (EC

3.1.1.1) using ethanol and acetate (Fredlund et al., 2004).

In S. cerevisiae, the alcohol acetyltransferases ATF1 and

ATF2 were associated with the formation of volatile

esters, in particular ethyl acetate and isoamyl acetate

(Verstrepen et al., 2003). A bioinformatics search with

the associated protein families (PFAM) database model

PF07247 was performed (E-value cutoff 10�35), to iden-

tify the potential candidates involved in the generation

of ethyl acetate. This search revealed six protein hits to

Table 1. Genome sequencing and assembly results of

Wickerhamomyces anomalus DSM 6766

Features of the genome sequencing project Result

Number of sequence reads 2 776 564

Number of sequenced bases 900 439 834

Size of assembled sequence (bp) 25 465 571

Number of all contigs 3311

Largest contig (bp) 144 422

N50 contig size (bp) 21 958

Number of scaffolds 356

Number of contigs in scaffolds 1735

Size of assembled scaffolds 23 358 999

N50 scaffold size (bp) 97 954

Mean G + C content (%) 33.1

Table 2. Results of the regional and functional annotation process

applying RAPYD

Features of the annotation Result

Number of protein-coding genes 11 512

Number of single-exon genes 9045

Number of multi-exon genes 2467

Genes with two exons 2072

Genes with three exons 331

Genes with four exons 49

Genes with five or more exons 15

Number of pseudo-genes 263

Genes with functional descriptions 7444

Genes with gene name abbreviations 3398

Genes with assigned EC numbers 2712

Number of hypothetical proteins 3288

Number of signal peptides 804

Thereof hypothetical proteins 366

Number of transmembrane proteins 1234

Thereof hypothetical proteins 412

FEMS Yeast Res 12 (2012) 382–386 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Draft genome sequence of Wickerhamomyces anomalus 383

potential alcohol acetyltransferases (WAN_10657,

WAN_10642, WAN_10373, WAN_11037, WAN_10494

and WAN_11039). Additionally, a search for esterases

using the PFAM model PF00135 resulted in eight candi-

date proteins (WAN_6336, WAN_6503, WAN_6449,

WAN_1455, WAN_6337, WAN_6502, WAN_6338 and

WAN_6501). These proteins represent the starting point

for further investigations to determine the functional

proteins associated with the production of ethyl acetate

by W. anomalus DSM 6766.

Besides the volatile compounds, potential killer pro-

teins also represent an interesting target for bioinformat-

ics analysis in W. anomalus yeasts. W. anomalus strain K

has been shown to encode the b-1,3-glucanases PAEXG1

(GI:4007653) and PAEXG2 (GI:4007667) involved in the

biological control activity against the apple pathogen

Botrytis cinerea (Friel et al., 2007). Wickerhamomyces

anomalus DBVPG 3003 secretes a b-1,6-glucanase killer

protein (Pikt) that exhibits antifungal activity against

Brettanomyces and Dekkera yeasts. The N-terminal

sequence of Pikt (MQIFVXTLTG) is identical to that of

the ubiquitin protein UBI2 (GI:450555) that was initially

described in S. cerevisiae. Both proteins have similar

molecular masses but differ in the sensitivity to proteo-

lytic enzymes (De Ingeniis et al., 2009). Nevertheless, a

complete nucleotide sequence of Pikt is not available.

Therefore, HMM searches for W. anomalus DSM 6766

were carried out using the PFAM models PF00150 and

PF00240 for b-1,3-glucanases and b-1,6-glucanases,respectively.

Fig. 1. Visualization of the comparative genome analysis of Wickerhamomyces anomalus DSM 6766. (a) Taxonomic distance tree based on the

computed core genome that covers 1020 orthologous proteins. The predicted gene set of W. anomalus DSM 6766 was reduced to a haploid set

consisting of 6593 genes by means of BLAT (Kent, 2002). Match ratio values (number of identical bases divided by the length of the gene) were

computed for each aligned gene pair. One gene of a gene pair was added to a haploid gene set, if the ratio values of both genes were larger

than 0.95. (b) Venn diagram showing the comparison of the proteomes of W. anomalus DSM 6766 and Saccharomyces cerevisiae S288c.

Fig. 2. Phylogenetic trees of putative glucanases that were identified by HMM searches. (a) Putative ß-1,3-glucanases and their relationship to

PAEXG1 and PAEXG2 of Wickerhamomyces anomalus strain K. (b) Putative ß-1,6-glucanases of W. anomalus DSM 6766 and UBI2 of

Saccharomyces cerevisiae. These proteins contain the N-terminal sequence motif (MQIFVXTLTG) determined for the Pikt protein of W. anomalus

DBVPG 3003 (De Ingeniis et al., 2009). (c) The location of this motif is visualized by gray lines within the associated proteins. The proteins

contain at least one conserved ubiquitin domain.

ª 2012 Federation of European Microbiological Societies FEMS Yeast Res 12 (2012) 382–386Published by Blackwell Publishing Ltd. All rights reserved

384 J. Schneider et al.

The search for b-1,3-glucanases revealed eight proteins,

of which two were orthologous each to PAEXG1

(WAN_362 and WAN_6658) and PAEXG2 (WAN_2708

and WAN_9541) of W. anomalus strain K (Fig. 2a).

Thereby, the length of these orthologous proteins is iden-

tical to that of PAEXG1 and PAEXG2 with a few amino

acid replacements (WAN_362: E67D, P163L, S306T,

T374S, P479S; WAN_2708: S259Y, E300D, A303T,

T418N; and WAN_9541: E146K, N198K, S259Y, D367N,

T418N). While the physiological function of the PAEXG

homologs is likely to be similar to that in W. anomalus

strain K, the function of the other potential killer proteins

(WAN_7786, WAN_5696, WAN_7540 and WAN_7517)

has to be further investigated and experimentally verified.

The search for b-1,6-glucanases resulted in the identifi-

cation of six proteins (Fig. 2b). All of them carry the

characteristic N-terminal amino acid motif

MQIFVXTLTG known from the Pikt protein of W.

anomalus DBVPG 3003 that is part of a conserved 76

amino acid ubiquitin domain of UBI2 from S. cerevisiae

(De Ingeniis et al., 2009). Thereby, WAN_5293,

WAN_8495, WAN_11511 and WAN_11512 carry one

amino acid motif known from Pikt, whereas WAN_2213

and WAN_2377 proteins contain multiple motifs

(Fig. 2c). Ubiquitin is a small and highly conserved pro-

tein facilitating the protein degradation in eukaryotic

organisms by reversible posttranslational modifications

(Li & Ye, 2008) and is related to various cellular processes

(Kimura & Tanaka, 2010). WAN_11511, WAN_11512

and UBI2 have an identical length of 128 amino acids.

Both W. anomalus proteins differ from UBI2 by only two

amino acid changes (D92E and V95I). Therefore, these

proteins are likely to represent the Pikt orthologs in W.

anomalus DSM 6766. The substitutions do not change the

side chain characteristics and the charge of the proteins.

However, these replacements might cause slightly differ-

ent protein function and different behavior of proteolytic

enzymes as recently suggested for UBI2 and Pikt (De In-

geniis et al., 2009). On the nucleotide level, WAN_11511

and WAN_11512 showed ten substitutions that do not

affect their amino acid sequences. Hence, with the detec-

tion of these genes, the complete nucleotide and the

derived amino acid sequences of potential Pikt candidates

were identified for the first time.

In conclusion, the generation of the draft genome

sequence of W. anomalus DSM 6766 is the first sequenc-

ing and analysis approach of a yeast within the genus

Wickerhamomyces and contributes to the understanding

of characteristic features of this newly described genus.

Our analysis clearly points out that the availability of

high-throughput sequencing data and a mostly auto-

mated sequence analysis enables fast and cost-efficient

insights into species-specific features. The draft genome

sequence presented in this study might contribute to fur-

ther investigations of this biotechnologically important

yeast with regard to fermentation processes and biocon-

trol aspects.

Acknowledgements

J.S. and E.T. acknowledge the receipt of a scholarship from

the CLIB Graduate Cluster Industrial Biotechnology. V.P.

was supported by the thematic research program

MicroDrive. The authors thank the sequencing team of the

Center for Biotechnology for their valuable contribution.

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