Insight into the Dekkera anomala YV396 genome

EurekaBrewing.wordpress.com • September 2015

Insight into the Dekkera anomalaYV396 genome

Samuel H. Aeschlimann*

*EurekaBrewing.wordpress.com (powered by Blackwell Brewery)

Abstract

The yeast Dekkera (anamorph Brettanomyces) is associated with various industrial fermentations andcurrently experiences an increase of interest in the Wild Ale beer brewing scene. The notorious spoilageyeast is capable of forming volatile phenols with flavor descripties like ’wet animal, musty’. Due to the highabundance of spoilage in the wine industry, so far only genomes of Brettanomyces bruxellensis have beenpublished and discussed. Other species from the Dekkera taxa include Dekkera anomala mainly associatedwith beers such as Belgian Lambics. The current draft Dekkera anomala YV396 genome assembly consistsof 30 contigs with an estimated genome size of 12.9 Mb. Addressing the degree of completeness of thegenome revealed the existance of a set of tRNAs capable of encoding the standard 20 amino acids includingtwo tRNAs encoding for selenocysteine. Furthermore the entire set of 248 CEGs could be found in theassembly indicating a rather complete draft genome. 4,000 genes were predicted of which the main parthas homologues in Brettanomyces bruxellensis. Two phenolic acid decarboxylses as well as one vinylphenolreductase - 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 associateswith flavor compounds in beer production.

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

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Contents

I Introduction 2

II Results and Discussion 3I Genome assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3II tRNA analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4III Gene prediction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4IV Gene annotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4V Carbohydrate metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6VI Phenolic acid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

VI.1 Phenolic acid decarboxylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7VI.2 Vinylphenol reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

III Methods 10I Genome assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10II Gene prediction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10III Gene annotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

I. Introduction

Dekkera (anamorph Brettanomyces) are notorious beverage spoilage yeasts associated with a varietyof 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 lotof research has been conducted on Dekkera to further understand the associated pathways leadingto 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 asthese compounds exhibit low detection thresholds [Curtin et al., 2012]. Beside its importance inthe wine industry, Dekkera is also present in Belgian beers like Lambics and Gueuzes where thevolatile 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 betterunderstand the organisms and metabolic pathways. This publication discusses the genomeassembly created by Vervoort et al. [2015], the gene prediction as well as certain pathways linkedto beer production.

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II. Results and Discussion

I. Genome assembly

The current D. anomala genome consists of 30 contigs with an estimated genome size of about12.9 Mb (Tab 1). The estimated genome is similar in size to the close relative D. bruxellensis orSaccharomyces cerevisiae [Goffeau et al., 1996; Curtin et al., 2012]. Looking at the GC content of theD. anomala draft genome in comparison to other Dekkera genomes reveales a similar distributionpattern (Fig 2).

Table 1: D. anomala YV396 genome statistics (accession number GCA_001005505.1)

Name

Total sequence length [bp] 12,880,761Number of contigs [n] 30Contig N50 [bp] 1,112,461

Figure 2: GC plots for D. bruxellensis CBS2499 [Piškur 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]

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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 tRNAssufficient 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 aparameter 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 inTab 2. 4,050 potential genes and 4,160 potential transcripts could be predicted. The average codingsequence length is about 1,600 bp with the longest transcript length of 14,904 bp (Fig 3a). Exonsare 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 ribosomalsubunit) whereas the longest 4,967 encodes for midasin (nuclear chaperone). RPS28A, the 40Sribosomal protein homologue in S. cerevisiae has a length of 67 aa as well (Uniprot). MDN1, themidasin homologue in S. cerevisiae has a length of 4,910 aa (Uniprot). The average protein lengthdistribution 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 reportedabout 3,000 genes for a D. bruxellensis assembly, Curtin et al., 2012 about 4,969 and Piškur et al.,2012 of about 5,600 genes.

To address the completeness of the assembly in terms of proteins, a set of 248 highly conservedcore 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 greaterthan 70% and e-value threshold of 1e-10. A tblastn analsyis further revealed hits for 28 of theremaining CEGs. The remaining CEG candidate (KOG3479 Mitochondrial import inner membranetranslocase, subunit TIM9 [Intracellular trafficking, secretion, and vesicular transport]) could befound in the initial blastp results (hit = coverage of 69% and a sequence identity of 92%) but couldnot be picked up by the blast-result parsing scripts due to the sequence coverage threshold set to70%. The hereby presented draft genome assembly of D. anomala therefore very likely contains theentire set of 248 CEG proteins.

All the investigations to address the completeness of the genome indicate a rather completegenome assembly of D. anomala strain YV396. The next chapters discuss certain pathways andenzymes of importance to beer production.

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0 2000 4000 6000 8000 10000 12000 14000 16000Transcript sequence length [bp]

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(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 4,050Average gene length [bp] 1,715Total predicted transcripts 4,160Average transcript length 1,742Introns [n] 1,277Average intron length [bp] 473Exons [n] 5,435Average exon length [bp] 1,221

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V. Carbohydrate metabolism

Various enzymes are part of the carbohydrate metabolism in yeasts (Fig 4). The most importantones 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 whichwas however not annotated by the Blast2GO pipeline (not colorized EC 3.2.1.20 in Fig 4). Alpha-glucosidase is capable of cleaving terminal 1>4 linked sugars of higher sugars such as sucrose andstarch. The predicted D. anomala alpha-glucosidase is 949 amino acids in length and shows highsequence 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 inthe D. anomala genome. This enzyme can cleave 1>4 beta-D-glucose from polysaccharides likecellulose. It is 841 amino acids in length and shows high sequence identity to a D. bruxellensisbeta-glucosidase (90% sequence identity, total length 841 amino acids, EIF45415.1, deposited byCurtin et al., 2012). The sequence of the predicted D. anomala beta-glucosidase from this genomeassembly was already deposited on NCBI by Vervoort et al., 2015 with the accession numberAKS48904.1.

Figure 4: Enzymes linked to starch and sucrose metabolism - Enzymes highlighted in color could be predicted andannotated 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 compoundshave 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 hydroxycinnamicacids into volatile phenols (Fig 5). The hydroxycinnamic acids originate from cinnamic acidswhich are metabolized by an esterase [Kheir et al., 2013]. Cinnamic acids are part of plant cellwalls and are associated with antimicrobial function Godoy et al., 2009.

The first enzyme, a phenolic acid decarboxylase (padc), is responsible for the conversion ofhydroxycinnamic acids into vinyl derivatives. Another enzyme (vinylphenol reductase (vpr)) thenreduces the vinyl derivatives into ethyl phenols. The following two chapters discuss PADC andVPR in more detail.

Figure 5: Phenolic acid metabolism - based on two enzymes. First row of compounds (top-bottom) represent hydrox-ycinnamic 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 oftransforming p-coumaric acids into 4-vinyl derivaties: phenolic acid decarboxylase (padc). PADChave homologues in other species such as S. cerevisiae, Lactobacillus plantarum, Pediococcus pentosaceusor Pseudomonas fluorescens [Harris et al., 2009]. The first sequencing projects of D. bruxellensis byWoolfit 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 metabolismup 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 usinghttp://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 orPADC (Fig 6a). The PADC homologue is 176 amino acids in length with close sequence identitiesto a predicted protein from a fungi called Nectria haematococca (XP_003042417) and contains aphenolic acid decarboxylase domain (IPR008729). The PST2 homologue is around 258 amino acidsin 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 twoproteins 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 wasperformed using SWISSPROT [Biasini et al., 2014; Arnold et al., 2006]. The D. anomala PADChomologue revealed a structre modeled on a ferulic acid decarboxylase model working as homo-dimers (Fig 6b). Indicating a possible function as a decarboxylase like PADC. The structure of thePST2 homologue was modeled based on a flavoprotein wrbA and resulted in a homo-tetramerstructure (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 acidmetabolism.

VI.2 Vinylphenol reductase

Vinylphenol reductase (VPR) is a very unique enzyme present only in yeasts like Dekkera [Tchobanovet al., 2008]. The enzyme can turn vinyl derivatives such as 4-vinyl guaiacol, 4-vinylphenol or 4-vinylcatechol 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 proteinweight was around 26 kDa (SDS-PAGE) and a length of 210 amino acids was determined bypeptide sequencing. The authors then generated an in silico DNA sequence from the peptidesequence. 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 sequencesimilarity to the in silico sequence provided by Tchobanov et al., 2008. The retreived sequence is210 amino acids in length and shows about 77.6% sequence identity compared to the predictedin-silico sequence (Fig 7). Although the VPR activity is reported to be NADH dependent [Kheiret 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 sequencefrom Tchobanov et al., 2008 (bottom)

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III. Methods

I. Genome assembly

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

II. Gene prediction

Gene prediction on contigs was performed using the AUGUSTUS web-service (AUGUSTUSparameter 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 statisticsare summarized in Tab. 2.

III. Gene annotation

Gene annotation was performed by Blast2GO including remote blastx on NCBI and InterProScanfor domain predictions [Conesa et al., 2005]. GO-term mapping and annotation performed byBlast2GO pipeline. Close to 3,000 out of the predicted 4,160 sequences could be annotated byBlast2GO (Fig 8). Another subset of about 600 sequences could be mapped to a biological functionwithout a GO term and about 460 sequences only resulted in BLAST hits which could not befurther associated with a protein function.

Most abundant species associated with the best blastx hits were Dekkera bruxellensis, Ogataeapolymorpha and Pichia kudriavzevi (not shown).

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

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References

Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006). The SWISS-MODEL workspace: aweb-based environment for protein structure homology modelling. Bioinformatics, 22:195–201.

Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., Kiefer, F., Cassarino, T.,Bordoli, L., and Schwede, T. (2014). SWISS-MODEL: modelling protein tertiary and quaternarystructure using evolutionary information. Nucleic Acids Research, 42.

Campolongo, S., Lamberti, C., Rantsiou, K., Pessione, E., and Cocolin, L. (2009-2010). Brettanomycesbruxellensis and off-odours: Genetic and proteomic approaches to unravel the molecular mecha-nism of ethyl-phenol production. QUAD. VITIC. ENOL. UNIV. TORINO, 31.

Conesa, A., Götz, S., García-Gómez, J. M., Terol, J., Talón, M., and Robles, M. (2005). Blast2GO:a universal tool for annotation, visualization and analysis in functional genomics research.Bioinformatics, 21(18):3674–3676.

Curtin, C., Borneman, A., Chambers, P., and Pretorius, I. (2012). De-novo assembly and analysisof the heterozygous triploid genome of the wine spoilage yeast Dekkera bruxellensis AWRI1499.PLoS One, 7(3).

Doss, G. (2008). Brettanomyces: Flavors and performance of single and multiple strain fermentationswith respect to time. AHA NHC June 2008 lecture slides.

Edlin, D., Narbad, A., J.R., D., and Lloyd, D. (1995). The biotransformation of simple phenoliccompounds by Brettanomyces anomalus. FEMS Microbiology Letters, 125:311–316.

Godoy, L., Garrido, D., Martinez, C., Saavedra, J., Combina, M., and Ganga, M. (2009). Study of thecoumarate decarboxylase and vinylphenol reductase activities of Dekkera bruxellensis (anamorphBrettanomyces bruxellensis) isolates. Letters in Applied Microbiology, 48:452–4557.

Goffeau, A., Barrell, B. G., Bussey, H., Davis, R. W., Dujon, B., Feldmann, H., Galibert, F., Hoheisel,J. D., Jacq, C., Johnston, M., Louis, E. J., Mewes, H. W., Murakami, Y., Philippsen, P., Tettelin, H.,and Oliver, S. G. (1996). Life with 6000 Genes. Science, 274(5287):546–567.

Harris, V., Ford, C., Jiranek, V., and Grbin, P. (2009). Survey of enzyme activity responsible forphenolic off-flavour production by Dekkera and Brettanomyces yeast. Appl Microbiol Biotechnol,81:1117–1127.

Kheir, J., Salameh, D., Strehaiano, P., Brandman, C., and Lteif, R. (2013). Impact of volatile phenolsand their precursors on wine quality and control measures of Brettanomyces/Dekkera yeasts. EurFood Res Technol.

Lowe, T. M. and Eddy, S. R. (1997). tRNAscan-SE: A Program for Improved Detection of TransferRNA Genes in Genomic Sequence. Nucleic Acids Research, 25(5):0955–964.

Parra, G., Bradnam, K., Ning, Z., Keane, T., and Korf, I. (2009). Assessing the gene space in draftgenomes. Nucleic Acids Research, 37(1):289–297.

Piškur, J., Ling, Z., Marcet-Houben, M., Ishchuk, O., Aerts, A., LaButti, K., Copeland, A., Lindquist,E., Barry, K., Compagno, C., Bisson, L., Grigoriev, I., Gabaldón, T., and Phister, T. (2012). Thegenome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties.International Journal of Food Microbiology, 157:202–209.

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Spitaels, F., D., W. A., Maarten, J., Maarten, A., Heide-Marie, D., Anita, V. L., Luc, D. V., and Peter,V. (2014). The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer. PLoSONE, 9(4):e95384.

Stanke, M., Diekhans, M., Baertsch, R., and Haussler, D. (2008). Using native and syntenicallymapped cDNA alignments to improve de novo gene finding. Bioinformatics, 24(5):637–644.

Stanke, M., Schoffmann, O., Morgenstern, B., and Waack, S. (2006). Gene prediction in eukary-otes with a generalized hidden Markov model that uses hints from external sources. BMCBioinformatics, 7(1):62.

Tchobanov, I., Gal, L., Guilloux-Benatier, M., Remize, F., Nardi, T., Guzzo, J., Serpaggi, V., andAlexandre, H. (2008). Partial vinylphenol reductase purification and characterization fromBrettanomyces bruxellensis. FEMS Microbiol. Lett., 284:213–217.

Valdes, J., Tapia, P., Cepeda, V., Varela, J., Godoy, L., Cubillos, F., Silva, E., Martinez, C., andGanga, M. (2014). Draft genome sequence and transcriptome analysis of the wine spoilageyeast Dekkera bruxellensis LAMAP2480 provides insights into genetic diversity, metabolism andsurvival. FEMS Microbiology Letters, 361(2).

Vervoort, Y., Herrera-Malaver, B., Mertens, S., Guadalupe Medina, V., Duitama, J., Michiels, L.,Derdelinckx, G., Voordeckers, K., and Verstrepen, K. (2015). Purification and characterizationof a novel Brettanomyces anomalus beta-glucosidase enzyme suitable for food bioflavoring.unpublished.

White, C. and Zainasheff, J. (2010). Yeast: the practical guide to beer fermentation. Brewers Publication.

Woolfit, M., Rozpedowska, E., Piškur, J., and Wolfe, K. (2007). genome Survey Sequencing of theWine Spoilage Yeast Dekkera (Brettanomyces) bruxellensis. Eukaryotic Cell, 6(4):721–733.

Yakobson, C. (2010). Brettanomyces project.

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Insight into the Dekkera anomala YV396 genome

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