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Genetics and Molecular Biology

Print version ISSN 1415-4757On-line version ISSN 1678-4685

Genet. Mol. Biol. vol.43 no.3 Ribeirão Preto  2020  Epub June 08, 2020

https://doi.org/10.1590/1678-4685-gmb-2019-0122 

Genomics and Bioinformatics

Draft genome sequence of Wickerhamomyces anomalus LBCM1105, isolated from cachaça fermentation

Aureliano C. Cunha1 
http://orcid.org/0000-0003-3437-6105

Renato A. Corrêa dos Santos2 
http://orcid.org/0000-0003-0826-5479

Diego M. Riaño-Pachon3 
http://orcid.org/0000-0001-9803-3465

Fábio M. Squina4 

Juliana V. C. Oliveira2 

Gustavo H. Goldman5 

Aline T. Souza2 

Lorena S. Gomes1 

Fernanda Godoy-Santos1 

Janaina A. Teixeira1 

Fábio Faria-Oliveira1 
http://orcid.org/0000-0003-0723-7695

Izinara C. Rosse1 

Ieso M. Castro1 
http://orcid.org/0000-0002-1319-1251

Cândida Lucas6 

Rogelio L. Brandão1 
http://orcid.org/0000-0002-8116-5979

1Universidade Federal de Ouro Preto, Laboratório de Biologia Molecular e Celular, MG, Brazil.

2Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Campinas, SP, Brazil.

3Universidade de São Paulo, Centro de Energia Nuclear na Agricultura, Laboratório de Biologia Computacional, Evolutiva e de Sistemas, Piracicaba, SP, Brazil.

4Universidade de Sorocaba, Programa de Pós-Graduação em Processos Tecnológicos e Ambientais, Sorocaba, SP, Brazil.

5Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Ribeirão Preto, SP, Brazil.

6Universidade do Minho, Centro de Pesquisa Molecular e Ambiental (CBMA), Instituto de Ciência e Inovação para a Bio-Sustentabilidade (IB - S), Braga, Portugal.


Abstract

Wickerhamomyces anomalus LBCM1105 is a yeast isolated from cachaça distillery fermentation vats, notable for exceptional glycerol consumption ability. We report its draft genome with 20.5x in-depth coverage and around 90% extension and completeness. It harbors the sequences of proteins involved in glycerol transport and metabolism.

Keywords: Non-conventional yeast; glycerol; “de novo” assembly; glycerol

Wickerhamomyces anomalus (synonyms Pichia anomala, Hansenula anomala and Candida pelliculosa) are found in several diverse natural habitats, frequently associated with spoilage or processing of food and grain products (Passoth et al., 2006). Different strains of W. anomalus were reported (i) to be able to grow on a wide variety of conditions, including different carbon and nitrogen sources (Conceição et al., 2015; Cunha et al., 2019), at both low and high pH (2.0 to 12.4) and from 3 to 37 °C (Fredlund et al., 2002), (ii) to be highly tolerant to different stress conditions, like osmotic stress (salt), high concentrations of ethanol, and the presence of heavy metals, and (iii) to produce ethanol from glucose, sucrose or xylose. W. anomalus strains have also been reported to display constitutive cyanide-resistant alternative oxidase (Cunha et al., 2019). W. anomalus has been used as a cell factory for the production, among others, of enzymes (Díaz-Rincón et al., 2017), biosurfactants (Teixeira Souza et al., 2018) and fermented-beverages (Aplin et al., 2019). Although W. anomalus strains show a high industrial versatility, only two strains have its genome sequenced to date (Schneider et al., 2012; Riley et al., 2016).

W. anomalus strain LBCM1105 (previously LBCM105) was isolated from sugarcane fermentation vats in a cachaça distillery in Brazil (Conceição et al., 2015), (S22.099694, W41.511090). Extraction of DNA was carried out using the phenol/chloroform method, and purification was performed using the PowerClean DNA Clean-UP kit (MoBio, QIAGEN, Carlsbad, US). The genome size was determined by flow cytometry as previously described (Hare and Johnston, 2011). Cell samples were stained with 2 μM Sytox Green (Thermo Fisher Scientific, MA, US) and the assessment was made in triplicate. The genomic library for sequencing was prepared with the Nextera DNA Library kit (Illumina, San Diego, California, US). Genome sequencing (1.0 million paired-end reads of 151 bp) was performed with an Illumina HiSeq 2500. Quality trimming, and the removal of reads shorter than 90 nucleotides, were carried out using Trimommatic v.0.32 (Bolger et al., 2014). The genome was assembled into contigs (20.5 x in depth coverage, ≥ 1 kb) using SPAdes v.3.11.1, dipSPAdes mode (Bankevich et al., 2012). The completeness was evaluated by BUSCO v.3.0 (Simão et al., 2015), using the Fungi and Saccharomycetales datasets. Genome statistics were computed with QUAST v5.0.2 (Gurevich et al., 2013). A multilocus phylogenetic analysis was performed using RAxML v.8 (Stamatakis, 2014) building a Maximum Likelihood tree based on DNA sequences from the Internal Transcribed Spacers 1 and 2 (ITS1, ITS2), the large and small ribosomal subunits (LSU, SSU), and the Elongation Factor-1α (EF-1α) from species within the genus Barnettozyma, Wickerhamomyces and Candida. The species and the accession numbers of loci LSU, SSU and EF-1α of the related microorganism were previously described (Kobayashi et al., 2017). The accession numbers for ITS are listed in Figure S1). Saccharomyces cerevisiae S288c was used as the outgroup. The sequences of the loci SSU, LSU and EF-1α of the LBCM1105 strain were identified via Blast searches using the proper sequences from W. anomalus NRRL Y-366 as baits (SSU- EF550479.1, LSU- EF550341.1 and EF-1α- EF552565.1). ITS1 and ITS2 sequences from W. anomalus LBCM1105 was extracted using ITSx v.1.0.11 (Bengtsson-Palme et al., 2013). The sequences of ITS1, ITS2, LSU and SSU were aligned using MXSCARNA v.2.1 (Tabei et al., 2008), and of EF-1α protein using MAFFT v.7 (Katoh et al., 2017). rtREV was selected using IQ-TREE v1.6 (Nguyen et al., 2015) as the best evolutionary model for the EF-1α phylogenetic analysis. All the alignments were concatenated in a supermatrix using FASconCAT v.1.04 (Kuck and Meusemann, 2010), which was used to conduct a partitioned phylogenetic analysis. A phylogenetic tree based on the alignments and in the evolutionary model (rtREV for EF-1α and GTR for the others – ITS1, ITS2, LSU and SSU), was inferred using RAxML v.8.4 (Stamatakis, 2014), with 1,000 bootstrap replicates. Genome annotation was done using Augustus v3.3.1 (Stanke et al., 2008) and BRAKER2 v2.1.2 (Hoff et al., 2019), using as extrinsic evidence for training the proteins of W. anomalus deposited in GenBank. Proteins related to glycerol transport and metabolism were identified in the LBCM1105 genome using Blastx.

The GC content of the genome was 34.51%. The phylogenetic analysis (Figure S1) confirmed that LBCM1105 is, in fact, a strain within W. anomalus, in the same clade with the W. anomalus NRRL Y-366-8, with a bootstrap of 100%. Moreover, according to flow cytometry analyses, the genome of strain LBCM1105 is 13.93 ± 0.11 Mb. The total genome assembly corresponds to 12.72 Mb, i.e., 91.31% of the expected size, and 89.89% in relation to the genome of the W. anomalus strain NRRL Y-366-8 (GCA_001661255.1) which has a genome size of 14.15 Mb. The completeness of the genome assembly, as evaluated on the gene space by BUSCO, was 88.6% for the fungi dataset (290 genes) and 85.5% for the Saccharomycetales dataset (1711 genes). Half of the data is present in 51 scaffolds (L50) larger than 76 kb (N50), the largest being 229 kb. The total number of contigs was 389 with 6,812 predicted protein-coding genes. This number is similar to the 6,421 ORFs previously reported from the genome of W. anomalus NRRL Y-366-8 (Riley et al., 2016), and to the 5,885 ORFs of Saccharomyces cerevisiae (Goffeau et al., 1996). We compared the genome annotation of LBCM1105 (Augustus and BRAKER2) to that of NRRL Y-366-8, S. cerevisiae S288c and W. ciferrii using OrthoFinder (Emms and Kelly, 2015). This comparison clearly showed that most predicted genes in LBCM1105 can be assigned to orthologous groups and are shared with the other genomes in the analysis (Figure S2 and Table 1). This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession SHLV00000000. The version described in this paper is version SHLV01000000.

Table 1 Comparison of groups of orthologous genes between W. anomalus LBCM1105 with two annotation strategies A) Augustus, B) BRAKER2, W. anomalus NRRL Y-366-8, W. ciferrii NRRL Y-1031 and S. cerevisiae S288c. 

Groups of orthologous genes LBCM1105-A LBCM1105-B S288c NRRL Y-366-8 NRRL Y-1031
Number of genes in strains/species 6812 6159 6002 6421 6702
Number of genes in orthogroups 5965 6106 4651 6227 5936
Number of unassigned genes 847 53 1351 194 766
Percentage of genes in orthogroups 87,6 99,1 77,5 97,0 88,6
Number of species-specific orthogroups 0 0 7 0 7
Number of genes in species-specific orthogroups 0 0 17 0 79

DNA sequences from S. cerevisiae S288c encoding the proteins that perform glycerol transport (the channel Fps1p and the high affinity transporter Stl1p) and metabolism (the consumption Gut1p/Gut2p, the production Gpd1p/Gpd2p and Gpp1p/Gpp2p, as well as the putative pathway Gcy1p, Ypr1p and Dak1p/Dak2p) (Figure 1, and Table 2) were obtained from SGD (https://www.yeastgenome.org) and used to identify the correspondent putative ORFs in the W. anomalus LBCM1105 genome. Homologous sequences to the proteins were found (Table 2), in some cases different S. cerevisiae proteins aligned to the same protein in the W. anomalus LBCM1105 genome, it is not clear which will be the exact function of the LBCM1105's protein, more studies are need to elucidate this. The W. anomalus Stl1p was previously studied in detail, showing very high affinity for glycerol (Cunha et al., 2019). The genome sequence presented here provides evidence for the existence of the genes needed to ensure the two glycerol consumption and production pathways known in S. cerevisiae. Further studies are required to verify how intrinsic characteristics of these proteins and their expression and regulation are the cause underlying the LBCM1105's extraordinary ability to grow on glycerol as single a carbon source (Conceição et al., 2015).

Figure 1 Global yeast metabolism overview focusing on glycerol transport, consumption and production pathways. Red: main metabolic pathway. Blue: alternative pathway with unclear physiological relevance in S. cerevisiae

Table 2 Similarity between the S. cerevisiae genes encoding the proteins responsible for glycerol transport and metabolism as in Figure 1, and the corresponding sequences identified in the genome of W. anomalus LBCM1105. Protein Sequences are available at https://doi.org/10.6084/m9.figshare.11441061.v1 

Protein role S. cerevisiae - SGD database Gene Percentage target aligned Similarity
Gene ID
Regular pathway Transport Glycerol channel FPS1 S000003966 g1373.t1 45.3 56%
Glycerol active permease/ H+ symporter STL1 S000002944 g4293.t1 85.4 57%
Consumption Glycerol kinase GUT1 S000001024 g1371.t1 91.2 72%
Glycerol 3P GUT2 S000001417 g5045.t1 98.8 72%
dehydrogenase/mitochondria
Production Glycerol 3P dehydrogenase GPD1 S000002180 g1302.t1 100 78%
Glycerol 3P dehydrogenase GPD2 S000005420 g1302.t1 81.1 82%
Glycerol 3P phosphatase GPP1 S000002180 g4575.t1 99.2 71%
Glycerol 3P phosphatase GPP2 S000005420 g4575.t1 99.2 71%
Alternative pathway Consumption/Production Glycerol dehydrogenase GCY1 S000005646 g1045.t1 98.7 79%
Glycerol dehydrogenase YPR1 S000002776 g1045.t1 98.7 78%
Consumption Dihydroxyacetone kinase DAK1 S000004535 g4297.t1 98.5 56%
Dihydroxyacetone kinase DAK2 S000001841 g4297.t1 97.8 52%

Acknowledgments

The authors gratefully acknowledge Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE) and the Centro Nacional de Pesquisa em Energia e Materiais (CNPEM) for support with the sequencing of LBCM1105. This work was supported by CAPES/Brazil (PNPD 2755/2011; PCF-PVE 021/2012), by CNPq (Brazil), processes 304815/2012 (research grant) and 305135/2015-5, and by AUXPE-PVES 1801/2012 (Process 23038.015294/2016-18) from Brazilian Government and by UFOP. C.L. is supported by the strategic program UID/BIA/04050/2013 [POCI-01-0145-FEDER-007569] funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional de Competitividade e Internacionalização (POCI). DMRP is a fellow from the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) – Brazil (310080/2018-5).

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Associate editor: Ana Tereza Vasconcelos

Received: April 09, 2019; Accepted: April 06, 2020

Send correspondence to Diego M. Riaño-Pachon. Universidade de São Paulo, Centro de Energia Nuclear na Agricultura, Laboratório de Biologia Computacional, Evolutiva e de Sistemas, Piracicaba, SP, Brazil. E-mail: email. diego.riano@cena.usp.br

Conflict of Interest

The authors declare that there is no conflict of interest that could be perceived as prejudicial to the impartiality of the reported research.

Authors Contributions

ACC, LSG, FGS, JAT, FFO, IMC, CL, RLB contributed to project conceptualization; ACC, RACS, DMRP, FMS, JVCO, GHG, ATS, FGS, CL, RLB were responsible for data curation; ACC, RACS, DMRP, FMS, JVCO, GHG, ATS, LSG, FGS, JAT, FFO, ICR, IMC, CL, RLB carried out formal data analysis; DMRP, IMC, CL, RLB were responsible for funding acquisition; ACC, RACS, DMRP, FMS, JVCO, GHG, ATS, LSG, FGS, JAT, FFO, ICR, IMC, CL, RLB performed the experiments, and data collection; ACC, RACS, DMRP, LSG, FGS, JAT, FFO, ICR, CL, RLB designed the methodology; DMRP, IMC, CL, RLB managed and coordinated the project; DMRP, FGS, JAT, FFO, CL, RLB supervised the project; ACC, RACS, DMRP, FGS, FFO, IMC, CL, RLB wrote the original draft; all authors participated in revising and editing the final version of the manuscript.

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