Date Published: May 1, 2019
Publisher: Public Library of Science
Author(s): Ievgeniia A. Tiukova, Mats E. Pettersson, Marc P. Hoeppner, Remi-Andre Olsen, Max Käller, Jens Nielsen, Jacques Dainat, Henrik Lantz, Jonas Söderberg, Volkmar Passoth, Joseph Schacherer.
Here, we present the genome of the industrial ethanol production strain Brettanomyces bruxellensis CBS 11270. The nuclear genome was found to be diploid, containing four chromosomes with sizes of ranging from 2.2 to 4.0 Mbp. A 75 Kbp mitochondrial genome was also identified. Comparing the homologous chromosomes, we detected that 0.32% of nucleotides were polymorphic, i.e. formed single nucleotide polymorphisms (SNPs), 40.6% of them were found in coding regions (i.e. 0.13% of all nucleotides formed SNPs and were in coding regions). In addition, 8,538 indels were found. The total number of protein coding genes was 4897, of them, 4,284 were annotated on chromosomes; and the mitochondrial genome contained 18 protein coding genes. Additionally, 595 genes, which were annotated, were on contigs not associated with chromosomes. A number of genes was duplicated, most of them as tandem repeats, including a six-gene cluster located on chromosome 3. There were also examples of interchromosomal gene duplications, including a duplication of a six-gene cluster, which was found on both chromosomes 1 and 4. Gene copy number analysis suggested loss of heterozygosity for 372 genes. This may reflect adaptation to relatively harsh but constant conditions of continuous fermentation. Analysis of gene topology showed that most of these losses occurred in clusters of more than one gene, the largest cluster comprising 33 genes. Comparative analysis against the wine isolate CBS 2499 revealed 88,534 SNPs and 8,133 indels. Moreover, when the scaffolds of the CBS 2499 genome assembly were aligned against the chromosomes of CBS 11270, many of them aligned completely, some have chunks aligned to different chromosomes, and some were in fact rearranged. Our findings indicate a highly dynamic genome within the species B. bruxellensis and a tendency towards reduction of gene number in long-term continuous cultivation.
The yeast, Brettanomyces bruxellensis (syn. Dekkera bruxellensis- the last issue of the taxonomic monography of the yeasts  mentioned D. bruxellensis as the valid name of this species, however, according to the recently introduced principle “one species, one name”  we use the older name B. bruxellensis in this study), is regarded as a major contaminant in wine [3, 4] and bioethanol production [5, 6]. However, it is also involved in certain economically relevant, spontaneous fermentations, such as the production of Belgian Lambic beer [7–9]. It has also been found to be the production yeast in a continuous ethanol production process with cell recirculation, after outcompeting the initially inoculated Saccharomyces cerevisiae . B. bruxellensis has an ethanol tolerance similar to S. cerevisiae, and has the ability to grow at low sugar concentrations. This explains why it usually becomes important in the later stages of wine or beer production, or in sugar limited continuous fermentations . The mechanism of outcompeting S. cerevisiae is not completely known at present. It has been speculated that the ability of B. bruxellensis to assimilate nitrate may play a role, such as in some Brazilian ethanol production plants, where nitrate can come into the fermentation with the substrate, sucrose from sugarcane . However, outcompetition of S. cerevisiae by B. bruxellensis has been observed in nitrate-free, glucose-limited fermentations, and thus, the competitiveness of the yeast could rather be due to a higher affinity for the substrate and/or a more efficient energy metabolism .
This study represents the first genomic investigation of a B. bruxellensis-strain that functions as an ethanol production strain [12, 16]. Using the recently developed assembly of the CBS 11270 genome to scaffolds of chromosome size  we could associate a major part, 86.4% of the genome sequences, to the assembled four chromosomes.