Biology subjects

van den Broek, M.

Publications and source records attributed to van den Broek, M..

3 recordsLinked to original sources

bioRxiv2018

In vivo recombination of Saccharomyces eubayanus maltose-transporter genes yields a chimeric transporter that enables maltotriose fermentation

Saccharomyces pastorianus lager-brewing yeasts are aneuploid S. cerevisiae x S. eubayanus hybrids, whose genomes have been shaped by domestication in brewing-related contexts. In contrast to most S. cerevisiae and S. pastorianus strains, S. eubayanus cannot utilize maltotriose, a major carbohydrate in brewers wort. Accordingly, S. eubayanus CBS 12357{top} harbors four SeMALT maltose-transporter genes, but no genes resembling the S. cerevisiae maltotriose-transporter gene ScAGT1 or the S. pastorianus maltotriose-transporter gene SpMTY1. To study the evolvability of maltotriose utilization in S. eubayanus CBS 12357{top}, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation, as well as an improved flavor profile, in 7-L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalt413 was comparable to the original SeMalt transporters, but overexpression of SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-transporter gene SpMTY1, which has sequences with high similarity to alternatingly ScMALx1 and SeMALT3. Evolution of the maltotriose-transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study.\n\nAuthor SummaryFermentation of the wort sugar maltotriose is critical for the flavor profile obtained during beer brewing. The recently discovered yeast Saccharomyces eubayanus is gaining popularity as an alternative to S. pastorianus and S. cerevisiae for brewing, however it is unable to utilize maltotriose. Here, a combination of non-GMO mutagenesis and laboratory evolution of the S. eubayanus type strain CBS 12357{top} was used to enable maltotriose fermentation in brewers wort. A resulting S. eubayanus strain showed a significantly improved brewing performance, including improved maltose and maltotriose consumption and a superior flavor profile. Whole genome sequencing identified a novel transporter gene, SeMALT413, which was formed by recombination between three different SeMALT maltose-transporter genes. Overexpression of SeMALT413 in CBS 12357{top} confirmed its neofunctionalization as a maltotriose transporter. The mosaic structure of the maltotriose transporter SpMty1 in S. pastorianus resembles that of SeMalt413, suggesting that maltotriose utilization likely emerged through similar recombination events during the domestication of current lager brewing strains.

evolutionary biology↗

bioRxiv2018

Allele-specific genome editing using CRISPR-Cas9 causes off-target mutations in diploid yeast

Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when editing individual heterozygous loci in the yeast genome. Moreover, Cas9-mediated introduction of a DSB resulted in large scale loss of heterozygosity affecting DNA regions up to 360 kb that resulted in introduction of nearly 1700 off-target mutations, due to replacement of sequences on the targeted chromosome by corresponding sequences from its non-targeted homolog. The observed patterns of loss of heterozygosity were consistent with homology directed repair. The extent and frequency of loss of heterozygosity represent a novel mutagenic side-effect of Cas9-mediated genome editing, which would have to be taken into account in eukaryotic gene editing. In addition to contributing to the limited genetic amenability of heterozygous yeasts, Cas9-mediated loss of heterozygosity could be particularly deleterious for human gene therapy, as loss of heterozygous functional copies of anti-proliferative and pro-apoptotic genes is a known path to cancer.

molecular biology↗

bioRxiv2017

Nanopore sequencing enables near-complete de novo assembly of Saccharomyces cerevisiae reference strain CEN.PK113-7D

The haploid Saccharomyces cerevisiae strain CEN.PK113-7D is a popular model system for metabolic engineering and systems biology research. Current genome assemblies are based on short-read sequencing data scaffolded based on homology to strain S288C. However, these assemblies contain large sequence gaps, particularly in subtelomeric regions, and the assumption of perfect homology to S288C for scaffolding introduces bias.\n\nIn this study, we obtained a near-complete genome assembly of CEN.PK113-7D using only Oxford Nanopore Technologys MinION sequencing platform. 15 of the 16 chromosomes, the mitochondrial genome, and the 2-micron plasmid are assembled in single contigs and all but one chromosome starts or ends in a telomere cap. This improved genome assembly contains 770 Kbp of added sequence containing 248 gene annotations in comparison to the previous assembly of CEN.PK113-7D. Many of these genes encode functions determining fitness in specific growth conditions and are therefore highly relevant for various industrial applications. Furthermore, we discovered a translocation between chromosomes III and VIII which caused misidentification of a MAL locus in the previous CEN.PK113-7D assembly. This study demonstrates the power of long-read sequencing by providing a high-quality reference assembly and annotation of CEN.PK113-7D and places a caveat on assumed genome stability of microorganisms.

genomics↗