Biology subjects

van den Broek, M.

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

26 records · Page 2Linked to original sources

bioRxiv2020

Adaptive laboratory evolution and reverse engineering of single-vitamin prototrophies in Saccharomyces cerevisiae.

Quantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B-vitamins. Previous work demonstrated that, in S. cerevisiae CEN.PK113-7D, requirements for biotin could be eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA) or thiamine were omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B-vitamins. In all evolution lines, specific growth rates reached at least 90 % of the growth rate observed in SM supplemented with a complete B-vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid or pABA. Reaching similar results in SM lacking thiamine, pyridoxine or pantothenate required over 300 generations of selective growth. The genomes of evolved single-colony isolates were re-sequenced and, for each B-vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth were selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B-vitamin, introduction of a small number of mutations sufficed to achieve substantially a increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM. ImportanceMany strains of Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, nicotinic acid and inositol. However, omission of these B-vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B-vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotype by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step towards development of S. cerevisiae strains that exhibit fast growth on cheap, fully mineral media that only require complementation with a carbon source, thereby reducing costs, complexity and contamination risks in industrial yeast fermentation processes.

microbiology↗

bioRxiv2019

Maltotriose consumption by hybrid Saccharomyces pastorianus is heterotic and results from regulatory cross-talk between parental sub-genomes

S. pastorianus strains are hybrids of S. cerevisiae and S. eubayanus that have been domesticated for several centuries in lager-beer brewing environments. As sequences and structures of S. pastorianus genomes are being resolved, molecular mechanisms and evolutionary origin of several industrially relevant phenotypes remain unknown. This study investigates how maltotriose metabolism, a key feature in brewing, may have arisen in early S. eubayanus x S. cerevisiae hybrids. To address this question, we generated a near-complete genome assembly of Himalayan S. eubayanus strains of the Holarctic subclade. This group of strains have been proposed to be the origin of the S. eubayanus subgenome of current S. pastorianus strains. The Himalayan S. eubayanus genomes harbored several copies of a SeAGT1 -oligoglucoside transporter gene with high sequence identity to genes encountered in S. pastorianus. Although Himalayan S. eubayanus strains are unable to grown on maltose and maltotriose, their maltose-hydrolase and SeMALT1 and SeAGT1 maltose-transporter genes complemented the corresponding null mutants of S. cerevisiae. Expression, in a Himalayan S. eubayanus strain, of a functional S. cerevisiae maltose-metabolism regulator gene (MALx3) enabled growth on oligoglucosides. The hypothesis that the maltotriose-positive phenotype in S. pastorianus is a result of heterosis was experimentally tested by constructing a S. cerevisiae x S. eubayanus laboratory hybrid with a complement of maltose-metabolism genes that resembles that of current S. pastorianus strains. The ability of this hybrid to consume maltotriose in brewers wort demonstrated regulatory cross talk between sub-genomes and thereby validated this hypothesis. These results provide experimental evidence of the evolutionary origin of an essential phenotype of lager-brewing strains and valuable knowledge for industrial exploitation of laboratory-made S. pastorianus-like hybrids.\n\nImportanceS.pastorianus, a S.cerevisiae X S.eubayanus hybrid, is used for production of lager beer, the most produced alcoholic beverage worldwide It emerged by spontaneous hybridization and have colonized early lager-brewing processes. Despite accumulation and analysis of genome sequencing data of S.pastorianus parental genomes, the genetic blueprint of industrially relevant phenotypes remains unknown. Assimilation of wort abundant sugar maltotriose has been postulated to be inherited from S.cerevisiae parent. Here, we demonstrate that although Asian S.eubayanus isolates harbor a functional maltotriose transporter SeAGT1 gene, they are unable to grow on -oligoglucosides, but expression of S. cerevisae regulator ScMAL13 was sufficient to restore growth on trisaccharides. We hypothesized that S. pastorianus maltotriose phenotype results from regulatory interaction between S.cerevisae maltose transcription activator and the promoter of SeAGT1. We experimentally confirmed the heterotic nature of the phenotype and thus this results provide experimental evidence of the evolutionary origin of an essential phenotype of lager-brewing strains.

microbiology↗

bioRxiv2019

Nanopore sequencing and comparative genome analysis confirm lager-brewing yeasts originated from a single hybridization

BackgroundThe lager brewing yeast, S. pastorianus, is a hybrid between S. cerevisiae and S. eubayanus with extensive chromosome aneuploidy. S. pastorianus is subdivided into Group 1 and Group 2 strains, where Group 2 strains have higher copy number and a larger degree of heterozygosity for S. cerevisiae chromosomes. As a result, Group 2 strains were hypothesized to have emerged from a hybridization event distinct from Group 1 strains. Current genome assemblies of S. pastorianus strains are incomplete and highly fragmented, limiting our ability to investigate their evolutionary history.\n\nResultsTo fill this gap, we generated a chromosome-level genome assembly of the S. pastorianus strain CBS 1483 using MinION sequencing and analysed the newly assembled subtelomeric regions and chromosome heterozygosity. To analyse the evolutionary history of S. pastorianus strains, we developed Alpaca: a method to compute sequence similarity between genomes without assuming linear evolution. Alpaca revealed high similarities between the S. cerevisiae subgenomes of Group 1 and 2 strains, and marked differences from sequenced S. cerevisiae strains.\n\nConclusionsOur findings suggest that Group 1 and Group 2 strains originated from a single hybridization involving a heterozygous S. cerevisiae strain, followed by different evolutionary trajectories. The clear differences between both groups may originate from a severe population bottleneck caused by the isolation of the first pure cultures. Alpaca provides a computationally inexpensive method to analyse evolutionary relationships while considering non-linear evolution such as horizontal gene transfer and sexual reproduction, providing a complementary viewpoint beyond traditional phylogenetic approaches.

bioinformatics↗

bioRxiv2018

Laboratory evolution of a Saccharomyces cerevisiae x S. eubayanus hybrid under simulated lager-brewing conditions: genetic diversity and phenotypic convergence

Saccharomyces pastorianus lager-brewing yeasts are domesticated hybrids of S. cerevisiae x S. eubayanus that display extensive inter-strain chromosome copy number variation and chromosomal recombinations. It is unclear to what extent such genome rearrangements are intrinsic to the domestication of hybrid brewing yeasts and whether they contribute to their industrial performance. Here, an allodiploid laboratory hybrid of S. cerevisiae and S. eubayanus was evolved for up to 418 generations on wort under simulated lager-brewing conditions in six independent sequential batch bioreactors. Characterization of 55 single-cell isolates from the evolved cultures showed large phenotypic diversity and whole-genome sequencing revealed a large array of mutations. Frequent loss of heterozygosity involved diverse, strain-specific chromosomal translocations, which differed from those observed in domesticated, aneuploid S. pastorianus brewing strains. In contrast to the extensive aneuploidy of domesticated S. pastorianus strains, the evolved isolates only showed limited (segmental) aneuploidy. Specific mutations could be linked to calcium-dependent flocculation, loss of maltotriose utilisation and loss of mitochondrial activity, three industrially relevant traits that also occur in domesticated S. pastorianus strains. This study indicates that fast acquisition of extensive aneuploidy is not required for genetic adaptation of S. cerevisiae x S. eubayanus hybrids to brewing environments. In addition, this work demonstrates that, consistent with the diversity of brewing strains for maltotriose utilization, domestication under brewing conditions can result in loss of this industrially relevant trait. These observations have important implications for the design of strategies to improve industrial performance of novel laboratory-made hybrids.

evolutionary biology↗

bioRxiv2018

Topical cream with live lactobacilli modulates the skin microbiome and reduce acne symptoms

The skin is home to an important part of our commensal microbiota, despite it being a cool, acidic and desiccated environment. Tailored microbiome modulation approaches with, for example probiotics, are highly challenging for this body site. Here we show by next-generating sequencing that Lactobacillus taxa -especially those known to be dominant in the human vagina- are underestimated members of the skin microbiota. Specific Lactobacillus strains were selected in the lab and formulated in a viable form in an oil in water-based topical cream. Facial application by patients with mild-to-moderate acne symptoms was able to reduce inflammatory lesions and comedone formation. This was associated with a temporary modulation of the skin microbiome, including a reduction in relative abundance of staphylococci and an increase in lactobacilli. Skin microbiome modulation by addition of carefully formulated lactobacilli seems to be new therapeutic option to reduce antibiotic use for common acne symptoms.

clinical trials↗

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↗