July 19, 2019  |  

Population structure of mitochondrial genomes in Saccharomyces cerevisiae.

Rigorous study of mitochondrial functions and cell biology in the budding yeast, Saccharomyces cerevisiae has advanced our understanding of mitochondrial genetics. This yeast is now a powerful model for population genetics, owing to large genetic diversity and highly structured populations among wild isolates. Comparative mitochondrial genomic analyses between yeast species have revealed broad evolutionary changes in genome organization and architecture. A fine-scale view of recent evolutionary changes within S. cerevisiae has not been possible due to low numbers of complete mitochondrial sequences.To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS). Using de novo assemblies, we generated highly accurate complete mtDNA sequences. These mtDNA sequences were compared with 98 additional mtDNA sequences gathered from various published collections. Phylogenies based on mitochondrial coding sequences and intron profiles revealed that intraspecific diversity in mitochondrial genomes generally recapitulated the population structure of nuclear genomes. Analysis of intergenic sequence indicated a recent expansion of mobile elements in certain populations. Additionally, our analyses revealed that certain populations lacked introns previously believed conserved throughout the species, as well as the presence of introns never before reported in S. cerevisiae.Our results revealed that the extensive variation in S. cerevisiae mtDNAs is often population specific, thus offering a window into the recent evolutionary processes shaping these genomes. In addition, we offer an effective strategy for sequencing these challenging AT-rich mitochondrial genomes for small scale projects.


July 19, 2019  |  

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast.

BAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals.For the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters. Similarly, we achieved for the first time the microbial production of polyamine hydroxycinnamate amides; monolignol, malate and fatty alcohol hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate.We demonstrate in this study the effectiveness of expressing members of the plant BAHD acyltransferase family in yeast for the synthesis of numerous valuable hydroxycinnamate and benzoate conjugates.


July 19, 2019  |  

Contrasting evolutionary genome dynamics between domesticated and wild yeasts.

Structural rearrangements have long been recognized as an important source of genetic variation, with implications in phenotypic diversity and disease, yet their detailed evolutionary dynamics remain elusive. Here we use long-read sequencing to generate end-to-end genome assemblies for 12 strains representing major subpopulations of the partially domesticated yeast Saccharomyces cerevisiae and its wild relative Saccharomyces paradoxus. These population-level high-quality genomes with comprehensive annotation enable precise definition of chromosomal boundaries between cores and subtelomeres and a high-resolution view of evolutionary genome dynamics. In chromosomal cores, S. paradoxus shows faster accumulation of balanced rearrangements (inversions, reciprocal translocations and transpositions), whereas S. cerevisiae accumulates unbalanced rearrangements (novel insertions, deletions and duplications) more rapidly. In subtelomeres, both species show extensive interchromosomal reshuffling, with a higher tempo in S. cerevisiae. Such striking contrasts between wild and domesticated yeasts are likely to reflect the influence of human activities on structural genome evolution.


July 19, 2019  |  

Iterative optimization of xylose catabolism in Saccharomyces cerevisiae using combinatorial expression tuning.

A common challenge in metabolic engineering is rapidly identifying rate-controlling enzymes in heterologous pathways for subsequent production improvement. We demonstrate a workflow to address this challenge and apply it to improving xylose utilization in Saccharomyces cerevisiae. For eight reactions required for conversion of xylose to ethanol, we screened enzymes for functional expression in S. cerevisiae, followed by a combinatorial expression analysis to achieve pathway flux balancing and identification of limiting enzymatic activities. In the next round of strain engineering, we increased the copy number of these limiting enzymes and again tested the eight-enzyme combinatorial expression library in this new background. This workflow yielded a strain that has a ~70% increase in biomass yield and ~240% increase in xylose utilization. Finally, we chromosomally integrated the expression library. This library enriched for strains with multiple integrations of the pathway, which likely were the result of tandem integrations mediated by promoter homology. Biotechnol. Bioeng. 2017;114: 1301-1309. © 2017 Wiley Periodicals, Inc.© 2017 Wiley Periodicals, Inc.


July 7, 2019  |  

Genome sequences of Cyberlindnera fabianii 65, Pichia kudriavzevii 129, and Saccharomyces cerevisiae 131 isolated from fermented masau fruits in Zimbabwe.

Cyberlindnera fabianii 65, Pichia kudriavzevii 129, and Saccharomyces cerevisiae 131 have been isolated from the microbiota of fermented masau fruits. C. fabianii and P. kudriavzevii especially harbor promising features for biotechnology and food applications. Here, we present the draft annotated genome sequences of these isolates. Copyright © 2017 van Rijswijck et al.


July 7, 2019  |  

Construction of efficient xylose-fermenting Saccharomyces cerevisiae through a synthetic isozyme system of xylose reductase from Scheffersomyces stipitis.

Engineered Saccharomyces cerevisiae has been used for ethanol production from xylose, the abundant sugar in lignocellulosic hydrolyzates. Development of engineered S. cerevisiae able to utilize xylose effectively is crucial for economical and sustainable production of fuels. To this end, the xylose-metabolic genes (XYL1, XYL2 and XYL3) from Scheffersomyces stipitis have been introduced into S. cerevisiae. The resulting engineered S. cerevisiae strains, however, often exhibit undesirable phenotypes such as slow xylose assimilation and xylitol accumulation. This work was undertaken to construct an improved xylose-fermenting strain by developing a synthetic isozyme system of xylose reductase (XR). The DXS strain having both wild XR and mutant XR showed low xylitol accumulation and fast xylose consumption compared to the engineered strains expressing only one type of XRs, resulting in improved ethanol yield and productivity. These results suggest that the introduction of the XR-based synthetic isozyme system is a promising strategy to develop efficient xylose-fermenting strains. Copyright © 2017 Elsevier Ltd. All rights reserved.


July 7, 2019  |  

The dynamic three-dimensional organization of the diploid yeast genome.

The budding yeast Saccharomyces cerevisiae is a long-standing model for the three-dimensional organization of eukaryotic genomes. However, even in this well-studied model, it is unclear how homolog pairing in diploids or environmental conditions influence overall genome organization. Here, we performed high-throughput chromosome conformation capture on diverged Saccharomyces hybrid diploids to obtain the first global view of chromosome conformation in diploid yeasts. After controlling for the Rabl-like orientation using a polymer model, we observe significant homolog proximity that increases in saturated culture conditions. Surprisingly, we observe a localized increase in homologous interactions between the HAS1-TDA1 alleles specifically under galactose induction and saturated growth. This pairing is accompanied by relocalization to the nuclear periphery and requires Nup2, suggesting a role for nuclear pore complexes. Together, these results reveal that the diploid yeast genome has a dynamic and complex 3D organization.


July 7, 2019  |  

Whole-genome sequence and variant analysis of W303, a widely-used strain of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has emerged as a superior model organism. Selection of distinct laboratory strains of S. cerevisiae with unique phenotypic properties, such as superior mating or sporulation efficiencies, has facilitated advancements in research. W303 is one such laboratory strain that is closely related to the first completely sequenced yeast strain, S288C. In this work, we provide a high-quality, annotated genome sequence for W303 for utilization in comparative analyses and genome-wide studies. Approximately 9500 variations exist between S288C and W303, affecting the protein sequences of ~700 genes. A listing of the polymorphisms and divergent genes is provided for researchers interested in identifying the genetic basis for phenotypic differences between W303 and S288C. Several divergent functional gene families were identified, including flocculation and sporulation genes, likely representing selection for desirable laboratory phenotypes. Interestingly, remnants of ancestor wine strains were found on several chromosomes. Finally, as a test of the utility of the high-quality reference genome, variant mapping revealed more accurate identification of accumulated mutations in passaged mismatch repair-defective strains. Copyright © 2017 Matheson et al.


July 7, 2019  |  

Genome sequence of Saccharomyces cerevisiae strain Kagoshima No. 2, used for Brewing the Japanese distilled spirit Shochu.

Here, we report a draft genome sequence of Saccharomyces cerevisiae strain Kagoshima no. 2, which is used for brewing shochu, a traditional distilled spirit in Japan. The genome data will facilitate an understanding of the evolutional traits and genetic background related to the characteristic features of strain Kagoshima no. 2. Copyright © 2017 Mori et al.


July 7, 2019  |  

Meeting report on experimental approaches to evolution and ecology using yeast and other model systems.

The fourth EMBO-sponsored conference on Experimental Approaches to Evolution and Ecology Using Yeast and Other Model Systems (https://www.embl.de/training/events/2016/EAE16-01/), was held at the EMBL in Heidelberg, Germany, October 19-23, 2016. The conference was organized by Judith Berman (Tel Aviv University), Maitreya Dunham (University of Washington), Jun-Yi Leu (Academia Sinica), and Lars Steinmetz (EMBL Heidelberg and Stanford University). The meeting attracted ~120 researchers from 28 countries and covered a wide range of topics in the fields of genetics, evolutionary biology, and ecology with a unifying focus on yeast as a model system. Attendees enjoyed the Keith Haring inspired yeast florescence microscopy artwork (Figure 1), a unique feature of the meeting since its inception, and the one-minute flash talks that catalyzed discussions at two vibrant poster sessions. The meeting coincided with the 20th anniversary of the publication describing the sequence of the first eukaryotic genome, Saccharomyces cerevisiae (Goffeau et al. 1996). Many of the conference talks focused on important questions about what is contained in the genome, how genomes evolve, and the architecture and behavior of communities of phenotypically and genotypically diverse microorganisms. Here, we summarize highlights of the research talks around these themes. Nearly all presentations focused on novel findings, and we refer the reader to relevant manuscripts that have subsequently been published. Copyright © 2017, G3: Genes, Genomes, Genetics.


July 7, 2019  |  

Single molecule sequencing-guided scaffolding and correction of draft assemblies.

Although single molecule sequencing is still improving, the lengths of the generated sequences are inevitably an advantage in genome assembly. Prior work that utilizes long reads to conduct genome assembly has mostly focused on correcting sequencing errors and improving contiguity of de novo assemblies.We propose a disassembling-reassembling approach for both correcting structural errors in the draft assembly and scaffolding a target assembly based on error-corrected single molecule sequences. To achieve this goal, we formulate a maximum alternating path cover problem. We prove that this problem is NP-hard, and solve it by a 2-approximation algorithm.Our experimental results show that our approach can improve the structural correctness of target assemblies in the cost of some contiguity, even with smaller amounts of long reads. In addition, our reassembling process can also serve as a competitive scaffolder relative to well-established assembly benchmarks.


July 7, 2019  |  

Genome sequence and analysis of a stress-tolerant, wild-derived strain of Saccharomyces cerevisiae used in biofuels research

The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics. Copyright © 2016 McIlwain et al.


July 7, 2019  |  

Ploidy influences the functional attributes of de novo lager yeast hybrids.

The genomes of hybrid organisms, such as lager yeast (Saccharomyces cerevisiae × Saccharomyces eubayanus), contain orthologous genes, the functionality and effect of which may differ depending on their origin and copy number. How the parental subgenomes in lager yeast contribute to important phenotypic traits such as fermentation performance, aroma production, and stress tolerance remains poorly understood. Here, three de novo lager yeast hybrids with different ploidy levels (allodiploid, allotriploid, and allotetraploid) were generated through hybridization techniques without genetic modification. The hybrids were characterized in fermentations of both high gravity wort (15 °P) and very high gravity wort (25 °P), which were monitored for aroma compound and sugar concentrations. The hybrid strains with higher DNA content performed better during fermentation and produced higher concentrations of flavor-active esters in both worts. The hybrid strains also outperformed both the parent strains. Genome sequencing revealed that several genes related to the formation of flavor-active esters (ATF1, ATF2¸ EHT1, EEB1, and BAT1) were present in higher copy numbers in the higher ploidy hybrid strains. A direct relationship between gene copy number and transcript level was also observed. The measured ester concentrations and transcript levels also suggest that the functionality of the S. cerevisiae- and S. eubayanus-derived gene products differs. The results contribute to our understanding of the complex molecular mechanisms that determine phenotypes in lager yeast hybrids and are expected to facilitate targeted strain development through interspecific hybridization.


July 7, 2019  |  

Complete genomic and transcriptional landscape analysis using third-generation sequencing: a case study of Saccharomyces cerevisiae CEN.PK113-7D.

Completion of eukaryal genomes can be difficult task with the highly repetitive sequences along the chromosomes and short read lengths of second-generation sequencing. Saccharomyces cerevisiae strain CEN.PK113-7D, widely used as a model organism and a cell factory, was selected for this study to demonstrate the superior capability of very long sequence reads for de novo genome assembly. We generated long reads using two common third-generation sequencing technologies (Oxford Nanopore Technology (ONT) and Pacific Biosciences (PacBio)) and used short reads obtained using Illumina sequencing for error correction. Assembly of the reads derived from all three technologies resulted in complete sequences for all 16 yeast chromosomes, as well as the mitochondrial chromosome, in one step. Further, we identified three types of DNA methylation (5mC, 4mC and 6mA). Comparison between the reference strain S288C and strain CEN.PK113-7D identified chromosomal rearrangements against a background of similar gene content between the two strains. We identified full-length transcripts through ONT direct RNA sequencing technology. This allows for the identification of transcriptional landscapes, including untranslated regions (UTRs) (5′ UTR and 3′ UTR) as well as differential gene expression quantification. About 91% of the predicted transcripts could be consistently detected across biological replicates grown either on glucose or ethanol. Direct RNA sequencing identified many polyadenylated non-coding RNAs, rRNAs, telomere-RNA, long non-coding RNA and antisense RNA. This work demonstrates a strategy to obtain complete genome sequences and transcriptional landscapes that can be applied to other eukaryal organisms.


July 7, 2019  |  

Rationally designed perturbation factor drives evolution in Saccharomyces cerevisiae for industrial application.

Saccharomyces cerevisiae strains with favorable characteristics are preferred for application in industries. However, the current ability to reprogram a yeast cell on the genome scale is limited due to the complexity of yeast ploids. In this study, a method named genome replication engineering-assisted continuous evolution (GREACE) was proved efficient in engineering S. cerevisiae with different ploids. Through iterative cycles of culture coupled with selection, GREACE could continuously improve the target traits of yeast by accumulating beneficial genetic modification in genome. The application of GREACE greatly improved the tolerance of yeast against acetic acid compared with their parent strain. This method could also be employed to improve yeast aroma profile and the phenotype could be stably inherited to the offspring. Therefore, GREACE method was efficient in S. cerevisiae engineering and it could be further used to evolve yeast with other specific characteristics.


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