Gain and loss : a phylogenetic study of putative chloroperoxidases found within budding yeasts

Knoppjästsvampar är en typ av svampar med encellig morfologi. Knoppjästsvampar utgör Saccharomycotina, en understam till Ascomycota och innefattar mer än 1000 idag beskrivna arter, bland annat bakjästen Saccharomyces cerevisiae. Trots att S. cerevisiae är en av de mest studerade eukaryota organismerna förekommer ett stort antal andra arter av knoppjästsvampar som först nyligen sekvenserats och där ytterst få funktioner hos gener är kartlagda. Gener utan klarlagda funktioner brukar kallas ”orphan genes”. Som grund för den fylogenetiska analysen i detta projekt ligger en icke-karaktäriserad genfamilj hos knoppjästsvampar som kodar för ett förmodat kloroperoxidas. Syftet var att försöka upprätta en hypotes om enzymets funktion baserat på de ekologiska förhållanden där arter av knoppjästsvampar med denna typ av gen återfinns. Sökningar gjordes efter homologa sekvenser till den valda genen, både inom gruppen knoppjästsvampar och inom andra taxonomiska grupper. De påträffade homologerna låg till grund för konstruktionen av ett fylogenetiskt träd som sedan användes som underlag för en hypotes om relationen mellan de kloroperoxidas-liknande gener som påträffades. Undersökningen resulterade i ett fylogenetiskt träd där endast en del av alla arter av knoppjästsvampar bildade en monofyletisk grupp. Cirka två tredjedelar av de påträffade sekvenserna bildade en större monofyletisk grupp, medan de andra sekvenserna bildade mindre grupper som visade närmre släktskap med bakteriella kloroperoxidaser. Denna polyfyletiska fördelning av sekvenser från knoppjästsvamparna tyder på att nära evolutionärt släktskap finns mellan dessa och de bakteriella sekvenserna i analysen. Detta släktskap kan bero på en eller flera fall av horisontell genöverföring från bakterie till knoppjästsvamp.Budding yeasts are a group of predominantly unicellular fungi that together form the subphylum Saccharomycotina of phylum Ascomycota. Subphylum Saccharomycotina consists of over 1000 described species, which include the common baker’s yeast Saccharomyces cerevisiae. S. cerevisiae is one of the most studied eukaryotic organisms but until recently only a few other species of budding yeasts have had their genomes sequenced. Despite the recent increase in sequenced budding yeast genomes, genome annotation has lagged behind resulting in many so-called “orphan genes”, genes without a described function, which have yet to be examined. The focus of this project was a non-characterized gene family within budding yeasts that encode a putative chloroperoxidase. The purpose was to construct a hypothesis concerning enzyme function based on ecological or metabolic properties of the species where chloroperoxidase-like homologues were found. Searches for homologues for the chosen gene were performed both within Saccharomycotina and in other taxonomic groups. The found homologues formed a dataset which was used to construct a phylogenetic tree. This tree functioned as support for a hypothesis about possible relationships between species where homologous were found. The analysis generated a phylogenetic tree where only a part of the sequences from budding yeasts constituted a monophyletic group. Two thirds of sequences found in budding yeasts formed a larger monophyletic group, while the remaining third formed smaller groups that exhibited closer relationships with bacterial chloroperoxidase than with the yeast sequences. This polyphyletic distribution of budding yeast sequences indicates a closer evolutionary relationship to bacterial sequences. This relationship could be due to one or several instances of horizontal gene transfer from bacteria to yeast

Chromosome-level assemblies from diverse clades reveal limited structural and gene content variation in the genome of Candida glabrata

Background Candida glabrata is an opportunistic yeast pathogen thought to have a large genetic and phenotypic diversity and a highly plastic genome. However, the lack of chromosome-level genome assemblies representing this diversity limits our ability to accurately establish how chromosomal structure and gene content vary across strains. Results Here, we expanded publicly available assemblies by using long-read sequencing technologies in twelve diverse strains, obtaining a final set of twenty-one chromosome-level genomes spanning the known C. glabrata diversity. Using comparative approaches, we inferred variation in chromosome structure and determined the pan-genome, including an analysis of the adhesin gene repertoire. Our analysis uncovered four new adhesin orthogroups and inferred a rich ancestral adhesion repertoire, which was subsequently shaped through a still ongoing process of gene loss, gene duplication, and gene conversion. Conclusions C. glabrata has a largely stable pan-genome except for a highly variable subset of genes encoding cell wall-associated functions. Adhesin repertoire was established for each strain and showed variability among clades.TG group acknowledges support from the Spanish Ministry of Science and Innovation (MCIN) for grant PGC2018-099921-B-I00, cofounded by European Regional Development Fund (ERDF); from the Catalan Research Agency (AGAUR) SGR423; from the European Union’s Horizon 2020 research and innovation programme (ERC-2016-724173); from the Gordon and Betty Moore Foundation (Grant GBMF9742); from the “La Caixa” foundation (Grant LCF/PR/HR21/00737), and from the Instituto de Salud Carlos III (IMPACT Grant IMP/00019 and CIBERINFEC CB21/13/00061- ISCIII-SGEFI/ERDF). PWJG acknowledges support by grants SBPLY/19/180501/000114 and SBPLY/19/180501/000356 funded by the Regional government of Castilla-La Mancha and grants SAF2013-47570-P and PID2020-117983RB-I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF a way of making Europe.Peer ReviewedPostprint (author's final draft

EVOLUTION VIA GENE DUPLICATION AND ALTERNATIVE SPLICING IN THE EUKARYOTIC SKI7 AND HBS1 GENES

Gene duplication and alternative splicing are both recognized as important drivers of proteomic diversity and innovation during evolution, but the evolutionary changes over long periods of time or the interrelations of the two processes has not been extensively studied. Here I study these phenomena for the SKI7 and HBS1 gene pair. These Saccharomyces cerevisiae genes were created as part of a whole genome duplication (WGD) event and have since functionally diverged. Although both genes function in mRNA surveillance pathways, the two genes act on different RNAs and have different effects on the target mRNAs. Ski7 brings the Ski complex and exosome together to perform degradation of cytoplasmic mRNAs, but has a specific, poorly understood function in the nonstop decay pathway. In nonstop decay, Ski7 is thought to interact with a ribosome that has stalled while translating through the poly-A tail of a nonstop mRNA. Hbs1 disassembles ribosomes stalled within the coding region and may trigger endonuclease cleavage of some target mRNAs. In order to better understand their functions in mRNA surveillance, this dissertation focuses on dissecting their evolutionary relationship. I show that the pre-WGD SKI7/HBS1 gene produces two distinct proteins via alternative splicing. One of these proteins functions as Ski7, while the other function as Hbs1. Further examination of SKI7/HBS1 genes via transcriptome sequencing demonstrates that alternative splicing in this gene is extremely ancient and widespread among eukaryotes. Duplication of the SKI7 and HBS1 genes has also occurred in six independent instances. Changes in the alternative splicing pattern and in the genes following duplication has led to a variety of Ski7-like proteins that likely have an impact on Ski7 function. Post-duplication changes include loss of an Hbs1 N-terminal motif and changes in the conserved GTPase domain. When these changes are introduced into the Lachancea SKI7/HBS1 gene they disrupt Hbs1 function but not Ski7 function, consistent with the idea that they were important for functional divergence following duplication. Additionally, I have found that the Lachancea SKI7 isoform performs nonstop decay suboptimally compared to Saccharomyces SKI7, indicating that duplication may have allowed SKI7 to specialize in nonstop mRNA decay

A pipeline for automated annotation of yeast genome sequences by a conserved-synteny approach

Abstract Background Yeasts are a model system for exploring eukaryotic genome evolution. Next-generation sequencing technologies are poised to vastly increase the number of yeast genome sequences, both from resequencing projects (population studies) and from de novo sequencing projects (new species). However, the annotation of genomes presents a major bottleneck for de novo projects, because it still relies on a process that is largely manual. Results Here we present the Yeast Genome Annotation Pipeline (YGAP), an automated system designed specifically for new yeast genome sequences lacking transcriptome data. YGAP does automatic de novo annotation, exploiting homology and synteny information from other yeast species stored in the Yeast Gene Order Browser (YGOB) database. The basic premises underlying YGAP's approach are that data from other species already tells us what genes we should expect to find in any particular genomic region and that we should also expect that orthologous genes are likely to have similar intron/exon structures. Additionally, it is able to detect probable frameshift sequencing errors and can propose corrections for them. YGAP searches intelligently for introns, and detects tRNA genes and Ty-like elements. Conclusions In tests on Saccharomyces cerevisiae and on the genomes of Naumovozyma castellii and Tetrapisispora blattae newly sequenced with Roche-454 technology, YGAP outperformed another popular annotation program (AUGUSTUS). For S. cerevisiae and N. castellii, 91-93% of YGAP's predicted gene structures were identical to those in previous manually curated gene sets. YGAP has been implemented as a webserver with a user-friendly interface at http://wolfe.gen.tcd.ie/annotation.</p

Elucidation of weak organic acid resistance mechanisms in non-Saccharomyces yeast: a case study of Zygosaccharomyces parabailii and Kluyveromyces marxianus

The efficient implementation of biorefineries to produce bio-based chemicals and fuels requires sustainable source of feedstock and robust microbial factories. Among others, lignocellulose and whey, which are residual wastes deriving from wood/agriculture and dairy industries, represent cheap, sugar-enriched feedstocks. The conversion of lignocellulose and whey into the desired products using microbial cell factories is a promising option to replace the fossil based petrochemical refinery. Different bacteria, algae and yeasts are currently used as microbial hosts, and their number is predicted to increase over next years. Minimum nutritional requirements and robustness have made yeasts a class of microbial hosts widely employed in industrial biotechnology, exploiting their natural abilities as well as genetically acquired pathways for production of natural and recombinant products, including bulk chemicals such as organic acids. However, efficient and economically viable production of organic acids has to face problems related to low productivity/titer and toxicity of the final product. Therefore, the exploration of yeast biodiversity to exploit unique native features and the understanding of mechanisms to endure harsh conditions are essential to develop ultraefficient and robust industrial yeast with novel properties. The aim of the research thesis is to evaluate the mechanism of weak acid stress response in the non-Saccharomyces yeasts Zygosaccharomyces parabailii and Kluyveromyces marxianus. To better understand the weak acid stress response of Z. parabailii, we summarized recent finding on the species. Knowing the relevant scientific reports, the next study was focused on the effect of lactic acid stress on Z. parabailii. This organic acid can be used as monomer for the production of biodegradable bioplastic polymers, such as poly lactic acid (PLA). The study revealed that cells are able to tolerate 40g/l of lactic acid without inducing a lag phase of growth and exhibit a negligible percentage of dead cells. More importantly, during lactic acid exposure, we observed structural modifications at the level of cell wall and membrane. These findings confirmed the peculiar ability of Z. parabailii to adapt to weak organic acids via remodeling of cellular components. The lack of a complete genome assembly and annotation encouraged us to perform a genome sequencing and genome study of our Z. parabailii strain. The results revealed that Z. parabailii is undergoing fertility restoration after interspecies hybridization event, which may shed a light to the process of whole genome duplication. The availability of Z. parabailii complete genome information allowed us to perform the first RNA-sequencing analysis on the species exposed to lactic acid stress. The results showed upregulation of mitochondrial and oxidative stress genes, and downregulation of a subset of cell wall genes, in addition to other specific regulation related to redox balance and ion homeostasis. Remarkably, several differentially regulated genes differ significantly from the S. cerevisiae counterpart or, in some cases, even seem not to have a homologue. Increased interest of K. marxianus application in industrial biotechnology led us to study its multidrug resistance transporters during acetic and lactic acid stress, the first being a contaminant related to the use of lignocellulose as feedstocks, while the second as final product of interest, as mentioned above. The results showed a strain-specific response to weak organic acid stress, and a possible involvement of KmPDR12 in acetic and lactic acid stress resistance, opening potential for future discoveries and novel studies. Overall, this work contributes to the vast array of studies that are shedding light on yeasts biodiversity, both as a way for understanding their natural potential and as an instrument for tailoring novel cell factories

Impact of high sugar content on metabolism and physiology of indigenous yeasts

This PhD project is part of an ARC Training Centre for Innovative Wine Production larger initiative to tackle the main challenges for the Australian wine industry. In particular, the aim is to address the implication of the increasing trend of sugar accumulation in ripe grapes that consequently results in high sugar musts and high ethanol wines. These increase the risk of sluggish and stuck fermentation, especially when only the indigenous microflora of yeast is exploited. At the beginning of fermentation, yeast cells must coordinate genome expression rapidly in response to external changes to maintain competitive fitness and cell survival. Understanding how cells modulate their adaptation strategies can be the key to predicting their capacity to survive in a harsh environment and consequently be able to influence wine composition. This project aims to give strategic advice to deal with fermentations by studying non-conventional yeast physiology in response to high sugar must and correlating it with growth and metabolism. Chapter 2 compares T. delbrueckii and S. cerevisiae oenological traits at a molecular level. The mechanisms behind the metabolic differences that exist between these two species were inspected using Next Generation Sequencing technology (ILLUMINA) and analysed by assembling RNA transcriptomes. In Chapter 3 two Australian indigenous yeast species genomes were sequenced with the newest Next Generation Sequencing (NGS) technology, Nanopore MinION. Chapter 4 further analyzed the global short-term stress adaptive response to grape must, implementing the technique previously used. The results, discussed in Chapter 5, summarize the improvements in high-throughput data analysis and reveal the genomic and physiological differences of these wine-related species.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 201

Genome diversity in Torulaspora microellipsoides and its contributions to the evolution os the Saccharomyces genus

En esta tesis presentamos la diversidad genómica y la implicación en diferentes transferencias horizontales de genes de la especie T. microellipsoides. Hipotetizamos que esta especie es la responsable de la transferencia tanto de amplias regiones subtelomericas (hasta 65kb) como de genes individuales a las especies del género Saccharomyces. Hemos descubierto que dos genes que codifican para dos transportadores, uno de fructosa de alta afinidad, FSY1 y otro de transporte al exterior celular de amonio, ATO3, han sido transferidos desde esta especie a algunas Saccharomyces. Por otro lado, la secuenciación del genoma de las cepas disponibles de T. microellipsoides ha revelado la existencia de dos especies híbridas cuyas especies parentales han sido también identificadas y para una de ellas, CBS 6762, hemos hipotetizado que se trata de una nueva especie cercana a la especie T. microellipsoides pero que no ha sido descrita hasta el momento