An Optimized Genotyping Workflow for Identifying Highly SCRaMbLEd Synthetic Yeasts.

Synthetic Sc2.0 yeast strains contain hundreds to thousands of loxPsym recombination sites that allow restructuring of the Saccharomyces cerevisiae genome by SCRaMbLE. Thus, a highly diverse yeast population can arise from a single genotype. The selection of genetically diverse candidates with rearranged synthetic chromosomes for downstream analysis requires an efficient and straightforward workflow. Here we present loxTags, a set of qPCR primers for genotyping across loxPsym sites to detect not only deletions but also inversions and translocations after SCRaMbLE. To cope with the large number of amplicons, we generated qTagGer, a qPCR genotyping primer prediction tool. Using loxTag-based genotyping and long-read sequencing, we show that light-inducible Cre recombinase L-SCRaMbLE can efficiently generate diverse recombination events when applied to Sc2.0 strains containing a linear or a circular version of synthetic chromosome III.

Methodological advances enabled by the construction of a synthetic yeast genome.

The international Synthetic Yeast Project (Sc2.0) aims to construct the first synthetic designer eukaryote genome. Over the past few years, the Sc2.0 consortium has achieved several significant milestones by synthesizing and characterizing all 16 nuclear chromosomes of the yeast Saccharomyces cerevisiae, as well as a 17thde novo neochromosome containing all nuclear tRNA genes. In this commentary, we discuss the recent technological advances achieved in this project and provide a perspective on how they will impact the emerging field of synthetic genomics in the future.

Genome biology and evolution of mating-type loci in four cereal rust fungi.

Permanent heterozygous loci, such as sex- or mating-compatibility regions, often display suppression of recombination and signals of genomic degeneration. In Basidiomycota, two distinct loci confer mating compatibility. These loci encode homeodomain (HD) transcription factors and pheromone receptor (Pra)-ligand allele pairs. To date, an analysis of genome level mating-type (MAT) loci is lacking for obligate biotrophic basidiomycetes in the Pucciniales, an order containing serious agricultural plant pathogens. Here, we focus on four species of Puccinia that infect oat and wheat, including P. coronata f. sp. avenae, P. graminis f. sp. tritici, P. triticina and P. striiformis f. sp. tritici. MAT loci are located on two separate chromosomes supporting previous hypotheses of a tetrapolar mating compatibility system in the Pucciniales. The HD genes are multiallelic in all four species while the PR locus appears biallelic, except for P. graminis f. sp. tritici, which potentially has multiple alleles. HD loci are largely conserved in their macrosynteny, both within and between species, without strong signals of recombination suppression. Regions proximal to the PR locus, however, displayed signs of recombination suppression and genomic degeneration in the three species with a biallelic PR locus. Our observations support a link between recombination suppression, genomic degeneration, and allele diversity of MAT loci that is consistent with recent mathematical modelling and simulations. Finally, we confirm that MAT genes are expressed during the asexual infection cycle, and we propose that this may support regulating nuclear maintenance and pairing during infection and spore formation. Our study provides insights into the evolution of MAT loci of key pathogenic Puccinia species. Understanding mating compatibility can help predict possible combinations of nuclear pairs, generated by sexual reproduction or somatic recombination, and the potential evolution of new virulent isolates of these important plant pathogens.

Implications of the three-dimensional chromatin organization for genome evolution in a fungal plant pathogen.

The spatial organization of eukaryotic genomes is linked to their biological functions, although it is not clear how this impacts the overall evolution of a genome. Here, we uncover the three-dimensional (3D) genome organization of the phytopathogen Verticillium dahliae, known to possess distinct genomic regions, designated adaptive genomic regions (AGRs), enriched in transposable elements and genes that mediate host infection. Short-range DNA interactions form clear topologically associating domains (TADs) with gene-rich boundaries that show reduced levels of gene expression and reduced genomic variation. Intriguingly, TADs are less clearly insulated in AGRs than in the core genome. At a global scale, the genome contains bipartite long-range interactions, particularly enriched for AGRs and more generally containing segmental duplications. Notably, the patterns observed for V. dahliae are also present in other Verticillium species. Thus, our analysis links 3D genome organization to evolutionary features conserved throughout the Verticillium genus.

Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

Genomic and metabolomic diversity within a familial population of Aspergillus flavus.

Aspergillus flavus is an agriculturally significant micro-fungus having potential to contaminate food and feed crops with toxic secondary metabolites such as aflatoxin (AF) and cyclopiazonic acid (CPA). Research has shown A. flavus strains can overcome heterokaryon incompatibility and undergo meiotic recombination as teleomorphs. Although evidence of recombination in the AF gene cluster has been reported, the impacts of recombination on genotype and metabolomic phenotype in a single generation are lacking. In previous studies, we paired an aflatoxigenic MAT1-1 A. flavus strain with a non-aflatoxigenic MAT1-2 A. flavus strain that had been tagged with green fluorescent protein and then 10 F1 progenies (a mix of fluorescent and non-fluorescent) were randomly selected from single-ascospore colonies and broadly examined for evidence of recombination. In this study, we determined four of those 10 F1 progenies were recombinants because they were not vegetatively compatible with either parent or their siblings, and they exhibited other distinctive traits that could only result from meiotic recombination. The other six progenies examined shared genomic identity with the non-aflatoxigenic, fluorescent, and MAT1-2 parent, but were metabolically distinct. This study highlights phenotypic and genomic changes that may occur in a single generation from the outcrossing of sexually compatible strains of A. flavus.

Large-scale genomic analyses with machine learning uncover predictive patterns associated with fungal phytopathogenic lifestyles and traits.

Invasive plant pathogenic fungi have a global impact, with devastating economic and environmental effects on crops and forests. Biosurveillance, a critical component of threat mitigation, requires risk prediction based on fungal lifestyles and traits. Recent studies have revealed distinct genomic patterns associated with specific groups of plant pathogenic fungi. We sought to establish whether these phytopathogenic genomic patterns hold across diverse taxonomic and ecological groups from the Ascomycota and Basidiomycota, and furthermore, if those patterns can be used in a predictive capacity for biosurveillance. Using a supervised machine learning approach that integrates phylogenetic and genomic data, we analyzed 387 fungal genomes to test a proof-of-concept for the use of genomic signatures in predicting fungal phytopathogenic lifestyles and traits during biosurveillance activities. Our machine learning feature sets were derived from genome annotation data of carbohydrate-active enzymes (CAZymes), peptidases, secondary metabolite clusters (SMCs), transporters, and transcription factors. We found that machine learning could successfully predict fungal lifestyles and traits across taxonomic groups, with the best predictive performance coming from feature sets comprising CAZyme, peptidase, and SMC data. While phylogeny was an important component in most predictions, the inclusion of genomic data improved prediction performance for every lifestyle and trait tested. Plant pathogenicity was one of the best-predicted traits, showing the promise of predictive genomics for biosurveillance applications. Furthermore, our machine learning approach revealed expansions in the number of genes from specific CAZyme and peptidase families in the genomes of plant pathogens compared to non-phytopathogenic genomes (saprotrophs, endo- and ectomycorrhizal fungi). Such genomic feature profiles give insight into the evolution of fungal phytopathogenicity and could be useful to predict the risks of unknown fungi in future biosurveillance activities.

Improving ribonucleic acid production in Saccharomyces pastorianus via in silico genome-scale metabolic network model.

Ribonucleic acid (RNA) and its degradation products are important biomolecules widely used in the food and pharmaceutical industries for their flavoring and nutritional functions. In this study, we used a genome-scale metabolic network model (GSMM) to explore genetic targets for nucleic acid synthesis in a Saccharomyces pastorianus strain (G03). Yeast 8.5.0 was used as the base model, which accurately predicted G03's growth. Using OptForce, we found that overexpression of ARO8 and ATP1 among six different strategies increased the RNA content of G03 by 58.0% and 74.8%, respectively. We also identified new metabolic targets for improved RNA production using a modified GSMM called TissueModel, constructed using the GIMME transcriptome constraint tool to remove low-expressed reactions in the model. After running OptKnock, the RNA content of G03-△BNA1 and G03-△PMA1 increased by 44.6% and 39.8%, respectively, compared to G03. We suggest that ATP1, ARO8, BNA1, and PMA1 regulate cell fitness, which affects RNA content. This study is the first to identify strategies for RNA overproduction using GSMM and to report that regulation of ATP1, ARO8, BNA1, and PMA1 can increase RNA content in S. pastorianus. These findings also provide valuable knowledge on model reconstruction for S. pastorianus.

Nuclear genome organization in fungi: from gene folding to Rabl chromosomes.

Comparative genomics has recently provided unprecedented insights into the biology and evolution of the fungal lineage. In the postgenomics era, a major research interest focuses now on detailing the functions of fungal genomes, i.e. how genomic information manifests into complex phenotypes. Emerging evidence across diverse eukaryotes has revealed that the organization of DNA within the nucleus is critically important. Here, we discuss the current knowledge on the fungal genome organization, from the association of chromosomes within the nucleus to topological structures at individual genes and the genetic factors required for this hierarchical organization. Chromosome conformation capture followed by high-throughput sequencing (Hi-C) has elucidated how fungal genomes are globally organized in Rabl configuration, in which centromere or telomere bundles are associated with opposite faces of the nuclear envelope. Further, fungal genomes are regionally organized into topologically associated domain-like (TAD-like) chromatin structures. We discuss how chromatin organization impacts the proper function of DNA-templated processes across the fungal genome. Nevertheless, this view is limited to a few fungal taxa given the paucity of fungal Hi-C experiments. We advocate for exploring genome organization across diverse fungal lineages to ensure the future understanding of the impact of nuclear organization on fungal genome function.

Improving CRISPR-Cas9 mediated genome integration in interspecific hybrid yeasts.

Saccharomyces pastorianus is not a classical taxon, it is an interspecific hybrid resulting from the cross of Saccharomyces cerevisiae and Saccharomyces eubayanus. Exhibiting heterosis for phenotypic traits such as wort α-oligosaccharide consumption and fermentation at low temperature, it has been domesticated to become the main workhorse of the brewing industry. Although CRISPR-Cas9 has been shown to be functional in S. pastorianus, repair of CRISPR-induced double strand breaks is unpredictable and preferentially uses the homoeologous chromosome as template, preventing targeted introduction of the desired repair construct. Here, we demonstrate that lager hybrids can be edited with near 100% efficiency at carefully selected landing sites on the chimeric SeScCHRIII. The landing sites were systematically selected and evaluated for (i) absence of loss of heterozygosity upon CRISPR-editing, (ii) efficiency of the gRNA, and (iii) absence of effect on strain physiology. Successful examples of highly efficient single and double gene integration illustrated that genome editing can be applied in interspecies hybrids, paving the way to a new impulse to lager yeast strain development.

Beyond the genomes of Fulvia fulva (syn. Cladosporium fulvum) and Dothistroma septosporum: New insights into how these fungal pathogens interact with their host plants.

Fulvia fulva and Dothistroma septosporum are closely related apoplastic pathogens with similar lifestyles but different hosts: F. fulva is a pathogen of tomato, whilst D. septosporum is a pathogen of pine trees. In 2012, the first genome sequences of these pathogens were published, with F. fulva and D. septosporum having highly fragmented and near-complete assemblies, respectively. Since then, significant advances have been made in unravelling their genome architectures. For instance, the genome of F. fulva has now been assembled into 14 chromosomes, 13 of which have synteny with the 14 chromosomes of D. septosporum, suggesting these pathogens are even more closely related than originally thought. Considerable advances have also been made in the identification and functional characterization of virulence factors (e.g., effector proteins and secondary metabolites) from these pathogens, thereby providing new insights into how they promote host colonization or activate plant defence responses. For example, it has now been established that effector proteins from both F. fulva and D. septosporum interact with cell-surface immune receptors and co-receptors to activate the plant immune system. Progress has also been made in understanding how F. fulva and D. septosporum have evolved with their host plants, whilst intensive research into pandemics of Dothistroma needle blight in the Northern Hemisphere has shed light on the origins, migration, and genetic diversity of the global D. septosporum population. In this review, we specifically summarize advances made in our understanding of the F. fulva-tomato and D. septosporum-pine pathosystems over the last 10 years.

MycoCosm, the JGI's Fungal Genome Portal for Comparative Genomic and Multiomics Data Analyses.

MycoCosm ( https://mycocosm.jgi.doe.gov/ ) is an integrated fungal genomics portal that currently includes over 2000 fungal genomes. Efficiently exploring these genomes allows the scientific community to address challenges associated with energy and the environment. Here, we provide examples and guidelines for navigating around MycoCosm, and for using a variety of analysis tools to compare genomics and other "omics" data from the fungus Neocallimastix californiae with its relatives.

Divergent genomic trajectories predate the origin of animals and fungi.

Animals and fungi have radically distinct morphologies, yet both evolved within the same eukaryotic supergroup: Opisthokonta1,2. Here we reconstructed the trajectory of genetic changes that accompanied the origin of Metazoa and Fungi since the divergence of Opisthokonta with a dataset that includes four novel genomes from crucial positions in the Opisthokonta phylogeny. We show that animals arose only after the accumulation of genes functionally important for their multicellularity, a tendency that began in the pre-metazoan ancestors and later accelerated in the metazoan root. By contrast, the pre-fungal ancestors experienced net losses of most functional categories, including those gained in the path to Metazoa. On a broad-scale functional level, fungal genomes contain a higher proportion of metabolic genes and diverged less from the last common ancestor of Opisthokonta than did the gene repertoires of Metazoa. Metazoa and Fungi also show differences regarding gene gain mechanisms. Gene fusions are more prevalent in Metazoa, whereas a larger fraction of gene gains were detected as horizontal gene transfers in Fungi and protists, in agreement with the long-standing idea that transfers would be less relevant in Metazoa due to germline isolation3-5. Together, our results indicate that animals and fungi evolved under two contrasting trajectories of genetic change that predated the origin of both groups. The gradual establishment of two clearly differentiated genomic contexts thus set the stage for the emergence of Metazoa and Fungi.

Diploid-dominant life cycles characterize the early evolution of Fungi.

Most of the described species in kingdom Fungi are contained in two phyla, the Ascomycota and the Basidiomycota (subkingdom Dikarya). As a result, our understanding of the biology of the kingdom is heavily influenced by traits observed in Dikarya, such as aerial spore dispersal and life cycles dominated by mitosis of haploid nuclei. We now appreciate that Fungi comprises numerous phylum-level lineages in addition to those of Dikarya, but the phylogeny and genetic characteristics of most of these lineages are poorly understood due to limited genome sampling. Here, we addressed major evolutionary trends in the non-Dikarya fungi by phylogenomic analysis of 69 newly generated draft genome sequences of the zoosporic (flagellated) lineages of true fungi. Our phylogeny indicated five lineages of zoosporic fungi and placed Blastocladiomycota, which has an alternation of haploid and diploid generations, as branching closer to the Dikarya than to the Chytridiomyceta. Our estimates of heterozygosity based on genome sequence data indicate that the zoosporic lineages plus the Zoopagomycota are frequently characterized by diploid-dominant life cycles. We mapped additional traits, such as ancestral cell-cycle regulators, cell-membrane- and cell-wall-associated genes, and the use of the amino acid selenocysteine on the phylogeny and found that these ancestral traits that are shared with Metazoa have been subject to extensive parallel loss across zoosporic lineages. Together, our results indicate a gradual transition in the genetics and cell biology of fungi from their ancestor and caution against assuming that traits measured in Dikarya are typical of other fungal lineages.

Modelling the fitness landscapes of a SCRaMbLEd yeast genome.

The use of microorganisms for the production of industrially important compounds and enzymes is becoming increasingly important. Eukaryotes have been less widely used than prokaryotes in biotechnology, because of the complexity of their genomic structure and biology. The Yeast2.0 project is an international effort to engineer the yeast Saccharomyces cerevisiae to make it easy to manipulate, and to generate random variants using a system called SCRaMbLE. SCRaMbLE relies on artificial evolution in vitro to identify useful variants, an approach which is time consuming and expensive. We developed an in silico simulator for the SCRaMbLE system, using an evolutionary computing approach, which can be used to investigate and optimize the fitness landscape of the system. We applied the system to the investigation of the fitness landscape of one of the S. saccharomyces chromosomes, and found that our results fitted well with those previously published. We then simulated directed evolution with or without manipulation of SCRaMbLE, and revealed that controlling the SCRaMbLE process could effectively impact directed evolution. Our simulator can be applied to the analysis of the fitness landscapes of any organism for which SCRaMbLE has been implemented.

Construction of a synthetic Saccharomyces cerevisiae pan-genome neo-chromosome.

The Synthetic Yeast Genome Project (Sc2.0) represents the first foray into eukaryotic genome engineering and a framework for designing and building the next generation of industrial microbes. However, the laboratory strain S288c used lacks many of the genes that provide phenotypic diversity to industrial and environmental isolates. To address this shortcoming, we have designed and constructed a neo-chromosome that contains many of these diverse pan-genomic elements and which is compatible with the Sc2.0 design and test framework. The presence of this neo-chromosome provides phenotypic plasticity to the Sc2.0 parent strain, including expanding the range of utilizable carbon sources. We also demonstrate that the induction of programmable structural variation (SCRaMbLE) provides genetic diversity on which further adaptive gains could be selected. The presence of this neo-chromosome within the Sc2.0 backbone may therefore provide the means to adapt synthetic strains to a wider variety of environments, a process which will be vital to transitioning Sc2.0 from the laboratory into industrial applications.

Comparative Genomics of Microsporidia.

The microsporidia are a phylum of intracellular parasites that represent the eukaryotic cell in a state of extreme reduction, with genomes and metabolic capabilities embodying eukaryotic cells in arguably their most streamlined state. Over the past 20 years, microsporidian genomics has become a rapidly expanding field starting with sequencing of the genome of Encephalitozoon cuniculi, one of the first ever sequenced eukaryotes, to the current situation where we have access to the data from over 30 genomes across 20+ genera. Reaching back further in evolutionary history, to the point where microsporidia diverged from other eukaryotic lineages, we now also have genomic data for some of the closest known relatives of the microsporidia such as Rozella allomycis, Metchnikovella spp. and Amphiamblys sp. Data for these organisms allow us to better understand the genomic processes that shaped the emergence of the microsporidia as a group. These intensive genomic efforts have revealed some of the processes that have shaped microsporidian cells and genomes including patterns of genome expansions and contractions through gene gain and loss, whole genome duplication, differential patterns of invasion and purging of transposable elements. All these processes have been shown to occur across short and longer time scales to give rise to a phylum of parasites with dynamic genomes with a diversity of sizes and organisations.

Impact of Genome Reduction in Microsporidia.

Microsporidia represent an evolutionary outlier in the tree of life and occupy the extreme edge of the eukaryotic domain with some of their biological features. Many of these unicellular fungi-like organisms have reduced their genomic content to potentially the lowest limit. With some of the most compacted eukaryotic genomes, microsporidia are excellent model organisms to study reductive evolution and its functional consequences. While the growing number of sequenced microsporidian genomes have elucidated genome composition and organization, a recent increase in complementary post-genomic studies has started to shed light on the impacts of genome reduction in these unique pathogens. This chapter will discuss the biological framework enabling genome minimization and will use one of the most ancient and essential macromolecular complexes, the ribosome, to illustrate the effects of extreme genome reduction on a structural, molecular, and cellular level. We outline how reductive evolution in microsporidia has shaped DNA organization, the composition and function of the ribosome, and the complexity of the ribosome biogenesis process. Studying compacted mechanisms, processes, or macromolecular machines in microsporidia illuminates their unique lifestyle and provides valuable insights for comparative eukaryotic structural biology.