CAZyme prediction in ascomycetous yeast genomes guides discovery of novel xylanolytic species with diverse capacities for hemicellulose hydrolysis

Background Ascomycetous yeasts from the kingdom fungi inhabit every biome in nature. While filamentous fungi have been studied extensively regarding their enzymatic degradation of the complex polymers comprising lignocellulose, yeasts have been largely overlooked. As yeasts are key organisms used in industry, understanding their enzymatic strategies for biomass conversion is an important factor in developing new and more efficient cell factories. The aim of this study was to identify polysaccharide-degrading yeasts by mining CAZymes in 332 yeast genomes from the phylum Ascomycota. Selected CAZyme-rich yeasts were then characterized in more detail through growth and enzymatic activity assays. Results The CAZyme analysis revealed a large spread in the number of CAZyme-encoding genes in the ascomycetous yeast genomes. We identified a total of 217 predicted CAZyme families, including several CAZymes likely involved in degradation of plant polysaccharides. Growth characterization of 40 CAZyme-rich yeasts revealed no cellulolytic yeasts, but several species from the Trichomonascaceae and CUG-Ser1 clades were able to grow on xylan, mixed-linkage β-glucan and xyloglucan. Blastobotrys mokoenaii, Sugiyamaella lignohabitans, Spencermartinsiella europaea and several Scheffersomyces species displayed superior growth on xylan and well as high enzymatic activities. These species possess genes for several putative xylanolytic enzymes, including ones from the well-studied xylanase-containing glycoside hydrolase families GH10 and GH30, which appear to be attached to the cell surface. B. mokoenaii was the only species containing a GH11 xylanase, which was shown to be secreted. Surprisingly, no known xylanases were predicted in the xylanolytic species Wickerhamomyces canadensis, suggesting that this yeast possesses novel xylanases. In addition, by examining non-sequenced yeasts closely related to the xylanolytic yeasts, we were able to identify novel species with high xylanolytic capacities. Conclusions Our approach of combining high-throughput bioinformatic CAZyme-prediction with growth and enzyme characterization proved to be a powerful pipeline for discovery of novel xylan-degrading yeasts and enzymes. The identified yeasts display diverse profiles in terms of growth, enzymatic activities and xylan substrate preferences, pointing towards different strategies for degradation and utilization of xylan. Together, the results provide novel insights into how yeast degrade xylan, which can be used to improve cell factory design and industrial bioconversion processes.

CAZyme Prediction in Ascomycetous Yeast Genomes Guides Discovery of Novel Xylanolytic Species with Diverse Capacities for Hemicellulose Hydrolysis

Background Ascomycetous yeasts from the kingdom fungi inhabit every biome in Nature. While filamentous fungi have been studied extensively regarding their enzymatic degradation of the complex polymers comprising lignocellulose, yeasts have been largely overlooked. As yeasts are key organisms used in industry, understanding their enzymatic strategies for biomass conversion is an important factor in developing new and more efficient cell factories. The aim of this study was to identify polysaccharide-degrading yeasts by mining CAZymes in 332 yeast genomes from the phylum Ascomycota. Selected CAZyme-rich yeasts were then characterized in more detail through growth and enzymatic activity assays. Results The CAZyme analysis revealed a large spread in the number of CAZyme-encoding genes in the Ascomycetous yeast genomes. We identified a total of 224 predicted CAZyme families, including several CAZymes likely involved in degradation of plant polysaccharides. Growth characterization of 40 CAZyme-rich yeasts revealed no cellulolytic yeasts, but several species from the Trichomonascaceae and CUG-Ser1 clades were able to grow on xylan, β-glucan and xyloglucan. Blastobotrys mokoenaii, Sugiyamaella lignohabitans, Spencermartinsiella europaea and several Scheffersomyces species displayed superior growth on xylan and well as high enzymatic activities. These species contained several putative xylanolytic enzymes, including the well-studied xylanase-containing glycoside hydrolase families GH10 and GH30 that appear attached to the cell surface. B. mokoenaii was the only species containing a GH11 xylanase, which was shown to be secreted. Surprisingly, no known xylanases were predicted in the xylanolytic species Wickerhamomyces canadensis, suggesting that this yeast possess novel xylanases. In addition, by examining non-sequenced yeasts closely related to the xylanolytic yeasts, we were able to identify novel species with high xylanolytic capacities. Conclusions Our approach of combining high-throughput bioinformatic CAZyme-prediction with growth and enzyme characterization proved to be a powerful pipeline for discovery of novel xylan-degrading yeasts and enzymes. The identified yeasts display diverse profiles in terms of growth, enzymatic activities and xylan substrate preferences, pointing towards different strategies for degradation and utilization of xylan. Together, the results provide novel insights into how yeast degrade xylan, which can be used to improve cell factory design and industrial bioconversion processes.

Black Yeast Genomes Assembled from Plastic Fabric Metagenomes Reveal an Abundance of Hydrocarbon Degradation Genes

ABSTRACT We report the assembly and annotation of 10 different black yeast genomes from microbiome metagenomic data derived from biofouled plastic fabrics. The draft genomes are estimated to be 9 to 33.2 Mb, with 357 to 5,108 contigs and G+C contents of 43.9% to 57.4%, and they harbor multiple genes for hydrocarbon adaptation and degradation.

Engineered yeast genomes accurately assembled from pure and mixed samples

Yeast whole genome sequencing (WGS) lacks end-to-end workflows that identify genetic engineering. Here we present Prymetime, a tool that assembles yeast plasmids and chromosomes and annotates genetic engineering sequences. It is a hybrid workflow—it uses short and long reads as inputs to perform separate linear and circular assembly steps. This structure is necessary to accurately resolve genetic engineering sequences in plasmids and the genome. We show this by assembling diverse engineered yeasts, in some cases revealing unintended deletions and integrations. Furthermore, the resulting whole genomes are high quality, although the underlying assembly software does not consistently resolve highly repetitive genome features. Finally, we assemble plasmids and genome integrations from metagenomic sequencing, even with 1 engineered cell in 1000. This work is a blueprint for building WGS workflows and establishes WGS-based identification of yeast genetic engineering.

Chromosome-level genome assembly and structural variant analysis of two laboratory yeast strains from the Peterhof Genetic Collection lineage

Thousands of yeast genomes have been sequenced with both traditional and long-read technologies, and multiple observations about modes of genome evolution for both wild and laboratory strains have been drawn from these sequences. In our study, we applied Oxford Nanopore and Illumina technologies to assemble complete genomes of two widely used members of a distinct laboratory yeast lineage, the Peterhof Genetic Collection (PGC), and investigate the structural features of these genomes including transposable element content, copy number alterations, and structural rearrangements. We identified numerous notable structural differences between genomes of PGC strains and the reference S288C strain. We discovered a substantial enrichment of mid-length insertions and deletions within repetitive coding sequences, such as in the SCH9 gene or the NUP100 gene, with possible impact of these variants on protein amyloidogenicity. High contiguity of the final assemblies allowed us to trace back the history of reciprocal unbalanced translocations between chromosomes I, VIII, IX, XI, and XVI of the PGC strains. We show that formation of hybrid alleles of the FLO genes during such chromosomal rearrangements is likely responsible for the lack of invasive growth of yeast strains. Taken together, our results highlight important features of laboratory yeast strain evolution using the power of long-read sequencing.

State-of-the-art structural variant calling: What went conceptually wrong and how to fix it?

Structural variant (SV) calling belongs to the standard tools of modern bioinformatics for identifying and describing alterations in genomes. Initially, this work presents several complex genomic rearrangements that reveal conceptual ambiguities inherent to the SV representations of state-of-the-art SV callers. We contextualize these ambiguities theoretically as well as practically and propose a graph-based approach for resolving them. Our graph model unifies both genomic strands by using the concept of skew-symmetry; it supports graph genomes in general and pan genomes in specific. Instances of our model are inferred directly from seeds instead of the commonly used alignments that conflict with various types of SV as reported here. For yeast genomes, we practically compute adjacency matrices of our graph model and demonstrate that they provide highly accurate descriptions of one genome in terms of another. An open-source prototype implementation of our approach is available under the MIT license at https://github.com/ITBE-Lab/MA.

Life as a Machine

Bacteria are a source of many of the tools used in biotechnology. A technique called the polymerase chain reaction, or PCR, made it possible for the first time to amplify tiny starting amounts of DNA and has revolutionised medical diagnosis, testing of IVF embryos for mutations, and forensic science. PCR involves the repeated generation of DNA from a starting sequence in a cycle, one stage of which occurs at boiling point. Because of this PCR uses a DNA polymerase enzyme purified from an ‘extremophile’ bacterium that lives in hot springs. More recently scientists have constructed artificial bacterial or yeast genomes from scratch. The next step will be to create reconfigured bacteria and yeast with enhanced characteristics for use in agriculture, energy production, or generation of new materials. Some scientists are now seeking to expand the genetic code itself. The DNA code that human beings share with all other species on the planet has four ‘letters’, A, C, G, and T, which pair as A:T and C:G to join the two strands of the DNA double helix. And each particular triplet of DNA letters, for instance CGA, or TGC, codes for a specific amino acid, the 20 different amino acids joining together in a specific sequence to make up a particular protein. Scientists have now developed a new DNA letter pair, X:Y. By introducing this into an artificial bacterial genome, it is becoming possible to create many more amino acids than the current 20 naturally occurring ones, and thereby allowing many new types of proteins.