Comparative genomics of biotechnologically important yeasts Supplementary Appendix Contents Note 1 – Summary of literature on ascomycete yeasts used in this study ............................... 3 CUG-Ser yeasts ................................................................................................................................................................ 3 Other Saccharomycotina ............................................................................................................................................. 5 Taphrinomycotina ....................................................................................................................................................... 10 Note 2 – Genomes overview .................................................................................................11 Yeast culturing, identification, DNA and total RNA extraction ................................................................. 12 Genome sequencing and assembly ....................................................................................................................... 12 Transcriptome sequencing and assembly ......................................................................................................... 13 Table S1. Genome statistics ..................................................................................................................................... 14 Table S2. Annotation statistics ............................................................................................................................... 15 Figure S1. Genome size, repeat content, and protein conservation measures for the yeasts. ..... 16 Figure S2. Intron occurrence in 303 predicted orthologs in the yeasts. ............................................... 17 Figure S3. Distribution of TY-LTR elements in the yeasts. ......................................................................... 18 Figure S4. Organization of rDNA genes in yeast species.............................................................................. 19 Note 3 – Organism phylogeny ...............................................................................................20 Figure S5. Phylogenetic tree inferred with ExaML ........................................................................................ 21 Figure S6. Phylogenetic tree inferred with FastME ....................................................................................... 22 Note 4 – Alternative genetic codes: CUG coding for Ser and Ala...........................................23 Figure S7. Translation of CUG codons to Ser or Ala ....................................................................................... 24 Table S3. Results of Bagheera predictions of codon usage for selected yeasts. ................................. 25 Dataset S1. Genetic code of P. tannophilus ........................................................................................................ 25 Note 5 – Correlation of genomically encoded enzymes to metabolic traits .............................26 Assigning genes to enzyme functions .................................................................................................................. 27 Figure S8. Fermentative, metabolic, osmotolerance, and temperature-dependent growth of yeasts. ............................................................................................................................................................................... 28 Figure S9. Loss of Complex I genes and evolution of fermentative lifestyle ....................................... 29 Figure S10. Correlation between disaccharide metabolism and genome content ........................... 30 1 Note 6 – Gene clusters ..........................................................................................................31 Dataset S2. Phylogenetic distribution of gene clusters ................................................................................ 32 Dataset S3. Pairwise associations of ECs ............................................................................................................ 32 Note 7 – MAT locus organization and mating-type switching .................................................33 Pan-ascomycete synteny at the MAT locus. ...................................................................................................... 33 Homothallism and heterothallism. ....................................................................................................................... 33 Origin of mating type switching in Saccharomycotina. ................................................................................ 34 Loss of H3K9me2/3 heterochromatin. ............................................................................................................... 34 Figure S11. Mating-type locus organization and synteny in Ascomycota. ........................................... 37 Figure S12. MAT locus isomeric structures in Pachysolen tannophilus and Ascoidea rubescens. ............................................................................................................................................................................................. 38 References ............................................................................................................................40 2 Note 1 – Summary of literature on ascomycete yeasts used in this study The organisms used in this study were selected to characterize the genomes of yeasts important or potentially important from a biotechnological perspective, and to fill in gaps in our understanding of phylogenetic relationships. Many of the yeasts were selected because of their abilities to ferment or metabolize xylose, cellobiose, arabinose, galactose, maltose and other substrates such as methanol or to produce useful fuels and advanced chemicals. Even though many yeasts and fungi will grow on various carbon sources aerobically, fewer will convert these substrates into ethanol or other chemicals with meaningful yields at significant rates. Some species were selected for their capacities to make lipids and others were selected for their acid- thermo- cryo- or osmotolerance. Even though we understand some of the mechanisms determining these traits, we do not know how widespread they are, nor do we know the constellation of metabolic activities supporting these properties. By sequencing the genomes of several species having the traits of interest, and by comparing those genomes to other species without those traits, we expected to discover new mechanisms. We also sought to better understand diverse species that occupy habitats such as insect guts, soil or exudates of plants. Finally, we wanted to broaden knowledge about yeast species that could be adapted to new uses. Increasingly, biotechnology is seeking new yeasts as “platform organisms” for the production of natural or engineered metabolites. Synthetic biology and metabolic engineering are increasing the capacities of microbes to make specialized, rapid xylose fermentation could provide novel routes for the bioconversion of agricultural and wood harvest residues into renewable fuels and chemicals. This literature summary provides a summary of the major physiological or genetic properties, and growth habits as documented in the existing literature. The text is organized to follow our overall taxonomic structure as determined by whole genome comparisons. Selected members of Pezizomycotina and Basidiomycota have been included in our study for phylogenetic comparisons, but they are not covered in this literature summary. CUG-Ser yeasts Scheffersomyces stipitis Scheffersomyces stipitis(1) is a predominantly haploid, heterothallic yeast related to Candida shehatae, Spathaspora passalidarum and several other pentose metabolizing ascomycetous yeast species(2, 3). Strains of Sch. stipitis are among the best xylose-fermenting yeasts in type culture collections(4-7). Fed batch cultures of Sch. stipitis produce up to 47 g/L of ethanol from xylose at 30°C under low aeration conditions(5). Sch. stipitis belongs to a group of yeasts that uses code 12 in which CUG codes for serine rather than leucine(8). This makes correct translation of many heterologous genes problematic. Sch. stipitis CBS 6054 is related to several yeasts found as endosymbionts of beetles that inhabit and degrade white-rotted wood(9-11). Unlike Saccharomyces cerevisiae, which regulates fermentation by sensing the presence of fermentable sugars such as glucose, Sch. stipitis induces fermentative activity in response to oxygen limitation(12). A description of this genome was published in 2007(13). The comparative 3 analysis presented in the current publication helps to identify the genomic features underlying its physiological traits. Spathaspora passalidarum Spathaspora passalidarum(10) like P. stipitis and Candida tenuis (see below), can ferment and assimilate xylose, cellobiose, glucose and maltose. Efficient fermentation of xylose, a five- carbon sugar that is a major component of plant cell walls, is a major step towards effective biofuel production from plant materials. These three yeasts are also unusual in that they are found in symbiotic association with wood-boring beetles, and comparative study may give new insight into the genetic underpinnings of symbiotic relationships(9, 14). A number of other species of Spathaspora have been described with similar properties(15-17). However, not all related species