Summary Budding yeasts (subphylum Saccharomycotina) are found in every biome and are as genetically diverse as plants or animals. To understand budding yeast evolution, we analyzed the genomes of 332 yeast species, including 220 newly sequenced ones, which represent nearly a third of all known budding yeast diversity. Here we establish a robust genus-level phylogeny comprised of 12 major clades, infer the timescale of diversification from the Devonian Period to the present, quantify horizontal gene transfer (HGT), and reconstruct the evolution of 45 metabolic traits and the metabolic toolkit of the Budding Yeast Common Ancestor (BYCA). We infer that BYCA was metabolically complex and chronicle the tempo and mode of genomic and phenotypic evolution across the subphylum, which is characterized by very low HGT levels and widespread losses of traits and the genes that control them. More generally, our results argue that reductive evolution is a major mode of evolutionary diversification.

The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.

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e Actinobacteria in the genus Streptomyces are critical players in microbial communities that decompose complex carbohydrates in the soil, and these bacteria have recently been implicated in the deconstruction of plant polysaccharides for some herbivorous insects. Despite the importance of Streptomyces to carbon cycling, the extent of their plant biomass-degrading ability remains largely unknown. In this study, we compared four strains of Streptomyces isolated from insect herbivores that attack pine trees: DpondAA-B6 (SDPB6) from the mountain pine beetle, SPB74 from the southern pine beetle, and SirexAA-E (SACTE) and SirexAA-G from the woodwasp, Sirex noctilio. Biochemical analysis of secreted enzymes demonstrated that only two of these strains, SACTE and SDPB6, were efficient at degrading plant biomass. Genomic analyses indicated that SACTE and SDPB6 are closely related and that they share similar compositions of carbohydrate-active enzymes. Genome-wide proteomic and transcriptomic analyses revealed that the major exocellulases (GH6 and GH48), lytic polysaccharide monooxygenases (AA10), and mannanases (GH5) were conserved and secreted by both organisms, while the secreted endocellulases (GH5 and GH9 versus GH9 and GH12) were from diverged enzyme families. Together, these data identify two phylogenetically related insect-associated Streptomyces strains with high biomass-degrading activity and characterize key enzymatic similarities and differences used by these organisms to deconstruct plant biomass.

21 2 22 42complexes. Nascent mRNAs are co-transcriptionally processed by adding 3' polyadenosine 43 (poly(A)) tails and 5' caps of 7-methyl-guanosine (m 7 G) before they are trafficked out of the 44 3 nucleus for translation. In bacteria, transcription is tightly coupled with translation, and both 45 occur inside the cytosol. Furthermore, bacterial transcription often operates on clusters of genes, 46 known as operons, where a single regulatory region regulates the expression of physically-linked 47

Current genome editing techniques available for Saccharomyces yeast species rely on auxotrophic markers, limiting their use in wild and industrial strains and species. Taking advantage of the ancient loss of thymidine kinase in the fungal kingdom, we have developed the herpes simplex virus thymidine kinase gene as a selectable and counterselectable marker that forms the core of novel genome engineering tools called the Haploid Engineering and Replacement Protocol (HERP) cassettes. Here we show that these cassettes allow a researcher to rapidly generate heterogeneous populations of cells with thousands of independent chromosomal allele replacements using mixed PCR products. We further show that the high efficiency of this approach enables the simultaneous replacement of both alleles in diploid cells. Using these new techniques, many of the most powerful yeast genetic manipulation strategies are now available in wild, industrial, and other prototrophic strains from across the diverse Saccharomyces genus.G ENOME editing is a precise and powerful tool to investigate basic genetic processes or to reprogram an organism's metabolism. Techniques to precisely manipulate genomes exist for many model organisms (Storici et al. 2003;Gratz et al. 2013;Hwang et al. 2013;Jiang et al. 2013;Tzur et al. 2013), but not all features of these approaches are easily portable to closely related species. The genus Saccharomyces is highly experimentally tractable, and laboratory strains of all seven natural species can be genetically manipulated Liti et al. 2013). Saccharomyces cerevisiae is the most well-known member of the genus due to its role in brewing (Lodolo et al. 2008), biofuels (Steen et al. 2008;Matsushika et al. 2009), winemaking (Peris et al. 2012, and baking, as well as a model system for the biological sciences (Botstein and Fink 2011). Other members of the genus are also used by humans in the form of interspecies hybrids, such as the S. cerevisiae 3 Saccharomyces kudriavzevii hybrids used to ferment some wines and Belgian beers (Peris et al. 2012) and the S. cerevisiae 3 Saccharomyces eubayanus (Libkind et al. 2011) hybrids found in the brewing of lager-style beers around the world. The Saccharomyces genus is also an emerging "model genus" for molecular evolution, and several experimentally tractable species are now used routinely in evolutionary genetics research (Hittinger 2013). Efficient genome editing of these diverse Saccharomyces yeasts would therefore provide new avenues of investigation for basic and applied research.One major reason for the popularity of S. cerevisiae as a model system is the availability of powerful genetic manipulation tools. One of these tools is the URA3 selection/counterselection system (Boeke et al. 1984). URA3 is a gene required for the de novo synthesis of uracil. Thus, the URA3 gene can be used as a selectable marker in ura3 strains by selecting for the ability to grow on synthetic media without uracil. The deactivation or replacement of URA3 can also be selected for using synt...

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