Project description:tRNAs are encoded by a large gene family, usually with several isogenic tRNAs interacting with the same codon. Mutations in the anticodon region of other tRNAs can overcome specific tRNA deficiencies. Phylogenetic analysis suggests that such mutations have occurred in evolution, but the driving force is unclear. We show that in yeast suppressor mutations in other tRNAs are able to overcome deficiency of the essential TRT2-encoded tRNAThrCGU at high temperature (40°C). Surprisingly, these tRNA suppressor mutations were obtained after whole-genome transformation with DNA from thermotolerant Kluyveromyces marxianus or Ogataea polymorpha strains, but from which the mutations did apparently not originate. We suggest that transient presence of donor DNA in the host facilitates proliferation at high temperature and thus increases the chances for occurrence of spontaneous mutations suppressing defective growth at high temperature. Whole-genome sequence analysis of three transformants revealed only four to five non-synonymous mutations of which one causing TRT2 anticodon stem stabilization and two anticodon mutations in non-threonyl-tRNAs, tRNALysCUU and tRNAeMetCAU, were causative. Both anticodon mutations suppressed lethality of TRT2 deletion and apparently caused the respective tRNAs to become novel substrates for threonyl-tRNA synthetase. LC-MS/MS data could not detect any significant mistranslation and RT-qPCR results contradicted induction of the unfolded protein response. We suggest that stress conditions have been a driving force in evolution for the selection of anticodon-switching mutations in tRNAs as revealed by phylogenetic analysis. Importance of the work In this work we have identified for the first time the causative elements in a eukaryotic organism introduced by applying whole-genome transformation and responsible for the selectable trait of interest, i.e. high temperature tolerance. Surprisingly, the whole-genome transformants contained just a few SNPs, which were unrelated to the sequence of the donor DNA. In each of three independent transformants, we have identified a SNP in a tRNA, either stabilizing the essential tRNAThrCGU at high temperature or switching the anticodon of tRNALysCUU or tRNAeMetCAU into CGU, which is apparently enough for in vivo recognition by threonyl-tRNA synthetase. LC-MS/MS analysis indeed indicated absence of significant mistranslation. Phylogenetic analysis showed that similar mutations have occurred throughout evolution and we suggest that stress conditions may have been a driving force for their selection. The low number of SNPs introduced by whole-genome transformation may favor its application for improvement of industrial yeast strains.

Project description:This is genome-scale metabolic model of Komagataella pastoris as the representative yeast species for the clade Pichiaceae. This model was generated through homology search using a fungal pan-GEM largely based on Yeast8 for Saccharomyces cerevisiae, in addition to manual curation. This model has been produced by the Yeast-Species-GEMs project from Sysbio (www.sysbio.se). This is model version 1.0.0 accompanying the publication (DOI: 10.15252/msb.202110427), currently hosted on BioModels Database and identified by MODEL2109130009. Further curations of this model will be tracked in the GitHub repository: https://github.com/SysBioChalmers/Yeast-Species-GEMs Models for species of the same clade includes: Ambrosiozyma kashinagacola; Ambrosiozyma monospora; Brettanomyces anomalus; Candida arabinofermentans; Candida boidinii; Candida sorboxylosa; Candida succiphila; Brettanomyces bruxellensis; Komagataella pastoris; Kuraishia capsulata; Ogataea methanolica; Ogataea parapolymorpha; Ogataea polymorpha; Pichia membranifaciens; Ogataea henricii; Ambrosiozyma ambrosiae; Citeromyces matritensis; Ambrosiozyma vanderkliftii; Brettanomyces custersianus; Komagataella populi; Saturnispora hagleri; Saturnispora mendoncae; Saturnispora saitoi; Saturnispora serradocipensis; Saturnispora silvae; Saturnispora zaruensis; Pichia occidentalis; Pichia norvegensis; Pichia nakasei; Pichia kudriavzevii; Pichia heedii; Pichia exigua; Martiniozyma abiesophila; Ogataea nitratoaversa; Ogataea populialbae; Ogataea zsoltii; Ogataea trehalophila; Ogataea trehaloabstinens; Ogataea ramenticola; Ogataea pini; Ogataea pilisensis; Ogataea philodendri; Ogataea glucozyma; Ogataea kodamae; Ogataea methylivora; Ogataea minuta; Ogataea naganishii; Ogataea nonfermentans; Kuraishia ogatae; Kuraishia molischiana; Komagataella pseudopastoris; Ambrosiozyma oregonensis; Ambrosiozyma philentoma; Citeromyces hawaiiensis; Citeromyces siamensis; Ambrosiozyma maleeae; Ambrosiozyma pseudovanderkliftii; Pichia terricola; Saturnispora dispora; Kregervanrija delftensis; Kregervanrija fluxuum. These models are available in the zip file. To cite BioModels, please use: V Chelliah et al; BioModels: ten-year anniversary. Nucleic Acids Res 2015; 43 (D1): D542-D548. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to MIT License for more information.

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Whole-genome sequencing of yeast strains

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