Posted on Authorea 24 Sep 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.163250208.83761439/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. A glimpse at an early stage of microbe domestication revealed in the variable genome of Torulaspora delbrueckii, an emergent industrial yeast Margarida Silva1 , Ana Pontes1 , Ricardo Franco-Duarte1 , Pedro Soares2 , Jose Paulo Sampaio3 , Maria Sousa2 , and Patrı́cia Brito3 1 Affiliation not available CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Braga, Portugal 3 UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 2 September 24, 2021 Abstract The yeast Torulaspora delbrueckii is gaining importance for biotechnology due to its ability to increase wine sensorial complexity and for enhancing pre-frozen bread dough leavening. However, little is known about its population structure, variation in gene content, or possible domestication routes. Here, we address these issues and update the delimitation of T. delbrueckii along five major clades. Among the three European clades, a basal lineage is associated with the wild arboreal niche, while the two other lineages are linked with anthropic environments, one to wine fermentations and the other to diverse sources including dairy products and bread dough (Mix- Anthropic clade). Using 62 genomes we identified 5629 genes in the pangenome of T. delbrueckii and 270 genes in the cloud genome. A pangenome tree analysis showed that wine strains have a genome composition more similar to European wild arboreal strains than to those of the Mix Anthropic clade, in contradiction with the phylogenetic analysis. An association of gene content and ecology gave further support to the hypothesis that the Mix - Anthropic clade has the most specialized genome content and indicated that some of the exclusive genes were implicated in galactose and maltose utilization. More detailed analyses traced the acquisition of a cluster of GAL genes in strains associated with dairy products and the expansion and functional diversification of MAL genes in strains isolated from bread dough. Contrary to S. cerevisiae, domestication in T. delbrueckii is not primed by alcoholic fermentation and appears to be a recent event. Hosted file Tdelbrueckii genomics_v10.doc available at https://authorea.com/users/434956/articles/538309a-glimpse-at-an-early-stage-of-microbe-domestication-revealed-in-the-variable-genome-oftorulaspora-delbrueckii-an-emergent-industrial-yeast 1 Posted on Authorea 24 Sep 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.163250208.83761439/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. A T. delbrueckii - DEL Torulaspora sp. I T. pretoriensis - PRT T. franciscae - FRC Torulaspora sp. II Torulaspora sp. III Torulaspora sp. IV Torulaspora sp. V T. globosa - GLO T. maleeae - MAL Torulaspora sp. VI T. microellipsoides - MCE B DEL I 98.3 85.1 100.0 PRT 85.4 87.7 96.7 FRC 85.0 87.7 88.0 100.0 II 84.5 84.1 84.2 84.4 NA III 84.1 85.2 85.3 85.1 85.1 IV 86.2 86.6 86.8 86.3 86.8 86.4 V 85.7 86.4 86.4 86.6 86.0 86.3 GLO 86.4 86.6 86.7 86.7 86.7 85.8 83.9 86.8 MAL 86.3 86.6 86.7 86.7 86.7 86.1 84.9 85.5 84.7 VI 86.5 87.1 86.5 86.1 86.8 87.5 85.4 86.4 86.7 85.9 NA MCE 86.5 87.5 87.3 87.6 87.0 88.0 86.8 86.3 87.2 87.2 88.2 NA I PRT FRC II III IV V VI MCE DEL 99.2 99.6 84.0 NA NA NA GLO MAL L11 L19 L15 MTF 1142 L20 MTF 3799 N S- G-9 N S-PDC-169 EVN 1141 PYCC 4739 PYCC 8309 EVN 1155 EVN 1129 NS-G-62 NS- G-72 COFT1 L09 L10 SRCM101298 ISA 1229 L12 L13 MTF 3987 PYCC 5321 PYCC 5323 V187 V393 NCYC 4020 PYCC 3209 MTF 3985 NCYC 161 PYCC 2913 PYCC 6792 NCYC 3506 NCYC 2629 PYCC 2477 CBS 1146 PYCC 2478 PYCC 2999 NCYC 3024 L18 Zymaflore Alpha PYCC 7193 PYCC 8416 MTF 4303 PYCC 6819 PYCC 8419 MTF 4301 MTF 4307 PYCC 2713 PYCC 8420 PYCC 8413 PYCC 8415 NCYC 696 NRRL Y-50541 NCYC 140 PYCC 2916 NCYC 3877 ISA 1549 L16 PYCC 2844 V405 PYCC 8414 CBS 11121 CBS 11124 CBS 11100 CBS 11123 NRRL Y-17251 UWOPS 83-1046.2 CBS 9333 CBS 2785 CBS 2926 NRRL Y-17532 CBS 5080 PYCC 8099 PYCC 8100 PYCC 8101 PYCC 8417 PYCC 8418 CBS 2947 CBS 764 CBS 10694 N CYC 2473 NRRL Y-1549 DEL I PRT FRC II III IV V GLO MAL VI MCE Zygotorulaspora florentina NRRL Y-1560 Zygotorulaspora mrakii NRRL Y-12654 Zygosaccharomyces bailii CBS 680 0.08 2 Posted on Authorea 24 Sep 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.163250208.83761439/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 1.0 0.8 0.6 0.4 0.2 0.0 GLOBAL Wine WINE MIX ANTHROPIC L11 L19 L15 MTF 1142 L20 NS-G-9 NS-PDC-169 MTF 3799 EVN 1141 EVN 1129 NS-G-62 NS-G-72 COFT1 L09 PYCC 4739 PYCC 8309 EVN 1155 L10 SRCM101298 ISA 1229 L12 L13 MTF 3987 PYCC 5321 PYCC 5323 V187 V393 PYCC 2999 NCYC 3024 PYCC 3209 NCYC 4020 MTF 3985 NCYC 161 PYCC 2913 PYCC 6792 NCYC 3506 NCYC 2629 PYCC 2478 PYCC 2477 CBS 1146 L18 Zymaflore Alpha PYCC 7193 PYCC 8416 PYCC 6819 PYCC 8419 MTF 4301 MTF 4307 MTF 4303 PYCC 2713 PYCC 8420 Hyb EUROPE ARBOREAL NEW WORLD NCYC 140 NCYC 3877 PYCC 2916 ISA 1549 PYCC 8414 L16 PYCC 2844 V405 0.001 Mix - Anthropic Europe 3 Arboreal Ecology Geography Ecology Wine Alcoholic non-wine Fermentation Bread Dairy Plant Arboreal / Soil Unknwon / Human Europe New World Geography Asia Oceania Sp. III PYCC 8413 PYCC 8415 NCYC 696 NRRL Y-50541 Global No Information New World MTF 1142 MTF 3799 NS-G-9 NS-PDC-169 L11 L15 L19 L20 PYCC 4739 PYCC 8309 L09 EVN 1141 EVN 1155 NS-G-62 COFT1 NS-G-72 EVN 1129 L10 SRCM101298 ISA 1229 PYCC 2999 NCYC 3024 PYCC 3209 NCYC 3506 V187 V393 MTF 3985 MTF 3987 PYCC 2913 PYCC 6792 NCYC 4020 PYCC 5323 PYCC 5321 L12 L13 NCYC 161 PYCC 2478 CBS 1146 PYCC 2477 NCYC 2629 PYCC 8308 MTF 4301 MTF 4303 MTF 4307 Zymaflore Alpha PYCC 2713 PYCC 6819 PYCC 7193 PYCC 8416 PYCC 8419 PYCC 8420 L18 PYCC 8413 PYCC 8415 NCYC 696 NRRL Y-50541 ISA 1549 PYCC 8414 PYCC 2916 NCYC 3877 NCYC 140 PYCC 2844 V405 L16 PYCC 8099 PYCC 8100 PYCC 8101 A B Admixture Proportions MIX ANTHROPIC 2 3 NCYC 696 PYCC 8413 PYCC 8415 NCYC 3877 PYCC 2916 ISA1549 PYCC 8414 L16 PYCC 2844 V405 4 NEW WORLD GLOBAL * Galactose metabolism Drug resistance / Detoxification Maltose metabolism Oxidoreductase Sulfate metabolism Iron metabolism Meiosis and sporulation Nitrogen metabolism Unknown Pseudogene B. ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● OTHERPLANT ● ●● ● ● ● ●● ● ● BREAD ● ●● ● ● ● ●● ● ● DAIRY ● ●● ● ● NCYC 140 0.008 Cluster # ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● * 4 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 4968 1 ● ● ● ● ● ● ● ● ● ARBOREAL 25088 2929 4049 1561 1567 COFT1 L09 NS-G-9 NS-PDC-169 MTF 1142 MTF 3799 PYCC 4739 PYCC 8309 SRCM101298 EVN 1155 ISA 1229 MTF 4303 EVN 1129 NS-G-62 EVN 1141 NS-G-72 PYCC 2713 PYCC 8420 ZymafloreAlpha PYCC 6819 MTF 4301 MTF 4307 PYCC 7193 PYCC 8416 PYCC 8419 L10 L11 L15 L19 L20 L18 L12 L13 NCYC 3506 NCYC 161 PYCC 2913 NCYC 4020 PYCC 2999 PYCC 3209 NCYC 3024 PYCC 5321 PYCC 5323 MTF 3987 V187 MTF 3985 PYCC 6792 V393 CBS 1146 NCYC 2629 PYCC 2477 PYCC 2478 2922 73030 68242 134978 111849 134320 68243 75200 80255 46069 4520 28735 4522 4524 4525 4521 4114 28240 76 566 2899 3638 Posted on Authorea 24 Sep 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.163250208.83761439/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. WINE A. * ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● MTF4307 MTF4301 MTF4303 PYCC8419 PYCC6819 PYCC8416 PYCC7193 L18 PYCC8420 NS−G−62 EVN1129 NS−G−72 COFT1 NS−PDC−169 NS−G−9 MTF3799 EVN1141 EVN1155 ISA1229 V393 V187 PYCC5323 PYCC5321 PYCC6792 PYCC2478 GAL GAL GAL7 AGT2 AG2 AGT1 AG1 AG3 GAL1 GAL10 GAL4 MEL1 MAL chr V MAL chr VII chr I chr II chr IV chr V WINE GAL2 BREAD PGM1 MIX ANTHROPIC Pseudogene Relic MTF 3985 MTF 3987 PYCC 5321 PYCC 5323 PYCC 2913 OTHER (9 strains) DAIRY CBS 1146 PYCC 6792 PYCC 2478 NCYC 3506 Wild Domesticated Unknown ARBOREAL NEW WORLD GLOBAL 307 411 218 217 279 T. delbrueckii PYCC 5321 AG2 278 T. delbrueckii CBS 1146 Ψ 219 100 AG1 216 signature aminoacids B. 158 Posted on Authorea 24 Sep 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.163250208.83761439/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. A. Y A G G L M A D E Y A G G L M A D E C. T. delbrueckii PYCC 5321 AG3 Y A G G L M A D T. delbrueckii CBS 1146 AGT1 98 AGT2 100 T. delbrueckii PYCC 5321Ψ Ψ Pseudogene E 98 Ψ Pseudogene T. pretoriensis NRRL Y-17251 AG 97 100 T. pretoriensis NRRL Y-17251 AGT 98 T. franciscae CBS 2926 AG MALTASE ISOMALTASE T. delbrueckii PYCC 5321 T. pretoriensis NRRL Y-17251 AG T. franciscae CBS 2926 AGT 100 T. delbrueckii CBS 1146 100 79 100 96 T. delbrueckii PYCC 5321 Ψ V T. delbrueckii CBS 1146 100 T. delbrueckii PYCC 5321 100 T. delbrueckii PYCC 5321 Ψ IV T. pretoriensis NRRL Y-17251 AGT Y V G S L M Q D E 98 100 83 T. franciscae CBS 2926 AGT T. pretoriensis NRRL Y-17251 AG ISOMALTASE Saccharomyces cerevisiae S288C MAL11/AGT1 100 T. franciscae NRRL Y-17532 AG Saccharomyces cerevisiae MAL61 84 Y V G S L M Q D Saccharomyces cerevisiae S288C IMA1 Y V G S L M Q D E Saccharomyces cerevisiae S288C MAL32 F T A G L V A E D F T A G L V A D D F T A G L M G E D Lachancea thermotolerans CBS 6340 IMA A E Lachancea thermotolerans MAL61 100 100 Saccharomyces cerevisiae S288C MPH2 100 100 99 MALTASE Saccharomyces cerevisiae S288C MPH3 Lachancea thermotolerans CBS 6340 MAL2 Lachancea thermotolerans MAL31 Lachancea thermotolerans CBS 6340 IMA B Ogataea polymorpha MAL2 100 Ogataea polymorpha MAL1 100 0.2 Ogataea parapolymorpha AGT1 0.2 Ogataea parapolymorpha AG1 5