bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.974352; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 1/38 1 Strong effect of Penicillium roqueforti populations on volatile and metabolic compounds 2 responsible for aromas, flavour and texture in blue cheeses 3 4 Authors: 5 Thibault CARON1,4, Mélanie LE PIVER4, Anne-Claire PÉRON3, Pascale LIEBEN3, René 6 LAVIGNE2, Sammy BRUNEL4, Daniel ROUEYRE4, Michel PLACE4, Pascal 7 BONNARME3, Tatiana GIRAUD1*, Antoine BRANCA1*, Sophie LANDAUD3*, Christophe 8 CHASSARD2* 9 1: Ecologie Systematique Evolution, Université Paris Saclay, CNRS, AgroParisTech, 91400 10 Orsay, France 11 2: Université Clermont Auvergne, INRAE, Vetagro Sup, UMRF, 20 Côte de Reyne, 15000 12 Aurillac, France 13 3: Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, 78850 Thiverval- 14 Grignon, France 15 4: Laboratoire Interprofessionnel de Production – SAS L.I.P., 34 rue de Salers, 15 000 16 Aurillac, France 17 *These authors jointly supervised the study 18 19 Corresponding author: Antoine Branca [email protected] 20 Running title: Penicillium roqueforti population impact on cheeses 21 Keywords: Roquefort cheese, fungi, Penicillium, domestication, volatile compounds 22 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.974352; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 2 2/38 23 Abstract: The study of food microorganism domestication can bring important insights on 24 adaptation mechanisms and have industrial applications. The Penicillium roqueforti mold is 25 divided into four main populations, with two populations domesticated for blue-cheese 26 making and two populations thriving in other environments. While most blue cheeses 27 worldwide are made with the same P. roqueforti clonal lineage, the emblematic Roquefort 28 cheeses are inoculated with a specific population. To study the differences among P. 29 roqueforti populations in the context of domestication for cheesemaking, we compared blue 30 cheeses made with the four fungal populations following Roquefort-type production 31 specifications. We found that the P. roqueforti populations had a minor impact on the cheese 32 bacterial diversity and none on the main microorganism abundance. The cheese P. roqueforti 33 populations produced cheeses with higher percentages of blue area and with different sets and 34 higher quantities of desired volatile compounds. The Roquefort P. roqueforti population in 35 particular produced higher quantities of positive aromatic compounds in cheeses, which was 36 related due to its most efficient proteolysis and lipolysis, and also produced cheeses with 37 lower water activity, thus restricting spoiler microorganisms. Our results show the strong 38 influence of P. roqueforti populations on several important aspects of cheese safety, 39 appearance and flavour. The typical appearance and flavours of blue cheeses are therefore the 40 result of human selection on P. roqueforti, thus constituting domestication, and the two 41 cheese populations have acquired specificities. This has important implications for our 42 understanding of adaptation and domestication processes as well as for improving cheese 43 production. 44 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.974352; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 3 3/38 45 Importance: Several fungi have been domesticated for food fermentation, with selection for 46 traits beneficial for food production. The mold used for making blue cheeses, Penicillium 47 roqueforti, is subdivided into four genetically different populations, two being found in 48 cheese, one being specific of the Roquefort protected designation of origin, and two in other 49 environments. The cheese P. roqueforti populations produced bluer cheeses with higher 50 quantities of desired volatile compounds. The Roquefort P. roqueforti population in 51 particular produced higher quantities of positive aromatic compounds in cheeses, in relation 52 to its most efficient proteolysis and lipolysis, and also produced cheeses with lower water 53 activity, thus restricting spoiler microorganisms. Our results support that the blue cheese 54 typical aspect and flavors are the result of a selection by humans and show the strong 55 influence of P. roqueforti populations for several important aspects of cheese safety, aspect 56 and flavor, paving the way for improving cheese production. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.974352; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 4 4/38 57 Domestication is an evolutionary process that has been studied by many biologists since 58 Darwin. Indeed, domestication is an excellent model for understanding adaptation, being the 59 result of a strong and recent selection on traits that are often known and of interest for 60 humans (1.Larson 2014). In addition, studying domestication often has important applied 61 consequences for the improvement of cultivated organisms. Domesticated fungi have 62 however been little studied so far compared to crops, despite representing excellent models in 63 this field (2.Gladieux 2014; 3.Giraud 2017). Most fungi can be cultivated in Petri dishes, 64 stored alive for decades in freezers and propagated asexually, which facilitate experiments. 65 Their metabolisms are used to produce various compounds of interest such as fuels, enzymes 66 and antibiotics (4.Bigelis 2001). Their oldest and most frequent use by humans is for 67 fermentation, to preserve and mature food; for example, the yeast Saccharomyces cerevisiae 68 is used for bread, wine and beer fermentation and the filamentous fungus Aspergillus oryzae 69 for soy sauce and sake fermentation (5.Dupont 2017) and these models have provided 70 important insights into the mechanisms of adaptation and domestication (6.Almeida 2014; 71 7.Baker 2015; 8.Gallone 2016; 9.Gibbons 2012; 10.Gonçalves 2016; 11.Libkind 2011; 72 12.Sicard and Legras 2011). 73 74 The Penicillium genus contains more than 300 species, several of them being used by 75 humans; for example P. rubens led to the discovery of penicillin and P. nalgioviense and P. 76 salamii are used for the production of dry-cured meat (13.Fleming 1929; 14.Ludemann 2010, 77 15.Perrone 2015). For centuries, Penicillium roqueforti has been used for the maturation of 78 all the numerous varieties of blue cheeses worldwide (16,17.Labbe and Serres, 2004, 2009; 79 18.Vabre 2015), the fungus being responsible for the cheese blue veined aspect through the 80 production of melanized spores in cheese cavities, where oxygen is available (19.Moreau 81 1980). Penicillium roqueforti can be found in other environments than cheeses, thriving in bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.974352; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 5 5/38 82 spoiled food and silage (20.Pitt 2009; 21.Ropars 2012). Genomic and experimental 83 approaches have recently elucidated several important aspects of P. roqueforti domestication 84 (22.Cheeseman 2014; 23,24,25,26Ropars 2015, 2016 a and b, 2017; 27.Gillot 2015; 28.Gillot 85 2017; 29.Dumas 2020). Four main populations have been identified, two being used for 86 cheesemaking, and the two other populations thriving in silage, lumber or spoiled food 87 (30.Ropars 2014; 27.Gillot 2015; 29.Dumas 2020). Populations of P. roqueforti used to make 88 blue cheeses display characteristic features of domesticated organisms, with genetic and 89 phenotypic differences compared to non-cheese populations, and in particular for traits of 90 interest for cheese production (29.Ropars 2014; 27.Gillot 2015; 29.Dumas 2020). While both 91 cheese populations harbour lower genetic diversity than the two other populations, the two 92 cheese populations differ from each other, both genetically and phenotypically, and resulted 93 from independent domestication events (29.Dumas 2020). One of the cheese populations, 94 called the non-Roquefort population, is a single clonal lineage, used to produce most types of 95 blue cheeses worldwide; the second cheese population, called the Roquefort population, is 96 genetically more diverse and contains all the strains used to produce blue cheeses from the 97 Roquefort protected designation of origin (PDO) (29.Dumas 2020). Based on in vitro tests, 98 the non-Roquefort population was found to display faster tributyrin degradation (i.e. a certain 99 type of lipolysis) and higher salt tolerance, faster in vitro growth on cheese medium and 100 better exclusion of competitors, compared to the Roquefort population (30.Ropars 2014; 101 23.Ropars 2015; 29.Dumas 2020). Horizontally-transferred genes only present in the non- 102 Roquefort population are involved in the production of an antifungal peptide and in lactose 103 catabolism (30.Ropars 2014; 23.Ropars 2015; 22.Cheeseman 2014). Positive selection has 104 been detected in genes with predicted functions involved in flavor compound production in 105 each of the cheese populations (29.Dumas 2020).