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Yeast Culture Collections Yeast Culture Collections Kyria Boundy-Mills Curator, Phaff Yeast Culture Collection Food Science and Technology UC Davis Outline • Definition and importance of yeast • How yeasts are isolated and characterized • How yeasts are preserved and stored • Yeast collections of the world • Phaff Yeast Culture Collection history, holdings and activities What are yeasts? Single-celled fungi ~1700 species known Only 1% of species are named! Require C, N sources Basic life science research Model organisms 4 Nobel prizes in last 15 years Fermented foods and beverages, chemicals, biofuels, pharmaceuticals Products made using Yeasts • Amino acids • L-cystine, L-glutamic acid, L-serine, L-lysine, L-threonine, D and L-methionine; L-tryptophan, Nacetyl-glycine, L-phenylalanine • Carotenoids • Astaxanthin, carotene, lycopene • Acetoin, adipic acid, acetanilide, dicarboxylic acids, 1,3- • Chemicals propanediol, ethylacetate, 2,3-butanediol, glycerol, 1-ß- • Enzymes hydoxybutyric acid, !-ketoglutaric acid, lactic acid • Invertase, galactosidase, lipase, phytase, pectinases, trehalase, • Feed additives inulinase, glucoamylase, proteases, esterases • Fermented foods • SCP, SCO, phytase, ß-glucans, riboflavin, amino acids • Beer, bread, wine, miso, soy sauce, many other fermented foods • Flavors and beverages, ethanol • Food ingredients • 2-Phenlyethanol, nucleotides,ethylacetate, acetaldehyde, citric acid, furanones, methyl ketones • Heterologous and other proteins • Citric acid, acetic acid, lactic acid, ribonucleotides, biosurfactants- sophorolids and additives • Organic acids • Enzymes, pharmaceuticals-vaccines, immunoproteins • Nutraceuticals, pharmaceuticals • Acetic, lactic, citric, iso-citric, formic, pimelic, homogentisic • N-acetylglucosamine, inositol, S-adenosyl methionine, melanin, and cosmetics tumor necrosis factor, human serum, factors, ß-glucans • Polyols and sweeteners • Erythitol, glycerol, xylitol, D-arabitol, L-arabitol, D-mannitol • Xylulose, D-psicose, D-lyxose, mannans, phosphomannans, • Simple sugars and polysaccharides phosphogalactans, pullulans, mannoproteins, glucans, • Vitamins heteroxylans, exo-and acidic-polysaccharides • Riboflavin (B2), B12, thiamine, ergosterol, ubiquinone Abbas, C. A., Chapter X. Biotechnological applications of nonconventional yeasts. 2003. Kingdom Fungus: 7 Phyla plus four subphyla “incertae sedis” Phyla (?) (?) (?) Yeasts, molds, mushrooms, (?) bracket fungi, etc. (wikipedia.org) Phyla with some single celled forms Yeast ecology • No photosynthesis: heterotrophic • Varying specialization • GENERALISTS: Soil, plants, insects • SPECIALISTS: Gut of a specific insect, flower nectar, decaying plant, high sugar • Many species thrive best in moist, sugar-rich environments Rules for naming yeast: ICNAFP (Melbourne Code) • Scientific publication: species description • Type strain • Can’t name a species after yourself • Latinized name • Person: Phaffia, mrakii, Lachancea • Place: ontarioensis, californica, bruxellensis • Property: cellulolyticus, thermotolerans, arabinofermentans • Origin material: cerevisiae, alnii, How and why yeasts are isolated WHAT ARE YOU LOOKING FOR? • New species: look in unexplored habitats such as insects, plants • Anaerobic yeasts: look in anaerobic habitats • Specific nutrient utilization, pH or temperature tolerance: look in nature with those conditions • Brewing yeast: look at similar uninoculated fermentations Cultivating yeasts from nature • With or without enrichment (such as ethanol tolerance) • Plating most common; select appropriate media and growth conditions • Be aware of treaties, regulations, agreements and other regulations! How yeasts are identified • “Traditional” method: Panel of up to 100 tests • Carbon source assimilation • Carbon source fermentation • Temperatures • Enzymes • Stresses: high sugar, high salt, fungicides, etc. • Cell and colony morphology • Mating, sporulation Modern method: Ribotyping Ribosomal RNA Gene Repeat (DNA): ~140 sets in S. cerevisiae • Partial 26S rRNA, about 600 nt PRIMERS NL1 NL4 • Partial 26S sequences in 18S RNA ITS1 5.8S ITS2 26S RNA 5S Large Subunit GenBank for all (LSU) known yeast species • ITS sequences for NL1 many yeast species ¯ D1/D2 Domain (600 nt) NL4 Conserved among strains of the same species, variable between species How and why yeasts are characterized • Depends on application • Biotechnology: fermentation optimization, metabolites produced • Pathology: serotype, fungicide sensitivity • Food and beverage fermentations: attenuation, growth temperature, etc How yeasts are preserved and stored • Cryopreserved at -80oC or liquid nitrogen • Freeze dried • Agar slants • Water stocks • Filter paper Sources of yeasts • Nature • Other labs, researchers • Starter culture companies • White Labs, Wyeast, Lallemand, Lesaffre, etc. • Culture collections Importance of culture collections Three resources: Culture collection organizations establish best practice guidelines: Professionally preserved strains US Culture Collection Network Data World Federation of Culture Curator expertise Collections Public repositories • Large, diverse collections Examples: • Type strains of all known species • American Type Culture • Strains cited in publications Collection (ATCC) • Patent repository • USDA-ARS (NRRL) • May have research activities • Centraalbureau voor Schimmelcultures (CBS) • DSMZ “Boutique” collections Narrower range of species Examples: Historic specialty: wine, brewing, Phaff Yeast Culture Collection, ecology Food Science, UC Davis Broadening services to capture University of Perugia, Italy additional funding, such as (DBVPG) contract screening Institute of Food Research, UK (NCYC) Wine Yeast and Bacteria Collection, Viticulture and Enology, UC Davis Phaff collection VEN collection Phaff Yeast Culture Collection • 4th largest of its kind • 7,000 yeast strains • Over 800 species (half of the known yeast species), >200 novel species • phaffcollection.ucdavis.edu • Collected by Herman Phaff, colleagues (Starmer, Ganter, Lachance) • Yeast ecology, taxonomy, physiology • Decaying plant matter • Insects • Foods and beverages • Independent wild isolates • 80% of strains unique to this collection; owned by UC Diversity of yeasts • Oldest yeast isolated from UC Berkeley winery in 1893 • Yeasts from all over the world • Red: Phaff ’s travels • Flowers • Fruits • Blue: yeasts from others • Wood • Insects • Type strains of over 500 • Soil species • Foods and beverages • Food processing facilities Data “Traditional” chemotaxonomy data databased in 2000: • Assimilation of 40 carbon compounds • Fermentation of 6-8 sugars • Growth temperatures • High sugar, high salt • Enzymes • Cell, colony, spore morphology Activities at the Phaff collection • Maintenance • Distribution • Expansion – collecting expeditions • Research using the collection • Federal, state funded • Industry funded Maintenance Funding: NSF Collections in Support of Biological Research • Confirm viability, purity • Re-identify by ribosomal sequencing • New website, database (BioloMICS) • Remote storage at NCGRP Distribution • Academic: yeast ecology (insect, plant associations), taxonomy, transcription regulation, comparative genomics, • Industry: Wine, beer, biofuels, food and feed ingredients, hosts for heterologous protein expression • Government agency: Human and animal pathogens, biofuels (ethanol, lipids) Research at the Phaff collection Building the collection Food spoilage Microbiome of food processing facilities Insect-yeast associations In-house research tapping the collection Takes advantage of taxonomic, phenotypic diversity Screen hundreds/thousands of yeast for desired properties Screening example: 250 yeasts, 22 conditions • Ethanol producers, relatives • Generated heat map of OD in MATLAB • Can rank based on which tests are most pertinent to the process Carbon sources Nitrogen sources Inhibitors One research focus area: Oleaginous yeasts • Definition: Over 20% intracellular oil, stored as TAGs; can by up to 65% oil by dry weight • Most triggered by nutrient depletion (N) • Accumulate high concentrations of triacylglycerols Exponential • Ascomycetes, basidiomycetes • A few species commonly used in research and development: Yarrowia, Lipomyces, Rhodotorula, Rhodosporidium, Cryptococcus • Over 90 known oleaginous species Stationary Our recent contributions to oleaginous yeast knowledge Carbon Sources Inhibitors a i d d n i ) e o c ) L i / ) t M L A g / a ) ) l e L e UCDFST c e 5 g / i . e s u d Species s L L r g i n c 0 1 o e e / / o ( ( c 5 e Strain ID# F e s o c s n . g g ) n e l s r s i l l i o o o e s A 0 1 2 e o n i l o u L o b a a s b i ( ( ( n r / o t s t l r r b c a F a o e c c i g n o y r u u r o m m t F F F c r c a a l f f n a l a l e l 5 X A c r r u y A t a . l - - a a l c M M M e h i - u u u 2 ( G D L D C M G R S G G V F F A H H H Candida aff. tropicalis 10-1087 Candida diddensiae 10-168 Cryptococcus aerius 73-135 D D D D D D Cryptococcus aff. taibaiensis 73-750 D D D D • Cryptococcus albidus 63-203 D D D Discovery of 35 Cryptococcus cf. aureus 81-663.4 D D D D Cryptococcus curvatus 76-559 D D D Cryptococcus humicola 10-1004 D Cryptococcus humicola 12-717 D D Cryptococcus laurentii 12-803 D D oleaginous yeast species Cryptococcus oeirensis 05-864 D D Cryptococcus ramirezgomezianus 54-11.224 D D D Cryptococcus terreus 61-443 D D D Cryptococcus victoriae 10-939 D D D Cryptococcus wieringae 05-544 Cyberlindnera jadinii 76-80 D D D D D D D D D D Cyberlindnera saturnus 68-1113 D • Carbon source utilization Geotrichum fermentans
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