Culture Collections

Kyria Boundy-Mills Curator, Phaff Yeast Culture Collection Food Science and Technology UC Davis Outline

• Definition and importance of yeast • How 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

• 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 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, , • 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

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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 89-29 D D D Hannaella aff. zeae 92-112 D D D D D D Kodamaea ohmeri 54-7 D D Kurtzmaniella cleridarum 76-729.2 D D D Leucosporidiella creatinivora 62-1032 D D D D D Lipomyces lipofer 78-19 D D D D and inhibitor tolerance of 45 Lipomyces starkeyi 78-23 D D D D D D D D D Lipomyces tetrasporus 78-28 D D D D D D D D D Metschnikowia cf. gruessii 11-1106 D Metschnikowia cf. gruessii 11-1130 D Metschnikowia cf. pulcherrima 11-1039 Myxozyma melibiosi 52-87 D D D D oleaginous yeast species Pseudozyma aphidis 11-1358 D D D D D D Rhodosporidium paludigenum 09-163 D D D D Rhodosporidium babjevae 04-877 D D Rhodosporidium babjevae 05-775 D D Rhodosporidium diobovatum 04-830 D Rhodosporidium fluviale 81-485.4 D D D D D D • Conversion of corn stover Rhodosporidium toruloides 68-264 D D D D Rhodotorula glutinis 50-309 D D D D Rhodotorula graminis 04-862 D D D D D Rhodotorula minuta 78-281 D D Rhodotorula mucilaginosa 40-129 D D D Schwanniomyces occidentalis 73-1 hydrolysate to 15.5 g/L oi Tremella enchepala 68-887.2 D D D Trichosporon coremiiforme 88-108.4 D D Trichosporon dermatis 63-110 D D D Trichosporon guehoae 60-59 D D Trigonopsis variabilis 75-19 Wickerhamomyces ciferii 04-836 D Yarrowia lipolytica 51-30 PROBLEM:

• Single low value commodity products (such as ethanol, lipid or protein) Strategies to improve economics

• Reduce costs • Cheaper feedstocks • Faster process • Increase productivity • Higher density cultures • Multiple co-products Multiple co-products

Protein Food, feed

Lipids TAG

At least 3 Pigments carotenoids

Other Vitamins, sterols, etc.

Glycolipids Biosurfactant Secreted glycolipids

 Made by several Phaff collection yeast species  Glycolipids: Glycan, fatty acids. Most similar: Sophorolipids  Biosurfactant properties  Comparable uses to SL: household cleaning products, agricultural pest control, etc.  Our production technology is cheaper Status of technology

• IP: Filed patent March 2015; filing new patent later this year with updated chemical structures • Taking steps towards startup company and/or licensing out technology • Seeking investors, government funding (DOE BRDI pending) Ways to access Phaff collection yeasts

• Online catalog: phaffcollection.ucdavis.edu • Order strains • Assistance in strain selection • Contract screening at UC Davis; obtain top strains for further analysis and development Lessons

• Carefully select yeast • International treaties, IP strains based on desired issues, proprietary characteristics technologies influence • Use validated, strain selections characterized strains • Compare performance of many strains Acknowledgments

Culture Collection Community: WFCC, USCCN

Screening work: Ting Lin, Erin Cathcart, Jennifer Lincoln, Idelia Chandra, Joe Williams, Tina Jeoh

Metabolomics: Oliver Fiehn, Tom Cajka

Funding: Industrial partners Sloan Foundation NSF CSBR UC Davis STAIR program CONACYT NIH/USDA/NSF/DOE