Heterologous Protein Heterologous Protein Expression in YeastExpression in Yeast

Malcolm Stratford & Hazel SteelsMalcolm Stratford & Hazel Steels

MOLOGICMOLOGIC

Yeasts & Moulds – The BasicsYeasts & Moulds – The Basics

Yeasts – single-celled fungiYeasts – single-celled fungi > 800 species discovered to date> 800 species discovered to date Yeasts are generally regarded as safeYeasts are generally regarded as safe Moulds – multicelled hyphae, slower Moulds – multicelled hyphae, slower

growing than yeastsgrowing than yeasts Almost all aerobicAlmost all aerobic Form asexual spores for dispersalForm asexual spores for dispersal

= conidia= conidia

Some moulds form mycotoxinsSome moulds form mycotoxins

Yeast expression systemsYeast expression systems

S. cerevisiaeS. cerevisiae, usually based on 2μm plasmid, , usually based on 2μm plasmid, multicopy episomal, auxotrophic markersmulticopy episomal, auxotrophic markers

P. pastorisP. pastoris, integrating vector, zeocin , integrating vector, zeocin selection, AOX promoter induced by selection, AOX promoter induced by methanolmethanol

K. lactisK. lactis, integrating vector, selection based , integrating vector, selection based on use of an unorthodox nitrogen sourceon use of an unorthodox nitrogen source

Others, small scale work on a few other Others, small scale work on a few other yeasts (10) usually using G418-kanomycin yeasts (10) usually using G418-kanomycin selectionselection

Can we express Core protein in yeast?Can we express Core protein in yeast?Will it fold correctly? Will it fold correctly? Will GFP be active?Will GFP be active?

Initial experiments – Pichia pastoris expression under

AOX promoter induced by methanol

HeterotandemCore and HeterotandemCore and Core+GFP:Core+GFP:expression in expression in Pichia pastorisPichia pastoris

pPIC vectors for pPIC vectors for P. pastorisP. pastoris

pPICZ A3329 bp

6xHis

c-myc epitope

Zeo(R)

MCS

3' AOX1 primer

5' AOX1 primer

TEF1 promoter

AOX1 promoter

EM7 promoter

pUC origin

CYC1 transcription terminator

ResultsResults

Successful transformation of both Successful transformation of both Core protein and Core+GFP into Core protein and Core+GFP into P. P. pastorispastoris

After induction with methanol, After induction with methanol, detected Core and GFP by dot blots detected Core and GFP by dot blots (antibodies)(antibodies)

GFP cultures turned progressively GFP cultures turned progressively green, showing active, correctly-green, showing active, correctly-folded GFPfolded GFP

Induction of Core+GFP in Induction of Core+GFP in P. P. pastorispastoris

Time-dependent and aeration-Time-dependent and aeration-dependentdependent

Green forms progressively over timeGreen forms progressively over time Faster aeration results in faster Faster aeration results in faster

formation of greenformation of green

P.pastorisP.pastoris induction media induction media

Standard system = growth in any media; Standard system = growth in any media; induction in YNB + 0.5% methanolinduction in YNB + 0.5% methanol

Eden suggestion = growth on Basal salts; Eden suggestion = growth on Basal salts; induction in YNB + 0.5% methanolinduction in YNB + 0.5% methanol

Mologic system = growth on YEPD;Mologic system = growth on YEPD;induction on low level YNB + 0.7% induction on low level YNB + 0.7% methanol + 30mM succinate buffer pH methanol + 30mM succinate buffer pH 4.54.5

Methanol Effect:Methanol Effect:Core+GFP in Core+GFP in P. pastorisP. pastoris

0

100000

200000

300000

400000

500000

600000

0 0.1 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Methanol daily (%)

Gre

en

Buffer Effect II:Buffer Effect II:ConcentrationConcentration

0

100000

200000

300000

400000

500000

600000

Malic Buffer

Day 3

Day 7

P.pastorisP.pastoris induction media induction media

Standard system = growth in any media; Standard system = growth in any media; induction in YNB + 0.5% methanolinduction in YNB + 0.5% methanol

Eden suggestion = growth on Basal salts; Eden suggestion = growth on Basal salts; induction in YNB + 0.5% methanolinduction in YNB + 0.5% methanol

Mologic system = growth on YEPD;Mologic system = growth on YEPD;induction on low level YNB + 0.7% induction on low level YNB + 0.7% methanol + 30mM succinate buffer pH methanol + 30mM succinate buffer pH 4.54.5

Initial experiments –

expression vector under AOX promoter

Core and Core+GFP:Core and Core+GFP:expression in other yeast expression in other yeast speciesspecies

ResultsResults

Core and Core and Core+GFP also Core+GFP also transformed into transformed into other yeast speciesother yeast species

Expression tested Expression tested in 6 induction in 6 induction mediamedia

8 transformants of 8 transformants of each species each species testedtested

Candida glabrata (Nakaseomyces)

Candida norwegica

Lachancea cidri

Lachancea fermentati

Pichia galeiformis

Schizosaccharomyces pombe

Torulaspora delbrueckii

Torulaspora microellipsoides

Williopsis californica

Zygosaccharomyces rouxii

Induction MediaInduction Media

1. YNB Methanol1. YNB Methanol 2. YEPD Glucose2. YEPD Glucose 3. YNB Glucose3. YNB Glucose 4. YEPD Glycerol4. YEPD Glycerol 5. Malt extract5. Malt extract 6. YNB + YEP Methanol6. YNB + YEP Methanol

ResultsResults

In methanol, only green formed by In methanol, only green formed by P. P. pastorispastoris X33 and WT X33 and WT P. pastorisP. pastoris

Schiz pombe Schiz pombe formed moderate levels formed moderate levels on glucose and glycerolon glucose and glycerol

Z. rouxiiZ. rouxii, , Lachancea cidriLachancea cidri, and , and WilliopsisWilliopsis formed moderate levels on formed moderate levels on glycerolglycerol

Green Ring phenomenon!Green Ring phenomenon!

New PromotersNew Promoters

Core + GFP placed downstream of 5 Core + GFP placed downstream of 5 yeast promotersyeast promoters

ENO1, PGK1, PMA1, PYK1, RD25ENO1, PGK1, PMA1, PYK1, RD25

TransformantsTransformantsYeast ENO1 PGK1 PMA1 PYK1 RDN25Candida glabrata 8 8 8 8 8Candida norwegica 8 8 8 8 8Hanseniaspora meyerii 0 1 3 3 4Lachancea cidri 8 8 8 8 8Lachancea fermentati 8 8 8 8 8Pichia galeiformis 8 8 0 0 8Pichia pastoris 219 8 8 8 8 8Pichia pastoris X33 8 8 8 8 7Schizosaccharomyces pombe 8 8 2 0 8Torulaspora delbrueck ii 8 8 8 8 8Torulaspora microellipsoides 8 8 8 8 7Williopsis californica 8 8 6 8 8Z. kombuchaensis 0 0 0 0 1Zygosaccharomyces bailii 2 1 0 2 0Zygosaccharomyces rouxii 0 0 2 0 0

Induction Media for 5 PromotersInduction Media for 5 Promoters

1. YEPD Glucose pH 4.51. YEPD Glucose pH 4.5 2. YEPD Glycerol pH 4.52. YEPD Glycerol pH 4.5 3. YNB Glucose pH 4.53. YNB Glucose pH 4.5 4. Malt extract4. Malt extract

Results – 5 PromotersResults – 5 Promoters

Each yeast species (15), 5 promoters, 8 Each yeast species (15), 5 promoters, 8 transformants of each, in 4 mediatransformants of each, in 4 media

= 2400 experimental time courses= 2400 experimental time courses Work ongoing (50% complete)Work ongoing (50% complete) Results disappointing – highest levels Results disappointing – highest levels

only 5% of standard only 5% of standard P.pastorisP.pastoris Promoters generally not recognisedPromoters generally not recognised Need powerful promoter for each new Need powerful promoter for each new

speciesspecies

Where do we go from here?Where do we go from here?

Complete promoter testingComplete promoter testing Find new species specific promoters Find new species specific promoters

by random integrationby random integration Use existing knowledge of powerful Use existing knowledge of powerful

promoters, e.g. in germinating mould promoters, e.g. in germinating mould spores of spores of Aspergillus nigerAspergillus nigercapable of degrading several million capable of degrading several million times the mass of a spore in 2 days!times the mass of a spore in 2 days!