1. Heterologous Protein Expression in Yeast Malcolm Stratford & Hazel Steels MOLOGIC

2. Yeasts & Moulds – The Basics Yeasts – single-celled fungi Yeasts – single-celled fungi > 800 species discovered to date > 800 species discovered to date Yeasts are generally regarded as safe Yeasts are generally regarded as safe Moulds – multicelled hyphae, slower growing than yeasts Moulds – multicelled hyphae, slower growing than yeasts Almost all aerobic Almost all aerobic Form asexual spores for dispersal Form asexual spores for dispersal = conidia = conidia Some moulds form mycotoxins

3. Yeast expression systems S. cerevisiae, usually based on 2μm plasmid, multicopy episomal, auxotrophic markers S. cerevisiae, usually based on 2μm plasmid, multicopy episomal, auxotrophic markers P. pastoris, integrating vector, zeocin selection, AOX promoter induced by methanol P. pastoris, integrating vector, zeocin selection, AOX promoter induced by methanol K. lactis, integrating vector, selection based on use of an unorthodox nitrogen source K. lactis, integrating vector, selection based on use of an unorthodox nitrogen source Others, small scale work on a few other yeasts (10) usually using G418-kanomycin selection Others, small scale work on a few other yeasts (10) usually using G418-kanomycin selection

4. Can we express Core protein in yeast? Will it fold correctly? Will GFP be active? Initial experiments – Pichia pastoris expression under AOX promoter induced by methanol HeterotandemCore and Core+GFP: expression in Pichia pastoris

5. pPIC vectors for P. pastoris

6. Results Successful transformation of both Core protein and Core+GFP into P. pastoris Successful transformation of both Core protein and Core+GFP into P. pastoris After induction with methanol, detected Core and GFP by dot blots (antibodies) After induction with methanol, detected Core and GFP by dot blots (antibodies) GFP cultures turned progressively green, showing active, correctly- folded GFP GFP cultures turned progressively green, showing active, correctly- folded GFP

9. Induction of Core+GFP in P. pastoris Time-dependent and aeration- dependent Time-dependent and aeration- dependent Green forms progressively over time Green forms progressively over time Faster aeration results in faster formation of green Faster aeration results in faster formation of green

10. P.pastoris induction media Standard system = growth in any media; induction in YNB + 0.5% methanol Standard system = growth in any media; induction in YNB + 0.5% methanol Eden suggestion = growth on Basal salts; induction in YNB + 0.5% methanol Eden suggestion = growth on Basal salts; induction in YNB + 0.5% methanol Mologic system = growth on YEPD; induction on low level YNB + 0.7% methanol + 30mM succinate buffer pH 4.5 Mologic system = growth on YEPD; induction on low level YNB + 0.7% methanol + 30mM succinate buffer pH 4.5

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

12. Buffer Effect II: Concentration

13. P.pastoris induction media Standard system = growth in any media; induction in YNB + 0.5% methanol Standard system = growth in any media; induction in YNB + 0.5% methanol Eden suggestion = growth on Basal salts; induction in YNB + 0.5% methanol Eden suggestion = growth on Basal salts; induction in YNB + 0.5% methanol Mologic system = growth on YEPD; induction on low level YNB + 0.7% methanol + 30mM succinate buffer pH 4.5 Mologic system = growth on YEPD; induction on low level YNB + 0.7% methanol + 30mM succinate buffer pH 4.5

14. Initial experiments – expression vector under AOX promoter Core and Core+GFP: expression in other yeast species

15. Results Core and Core+GFP also transformed into other yeast species Core and Core+GFP also transformed into other yeast species Expression tested in 6 induction media Expression tested in 6 induction media 8 transformants of each species tested 8 transformants of each species tested Candida glabrata (Nakaseomyces) Candida norwegica Lachancea cidri Lachancea fermentati Pichia galeiformis Schizosaccharomyces pombe Torulaspora delbrueckii Torulaspora microellipsoides Williopsis californica Zygosaccharomyces rouxii

16. Induction Media 1. YNB Methanol 1. YNB Methanol 2. YEPD Glucose 2. YEPD Glucose 3. YNB Glucose 3. YNB Glucose 4. YEPD Glycerol 4. YEPD Glycerol 5. Malt extract 5. Malt extract 6. YNB + YEP Methanol 6. YNB + YEP Methanol

17. Results In methanol, only green formed by P. pastoris X33 and WT P. pastoris In methanol, only green formed by P. pastoris X33 and WT P. pastoris Schiz pombe formed moderate levels on glucose and glycerol Schiz pombe formed moderate levels on glucose and glycerol Z. rouxii, Lachancea cidri, and Williopsis formed moderate levels on glycerol Z. rouxii, Lachancea cidri, and Williopsis formed moderate levels on glycerol Green Ring phenomenon! Green Ring phenomenon!

19. New Promoters Core + GFP placed downstream of 5 yeast promoters Core + GFP placed downstream of 5 yeast promoters ENO1, PGK1, PMA1, PYK1, RD25 ENO1, PGK1, PMA1, PYK1, RD25

20. Transformants

21. Induction Media for 5 Promoters 1. YEPD Glucose pH 4.5 1. YEPD Glucose pH 4.5 2. YEPD Glycerol pH 4.5 2. YEPD Glycerol pH 4.5 3. YNB Glucose pH 4.5 3. YNB Glucose pH 4.5 4. Malt extract 4. Malt extract

22. Results – 5 Promoters Each yeast species (15), 5 promoters, 8 transformants of each, in 4 media = 2400 experimental time courses Each yeast species (15), 5 promoters, 8 transformants of each, in 4 media = 2400 experimental time courses Work ongoing (50% complete) Work ongoing (50% complete) Results disappointing – highest levels only 5% of standard P.pastoris Results disappointing – highest levels only 5% of standard P.pastoris Promoters generally not recognised Promoters generally not recognised Need powerful promoter for each new species Need powerful promoter for each new species

23. Where do we go from here? Complete promoter testing Complete promoter testing Find new species specific promoters by random integration Find new species specific promoters by random integration Use existing knowledge of powerful promoters, e.g. in germinating mould spores of Aspergillus niger capable of degrading several million times the mass of a spore in 2 days! Use existing knowledge of powerful promoters, e.g. in germinating mould spores of Aspergillus niger capable of degrading several million times the mass of a spore in 2 days!