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Evaluation of S. cerevisiae promoters during growth on xylose Presenter: L. Mande Supervisor: Dr D.C. La Grange Co-Supervisor: Prof I. Ncube & Prof W.H. van Zyl

Introduction Common name Baker’s yeast and Brewer’s yeast (Campbell, 2002)

Introduction • S.cerevisiae has been used for years in the production of recombinant proteins • S.cerevisiae is a widely used organism • - fast cell growth • - tolerate high ethanol concentration • - tolerate wide spectrum of inhibitors • - GRAS status • - well-characterised physiology & genetics

Introduction • Recombinant protein production linked to biomass • S.cerevisiae is a Crabtree-positive yeast and produces less biomass when cultivated on glucose • Xylose second most abundant sugar and does not cause Crabtree-effect • Wild-type strains of S.cerevisiae cannot utilize xylose as carbon source

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Aim& Objectives Aim: Evaluate six promoters commonly used for protein expression in S.cerevisiae, during growth on xylose Objectives: Construction of six plasmids with ENO1, PFK2, PGK1, ENO2 , ADH2, GAL10 with T. reesei xyn2 gene as a reporter gene Transformation of the plasmid in xylose utilizing strain S.cerevisiae (sXI, XYL3 , gre3::ZEO) - Xylanase activity assay using DNS assay - Growth curve and evaluation of growth in bioreactor

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Step 1: Construction of plasmids Project overview Step 2: Transformation and strain confirmation Step 3: xylanase activity assay Step 4: Growth curve, evaluation growth condition

Method & Results Step 1:Construction of six expression vectors - PGK1p and PGK1t isolated from yeast plasmid - G418 selection on geneticin - Target expression cassette to URA3 locus - Expression cassette cloned into pUC19 G418 PGK1 URA3 URA3 PGK1t

CONT…… • Isolate xyn2 with PCR from previous study XYN2

CONT…… • Isolate xyn2 with PCR from previous study • PCR product digested with PacI and Ascl cloned in uPGK1 plasmid to make uPGK1X plasmid XYN2 G418 URA3 PGK1t PGK1 URA3

Cont….. • Plasmid used as a base for construction of the five expression vectors

Cont….. Fig 1b: Confirmation of the uENO1X plasmid by restriction enzymes 5.183kb 10000 8000 6000 5000 0.51kb 4000 4.1kb 3500 3000 2500 2000 1.8kb 1500 1000 750 500 Fig 1a: Confirmation of the uPFK2X plasmid by restriction enzymes

Cont….. 5.183kb Fig 1b: Confirmation of the uENO1X, uGAL10Xplasmid by restriction enzymes 0.52kb 4.1kb 1.8kb Fig 1a: Confirmation of the uPFK2X plasmid by restriction enzymes

Cont….. 5.2kb Fig 1b: Confirmation of the uENO1X, uGAL10X and uENO2pXplasmid by restriction enzymes 0.57kb 4.1kb 1.8kb Fig 1a: Confirmation of the uPFK2pX plasmid by restriction enzymes

Cont…… 5.1kb 5.6kb 0.51kb 0.59kb Fig 2b: Confirmation of the uADH2X Plasmid with REN Fig 2a: Confirmation of the uPGK1X plasmid with REN

Method and Results Step 2:Transformation and Strain confirmation • Transformation • Transformation in S. cerevisiae (Cho et al.,1999) • Transformants selected on geneticin (G418)

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Cont….. • Strain confirmation - PCR confirmation - Using xyn2 primers 10000 8000 6000 5000 4000 3500 3000 2500 2000 1500 1000 610bp 750 500 250

Cont….. RBB-xylan..xylose RBB-xylan..glucose RBB-xylan..galactose

Acknowledgement • Many thanks Dr La grange • Van Zyl lab (Stellenbosch university) • BMBT department (University of Limpopo) • RSES for financial assistance

Thank you ………………… OUR GOAL

Introduction (Aristidou and Pentila,2000)

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