Introduction The Future

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

Introduction

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

Introduction GRE3

Project overview Step 1: Construction of plasmids 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 PGK1t URA3

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 PGK1t G418 URA3 PGK1 URA3

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

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

Cont….. 5.183kb 0.52kb 4.1kb Fig 1b: Confirmation of the uENO1X, uGAL10X plasmid by restriction enzymes 1.8kb Fig 1a: Confirmation of the uPFK2X plasmid by restriction enzymes

Cont….. 5.2kb 0.57kb 4.1kb 1.8kb Fig 1b: Confirmation of the uENO1X, uGAL10X and uENO2pX plasmid by restriction enzymes 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 5.6kb 0.59kb 5.1kb 0.51kb Fig 2a: Confirmation of the uPGK1X plasmid with REN

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

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

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

Cont….. NotI NotI

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

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

Many thanks Dr La grange Van Zyl lab (Stellenbosch university) 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)