Exploring genetic tools for the overexpression of the lactate permease Jen1p of Saccharomyces cerevisiae: constitutive expression in S. cerevisiae and heterologous expression in Pichia pastoris

Abstract

In Saccharomyces cerevisiae the active transport of monocarboxylic acids is dependent upon the expression of the permease Jen1p. Cells of S. cerevisiae jen1 were transformed with the plasmids p416GPD (centromeric) and p426GPD (multicopy) containing JEN1 gene under the control of the GPD constitutive promotor. Lactic acid uptake and JEN1 expression was found in cells grown in glucose, acetic acid, or glucose and acetic acid. Constitutive expression of the permease was achieved in the p416GPD-JEN1 transformants; the highest V_max (0.84 nmol sˉ¹ mgˉ¹ d.w.) was obtained in acetic acid-grown cells. In a second approach, Pichia pastoris strains X-33 (Mut+) and KM71H (Muts) were transformed with the vectors pPICZB-JEN1 (integrative) and pZPARS-JEN1 (episomal). Activity for the lactate permease was only obtained in JEN1-transformed cells, the highest level (V_max 0.86 nmol sˉ¹ mgˉ¹ d.w.) being detected in the KM71H strain containing genomic insertions of the vector pPICZB-JEN1, after 24 h of methanol induction. RT-PCR, Northern and Western-blotting analysis confirmed the expression of JEN1 in P. pastoris and S. cerevisiae. Both strategies described for JEN1 expression resulted in a two-fold increase for the activity of the lactate transport system when compared to the data previously found in lactic acid-grown cells of native S. cerevisiae strains.