Characterization of Cyberlindnera jadinii carboxylate permeases by heterologous expression in Saccharomyces cerevisiae

Abstract

Background: The wide applicability of organic acids for direct use as commodity chemicals and as polymer building blocks has evidenced their importance in diverse types of industries. In Saccharomyces cerevisiae, two permeases are responsible for the uptake of carboxylates (CA) at the plasma membrane, Jen1p a monocarboxylate proton symporter (Major Facilitator Superfamily) and Ady2p an acetate permease (AceTr Family). Objectives: In Cyberlindnera jadinii, different uptake systems for CAs were functionally characterized however until now the genes encoding these transporters remain unidentified. In this study, CA transporter homolog genes from C. jadinii were identified and expressed in S. cerevisiae. Methods: The S. cerevisiae strain W303-1A jen1Δ ady2Δ, lacking carboxylate uptake capacity, was used to express C. jadinii ScADY2 homologs. Genes were identified through sequence alignment and homology prediction and cloned in the p416GPD vector, under the control of a GPD constitutive promoter. GFP-fusions versions were used to determine protein expression and localization. Transport activity was determined through growth on different carbon sources and measurement of the uptake of labelled CAs, namely D,L-[U14C] lactic acid, [2,3-14C] succinic acid and [1-14C] acetic acid. Results: In C. jadinii, 4 genes homolog to ScADY2 were identified. These are functional carboxylate transporters in S. cerevisiae, localized at the plasma membrane, presenting different specificities for the mono- and di-carboxylates. Further studies are underway to fully characterize these four new plasma membrane transporters, including molecular docking of these transporters to unveil the amino acids that play a major role in the substrate binding of CAs tested.