Functional characterization of Cyberlindnera jadinii carboxylate transporters in Saccharomyces cerevisiae

Abstract

The wide applicability of organic acids for direct use as commodity chemicals and as polymer building blocks has evidenced their importance into diverse types of industries. As organic acids, carboxylic acids (CAs) are underlined as top value products by the US Department of Energy (DOE), which has included nine organic acids among the future top 12 value-added platform chemicals. In the yeast Saccharomyces cerevisiae, two permeases are responsible for the uptake of carboxylates at the plasma membrane. With affinity for lactate, pyruvate acetate and propionate, the JEN1 gene encodes a monocarboxylate proton symporter that belongs to the Major Facilitator Superfamily (TC 2.A.1.12.2). The ADY2 gene encodes an acetate permease, a member of the Acetate Uptake Transporter (AceTr) Family (TC 2.A.96.1.4). In the yeast Cyberlindnera jadinii, a close relative of Candida utilis, different uptake systems for CAs were functionally characterized however until now the genes encoding these transporters remain unidentified. In this work, carboxylic acid transporter homolog genes from C. jadinii were identified and expressed in Saccharomyces cerevisiae. The C. jadinii ScJEN1 and ScADY2 homologs were identified through sequence alignment, secondary structure prediction and phylogenetic analysis of putative transporters. In C. jadinii, 6 genes homolog to ScJEN1 (Cjj23088, Cjj21966, Cjj22358, Cjj21989, Cjj21602, Cjj25129) and 4 genes homolog to ScADY2 (Cja24587, Cja20823, Cja20690, Cja20822) were identified. These genes were expressed under the control of a GPD constitutive promoter, in a S. cerevisiae jen1Δady2Δ strain, that presents no activity for plasma membrane carboxylate permeases. GFP-fusions versions were performed revealing the localization of C. jadinii ScJEN1 and ScADY2 homolog transporters mainly at the plasma membrane. The complete characterization of these new plasma membrane transporters in C. jadinii is ongoing.