Exploring the Cyberlindnera jadinii yeast transportome to uncover novel carboxylate transporters for biotechnological applications

Abstract

Due to global problems of resource scarcity and environmental damage, new technologies based on renewable biological sources are required, as the current model of natural resource exploitation is unsustainable. Novel strategies to boost bio-based production of organic acids are based on the expression of carboxylate transporters in microbial cell factories. In this work we have focused on the identification and characterization of novel carboxylate transporters in the yeast Cyberlindnera jadinii. C. jadinii transportome was analysed by two approaches. First, the C. jadinii homologs of the carboxylate transporters Jen1p (Major Facilitator Superfamily) and Ady2p (AceTr Family) were identified and expressed in Saccharomyces cerevisiae. The S. cerevisiae strain W303 1A jen1Δ ady2Δ was used for the heterologous expression. Genes were identified through sequence alignment and homology prediction. In a parallel bioinformatic approach, the proteome from C. jadinii NRRL 1542 was downloaded from the NCBI database and explored using a pipeline. This tool was designed to retrieve data from a specific database: a) that contains a single representative genome/proteome on the species level; b) where multiple matches within a species directly reflect the presence of homologs within the same genome, and c) e-values from BLAST searches that are statistically more reliable [1]. A set of genes were selected using this tool and expressed in the IMX1000 strain, which is deleted in 25 genes related to carboxylic acid transport [2]. Protein expression and localization was determined by microscopy evaluation of GFP- fused transporter proteins. Transporter activity was evaluated through growth on different carbon sources and measurement of the uptake of several radiolabelled CAs. The full characterization of the Ady2 and Jen1 homologs as well as other candidate CAs transporters from C. jadinii is ongoing.