Author(s): Sousa-Silva, M.
Date: 2021
Persistent ID: https://hdl.handle.net/1822/75606
Origin: RepositóriUM - Universidade do Minho
Subject(s): Biotechnology; Carboxylic acids; Membrane transporters; Microbial cell factories; Ácidos carboxílicos; Biotecnologia; Microfábricas celulares; Transportadores de membrana
Organic acids are recognized as versatile chemical compounds with a vast variety of applications in sectors ranging from food and beverages, pharmaceutical, personal care, cosmetic products, consumer goods to the chemical industry. Due to the strong demand for these compounds, alternative approaches to non-sustainable processes, e.g. chemical synthesis from petroleum derivatives, are being developed. Sustainable strategies rely on the utilization of microbial cell factories, where transporter proteins play a crucial role through the control of substrate influx and product efflux. In particular, the expression of suitable carboxylic acid exporters avoids the internal cell toxicity due to the accumulation of these compounds, while facilitating its purification from the culture broth. The biodiversity of the microbial world is an excellent pool to uncover new organic acid transporters. In this study, wild yeast species isolated from acidic environments were explored regarding their ability to utilize organic acids. The yeast Cyberlindnera jadinii was selected for further studies due to its ability to utilize a vast range of organic acids as sole carbon and energy source, to synthesize a variety of valuable compounds for the food and pharmaceutical sectors, its intrinsic robust characteristics, and its capacity to use inexpensive media with high productivity levels. Using an in silico approach, we have explored the predicted transportome of C. jadinii and selected putative carboxylate transporters for functional characterization by heterologous expression in Saccharomyces cerevisiae. A total of 16 plasma membrane carboxylate transporters, members of the AceTr, SHS, SSS, TDT, DASS, and MCT families, were characterized in terms of transport activity and specificity, structural features, and evolutionary relationships. S. cerevisiae was also used as expression host to deepen the knowledge of the citrate exporter CexAp from Aspergillus niger, a member of the DHA1 transporter family. The structural characterization of AceTr family members Ato1 and SatP led to the identification of a conserved motif essential for protein function. In addition, the plasma membrane proteins Gpr1 from Yarrowia lipolytica and AceP from the archaea Methanosarcina acetivorans were functionally characterized as acetate transporters. The role of AceTr members from S. cerevisiae, Ato1, Ato2, and Ato3, in the transport of monocarboxylic acids was evaluated via a combination of directed evolution, whole-genome resequencing, and reverse engineering, leading to the discovery of Ato2 and Ato3 mutants as two novel lactic acid transport proteins. Structural insights were also provided using 3D structure predictions combined with molecular docking.
