Project Portugal 2030

Summary

Due to the increasing relevance of CAs, major chemical companies invested in smaller start-ups and a number of governments have promoted research and development on their bioproduction [20]. Advancements in fermentation technology, strain enhancement, and process refinement are essential to meet shifting market demands and maintain competitiveness. Focusing research and development efforts on enhancing fermentation yields, increasing productivity, and expanding the range of CA generated through fermentation can unlock new market opportunities. One of the main drawbacks in the CA microbial production is its toxicity, due to membrane disruption and perturbed metabolic pathway by anion accumulation inside the cell [21, 22]. The expression of membrane exporters in MCF allows overcoming this bottleneck, by exporting CA and improving the MCF’s tolerance to the produced compounds [5, 6]. As biotechnological applications expand, it is necessary to identify new membrane exporters, with improved features [23], as the current transporters expressed in MCF are very limited and do not cover the complete range of organic acids of interest for industrial biotechnology players. The presence of CATs in cellular membranes is ubiquitous and highly conserved among the different super-kingdoms, highlighting their ancient nature [7]. Thus, the biodiversity of the microbial world is an excellent pool to uncover relevant exporters for organic acids production. This project will exploit an innovative high-throughput bioinformatics tool and implement a novel phenotypic platform, developed by our group, designed to identify and characterize CA exporters encoded in microbial genomes. Through engineering plasma membrane exporters, one can improve expression levels, transport capacity, and substrate range [6]. Our aim is to provide efficient CA exporters to be used for the industrial bioproduction of organic acids. The work plan has the following objectives: 1. Establish a bioinformatics platform to screen for membrane transporters. By developing (i) an in-house well curated proteome database and (ii) specific software tools for the identification of transporter proteins we estimate to identify (at least 10.000) novel membrane transporters. 2. Functional analysis of new plasma membrane transporters. The most promising transporters associated with the bio-production of CAs will be functionally characterized. We will utilize well established cell models to heterologously express selected transporters. 3. Engineering membrane transporters for the improvement of cell factories. In silico approaches of phylogenetic analysis, 3D structure prediction, and molecular docking, combined with rational site-directed mutagenesis will unravel the structural features of CATs, including amino acid residues crucial for function, activity and substrate specificity. 4. Expression of CATs in microbial cell factories to improve CA bioproduction. By protein engineering approaches, CAT with improved features will be expressed in industrial CA producing stains. This methodology will be applied to the most promising transporters delivered from our pipeline. TransFactory lies in the core of the research developed for several years by the PI and senior researchers on CAT. With this project, we will take a step forward with the expression of plasma membrane exporters to increase the robustness of MCF, improve biotechnological processes and develop biosustainable solutions for CA production