PHA4FUTURE aims to study promising PHA-storing organisms. We are studying Pseudomonas mandelli (Pm), for its capability to produce SCL-MCL PHAs. Pm, which encodes three PhaC classes (I to III) in its genome, was previously identified by a bioinformatic study as a promising organism to produce mixed SCL-MCL PHAs.

In parallel, we will also focus at the molecular level and study Pm class I and class II PhaCs, performing biochemical and structural/functional characterizations. These data will be crucial to understand the catalytic mechanisms, specificity determinants aiming to produce variants with improved catalytic activity and tuned substrate specificity.

To achieve these objectives, we will use an interdisciplinary approach: (i) Pm will grow in controlled bioreactors and the process will be optimised to maximise the PHA storing capacity and the growth rate. PHA will be extracted from cells and its composition, molecular weight and thermal properties determined; (ii) At the same time, we will use a high- throughput approach to heterologously produce PhaCs, characterize specificity and determine kinetic parameters for the substrates identified in (i); (iii) We will use X-ray crystallography in combination with Small Angle X-ray Scattering and Cryo-Electron Microscopy for the structural studies; (iv) A structural based analysis will be performed to pinpoint key residues that will be mutated for further investigation on PHAs optimization.

The project will be accomplished by a multidisciplinary team composed of members from two research groups: the Biochemical Engineering group (BioEng - FCT NOVA) with proven expertise in PHA production process, assessing the Biotechnological application, and the Macromolecular Crystallography group (MX - FCT NOVA) with expertise in structural biology and X-ray crystallography, that will exploit the fundamental knowledge. The link between the two fields will enhance the success rate of the project.

Finally, we are confident that additional structural data on the different PhaC classes, mainly new structures of class II PhaCs, will provide valuable clues to understand MCL substrate specificity, design efficient enzyme variants, and in a near future, identify or engineer microorganisms capable of efficiently produce tailor-made PHA for industrial and medical applications, creating added value to the research field and ultimately to the society.

This project is funded by Fundação para a Ciência e Tecnologia (EXPL/BIA-BQM/1300/2021).