GENETICALLY ENGINEERED PICHIA PASTORIS FOR THE POLY 3-HYDROXYPROPIONATE BIOPLASTIC PRODUCTION

FRESCURA, JULIETA M; ROJAS, NATALIA L

Buzios

Simposio; Simposio Nacional de Fermentaciones; 2022

Plastics are essential materials in daily life because of their multiple applications. Nevertheless, they entail an environmental problem due their polluting production process and their non-biodegradability after use. In the last decades, biobased biodegradable bioplastics -polyhydroxyalkanoates (PHAs)- have arisen. In particular, poly 3-hydroxypropionate (p(3HP)), presents promising physical and mechanical properties -high stiffness, ductility and tensile strength- advantageous qualities for industrial processes, for the generation of capsules for controlled drugs release or as a scaffold for tissue engineering. These make p(3HP) competitive against plastics derived from fossil fuels such as polyethylene (PE). Although organisms capable to naturally produce p(3HP) have not been found to date, different biosynthesis pathways have been developed, such as the propionaldehyde dehydrogenase (pduP) metabolic pathway for the production of p(3HP) from glycerol. Particularly its relevance lies in the possibility of using crude glycerol from biodiesel production as carbon source, which means a biorefinery based process.In this work the pduP pathway was cloned in the methylotrophic yeast Pichia pastoris genome using a monocistronic strategy with T2A peptides, and the constitutive p(3HP) production was evaluated.A polyprotein composed by all the subunits involved in pduP pathway was designed, arranging its respective ORFs without stop codons alternated with different coding sequences for the T2A peptide. The pGap-polyprotein plasmid was inserted into the P. pastoris X-33 strain genome by electroporation. The heterologous expression was analyze by flow cytometry and it was observed a higher fluorescence level in the three analyzed clones, suggesting the presence of a biopolymer into the cell.