Resumen del poster presentado en: XIII Reunión del Grupo de Microbiología Molecular de La SEM. Granada, 7-9 septiembre (2022), This work was supported by grants PID2019-103972GA-I00 (to M.A.M.), PID2020-116261GBI00 (to J.A.G.) and PID2020-112612GB-I00 (to T.K.) from the Spanish Ministry for Science and
Innovation/Agencia Estatal de Investigación 10.13039/501100011033, grant P18-FR-1621 (to T.K.) from the Junta de Andalucía.

XXX simposio del Grupo Especializado de Cristalografía y Crecimiento Cristalino - GE3C, Supported the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033, grant PID2020-116261GB-I.

Resumen de la comunicación oral presentada en: XXIX Congreso de la Sociedad Española de Microbiología SEM. Microorganismos: un universo en continua evolución. Burgos, España. 25-28 junio (2023), Polyethylene terephthalate (PET) is a versatile, and low-cost polyester with low reactivity properties that has become a key element in modern society. It is used in a wide variety of applications, such as bottles, packaging, and textile fibers. However, the lack of an effective recycling system has led to an alarming accumulation of PET and other plastics in landfills and oceans, posing a significant threat to the environment and human health (1). The main goal of this work is to develop an optimal
biocatalyst for the efficient biodegradation of PET into its monomers (terephthalic acid (TPA) and ethylene glycol (EG)). The development of such a biocatalyst could offer a viable and environmentally friendly alternative to existing recycling processes that are either less specific or more contaminant.
Our approach involved the combination of two engineering strategies. First, we used Ancestral Sequence Reconstruction (ARS) to infer the protein sequence of a hypothetical ancient hydrolase, using the modern sequence of PET hydrolase from Ideonella sakaiensis (IsPETase) as a reference (2). This strategy allowed us to generate a new set of functional enzymes that retain the spatial structure of IsPETase, yet exhibit significant amino acid sequence changes. The resulting enzymes showed
increased robustness and improved stability properties (3). After a preliminary characterization, the putative ancestral enzyme with most promising features was selected as a parental template in a directed evolution campaign to improve its PET-degrading activity. This strategy required the development of a reliable High-Through Put Screening (HTPS) protocol to detect significant improvements among mutants and select the most suitable candidate for industrial applications.
The combination of both strategies allowed us to identify regions that have been highly conserved across evolution, as well as regions where novel mutations could potentially enhance PET-hydrolyzing activity., This study was supported through grants from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033 (PID2019-103972GA-I00 to M.A.M., grant
PID2020-112612GBI00 to T.K. and PID2020-116261GB-I00 to J.A.G.), the Junta de Andalucía (grant P18-FR-1621 to T.K.) and by NIH Grant 1R35GM131760 (to I.B.Z.)