title: Cell system engineering to produce extracellular polyhydroxyalkanoate depolymerase with targeted applications.
abstract: Novel platforms based on the application of bacterial cell systems as factories for production of new bioproducts open avenues and dramatically expand the catalogue of existing biomaterials. Herein, we designed the strategy based on in vivo production of extracellular Pseudomonas fluorescens GK13 (PhaZGK13) depolymerase to degrade previously biosynthesized polyhydroxyalkanotes (PHAs) or to obtain 3-hydroxyalkanoic acids (HAs). With this aim, extracellular PhaZGK13 was produced in recombinant strains and the optimal conditions for controlled release of HAs and oligomers by growing cells were set up with a particle suspension of (14)C-labelled PHA, being maximal after 24h of incubation. Genetic modification of key factors involved in fatty acids metabolism revealed the influence of an active β-oxidation pathway on the extracellular degradation of PHA and subsequent HAs isolation. The highest HAs production was obtained using Pseudomonas putida KT2442 fadB mutant (0.27mg/mL) due to the reduced ability of this strain to metabolize the degradation products. The system was applied to produce new added value HAs harboring thioester groups in the side chain from the functionalized mcl-PHA, PHACOS. Remarkably, hydrolyzed PHACOS showed greater potential to inhibit Staphylococcus aureus(T) growth when compared to that of degradation products of non functionalized polyhydroxyoctanoate-co-hexanoate P(HO-co-HH).
authors.affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Virginia
authors.initials: V
authors.lastname: Martínez
authors.affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Nina
authors.initials: N
authors.lastname: Dinjaski
authors.affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Laura I
authors.initials: LI
authors.lastname: de Eugenio
authors.affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Peña
authors.affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain. Electronic address: auxi@cib.csic.es.
authors.forename: María Auxiliadora
authors.initials: MA
authors.lastname: Prieto
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002849:Chromatography, Gas; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis* / Q000737:chemistry; D016958:Pseudomonas putida / Q000378:metabolism
chemical_list: D005227:Fatty Acids; D054813:Polyhydroxyalkanoates; D002265:Carboxylic Ester Hydrolases; C077226:poly(3-hydroxyalkanoic acid) depolymerase
keywords: 3-Hydroxyalkanoic acids; Antimicrobial activity; Polyhydroxyalkanoate depolymerase