Synthesis of functionalized polyhydroxyalkanoates in two Pseudomonas putida strains

GONZALEZ, GERMAN; DIAZ PEÑA, ROCIO; PELAZZO, LUCIANA; KOLENDER, ADRIANA A.; PETTINARI, MARÍA JULIA

Congreso; XVIII Congreso Argentino de Microbiología General, SAMIGE 2023; 2023

Sociedad Argentina de Microbiología General

Biobased polymers, including polyhydroxyalkanoates (PHAs), constitute an alternative to oil-derived polymers. PHAs are polyesters, composed of (R)-3-hydroxyacids of different chain lengths, which are synthesized by bacteria under appropriate conditions. These biopolymers are considered as promising candidates for packaging and biomedical applications, due to their mechanical and thermal properties but also to their biocompatibility, hydrophobicity, low oxygen permeability, water resistance, compostability and generation of non-toxic degradation products. However, conventional PHAs have restrictions in their applications that prompt the development of PHA-derivatives withenhanced physical and mechanical properties, in order to increase their potential applications. In this project, the synthesis of PHAs functionalized with terminal azide groups in Pseudomonas cultures is proposed. The azide groups can then be used in click reactions for the incorporation of different lateral substituents.The main goal was to use cultures of Pseudomonas putida KT 2440 and Pseudomonasputida KA strains in order to obtain functionalized PHAs with terminal azide groups, and to compare the incorporation yields of these groups in both strains.To obtain the functionalized PHA polymers, liquid cultures of KT and KA strains were grown in NE (low nitrogen) production medium supplemented with sodium octanoate and 6-azidohexanoic acid (synthesized in our laboratory) in a 50:50 ratio. The incorporation of the azide group was characterized by gas chromatography (GC) coupled to mass spectrometry, nuclear magnetic resonance and infrared spectroscopy. The quantification of the 3-hydroxyacids that constituted the obtained PHAs was performed by GC-FID.Both strains were able to grow and produce functionalized PHAs under these conditions,indicating that they could take up the modified substrates, metabolize them and incorporate them into the growing PHA chains. It is noteworthy that KT strain produced a higher amount of total PHA (composed by 3-OH-C8, 3-OH-C6 and 3-OH-C6N3 in a 72:2:26 ratio); while KA strain produced a higher percentage of functionalized 6-carbon PHA (composed by 3-OH-C8, 3-OH-C6 and 3-OH-C6N3 in a 52:0:48 ratio). These results could be due todifferences in the fatty acid metabolism of the strains or to differences in the specificity or activity of their PHA synthases.