Construction of recombinant pseudomonas strains harboring different PHA synthases in its genome to produce 3HB-co-3HAMCL

• Grant number: 14/15448-0
• Support Opportunities: Scholarships in Brazil - Master
• Start date: October 01, 2014
• End date: September 30, 2016
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Luiziana Ferreira da Silva
• Grantee: Edmar Ramos de Oliveira Filho
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Polyhydroxyalkanoates (PHA) are biopolymers produced and accumulated naturally by several bacterial strains. These biopolymers exhibit great potential to substitute petroleum-based plastics, being thermoplastic, biodegradable, biocompatible and produced from renewable sources. Due to their physical and mechanical characteristics which may vary with the composition of its monomeric chain, it has various applications. PHAs can be formed by short chain monomers (PHASCL) or medium (PHAMCL), that can form hybrid copolymers (HASCL-HAMCL) with intermediate properties, which are interesting for the materials industry. The PHA synthase is the key enzyme in the biosynthesis of PHA, responsible for catalyzing the polymerization of molecules of (R)-hydroxyacyl-CoA, units of the biopolymer. In a study conducted by our group, the production of copolymers HASCL-HAMCL in pseudomonas strains harboring Ralstonia eutropha PHA synthase gene in plasmid vectors was evaluated. In this proposal we aim the construction of four stable pseudomonas strains harboring Ralstonia eutropha and Aeromonas hydrophila PHA synthase genes in its genome. The insertion of this genes in pseudomonas genome exclude drawbacks inherent to the presence of a plasmid in a bacterial cell, as the gene gene multicopy state and the need for continuous selection using antibiotics. To this end, the mini-Tn7 system which is efficient and easy to handle will be used. The mini-Tn7 element can be readily transferred to the formation of mutants. This system's molecular biology is widely known and promotes a single insertion in the bacterium genome. The strains obtained will be tested phenotype and genotypically to confirm the presence of the gene of interest in the genomic DNA and the lack of resistance marker, resulting from genetic manipulations. To evaluate the ability of obtained recombinant strains regarding the accumulation of PHA, shaken flasks and bioreactor experiments will be conducted. The composition of produced PHA will be evaluated and compared with previously obtained results. In addition, metabolic flux analysis in the context of the biosynthesis of PHA will be performed. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

OLIVEIRA-FILHO, EDMAR RAMOS; GUAMAN, LINDA P.; MENDONCA, THATIANE TEIXEIRA; LONG, PAUL F.; TACIRO, MARILDA KEICO; CABRERA GOMEZ, JOSE GREGORIO; SILVA, LUIZIANA F.. Production of Polyhydroxyalkanoates Copolymers by Recombinant Pseudomonas in Plasmid- and Antibiotic-Free Cultures. JOURNAL OF MOLECULAR MICROBIOLOGY AND BIOTECHNOLOGY, v. 28, n. 5, p. 225-235, 2018. (12/51533-6, 10/51692-1, 14/15448-0)

CESPEDES, LUCAS G.; NAHAT, RAFAEL A. T. P. S.; MENDONCA, THATIANE T.; TAVARES, RAFAELA R.; OLIVEIRA-FILHO, EDMAR R.; SILVA, LUIZIANA F.; TACIRO, MARILDA K.; SANCHEZ, RUBEN J.; GOMEZ, JOSE G. C.. A non-naturally-occurring P(3HB-co-3HA(MCL)) is produced by recombinant Pseudomonas sp from an unrelated carbon source. International Journal of Biological Macromolecules, v. 114, p. 512-519, JUL 15 2018. (10/51692-1, 14/15448-0)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

OLIVEIRA FILHO, Edmar Ramos de. Construction of recombinant Pseudomonas strains harboring different PHA synthases in its genome to produce 3HB-co-3HAMCL.. 2017. Master's Dissertation - Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) São Paulo.