Evaluation of the role of Burkholderia sacchari genes involved in mobilization of poly-3-hydroxybutyrate in recombinant strains of Escherichia coli

• Grant number: 15/17399-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2015
• End date: October 31, 2016
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: José Gregório Cabrera Gomez
• Grantee: Arthur Shigueru Umeda
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Polyhydroxyalkanoates (PHA) are hydroxyalkanoates (HAs) polyesters synthesized by many bacteria as intracellular carbon and / or energy reserve components. Among the various PHA, the best known is poly-3-hydroxybutyrate (P3HB), a homopolymer with thermoplastic properties, biodegradable and biocompatible. The PHA mobilization process consists in the intracellular degradation of previously accumulated granules and occurs in situations of energy and/or carbon source demand for the microorganism, but little is known about how these mechanisms occur. One of the strategies used to better understand the metabolism of PHA is the cloning of genes which products are involved in the metabolism of these polymers in recombinant strains of Escherichia coli. This organism seems to be an interesting candidate for such constructions, since the genome has been well characterized, a well-known molecular machinery for genetic engineering, and do not have natural mechanisms for PHA synthesis and degradation. Several studies evaluated the genes which products are involved in the mobilization process in Ralstonia eutropha, the most studied organism in PHA studies. It has been shown that the expression of phaZa1 and lonA genes of R. eutropha in recombinant strains of E. coli, play an important role in the mobilization of P3HB when expressed simultaneously in the cell, indicating that there is an interaction between their products. The study of PHA metabolism in Burkholderia sacchari has proved interesting, since these bacteria is capable of producing high levels of P3HB from sucrose. The recent sequencing of the B. sacchari genome allows us the most detailed study of the products of genes supposedly involved in this metabolism. Given the above, this study aims to evaluate the role of the products of B. sacchari genes involved in P3HB mobilization process in recombinant strains of E. coli. From a recombinant E. coli strain capable of synthesizing P3HB will be built new strains harboring the phaZa1 and lonA B. sacchari genes expressed in two conditions: I) each of the genes being expressed singly in the cell; II) both genes, phaZa1 and lonA, expressed simultaneously. Thus, it is expected to evaluate the role of PhaZa1 and LonA in the P3HB mobilization, as well as to detect a possible interaction of these proteins during this process.