Construction of a recombinant library of Burkholderia sacchari for PHA production of mixed composition

• Grant number: 19/18742-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: December 01, 2019
• End date: January 31, 2021
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Luiziana Ferreira da Silva
• Grantee: Natália Andreoli Puerta Lopes
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Polyhydroxyalkanoates (PHA) are biodegradable, biocompatible bacterial polyesters with thermoplastic properties. Thus, they constitute an environmentally friendly alternative to the use of plastic of petrochemical origin. PHA include P (3HB-co-3HHx), 3-hydroxyhexanoate copolymerized poly-3-hydroxybutyrate, which has recently been studied as a scaffold or molds in tissue engineering and other medical applications. Two relevant points to develop application of this copolyester are: (i) control of the copolymer's 3HHx content, which determines its properties, and (ii) two increase the conversion of the carbon source supplied to 3HHx units. The bacterium B. sacchari accumulates high levels of PHA but low 3HHx, while Aeromonas has enzymes for high levels of 3HHx but accumulates low PHA content and uses few carbon sources. This work aims to overexpress the production of this biopolymer in Burkholderia sacchari with the insertion of the phaPCJ operon of Aeromonas sp. TSM81, already cloned into the suicide vector pBAMD1-6, thus creating a stable, plasmid-free, antibiotic-resistant bacterial strain for PHA production. Tn5 mini-transposon will be used for library construction of B. sacchari recombinants harboring PHA biosynthesis operon of Aeromonas sp. All recombinants will be evaluated in Petri dish for PHA production. PHA + strains will be further evaluated on a rotary shaker to confirm the phenotype and composition of the biopolymer produced. Recent work has indicated that, depending on the insertion site on the chromosome, the expression of heterologous genes is increased, so after sequencing the insertion site of the phaPCJ operon, we can indicate the appropriate gene loci for better gene expression in B. sacchari, contributing to the establishment of this strain as a biological platform for the production of bioproducts from renewable sources.