Estudo em biorreator e análise de fluxos metabólicos em cepas recombinantes de >i/ii/i<-3-hidroxihexanoato [P(3HB->i/i<-3HHx)] [thesis]

Jefferson Gonçalves Pinheiro Silva
The environmental impact of products of petrochemical origin, such as plastics, has led to the search for similar materials that are environmentally friendly and can be produced from renewable sources. An alternative is the polyhydroxyalkanoates (PHA), a family of thermoplastic or elastomeric polyesters, biodegradable and biocompatible, accumulated by some bacteria. The incorporation and modulation of variable monomers to the PHA structure present potential for the development of new materials
more » ... or medical applications. Recent literature and patents have reported poly-3-hydroxybutyrate-co-3-hydroxyhexanoate copolymers (3HB-co-3HHx) as products of great potential in this area. Burkholderia sacchari recombinants, harboring Aeromonas sp. genes, were constructed in the laboratory of Bioproducts of the Institute of Biomedical Sciences-USP, with the objective of having a safe organism capable of producing high levels of the copolymer. This work studied some aspects of the production of 3HB-co-3HHx from xylose and hexanoic acid, by these recombinants, to subsidize the production of this material with different levels of 3HHx, potentially conferring improved propertiesto the copolymer and widening its applications.Recombinants profiles in xylose and hexanoic were analyzed, to propose metabolic alternatives to improve xylose consumption based on metabolic flux analysis. Xylose was selected as the main carbon source due to the fact that, besides being a significant component of sugarcane bagasse hemicellulosic hydrolysates, it isa residue of second generation ethanol production. Six B. sacchari recombinants, harboring genes P, C and JfromAeromonas sp. or Aeromonas hydrophila, were tested shaken flasks; recombinant B. sacchari LFM344 pBBR1MCS-2 :: phaPCJAspwas selectedfor bioreactor experiments. The parameters delimited were: the maximum specific growth rate (µmaxX = 0,18 h -1 ), the efficiency in converting xylose to 3HB monomers (Y3HB / xil = 0,40 g.g -1 ),the efficiency in converting hexanoic acid to 3HHx monomers (Y3HHx / AHex = 0,89 g.g -1 ) in culture medium containing xylose plus hexanoic acid as carbon sources. Conditions were proposed and applied in order to modulate 3HHx content in the copolymer. Cells were able to accumulate from 35 to 64% of cell dry biomass as PHA. 3HHx content in the copolymers ranged from 10 mol% to 40 mol%, evidencing the possibility of controling monomers composition and thus polymer properties. Moreover, those figures were higher than previous values from the literature. Finally, Metatool and Matlab programs were tested as a preliminary analysis of metabolic fluxes, indicating the the need for the cell to improve reducing power production (NADPH).
doi:10.11606/d.42.2020.tde-23122021-183934 fatcat:fxznlxvpefc2fa2elblf2qmbki