Utilização dos recursos genéticos de Enterobacter aerogenes ATCC 13048 para o desenvolvimento de enzima quimérica glicerol desidrogenase-acetoína redutase

Abstract

2,3-butanediol (BDO) is an organic compound with formula C4H10O2 which has three stereoisomers, and that has been widely used in the manufacture of 1,3-butadiene, antifreeze additives, polyurethane foams, plastic polymers, liquid fuels and other chemicals usually produced from natural gas and crude petrol derivatives. Due to its diverse applications in the industry, the demand for BDO increases every year, and the overall potential market of BDO is around 32 million tonnes per year. All three stereoisomers of BDO can be produced by microorganisms consuming different reduced carbon sources as substrate. These microorganisms, especially E. aerogenes ATCC 13048, are capable of using other carbon sources for fermentation and BDO formation, with glycerol being of economic interest for it is the major residue of biodiesel production and currently is exported as a low-value compound. This project aims to identify genetic resources for consumption of glycerol and its conversion to dihydroxyacetone and production of 2,3-butanediol from pyruvate. The genes identified, present in the genome of Klebsiella aerogenes KCTC 2190 (synonym strain of E. aerogenes ATCC 13048) and its organization in operons and proximity to other related genes and metabolic pathways were used to develop a strategy of increasing the production of 2,3-butanediol from glycerol by constructing a fusion enzyme using synthetic biology. The fusion enzyme designed aims to confer an advantage on three bottlenecks for increasing the fermentative capacity of glycerol in BDO, the solutions for those bottlenecks being the increased consumption of glycerol in DHA, increased conversion of acetoin to BDO and the mutual recycle of NAD and NADH cofactors used in both pathways. The developed fusion enzyme was cloned in Escherichia coli DH5α to evaluate its effect on glycerol consumption and biomass formation and production of BDO from acetoin. The crude extracts of Escherichia coli containing the fusion enzyme were evaluated in an enzymatic reaction to investigate their activity on the conversion of glycerol and conversion of acetoin to BDO. The developed fusion enzyme can be used in future studies involving its cloning in bacterial strains what already produce acetoin and BDO as the species of the family Enterobacteriaceae. The plasmid constructed for the fusion enzyme expression also allows the cloning of genes in the operon with the fusion enzyme and under an individual promoter, expanding the possibilities of its use in the metabolic engineering of microorganisms for consumption of glycerol and production of BDO