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  • Introduction Klebsiella pneumoniae a Gram negative enteric b

    2020-08-03

    Introduction Klebsiella pneumoniae, a Gram-negative enteric bacterium, is an organism with significance in both biomedicine and biotechnology. Some of the strains of K. pneumoniae are opportunistic pathogens, which can cause suppurative infection, pneumoniae and urinary tract infection in humans (Bouza and Cercenado, 2002). On the other hand, it is also a widely used model organism for elucidating biochemical and genetic mechanisms with respect to citrate metabolism (Bott et al., 1995), nitrogen fixation (Jack et al., 1999) and microbial production of useful chemicals such as 1,3-propanediol (Nakamura and Whited, 2003, Zeng and Biebl, 2002). 1,3-Propanediol (1,3-PD) can be used as a monomer for the synthesis of novel polyesters and biodegradable plastics. Different strategies for the microbial production of 1,3-PD have been investigated in recent years by using microorganisms such as K. pneumoniae, Citrobacter freundii, Enterobacter agglomerans and Clostridium strains from substrates such as glycerol and more abundant renewable resources like glucose (Biebl et al., 1998, Hartlep et al., 2002, Nakamura and Whited, 2003, Papanikolaou et al., 2000, Zeng and Biebl, 2002, Gonzalez-Pajuelo et al., 2005). For the production of 1,3-PD from glucose in K. pneumoniae an extensive metabolic engineering of the metabolism is necessary. Since Klebsiella cannot directly convert dihydroxyacetone phosphate (DHAP) to glycerol, heterogeneous genes encoding Fosaprepitant dimeglumine salt such as glycerol 3-phosphate dehydrogenase (GPD) and glycerol 3-phosphate phosphatase (GPP2) from Saccharomyces cerevisiae need to be introduced to this end. Another key strategy is to knock-out the tpi gene encoding the triose phosphate isomerase (TPI, EC. 5.3.1.1) to direct the carbon flux towards the formation of glycerol and further to 1,3-propanediol after the split of fructose bisphosphate (Capitanio et al., 2002, Compagno et al., 2001). Triosephosphate isomerase (TPI) is the glycolytic enzyme that catalyzes the reversible interconversion of glyceroaldehyde 3-phosphate (GAP) and DHAP (Knowles, 1991). TPI plays an important role in several metabolic pathways such as glycolysis and gluconeogenesis and is essential for efficient energy production. It is a dimer of identical subunits, each of which is made up of about 250 amino-acid residues. A glutamic acid residue is involved in the catalytic mechanism. The sequence around the active site residue is well conserved in all known TPI\'s. Deficiencies of TPI in human being are associated with haemolytic anaemia coupled with a progressive, severe neurological disorder (Olah et al., 2002). Structure study has led to the renewed interest in this enzyme because it could serve as a drug target against many parasitic protozoans (Maithal et al., 2002). In order to increase the yield of glycerol from glucose in K. pneumoniae we set out to knockout the tpi1 gene. Surprisingly, the knockout of tpi1 gene did not result in the expected phenotype but led to the discovery of a new, additional tpi gene (tpi2) in this organism. The gene tpi2 was cloned and its functionality was demonstrated by enzyme activity assay. The phylogenetic relationship of this new gene was surveyed and its putative physiological function was discussed.