Introduction of novel artificial pathways in Escherichia coli with fructose 6-phosphate aldolase (FSA)

Abstract

Fructose 6-phosphate aldolase (FSA) enzymes are known to catalyse aldol and retroaldol reactions. These reactions have been shown in vitro by Schürmann and Sprenger (2001), Schürmann et al. (2002), Garrabou et al. (2009), Castillo et al. (2010), Sánchez-Moreno et al. (2012a and 2012b), among other groups. However, it was unclear whether these reactions would be possible in vivo. Since the discovery of FSA, encoded by the genes fsaA and fsaB in the Escherichia coli chromosome in 2001 (Schürmann and Sprenger, 2001), its true physiological function has not been reported yet (Samland and Sprenger, 2014). Due to the weak expression of the native promoters of fsaA and fsaB, the only enzymatic data reported so far are from recombinant FSA. To evaluate whether the cleavage of fructose 6-phosphate (F6P) in glycolysis, and the formation of arabinose 5-phosphate (A5P) in the synthesis of 2-keto-3-deoxymanno-octulosonic acid (KDO) could also be catalysed in vivo by FSA, E. coli mutant strains with numerous mutations in metabolic pathways were constructed. These mutant strains had blockades in central carbon metabolism or anabolism. Thus, these mutations impacted pleiotropic genes and, therefore, growth. Afterwards, it was examined if the activity of recombinant FSA in these mutant strains could restore deficiencies in growth. With the reactions catalysed by FSA, the blocked pathways were not restored, but new artificial pathways emerged. Through the F6P bypass a new route for the production of dihydroxyacetone (DHA) and glycerol was opened. Furthermore, a new approach for the provision of A5P was established.