Browsing by Author "Guitart Font, Emma"

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Open Access
Complementation of an Escherichia coli K-12 mutant strain deficient in KDO synthesis by forming D-arabinose 5-phosphate from glycolaldehyde with fructose 6-phosphate aldolase (FSA)
(2024) Guitart Font, Emma; Sprenger, Georg A.
KDO (2-keto-3-deoxy-D-manno-octulosonate) is a landmark molecule of the Gram-negative outer membrane. Mutants without KDO formation are known to be barely viable. Arabinose 5-phosphate (A5P) is a precursor of KDO biosynthesis and is normally derived from ribulose 5-phosphate by A5P isomerases, encoded by kdsD and gutQ genes in E. coli K-12. We created a kdsD gutQ-deficient double mutant of strain BW25113 and confirmed that these cells are A5P auxotrophs. Fructose 6-phosphate aldolase (FSA) is known to utilize (among other donors such as dihydroxyacetone or hydroxyacetone) glycolaldehyde (GoA) as a donor compound and to provide A5P in vitro when glyceraldehyde 3-phosphate is the acceptor. We show here that this FSA function in vivo fully reverses the growth defect and the A5P deficiency in kdsD gutQ double mutants. Expression of both plasmid-encoded fsaA, fsaAA129S, or fsaB genes as well as a chromosomally integrated form of fsaAA129S led to maximal OD600 values of >2.2 when GoA was added exogenously (together with glucose as a C source) at a concentration of 100 µM (Ks values in the range of 4-10 µM). Thus, a novel bio-orthogonal bypass to overcome an A5P deficiency was opened. Lower GoA concentrations led to lower growth yields. Interestingly, mutant strains with recombinant fsa genes showed considerable growth yields even without exogenous GoA addition, pointing to yet unknown endogenous GoA sources in E. coli metabolism. This is a further example of the usefulness of FSA in rewiring central metabolic pathways in E. coli.
Open Access
Introduction of novel artificial pathways in Escherichia coli with fructose 6-phosphate aldolase (FSA)
(2024) Guitart Font, Emma; Sprenger, Georg A. (Prof. Dr.)
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.
Open Access
Opening a novel biosynthetic pathway to dihydroxyacetone and glycerol in Escherichia coli mutants through expression of a gene variant (fsaAA129S) for fructose 6-phosphate aldolase
(2020) Guitart Font, Emma; Sprenger, Georg A.
Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h-1. A GL3 derivative with a chromosomally integrated copy of fsaAA129S (GL4) grew with 0.05 h-1 on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created.