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.
Keywords: Escherichia coli; dihydroxyacetone; fructose 6-phosphate aldolase; glycerol dehydrogenase; glycerol formation; metabolic bypass; new-to-nature pathway; pfkAB zwf triple deletion; phosphofructokinase; point mutation.