Opening a Novel Biosynthetic Pathway to Dihydroxyacetone and Glycerol in Mutants through Expression of a Gene Variant () for Fructose 6-Phosphate Aldolase.

Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes , (encoding the two PFK isoenzymes), and (glucose 6-phosphate dehydrogenase) in K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the wild type gene (encoding 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. A GL3 derivative with a chromosomally integrated copy of (GL4) grew with 0.05 h on glucose. A mutant strain from GL4 where genes were deleted (GL5) excreted DHA. By deletion of the gene (glycerol kinase) and overexpression of (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.