Regulating the molar fraction of 4-hydroxybutyrate in poly(3-hydroxybutyrate-4-hydroxybutyrate) biosynthesis by Ralstonia eutropha using propionate as a stimulator

The regulation of the molar fraction of 4-hydroxybutyrate (4-HB) in the pol y(3-hydroxybutyrate-4-hydroxybutyrate) [P(3HB-4HB)] biosynthesis by Ralston ia eutropha (formerly Alcaligenes eutrophus) was attempted by the supplemen tal addition of propionate. The molar fraction of 4-HB in P(3HB-4HB) was in creased significantly from 12.3 to 51.8 mol% by the addition of a small amo unt of propionate along with gamma-butyrolactone commonly used as a precurs or for the biosynthesis of P(3HB-4HB). The mechanism of regulation by propi onate was investigated by measuring the variation of enzyme activities rela ted to the biosynthesis of P(3HB-4HB) and the level of intermediate metabol ite acetyl-CoA. PHB synthase activity was induced significantly by propiona te, and the acetyl-CoA concentration also increased significantly due to th e additional supply of propionate. The overflowing acetyl-CoA seems to caus e an inhibitory effect on the ketolysis reaction catalysing the lysis of 4- hydroxybutyryl-CoA to two molecules of acetyl-CoA; consequently, the 4-HB f raction available for polymerization increased. Accordingly, the molar frac tion of 4-HB in P(3HB-4HB) biosynthesis seems to be regulated by both an in creased 4-HB fraction and an activated PHB synthase due to the supplemental addition of propionate as a stimulator.