FIG. 4.

Hypothetical pathway of MCL PHA biosynthesis of PHA polymerase- and ′thioesterase I-containing E. coli JMU193 grown on gluconate. Gluconate is degraded via the central carbohydrate metabolism (indicated by triple arrows), leading to acetyl-CoA. Via four conversions of the fatty acid synthesis pathway, acetyl-CoA is metabolized to acyl-ACP; acetyl-ACP, transferred from acyl-CoA, and malonyl-ACP are coupled to give 3-ketoacyl-ACP by the β-ketoacyl-ACP synthase (step 1). 3-ketoacyl-ACP is converted to (R)-3-hydroxyacyl-ACP by the β-ketoacyl-ACP reductase (step 2). The β-hydroxyacyl-ACP dehydrase (step 3) yields 2-trans-enoyl-ACP, which is metabolized by enoyl-ACP reductase (step 4) to acyl-ACP. Acyl-ACP is hydrolyzed by the ′thioesterase I (step 5), resulting in the corresponding fatty acid, which is activated by acyl-CoA synthase (step 6) to the corresponding acyl-CoA. Acyl-CoA is degraded in the β-oxidation cycle, resulting in 2-trans-enoyl-CoA by the acyl-CoA dehydrogenase (step 7) and yielding (S)-3-hydroxyacyl-CoA by the action of the enoyl-CoA hydratase (step 8). Because of the fadB mutation of E. coli JMU193 (indicated by double sticks), these latter intermediates accumulate and can be transferred by either an isomerase (step 9) or (R)-specific hydratase (step 10) into (R)-3-hydroxyacyl-CoA, which is converted by the PHA polymerase (step 11) into PHA. Reactions enclosed in ellipsoids are carried out by genes which were introduced into E. coli JMU193 encoding the ′thioesterase I and PhaC1 and PhaC2 proteins. Question marks indicate uncertainties about the actual pathway according to data from Pseudomonas.