Production of medium-chain-length poly(3-hydroxyalkanoates) from gluconateby recombinant Escherichia coli

It was shown recently that recombinant Escherichia coli, defective in the b eta-oxidation cycle and harboring a medium-chain-length (MCL) poly(3-hydrox yalkanoate) (PHA) polymerase-encoding gene of Pseudomonas, is able to produ ce MCL PHA from fatty acids but not from sugars or gluconate (S. Langenbach , B. H. A. Rehm, and A. Steinbuchel, FEMS Microbiol, Lett. 150:303-309, 199 7; Q, Ren, Ph.D. thesis, ETH Zurich, Zurich, Switzerland, 1997). In this st udy, we report the formation of MCL PNA from gluconate by recombinant E. co li. By introduction of genes coding for an MCL PHA polymerase and the cytos olic thioesterase I ('thioesterase I) into E. coli JMU193, we were able to engineer a pathway for the synthesis of MCL PNA from gluconate. We used two expression systems, i.e., the bad promoter and alk promoter, for the 'thio esterase I- and PHA polymerase-encoding genes, respectively, which enabled us to modulate their expression independently over a range of inducer conce ntrations, which resulted in a maximum MCL PHA accumulation of 2.3% of cell dry weight from gluconate. We found that the amount of PHA and the 'thioes terase I activity are directly correlated. Moreover, the polymer accumulate d in the recombinant E. coli consisted mainly of 3-hydroxyoctanoate monomer s, On the basis of our data, we propose an MCL PNA biosynthesis pathway sch eme for recombinant E. coli JMU193, harboring PHA polymerase and 'thioester ase I, when grown on gluconate, which involves both de novo fatty acid synt hesis and beta-oxidation.