Cloning of the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes and use of these genes for enhanced production of Poly(3-hydroxybutyrate) in Escherichia coli

Abstract

Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. Recombinant Escherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinant E. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, beta-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. Recombinant E. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinant E. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness.

FIG. 1

Organization of A. latus PHA biosynthesis genes. (A) Restriction map of the AL63 fragment. (B) Organization of phaC_Al, phaA_Al, phaB_Al, and ORF4. aa, amino acids.

FIG. 2

Construction of pJC1, pJC2, pJC3, and pJC4. Abbreviations: stb, parB locus of plasmid R1; ORI, origin of replication. Bold lines and arrows represent the DNA fragment from A. latus.

FIG. 3

Growth of recombinant E. coli strains harboring various plasmids and PHB production after cultivation at 30°C for 66 h in defined R medium containing 20 g of glucose per liter (L).

FIG. 4

Time profile of cell concentration, PHB concentration, and PHB content during fed-batch culturing of XL1-Blue(pJC4) in chemically defined medium. L, liter.