Enhanced Production of ( R )-3-Hydroxybutyrate Oligomers by Coexpression of Molecular Chaperones in Recombinant Escherichia coli Harboring a Polyhydroxyalkanoate Synthase Derived from Bacillus cereus YB-4.

The biodegradable polyester poly-( R )-3-hydroxybutyrate [P(3HB)] is synthesized by a polymerizing enzyme called polyhydroxyalkanoate (PHA) synthase and accumulates in a wide variety of bacterial cells. Recently, we demonstrated the secretory production of a ( R )-3HB oligomer (3HBO), a low-molecular-weight P(3HB), by using recombinant Escherichia coli expressing PHA synthases. The 3HBO has potential value as an antibacterial substance and as a building block for various polymers. In this study, to construct an efficient 3HBO production system, the coexpression of molecular chaperones and a PHA synthase derived from Bacillus cereus YB-4 (PhaRC YB4 ) was examined. First, genes encoding enzymes related to 3HBO biosynthesis ( phaRC YB4 , phaA and phaB derived from Ralstonia eutropha H16) and two types of molecular chaperones ( groEL , groES , and tig ) were introduced into the E. coli strains BW25113 and BW25113Δ adhE . As a result, coexpression of the chaperones promoted the enzyme activity of PHA synthase (approximately 2-3-fold) and 3HBO production (approximately 2-fold). The expression assay of each chaperone and PHA synthase subunit (PhaR YB4 and PhaC YB4 ) indicated that the combination of the two chaperone systems (GroEL-GroES and TF) supported the folding of PhaR YB4 and PhaC YB4 . These results suggest that the utilization of chaperone proteins is a valuable approach to enhance the formation of active PHA synthase and the productivity of 3HBO.