Combination of N149S and D171G mutations in Aeromonas caviae polyhydroxyalkanoate synthase and impact on polyhydroxyalkanoate biosynthesis

Abstract

Aeromonas caviae polyhydroxyalkanoate synthase (PhaCAc) is an important biocatalyst for the synthesis of practically useful two-component polyhydroxyalkanoate copolymer, poly[(R)-3-hydroxybutyrate-co-(R)-3- hydroxyhexanoate] [P(3HB-co-3HHx)]. In a previous study, two PhaCAc mutants that have a single amino acid substitution of either asparagine 149 by serine (N149S) or aspartate 171 by glycine (D171G) were isolated as higher active enzymes by means of evolutionary engineering. In this study, the synergistic effects of N149S and D171G double mutation (NSDG) in PhaC Ac on polyhydroxyalkanoate biosynthesis were investigated in recombinant Ralstonia eutropha. The PhaCAc NSDG mutant showed enhanced incorporation of longer 3-hydroxyalkanoate (3HA) units into the polyhydroxyalkanoate copolymer from octanoate (3HA fraction: 18.5 mol%) and soybean oil (5.4 mol%) as a carbon source. Besides, the NSDG mutant synthesized P(3HB) homopolymer with a very high molecular weight (Mw=368 × 104) when fructose was used as a carbon source. Thus, a combination of the beneficial mutations synergistically altered enzymatic properties, leading to synthesis of a polyhydroxyalkanoate copolymer with enhanced 3HA fraction and increased molecular weight. © 2007 Federation of European Microbiological Societies.