title: Combination of N149S and D171G mutations in Aeromonas caviae polyhydroxyalkanoate synthase and impact on polyhydroxyalkanoate biosynthesis.
abstract: Aeromonas caviae polyhydroxyalkanoate synthase (PhaC(Ac)) 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 PhaC(Ac) 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 PhaC(Ac) 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 (M(w)=368 x 10(4)) 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.
authors.forename: Takeharu
authors.initials: T
authors.lastname: Tsuge
authors.forename: Shinko
authors.initials: S
authors.lastname: Watanabe
authors.forename: Daisuke
authors.initials: D
authors.lastname: Shimada
authors.forename: Hideki
authors.initials: H
authors.lastname: Abe
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Doi
authors.forename: Seiichi
authors.initials: S
authors.lastname: Taguchi
mesh_terms: D000217:Acyltransferases / Q000235:genetics*; D000333:Aeromonas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics*; D020118:Cupriavidus necator / Q000235:genetics / Q000378:metabolism; D005632:Fructose / Q000378:metabolism; D008970:Molecular Weight; D020125:Mutation, Missense; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013024:Soybean Oil / Q000378:metabolism
chemical_list: D054813:Polyhydroxyalkanoates; D011994:Recombinant Proteins; D005632:Fructose; D013024:Soybean Oil; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase