Mutation Effects of a Conserved Alanine (Ala510) in Type I Polyhydroxyalkanoate Synthase from Ralstonia eutropha on Polyester Biosynthesis

Abstract

Type I polyhydroxyalkanoate (PHA) synthases, as represented by Ralstonia eutropha enzyme (PhaC(Re)), have narrow substrate specificity toward (R)-3-hydroxyacyl-coenzyme A with acyl chain length of C3-C5 to yield PHA polyesters. In this study, saturation point mutagenesis of a highly conserved alanine at position 510 (A510) in PhaC(Re) was carried out to investigate the effects on the polymerization activity and the substrate specificity for in vivo PHA biosynthesis in bacterial cells. A series of saturation mutants were first applied for poly[(R)-3-hydroxybutyrate] homopolymer synthesis in Escherichia coli and R. eutropha PHB(-)4 (PHA negative mutant) cells to assess the polymerization activity. All mutants showed quantitatively similar polymerization activities when R. eutropha PHB(-)4 was used for assay, whereas several mutants such as A510P showed low activities in E. coli. Further analysis has revealed that majority of mutants synthesize polyesters with higher molecular weights than the wild-type. In particular, substitution by acidic amino acids, A510D(E), led to remarkable increases in molecular weights. Subsequently, PHA copolymer synthesis from dodecanoate (C12 fatty acid) was examined. The copolymer compositions were varied depending on the mutants used. Significant increased fractions of long monomer units (C6 and C8) in PHA copolymers were observed for three mutants [A510M(Q,C)]. From these results, the mutations at this potion are beneficial to change the molecular weight of polyesters and the substrate specificity of PhaC(Re). Molecular weight distributions of PHA polymers synthesized by the wild-type enzyme (PhaC(Re)) and its mutants.

Mutations Derived from the Thermophilic Polyhydroxyalkanoate Synthase PhaC Enhance the Thermostability and Activity of PhaC from Cupriavidus necator H16

“…A G4D or F420S mutation results in more PHB accumulation in recombinant E. coli (26,41). Another beneficial mutation, A510D(E), leads PhaC H16 to synthesize a higher-molecular-weight polymer (43). The thermostability of PHA synthase has never been the aim of protein engineering.…”

Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase: In Vivo and In Vitro Studies of Enzymatic Activity and Substrate Specificity

“…Substitution with uncharged residues (G/M/S/T/Q) was concluded to be best for enhancement of both PHA accumulation and 3HHx fraction. Interestingly, it was found that A510M/Q in PhaC Re also improved PHA accumulation and 3HHx fraction in the resultant copolymer (12).…”

“…1) located at position 479 in PhaC Cs . Functional implications of amino acid substitutions at the position based on previous studies include alterations in substrate specificity, and in certain cases, variations in the molecular weight of the resultant polymer were observed (10,12,23). Position 479 in PhaC Cs , therefore, appears to be a suitable candidate for the mutagenesis approach in order to achieve similar positive effects.…”

“…Extensive efforts in this direction have yielded various mutant PHA synthases with dramatic improvements in their activities for polymerization, as well as novel substrate-binding properties (7,8). Engineered synthases with up to 5-fold enhancement in activity, increased efficiency of substrate incorporation, and extended substrate range have been documented in several studies (9)(10)(11)(12)(13). These effects, particularly those related to substrate specificity, are of industrial importance, as they are feasible strategies for efficient production of PHAs with desirable properties.…”

Polyhydroxyalkanoate (PHA) Synthesis by Class IV PHA Synthases EmployingRalstonia eutrophaPHB−4 as Host Strain

“…3,14) It has been reported that R. eutropha PHB À 4 expressing Aeromonas caviae PHA synthase (PhaC Ac ), which belongs to class I synthases but has exceptionally broad substrate specificity, syn- Fig. 1.…”