Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

Abstract

ABSTRACTThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolatedChromobacteriumsp. USM2 (PhaCCs). PhaCCsshowed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. Anin vitroassay of recombinant PhaCCsexpressed inEscherichia colishowed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strainC. necator(307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCswas 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC fromC. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation inEscherichia coliexpressing PhaCCsof up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCsis a naturally occurring, highly active PHA synthase with superior polymerizing ability.