Applied Microbiology and Biotechnology

, Volume 97, Issue 8, pp 3441–3447

Engineering of class I lactate-polymerizing polyhydroxyalkanoate synthases from Ralstonia eutropha that synthesize lactate-based polyester with a block nature

Biotechnologically relevant enzymes and proteins

DOI: 10.1007/s00253-012-4231-9

Cite this article as:
Ochi, A., Matsumoto, K., Ooba, T. et al. Appl Microbiol Biotechnol (2013) 97: 3441. doi:10.1007/s00253-012-4231-9

Abstract

Class I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (PhaCRe) was engineered so as to acquire an unusual lactate (LA)-polymerizing activity. To achieve this, the site-directed saturation mutagenesis of PhaCRe was conducted at position 510, which corresponds to position 481 in the initially discovered class II LA-polymerizing PHA synthase (PhaC1PsSTQK), a mutation in which (Gln481Lys) was shown to be essential to its LA-polymerizing activity (Taguchi et al., Proc Natl Acad Sci USA 105(45):17323–17327, 2008). The LA-polymerizing activity of the PhaCReA510X mutants was evaluated based on the incorporation of LA units into the P[3-hydroxybutyrate(3HB)] backbone in vivo using recombinant Escherichia coli LS5218. Among 19 PhaCRe(A510X) mutants, 15 synthesized P (LA-co-3HB), indicating that the 510 residue plays a critical role in LA polymerization. The polymer synthesized by PhaCReA510S was fractionated using gel permeation chromatography in order to remove the low molecular weight fractions. The 13C and 1H NMR analyses of the high molecular weight fraction revealed that the polymer was a P(7 mol% LA-co-3HB) copolymer with a weight-averaged molecular weight of 3.2 × 105 Da. Interestingly, the polymer contained an unexpectedly high ratio of an LA-LA*-LA triad sequence, suggesting that the polymer synthesized by PhaCRe mutant may not be a random copolymer, but presumably had a block sequence.

Keywords

Lactate-polymerizing enzyme Site-directed mutagenesis Polyhydroxybutyrate polymerase Block copolymer Cupriavidus necator 

Supplementary material

253_2012_4231_MOESM1_ESM.pdf (20 kb)
ESM 1(PDF 19 kb)

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  1. 1.Division of Biotechnology and Molecular Chemistry, Graduate School of EngineeringHokkaido UniversitySapporoJapan
  2. 2.Department of Innovative and Engineered MaterialsTokyo Institute of TechnologyYokohamaJapan

Personalised recommendations