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

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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.

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Acknowledgments

We thank Dr. Hideki Abe (RIKEN Institute) for critical discussion, and Dr. Nobuhito Kurono and Dr. Tomohiro Hirose (both Hokkaido University) for technical assistance with the NMR analysis. This work was partly supported by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science (To A. O.), a Grant-in-Aid for Scientific Research of Japan (no. 23310059) (to S.T.), and the Global COE Program (project no. B01: Catalysis as the Basis for Innovation in Materials Science), all from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Pacific Edit reviewed the manuscript prior to submission.

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Authors and Affiliations

  1. Division of Biotechnology and Molecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo, 060-8628, Japan

    Anna Ochi, Ken’ichiro Matsumoto, Takashi Ooba, Kohei Sakai & Seiichi Taguchi

  2. Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa, 226-8502, Japan

    Takeharu Tsuge

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  1. Anna Ochi
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  2. Ken’ichiro Matsumoto
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  3. Takashi Ooba
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  4. Kohei Sakai
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  5. Takeharu Tsuge
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  6. Seiichi Taguchi
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Correspondence to Ken’ichiro Matsumoto or Seiichi Taguchi.

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Ochi, A., Matsumoto, K., Ooba, T. et al. Engineering of class I lactate-polymerizing polyhydroxyalkanoate synthases from Ralstonia eutropha that synthesize lactate-based polyester with a block nature. Appl Microbiol Biotechnol 97, 3441–3447 (2013). https://doi.org/10.1007/s00253-012-4231-9

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  • DOI: https://doi.org/10.1007/s00253-012-4231-9

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