|
Hokkaido University Collection of Scholarly and Academic Papers >
Graduate School of Engineering / Faculty of Engineering >
Peer-reviewed Journal Articles, etc >
Engineering of polyhydroxyalkanoate synthase by Ser477X/Gln481X saturation mutagenesis for efficient production of 3-hydroxybutyrate-based copolyesters
| Title: | Engineering of polyhydroxyalkanoate synthase by Ser477X/Gln481X saturation mutagenesis for efficient production of 3-hydroxybutyrate-based copolyesters |
| Authors: | Shozui, Fumi Browse this author | | Matsumoto, Ken'ichiro Browse this author →KAKEN DB | | Sasaki, Takahiro Browse this author | | Taguchi, Seiichi Browse this author →KAKEN DB |
| Keywords: | PHA synthase | | Evolutionary engineering | | Monomer composition | | P(3HB-co-3HA) |
| Issue Date: | Oct-2009 |
| Publisher: | Springer Berlin / Heidelberg |
| Journal Title: | Applied Microbiology and Biotechnology |
| Volume: | 84 |
| Issue: | 6 |
| Start Page: | 1117 |
| End Page: | 1124 |
| Publisher DOI: | 10.1007/s00253-009-2052-2 |
| PMID: | 19506863 |
| Abstract: | Class II polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 (PhaC1_[Ps]) synthesizes 3-hydroxybutyrate (3HB)-based copolyesters, P[3HB-co-3-hydroxyalkanoate (3HA)]. Four sites (130, 325, 477, and 481) in PhaC1_[Ps] that affect the cellular content and 3HB fraction of P(3HB-co-3HA) produced have been identified. Simple combination of beneficial mutations at the sites successfully increased 3HB fraction in the copolymers (62 mol%). However, polymer content was often largely decreased (0.2 wt%) regardless of an enhancement in 3HB fraction, compared to the wild-type enzyme (14 mol% 3HB and 12 wt%) [Matsumoto et al. (2006) Biomacromolecules, 7:2436-2442]. In the present study, we attempted to explore residues combination at the four sites to overcome the problem. Here, pairwise saturation mutagenesis at the neighboring sites 477 and 481 of PhaC1_[Ps] was performed using single and double mutations at sites 130 and 325 as templates, to increase 3HB fraction in the copolymer without reducing the polymer content in recombinant Escherichia coli. These useful PhaC1_[Ps] mutants were screened based on enhanced P(3HB) content, and were subsequently applied to P(3HB-co-3HA) production. Among the mutants tested, the Ser325Cys/Ser477Lys/Gln481Leu mutant exhibited increased 3HB fraction in copolymer (63 mol%) and also polymer content (18 wt%), indicating that mutation scrambling was effective for obtaining the desired mutants. |
| Rights: | The original publication is available at www.springerlink.com |
| Type: | article (author version) |
| URI: | http://hdl.handle.net/2115/43941 |
| Appears in Collections: | 工学院・工学研究院 (Graduate School of Engineering / Faculty of Engineering) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
|
Submitter: 松本 謙一郎
|
