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Engineering of polyhydroxyalkanoate synthase by Ser477X/Gln481X saturation mutagenesis for efficient production of 3-hydroxybutyrate-based copolyesters

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タイトル: Engineering of polyhydroxyalkanoate synthase by Ser477X/Gln481X saturation mutagenesis for efficient production of 3-hydroxybutyrate-based copolyesters
著者: Shozui, Fumi 著作を一覧する
Matsumoto, Ken'ichiro 著作を一覧する
Sasaki, Takahiro 著作を一覧する
Taguchi, Seiichi 著作を一覧する
キーワード: PHA synthase
Evolutionary engineering
Monomer composition
P(3HB-co-3HA)
発行日: 2009年10月
出版者: Springer Berlin / Heidelberg
誌名: Applied Microbiology and Biotechnology
巻: 84
号: 6
開始ページ: 1117
終了ページ: 1124
出版社 DOI: 10.1007/s00253-009-2052-2
抄録: 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
資料タイプ: article (author version)
URI: http://hdl.handle.net/2115/43941
出現コレクション:雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

提供者: 松本 謙一郎

 

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