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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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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

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