2018年6月1日
Site-directed saturation mutagenesis of polyhydroxylalkanoate synthase for efficient microbial production of poly[(R)-2-hydroxybutyrate]
Journal of Bioscience and Bioengineering
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- 巻
- 125
- 号
- 6
- 開始ページ
- 632
- 終了ページ
- 636
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/j.jbiosc.2017.11.013
- 出版者・発行元
- Elsevier B.V.
In our previous study, artificial polyhydroxyalkanoate (PHA) poly[(R)-2-hydroxybutyrate] [P(2HB)] was successfully biosynthesized from racemic 2HB in recombinant Escherichia coli using an engineered PHA synthase, PhaC1Ps(S325T/Q481K). Although P(2HB) has promising material properties, the low level of polymer production was a drawback. In this study, we performed directed evolution of PhaC1Ps towards enhanced P(2HB) accumulation in E. coli by site-directed dual saturation mutagenesis at the positions 477 and 481, which was known for their potential in enhancing natural PHA accumulation. By using a screening on agar plates with Nile red, eight colonies were isolated which produced a greater amount of P(2HB) compared to a colony expressing the parent enzyme PhaC1Ps(S325T/Q481K). Among them, the cells expressing PhaC1Ps(S325T/S477R/Q481G) [ST/SR/QG] accumulated polymer at the highest level (up to 2.9-fold). As seen in PhaC1Ps(ST/SR/QG), glycine and basic amino acid residues (K or R) were frequently found at the two positions of the select mutated enzymes. The enzymatic activity of PhaC1Ps(ST/SR/QG) toward 2HB-CoA was approximately 3-fold higher than that of the parent enzyme. Additionally, expression levels of the select mutated enzymes were lower than the parent. These results indicated that PhaC1Ps mutagenesis at the positions 477 and 481 increased specific activity toward 2HB-CoA and it could result in the enhanced production of P(2HB).
- リンク情報
- ID情報
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- DOI : 10.1016/j.jbiosc.2017.11.013
- ISSN : 1347-4421
- ISSN : 1389-1723
- PubMed ID : 29573982
- SCOPUS ID : 85044267882