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Site-Directed Mutagenesis of Aeromonas hydrophila Enoyl Coenzyme A Hydratase Enhancing 3-Hydroxyhexanoate Fractions of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

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Abstract

The aim of this study is to enhance 3-hydroxyhexanoate (3HHx) fractions of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), abbreviated as PHBHHx, through site-directed mutagenesis of Aeromonas hydrophila enoyl Coenzyme A hydratase (PhaJAh). Two amino acids (Leu-65 and Val-130) were selected as a substitutional site based on the structural information of PhaJAh. The purified proteins from the wild-type enzyme and mutants were used to determine hydratase activities. Hydratase activities of four single-mutation enzymes were similar to those of the wild type PhaJAh, while hydratase activities of two double-mutation enzymes were much lower. In addition, the mutated phaJ Ah was individually co-transformed into E. coli BL21 (DE3) together with pFH21, which carried the PHA synthase (PhaCAh) gene from A. hydrophila. The recombinant E. coli harboring plasmid pETJ1 (L65A), pETJ2 (L65V) or plasmid pETJ3 (V130A) synthesized the enhanced 3HHx fractions of PHBHHx from dodecanoate, indicating that Leu-65 and Val-130 of PhaJAh play an important role in determining the acyl chain length substrate specificity. The mutated PhaJAh (L65A, L65V, or V130A) provided higher 3HHx precursors for PHA synthase, resulting in the enhanced 3HHx fractions of PHBHHx. It is possible to change the acyl chain length substrate specificity of PhaJ through site-directed mutagenesis and produce PHBHHx with a wider range of alterable monomer composition.

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Acknowledgments

This research was supported by the Province Nature Science Foundation of Liaoning, China (Grant No. 20062048).

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Authors and Affiliations

  1. School of Life Science, Liaoning University, 110036, Shenyang, China

    Fengqing Hu, Yan Cao, Fang Xiao, Jin Zhang & Hui Li

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  1. Fengqing Hu
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  2. Yan Cao
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  3. Fang Xiao
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  4. Jin Zhang
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  5. Hui Li
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Correspondence to Fengqing Hu.

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Hu, F., Cao, Y., Xiao, F. et al. Site-Directed Mutagenesis of Aeromonas hydrophila Enoyl Coenzyme A Hydratase Enhancing 3-Hydroxyhexanoate Fractions of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Curr Microbiol 55, 20–24 (2007). https://doi.org/10.1007/s00284-006-0490-y

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  • DOI: https://doi.org/10.1007/s00284-006-0490-y

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