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A novel and wide substrate specific polyhydroxyalkanoate (PHA) synthase from unculturable bacteria found in mangrove soil

  • Choon Pin Foong
  • Manoj Lakshmanan
  • Hideki Abe
  • Todd D. Taylor
  • Swee Yeok Foong
  • Kumar SudeshEmail author
ORIGINAL PAPER
Part of the following topical collections:
  1. Topical Collection on Bio-Based Polymers

Abstract

This study reports the discovery of a polyhydroxyalkanoate (PHA) synthase (PhaC) possessing very wide substrate specificity from a mangrove soil metagenome. For the first time, putative PhaCs were identified from a metagenome using next-generation sequencing (NGS) and bioinformatic approaches. High-throughput shotgun metagenomic sequencing was conducted using the Illumina HiSeq 2000 platform. Sequence annotation and bioinformatic analyses were performed using the MG-RAST metagenomic pipeline. Reads annotated as PhaC against the NCBI RefSeq database were retrieved using the MG-RAST RESTful API (Application Programming Interface). PhaC gene sequence assembly was accomplished using the SPAdes assembler. A total of two de novo assembled contigs were subjected to sequence verification. A putative PhaC sequence, “BP-M-CPF4”, was selected for functional assessment by in vivo PHA biosynthesis in a PHA-negative mutant. An artificial stop codon was added at the 3′-end of the incomplete coding gene sequence. This novel PhaC showed very broad substrate specificity with the ability to incorporate six types of PHA monomers, 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 3-hydroxy-4-methylvalerate (3H4MV), 5-hydroxyvalerate (5HV) and 3-hydroxyhexanoate (3HHx) in the presence of suitable precursors. This PHA synthase is suitable for the biosynthesis of PHAs that can be used in various biomedical applications due to its ability to incorporate the lipase-degradable monomer sequences of 4HB and 5HV. This study demonstrates that a functional metagenomic approach using next-generation sequencing can be used to mine novel PHA synthases with interesting substrate specificities from unculturable microorganisms.

Keywords

Functional metagenomics Mangrove soil MG-RAST Cupriavidus necator 

Notes

Acknowledgements

This study was supported by the Long-Term Research Grant Scheme (USM) and the USM-RIKEN Centre of Aging Sciences (URICAS). We are also grateful to Dr. Kovach ME for his kind gift of the pBBR1MCS broad-host-range vector derivatives used in this study. CPF gratefully acknowledges the MyPhD scholarship program from the Ministry of Higher Education Malaysia.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10965_2017_1403_MOESM1_ESM.docx (372 kb)
ESM 1 (DOCX 371 kb)

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© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  • Choon Pin Foong
    • 1
  • Manoj Lakshmanan
    • 1
    • 2
  • Hideki Abe
    • 2
    • 3
  • Todd D. Taylor
    • 2
    • 4
  • Swee Yeok Foong
    • 1
  • Kumar Sudesh
    • 1
    • 2
    Email author
  1. 1.School of Biological SciencesUniversiti Sains MalaysiaPenangMalaysia
  2. 2.USM-RIKEN Centre for Aging Sciences (URICAS)Universiti Sains MalaysiaPenangMalaysia
  3. 3.Bioplastic Research Team, Biomass Engineering Research DivisionRIKEN Center for Sustainable Resource ScienceSaitamaJapan
  4. 4.Laboratory for Integrated Bioinformatics, Core for Precise Measuring and ModelingRIKEN Center for Integrative Medical SciencesYokohamaJapan

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