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

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.