Diversity And Characterization Of Polyhydroxyalkanoate Synthase (Phac) In Seawater And Mangrove Metagenomes

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Date
2016-02
Authors
Foong, Choon Pin
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Universiti Sains Malaysia
Abstract
The microbial communities of two local Penang mangrove soils (Batu Maung and Balik Pulau) which are under anthropogenic influences were investigated using culture-independent shotgun metagenome sequencing approach. Two metagenome data sets (~250 GB) were generated from the Illumina HiSeq next-generation sequencing (NGS) platform and then deposited in Metagenomic-Rapid Annotations using Subsystems Technology (MG-RAST) public server. Microbial taxonomic analysis showed that both Penang mangrove soils were dominated by Bacteria (97 %), Proteobacteria (43 %) and Deltaproteobacteria (15 %) at the domain, phylum and class levels, respectively. At the genus level, predominance of anaerobic bacteria was observed and mostly belonged to Deltaproteobacteria. A large portion of the reads belonged to unknown or yet uncultured microbial species (70 %) and microbial phyla (32 %). Investigation on the PHA synthase (PhaC) diversity shown that about 21-23 % of the total detected microbial (bacteria and archaea) genera in the Penang mangrove soils contained PhaCs with putative lipase-box-like motif “(G/A/S)-X-C-X-G-(G/A/S)” based on the BLASTx results against National Center for Biotechnology Information Reference Sequence (NCBI RefSeq) database. These partial putative PhaCs predominantly (>80 %) belonged to the phylum Proteobacteria (Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria). About 27-37 % of the PhaCs potentially belonged to new microbial genus if a 70 % average amino acid identity (AAI) cutoff was applied. At the same time, a different PCR-based genotypic screening approach was employed in this study to investigate Class I and II PhaCs from shallow and deep-sea seawater metagenomes (24 m to 5373 m) which were collected from Nankai Trough and Japan Trench. A total of 20 partial PhaC genetic groups (GGs) were determined. All the GGs had closest organism matches to Proteobacteria and predominated by Alphaproteobacteria. Five PhaC GGs had AAI < 70 % and most probably belonged to new microbial genus from Alphaproteobacteria. Furthermore, phylogenetic analysis using all the PhaCs derived from metagenomic resources showed three new or undefined clusters of PhaC in addition to four existing known clusters of PhaC (Class I to IV). For functional verification, three complete DNA coding sequences were successfully obtained from Japan seawater metagenomes by genome walking approach. Only I-GG18 PhaC was functionally active and able to produce PHA in transformant Cupriavidus necator PHB¯4 (PHB-negative mutant). I-GG18 PhaC had very high protein sequence identity (97 %) to the PhaCs of new PHA producing genus Marinobacter. This I-GG18 PhaC had substrate specificity towards shortchain- length PHA (SCL-PHA) monomers such as 3-hydroxybutyryl-CoA and 4- hydroxybutyryl-CoA. The synthase activity of I-GG18 PhaC in transformant C. necator PHB¯4 was 10 folds lower than the wild-type C. necator H16 at 24th hour of incubation in nitrogen-limiting medium.
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Mangrove Metagenomes
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