Diversity And Characterization Of Polyhydroxyalkanoate Synthase (Phac) In Seawater And Mangrove Metagenomes
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Date
2016-02
Authors
Foong, Choon Pin
Journal Title
Journal ISSN
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Publisher
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.
Description
Keywords
Mangrove Metagenomes