Whole genome amplification approach reveals novel polyhydroxyalkanoate synthases (PhaCs) from Japan Trench and Nankai Trough seawater

Abstract

BackgroundSpecial features of the Japanese ocean include its ranges of latitude and depth. This study is the first to examine the diversity of Class I and II PHA synthases (PhaC) in DNA samples from pelagic seawater taken from the Japan Trench and Nankai Trough from a range of depths from 24 m to 5373 m. PhaC is the key enzyme in microorganisms that determines the types of monomer units that are polymerized into polyhydroxyalkanoate (PHA) and thus affects the physicochemical properties of this thermoplastic polymer. Complete putative PhaC sequences were determined via genome walking, and the activities of newly discovered PhaCs were evaluated in a heterologous host.ResultsA total of 76 putative phaC PCR fragments were amplified from the whole genome amplified seawater DNA. Of these 55 clones contained conserved PhaC domains and were classified into 20 genetic groups depending on their sequence similarity. Eleven genetic groups have undisclosed PhaC activity based on their distinct phylogenetic lineages from known PHA producers. Three complete DNA coding sequences were determined by IAN-PCR, and one PhaC was able to produce poly(3-hydroxybutyrate) in recombinant Cupriavidus necator PHBˉ4 (PHB-negative mutant).ConclusionsA new functional PhaC that has close identity to Marinobacter sp. was discovered in this study. Phylogenetic classification for all the phaC genes isolated from uncultured bacteria has revealed that seawater and other environmental resources harbor a great diversity of PhaCs with activities that have not yet been investigated. Functional evaluation of these in silico-based PhaCs via genome walking has provided new insights into the polymerizing ability of these enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0318-z) contains supplementary material, which is available to authorized users.

PHA Production and PHA Synthases of the Halophilic Bacterium Halomonas sp. SF2003

“…Km R . Foong et al 2014 [15] pBBR1-Pro Cn -phaC1 pBBR1MCS-2 derivatives with phaC1 promoter from C. necator and phaC1 of Halomonas sp. SF2003.…”

Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases

“…S. meliloti wild-type Rm1021 and previously isolated class I clones (CX92, pCX92M1, pCX92M3, and pCX92M5; Schallmey et al 2011) were used as controls breakage, and reduced melting point (Noda et al 2005). Most naturally occurring PhaC proteins polymerize either SCL or MCL monomers, but there are a few know examples such as the class I PhaC of Aeromonas caviae (Fukui and Doi 1997) and some Pseudomonas class II PhaC (Matsusaki et al 1998) that can synthesize SCL-MCL copolymers.…”

“…One of the isolated genes produced PHA copolymer when expressed in Pseudomonas putida. Moreover, partial phaC genes were also obtained from metagenomic DNA via direct PCR amplification (Foong et al 2014;Pärnänen et al 2015;Tai et al 2015). Building on these previous studies, we constructed a PHA − strain of P. putida KT2440 to use as a surrogate host and functionally identified phaC genes after screening millions of agricultural wheat soil metagenomic clones.…”

Effect of glucose and olive oil as potential carbon sources on production of PHAs copolymer and tercopolymer by Bacillus cereus FA11

“…poly-3-hydroxybutyrate-co-3-hydroxyvalerate-co-6-hydroxyhexanoate (PHBVHHx)]. This huge diversity of monomeric subunits is mainly dependent upon a type of microorganism, the type of substrate used, the substrate specificity of PHA synthases and the physico-chemical parameters used for PHA production (Tariq et al 2015;Foong et al 2014). The incorporation of diverse monomeric subunits on the main backbone of polymer has deeply influenced the physical properties of PHAs synthesized by microorganisms.…”