High natural PHA production from acetate in Cobetia sp. MC34 and Cobetia marina DSM 4741T and in silico analyses of the genus specific PhaC2 polymerase variant

Abstract

<b>Background</b>

<p>Several members of the bacterial <i>Halomonadacea</i> family are natural producers of polyhydroxyalkanoates (PHA), which are promising materials for use as biodegradable bioplastics. Type-strain species of <i>Cobetia</i> are designated PHA positive, and recent studies have demonstrated relatively high PHA production for a few strains within this genus. Industrially relevant PHA producers may therefore be present among uncharacterized or less explored members. In this study, we characterized PHA production in two marine <i>Cobetia</i> strains. We further analyzed their genomes to elucidate <i>pha</i> genes and metabolic pathways which may facilitate future optimization of PHA production in these strains.

<p><b>Results</b>

<i>Cobetia</i> sp. MC₃₄ and <i>Cobetia marina</i> DSM ₄₇₄₁^T were mesophilic, halotolerant, and produced PHA from four pure substrates. Sodium acetate with- and without co-supplementation of sodium valerate resulted in high PHA production titers, with production of up to 2.5 g poly(3-hydroxybutyrate) (PHB)/L and 2.1 g poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/L in <i>Cobetia</i> sp. MC₃₄, while <i>C. marina</i> DSM ₄₇₄₁^T produced 2.4 g PHB/L and 3.7 g PHBV/L. <i>Cobetia marina</i> DSM ₄₇₄₁^T also showed production of 2.5 g PHB/L from glycerol. The genome of <i>Cobetia</i> sp. MC₃₄ was sequenced and phylogenetic analyses revealed closest relationship to <i>Cobetia amphilecti</i>. PHA biosynthesis genes were located at separate loci similar to the arrangement in other <i>Halomonadacea</i>. Further genome analyses revealed some differences in acetate- and propanoate metabolism genes between the two strains. Interestingly, only a single PHA polymerase gene (<i>phaC</i>₂) was found in <i>Cobetia</i> sp. MC₃₄, in contrast to two copies (<i>phaC</i>₁ and <i>phaC</i>₂) in <i>C. marina</i> DSM ₄₇₄₁^T. In silico analyses based on <i>phaC</i> genes show that the PhaC₂ variant is conserved in <i>Cobetia</i> and contains an extended C-terminus with a high isoelectric point and putative DNA-binding domains.

<p><b>Conclusions</b>

<i>Cobetia</i> sp. MC₃₄ and <i>C. marina</i> DSM ₄₇₄₁^T are natural producers of PHB and PHBV from industrially relevant pure substrates including acetate. However, further scale up, optimization of growth conditions, or use of metabolic engineering is required to obtain industrially relevant PHA production titers. The putative role of the <i>Cobetia</i> PhaC₂ variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods.