Impact of Ralstonia eutropha's Poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB Storage in Recombinant Escherichia coli

Abstract

bThe model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay of these enzymes.

Recent strategies for efficient production of polyhydroxyalkanoates by micro-organisms

“…). In other studies, the lack of depolymerase system have found to be contributed to higher yields of PHAs (Eggers and Steinbuchel ), suggesting that phaZ can be a significant target for recombinant strains for PHA production. Engineered strains in attempts to broaden the substrate spectrum have also been manipulated (Liu et al .…”

“…For instance, the engineered Halomonas strains could overexpress the cell division inhibitor MinCD, which not only enhance PHB accumulation but also simplify the downstream processing, resulting in a decline of the production cost (Tan et al 2014). In other studies, the lack of depolymerase system have found to be contributed to higher yields of PHAs (Eggers and Steinbuchel 2014), suggesting that phaZ can be a significant target for recombinant strains for PHA production. Engineered strains in attempts to broaden the substrate spectrum have also been manipulated (Liu et al 2014a).…”

Phasin PhaP1 is involved in polyhydroxybutyrate granules morphology and in controlling early biopolymer accumulation in Azospirillum brasilense Sp7

“…Studies of PhaP2 Reu and PhaP4 Reu on PHB degradation conclude that both phasins, limit PHB degradation by interacting with the depolymerases PhaZ2, PhaZ3 and PhaZ7. Moreover, the expression of PhaP1 Reu reduced the PHB content in E. coli when expressing the phaCAB operon of R. eutropha (Eggers and Steinbüchel 2014).…”

Bradyrhizobium diazoefficiens USDA110 PhaR functions for pleiotropic regulation of cellular processes besides PHB accumulation

“…In addition, a blast search against the genome of R. eutropha strain H16 revealed that PhaZ3 shares a homology to PhaZ7 of H16 (E-value = 5e–16). It was demonstrated that induction of phaZ7 in a recombinant E. coli strain greatly reduced PHB accumulation [ 41 ]. Therefore, PhaZ3 may play an important role in PHB degradation in B. diazoefficiens .…”