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Pseudomonas strains have a variety of potential uses in bioremediation and biosynthesis of biodegradable plastics. Pseudomonas sp. strain phDV1, a Gram-negative phenol degrading bacterium, has been found to utilize monocyclic aromatic compounds as sole carbon source via the meta-cleavage pathway. The degradation of aromatic compounds comprises an important step in the removal of pollutants. The present study aimed to investigate the ability of the Pseudomonas sp. strain phDV1 to produce polyhydroxyalkanoates (PHAs) and examining the effect of phenol concentration on PHA production. The bacterium was cultivated in minimal medium supplemented with different concentrations of phenol ranging from 200–600 mg/L. The activity of the PHA synthase, the key enzyme which produces PHA, was monitored spectroscopically in cells extracts. Furthermore, the PHA synthase was identified by mass spectrometry in cell extracts analyzed by SDS-PAGE. Transmission electron micrographs revealed abundant electron-transparent intracellular granules. The isolated biopolymer was confirmed to be polyhydroxybutyrate (PHB) by FTIR, NMR and MALDI-TOF/TOF analyses. The ability of strain Pseudomonas sp. phDV1 to remove phenol and to produce PHB makes the strain a promising biocatalyst in bioremediation and biosynthesis of biodegradable plastics.

The Pseudomonas sp. strain phDV1 was found to utilize monocyclic aromatic compounds as a sole carbon source and has a variety of potential applications in the bioremediation and biosynthesis of biodegradable plastics. It was possible to produce polyhydroxybutyrate when cultivated in the presence of monocyclic aromatic compounds as the sole carbon source. This study provides the small-scale optimization for phenol bioremediation and polyhydroxybutyrate production. The bacterium was cultivated in minimal medium supplemented with different concentrations of phenol. The formation and localization of the polyhydroxybutyrate granules (carbonosomes) in the cell were determined after 72 h of cultivation using Nile Red stain in combination with fluorescence microscopy. Analytical HPLC was also used to quantify the PHB content in the cells and to optimize the production. Finally, comparative proteomic analysis of isolated carbonosomes was used to characterize of their protein composition.

Pseudomonas sp. phDV1 is a polyhydroxyalkanoate (PHA) producer. The presence of the endogenous PHA depolymerase (phaZ) responsible for the degradation of the intracellular PHA is one of the main shortages in the bacterial production of PHA. Further, the production of PHA can be affected by the regulatory protein phaR, which is important in accumulating different PHA-associated proteins. PHA depolymerase phaZ and phaR knockout mutants of Pseudomonas sp. phDV1 were successfully constructed. We investigate the PHA production from 4.25 mM phenol and grape pomace of the mutants and the wild type. The production was screened by fluorescence microscopy, and the PHA production was quantified by HPLC chromatography. The PHA is composed of Polydroxybutyrate (PHB), as confirmed by 1H-nuclear magnetic resonance analysis. The wildtype strain produces approximately 280 μg PHB after 48 h in grape pomace, while the phaZ knockout mutant produces 310 μg PHB after 72 h in the presence of phenol per gram of cells, respectively. The ability of the phaZ mutant to synthesize high levels of PHB in the presence of monocyclic aromatic compounds may open the possibility of reducing the costs of industrial PHB production.

Abstract The evolution of C4 photosynthesis required extensive modification of ancestral enzymes enabling the development of an efficient carbon concentrating mechanism. A key example is NADP-malic enzyme (NADP-ME), which, in maize and sorghum—members of the same C4 lineage—underwent gene duplication and neofunctionalization, resulting in 2 plastidic isoforms with distinct oligomeric states: a tetrameric C4-specific isoform and a dimeric housekeeping (nonC4) isoform. In this study, we resolve the structural basis of this oligomeric divergence using X-ray crystallography, cryo-electron microscopy, and molecular modeling combined with targeted biochemical analysis. Our findings demonstrate that the N-terminal region of nonC4-NADP-ME is involved in its oligomeric organization, whereas a suite of adaptive substitutions at the dimer interface drives the transition to the stable tetramer characteristic of the C4 isoform. Moreover, the C-terminal region stabilizes the oligomeric states of C4- and nonC4-NADP-ME through specific interactions with adaptive residues. We propose that tetramerization mitigates aggregation at the high expression levels demanded by the C4 cycle and likely creates a scaffold for the emergence of regulatory properties. Collectively, the data show that remodeling of terminal domains and inter-subunit interfaces rewires the quaternary architecture of the enzymes, illustrating how subtle structural changes can drive the evolution of complex innovations such as C4 photosynthesis.

Pseudomonas sp. phDV1 is a polyhydroxyalkanoate (PHA) producer. The presence of the endogenous PHA depolymerase (phaZ) responsible for the degradation of the intracellular PHA is one of the main shortages in the bacterial production of PHA. Further, the production of PHA can be affected by the regulatory protein phaR, which is important in accumulating different PHA-associated proteins. PHA depolymerase phaZ and phaR knockout mutants of Pseudomonas sp. phDV1 were successfully constructed. We investigate the PHA production from 4.25 mM phenol and grape pomace of the mutants and the wild type. The production was screened by fluorescence microscopy, and the PHA production was quantified by HPLC chromatography. The PHA is composed of Polydroxybutyrate (PHB), as confirmed by [sup.1] H-nuclear magnetic resonance analysis. The wildtype strain produces approximately 280 μg PHB after 48 h in grape pomace, while the phaZ knockout mutant produces 310 μg PHB after 72 h in the presence of phenol per gram of cells, respectively. The ability of the phaZ mutant to synthesize high levels of PHB in the presence of monocyclic aromatic compounds may open the possibility of reducing the costs of industrial PHB production.

The C4 carbon concentrating mechanism relies on specialized enzymes that have evolved unique expression patterns and biochemical properties distinct to their ancestral housekeeping forms. In maize and sorghum, the evolution of C4-NADP-malic enzyme (C4-NADP-ME) involved gene duplication and neofunctionalization, leading to the emergence of two plastidic isoforms: C4-NADP-ME and nonC4-NADP-ME, each with distinct kinetic and structural features. While C4-NADP-ME functions primarily as a tetramer, nonC4-NADP-ME exists in an equilibrium between dimeric and tetrameric forms, favoring the dimer in solution. This study shows which evolutionary changes in amino acid sequences influence the structure and function of these isoforms. By integrating X-ray crystallography, cryo-electron microscopy, computational molecular modeling and targeted biochemical analysis of mutant and truncated protein variants, we identify crucial roles for the N- and C-terminal regions and specific amino acid residues in governing isoform oligomerization. Our results reveal that the N-terminal region is essential for stabilizing the dimeric form of nonC4-NADP-ME, whereas specific adaptive substitutions and interactions with the C-terminal region enhance the stability of the tetrameric state characteristic of the C4-adapted isoform. We propose that differences in the N-terminal domain between the C4 and nonC4 isoforms reflect distinct selective pressures, which have driven their evolutionary divergence to fulfill specialized cellular functions.Competing Interest StatementThe authors have declared no competing interest.