Dataset Information

Prokaryotic Expression of Phosphoenolpyruvate Carboxylase Fragments from Peanut and Analysis of Osmotic Stress Tolerance of Recombinant Strains.

ABSTRACT: Phosphoenolpyruvate carboxylase (PEPC) is a ubiquitous cytosolic enzyme that catalyzes the irreversible ?-carboxylation of phosphoenolpyruvate (PEP) in presence of HCO₃^- to produce oxaloacetate (OAA) during carbon fixation and photosynthesis. It is well accepted that PEPC genes are expressed in plants upon stress. PEPC also supports the biosynthesis of biocompatible osmolytes in many plant species under osmotic stress. There are five isoforms of PEPC found in peanut (Arachis hypogaea L.), namely, AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the gene expression patterns of these AhPEPC genes were different in mature seeds, stems, roots, flowers, and leaves. The expression of all the plant type PEPC (PTPCs) (AhPEPC1, AhPEPC2, AhPEPC3, and AhPEPC4) was relatively high in roots, while the bacterial type PEPC (BTPC) (AhPEPC5) showed a remarkable expression level in flowers. Principal component analysis (PCA) result showed that AhPEPC3 and AhPEPC4 are correlated with each other, indicating comparatively associations with roots, and AhPEPC5 have a very close relationship with flowers. In order to investigate the function of these AhPEPCs, the fragments of these five AhPEPC cDNA were cloned and expressed in Escherichia coli (E. coli). The recombinant proteins contained a conserved domain with a histidine site, which is important for enzyme catalysis. Results showed that protein fragments of AhPEPC1, AhPEPC2, and AhPEPC5 had remarkable expression levels in E. coli. These three recombinant strains were more sensitive at pH 9.0, and recombinant strains carrying AhPEPC2 and AhPEPC5 fragments exhibited more growth than the control strain with the presence of PEG6000. Our findings showed that the expression of the AhPEPC fragments may enhance the resistance of transformed E. coli to osmotic stress.

SUBMITTER: Tu J

PROVIDER: S-EPMC7917721 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Prokaryotic Expression of Phosphoenolpyruvate Carboxylase Fragments from Peanut and Analysis of Osmotic Stress Tolerance of Recombinant Strains.

Tu Jiaqi J Feng Lanlan L Hong Yanbin Y Liu Qiuyun Q Huang Xia X Li Yin Y

Plants (Basel, Switzerland) 20210214 2

Phosphoenolpyruvate carboxylase (PEPC) is a ubiquitous cytosolic enzyme that catalyzes the irreversible β-carboxylation of phosphoenolpyruvate (PEP) in presence of HCO3- to produce oxaloacetate (OAA) during carbon fixation and photosynthesis. It is well accepted that PEPC genes are expressed in plants upon stress. PEPC also supports the biosynthesis of biocompatible osmolytes in many plant species under osmotic stress. There are five isoforms of ...[more]

PMID: 33672856

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Project description:BackgroundCyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium.ResultsWe were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium.ConclusionsHeterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

Project description:Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme in plants, which regulates carbon flow through the TCA cycle and controls protein and oil biosynthesis. Although it is important, there is little research on PEPC in cotton, the most important fiber crop in the world. In this study, a total of 125 PEPCs were identified in 15 Gossypium genomes. All PEPC genes in cotton are divided into six groups and each group generally contains one PEPC member in each diploid cotton and two in each tetraploid cotton. This suggests that PEPC genes already existed in cotton before their divergence. There are additional PEPC sub-groups in other plant species, suggesting the different evolution and natural selection during different plant evolution. PEPC genes were independently evolved in each cotton sub-genome. During cotton domestication and evolution, certain PEPC genes were lost and new ones were born to face the new environmental changes and human being needs. The comprehensive analysis of collinearity events and selection pressure shows that genome-wide duplication and fragment duplication are the main methods for the expansion of the PEPC family, and they continue to undergo purification selection during the evolutionary process. PEPC genes were widely expressed with temporal and spatial patterns. The expression patterns of PEPC genes were similar in G. hirsutum and G. barbadense with a slight difference. PEPC2A and 2D were highly expressed in cotton reproductive tissues, including ovule and fiber at all tested developmental stages in both cultivated cottons. However, PEPC1A and 1D were dominantly expressed in vegetative tissues. Abiotic stress also induced the aberrant expression of PEPC genes, in which PEPC1 was induced by both chilling and salinity stresses while PEPC5 was induced by chilling and drought stresses. Each pair (A and D) of PEPC genes showed the similar response to cotton development and different abiotic stress, suggesting the similar function of these PEPCs no matter their origination from A or D sub-genome. However, some divergence was also observed among their origination, such as PEPC5D was induced but PEPC5A was inhibited in G. barbadense during drought treatment, suggesting that a different organized PEPC gene may evolve different functions during cotton evolution. During cotton polyploidization, the homologues genes may refunction and play different roles in different situations.

Dataset Information

Prokaryotic Expression of Phosphoenolpyruvate Carboxylase Fragments from Peanut and Analysis of Osmotic Stress Tolerance of Recombinant Strains.

Publications

Prokaryotic Expression of Phospho<i>enol</i>pyruvate Carboxylase Fragments from Peanut and Analysis of Osmotic Stress Tolerance of Recombinant Strains.

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