Overexpression in Escherichia coli and Characterization of the Chloroplast Triosephosphate Isomerase from Spinach

Tang, Gong‐Li; Wang, Yanfang; Bao, Jian‐Shao; Chen, Hai-Bao

doi:10.1006/prep.1999.1087

Cited by 14 publications

(12 citation statements)

References 24 publications

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“…In one construct, the N-terminal 59 amino acids, encoding a putative plastid targeting peptide, was deleted (DpdTPI); the other construct expressed the full-length open reading frame. Both purified recombinant proteins showed TPI activity using an enzyme-linked spectrophotometric assay (388 6 32.2 nmol min 21 mg 21 for pdTPI and 469 6 43.9 nmol min 21 mg 21 for DpdTPI) with values close or exceeding those reported previously (Tomlinson and Turner, 1979;Tang et al, 1999).…”

Section: Mutation In Pdtpi Results In Stunted Growth and Abnormal Chlsupporting

confidence: 74%

“…Since a functional cytoTPI would equilibrate cytosolic DHAP and GAP, the plastid GAP pool in the pdtpi mutant is probably >60% reduced. However, additional experiments are necessary to determine if the increase in DHAP pool size is principally within the plastid, since the cytoTPI appears to have a higher affinity for GAP than DHAP (Tomlinson and Turner, 1979;Tang et al, 1999). This metabolic imbalance could profoundly affect the development of other biochemical pathways in a plastid transitioning to a mature chloroplast ( Figure 8).…”

Section: Reduced Expression Of Pdtpi Disrupts Metabolic Pathways In Cmentioning

confidence: 99%

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The Plastid Isoform of Triose Phosphate Isomerase Is Required for the Postgerminative Transition from Heterotrophic to Autotrophic Growth in Arabidopsis

2010

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During postgerminative seedling establishment, reserves stored during seed filling are mobilized to provide energy and carbon for the growing seedling until autotrophic growth is possible. A plastid isoform of triose phosphate isomerase (pdTPI) plays a crucial role in this transition from heterotrophic to autotrophic growth. A T-DNA insertion in Arabidopsis thaliana pdTPI resulted in a fivefold reduction in transcript, reduced TPI activity, and a severely stunted and chlorotic seedling that accumulated dihydroxyacetone phosphate (DHAP), glycerol, and glycerol-3-phosphate. Methylglyoxal (MG), a by-product of DHAP, also accumulated in the pdtpi mutant. Wild-type seed sown in the presence of any of these four metabolites resulted in a phenocopy of this pdtpi mutant, although MG and DHAP were the most effective based upon dosage. These metabolites (except MG) are by-products of triacylglycerol mobilization and precursors for glycerolipid synthesis, suggesting that lipid metabolism may also be affected. Lipid profiling revealed lower monogalactosyl but higher digalactosyl lipids. It is unclear whether the change in lipid composition is a direct or indirect consequence of the pdtpi mutation, as ribulose-1,5-bis-phosphate carboxylase/oxygenase expression, chloroplast morphology, and starch synthesis are also defective in this mutant. We propose that DHAP and MG accumulation in developing plastids delays the transition from heterotrophic to autotrophic growth, possibly due to MG toxicity.

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Section: Mutation In Pdtpi Results In Stunted Growth and Abnormal Chlsupporting

confidence: 74%

Section: Reduced Expression Of Pdtpi Disrupts Metabolic Pathways In Cmentioning

confidence: 99%

The Plastid Isoform of Triose Phosphate Isomerase Is Required for the Postgerminative Transition from Heterotrophic to Autotrophic Growth in Arabidopsis

2010

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“…Second, this approach can reduce the metabolic burden in cells that is observed as a result of constitutive overexpression of multiple proteins. Although our model cannot capture these effects, it has been well established that consistently overexpressed proteins can become a large metabolic burden on the cell representing as much as 15–40% of the total cellular protein produced by recombinant cells [47]–[49]. Third, oscillations could be valuable in situations where there are growth tradeoffs in producing the final product.…”

Section: Discussionmentioning

confidence: 99%

Optimizing Metabolite Production Using Periodic Oscillations

2014

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Methods for improving microbial strains for metabolite production remain the subject of constant research. Traditionally, metabolic tuning has been mostly limited to knockouts or overexpression of pathway genes and regulators. In this paper, we establish a new method to control metabolism by inducing optimally tuned time-oscillations in the levels of selected clusters of enzymes, as an alternative strategy to increase the production of a desired metabolite. Using an established kinetic model of the central carbon metabolism of Escherichia coli, we formulate this concept as a dynamic optimization problem over an extended, but finite time horizon. Total production of a metabolite of interest (in this case, phosphoenolpyruvate, PEP) is established as the objective function and time-varying concentrations of the cellular enzymes are used as decision variables. We observe that by varying, in an optimal fashion, levels of key enzymes in time, PEP production increases significantly compared to the unoptimized system. We demonstrate that oscillations can improve metabolic output in experimentally feasible synthetic circuits.

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“…A 1990 bp pUC backbone containing the origin of replication, an AmpR marker and a lac promoter was PCR amplified. A consensus E.coli Shine–Dalgarno sequence (13–15) and a 7 bp spacer were introduced using a 5′ tail on the reverse primer. All vector construction PCRs were performed using Pfu Turbo (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting

Choe

Guo²,

Engh³

2005

Nucleic Acids Research

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Molecular biology critically depends upon the isolation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50 000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic heterogeneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controlling for expression variability. We demonstrate a dual-reporter system using the green fluorescent protein (GFP) gene fused to the Discosoma red fluorescent protein (DsRed) gene. The system reports the successful insertion of foreign DNA with the loss of DsRed fluorescence and the maintenance of GFP fluorescence. Single cells containing inserts are readily recognized by their altered ratios of green to red fluorescence and separated using a high-speed cell-sorter for further processing. This novel reporter system and vector were successfully validated by shotgun library construction, cloned sequence isolation, PCR amplification and DNA sequencing of cloned inserts from bacteria after cell-sorting. This simple, robust system can also be adapted for diverse biosensor assays and is amenable to miniaturization. We demonstrated that dual-fluorescence reporting coupled with high-speed cell-sorting provides a more efficient alternative to traditional methods of clone isolation.

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Overexpression in Escherichia coli and Characterization of the Chloroplast Triosephosphate Isomerase from Spinach

Cited by 14 publications

References 24 publications

The Plastid Isoform of Triose Phosphate Isomerase Is Required for the Postgerminative Transition from Heterotrophic to Autotrophic Growth in Arabidopsis

The Plastid Isoform of Triose Phosphate Isomerase Is Required for the Postgerminative Transition from Heterotrophic to Autotrophic Growth in Arabidopsis

Optimizing Metabolite Production Using Periodic Oscillations

A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting

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