Overexpression Of Phosphoenolpyruvate Carboxylase Research Articles

Metabolic pathway engineering for the non-growth-associated succinate production in Escherichia coli based on flux solution space

Non-growth-associated bio-production using microorganisms has the potential to achieve a higher target yield than growth-associated production since the latter approach does not waste the substrate for cell growth. We previously proposed a metabolic pathway engineering method (SSDesign) for non-growth-associated target production based on metabolic flux solution space using elementary mode analysis. SSDesign predicts gene knockout combinations for enforcing cells to produce a target compound under non-growing conditions. For succinate production from glucose in Escherichia coli, gene knockouts of pykA-pykF-sfcA-maeB-zwf and pykA-pykF-sfcA-pntAB-sthA were predicted as candidates. In the present study, to verify the predictions of SSDesign, succinate productivities of these multiple knockout strains were evaluated in the stationary phase under microaerobic conditions. Succinate yields of the BW25113ΔpykAΔpykFΔsfcAΔmaeBΔzwf and BW25113ΔpykAΔpykFΔsfcAΔpntABΔsthA strains were 0.48 and 0.52mol/mol, respectively, and were higher than that of wild type strain (0.20mol/mol). The succinate yield of BW25113ΔpykAΔpykFΔsfcAΔpntABΔsthA strain was further improved to 0.66mol/mol by overexpression of phosphoenolpyruvate carboxylase as a potential bottleneck step in the metabolic pathway.

Metabolic engineering of threonine catabolism enables Saccharomyces cerevisiae to produce propionate under aerobic conditions.

Propionate is widely used as a preservative in the food and animal feed industries. Propionate is currently produced by petrochemical processes, and fermentative production of propionate remains challenging. In this study, a synthetic propionate pathway was constructed in the budding yeast Saccharomyces cerevisiae, for propionate production under aerobic conditions. Through expression of tdcB and aldH from Escherichia coli and kivD from Lactococcus lactis, L-threonine was converted to propionate via 2-ketobutyrate and propionaldehyde. The resulting yeast aerobically produced 0.21 g L-1 propionate from glucose in a shake flask. Subsequent overexpression of pathway genes and elimination of competing pathways increased propionate production to 0.37 g L-1 . To further increase propionate production, carbon flux was pulled into the propionate pathway by weakened expression of pyruvate kinase (PYK1), together with overexpression of phosphoenolpyruvate carboxylase (ppc). The final propionate production reached 1.05 g L-1 during fed-batch fermentation in a fermenter. In this work, a yeast cell factory was constructed using synthetic biology and metabolic engineering strategies to enable propionate production under aerobic conditions. Our study demonstrates engineered S. cerevisiae as a promising alternative for the production of propionate and its derivatives.

13 C Metabolic Flux Analysis of Escherichia coli Engineered for Gamma-Aminobutyrate Production.

Escherichia coli is engineered for γ-aminobutyrate (GABA) production in glucose minimal medium. For this, overexpression of mutant glutamate decarboxylase (GadB) and mutant glutamate/GABA antiporter (GadC), as well as deletion of GABA transaminase (GabT), are accomplished. In addition, the carbon flux to the tricarboxylic acid cycle is engineered by the overexpression of gltA, ppc, or both. The overexpression of citrate synthase (CS), encoded by gltA, increases GABA productivity, as expected. Meanwhile, the overexpression of phosphoenolpyruvate carboxylase (PPC) causes a decrease in the rate of glucose uptake, resulting in a decrease in GABA production. The phenotypes of the strains are characterized by 13 C metabolic flux analysis (13 C MFA). The results reveal that CS overexpression increases glycolysis and anaplerotic reaction rates, as well as the citrate synthesis rate, while PPC overexpression causes little changes in metabolic fluxes, but reduces glucose uptake rate. The engineered strain produces 1.2gL-1 of GABA from glucose. Thus, by using 13 C MFA, important information is obtained for designing metabolically engineered strains for efficient GABA production.

Towards efficient photosynthesis: overexpression of Zea mays phosphoenolpyruvate carboxylase in Arabidopsis thaliana.

Plants with C4 photosynthesis are efficient in carbon assimilation and have an advantage over C3 photosynthesis. In C4 photosynthesis, the primary CO2 fixation is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Here, we show that overexpression of Zea mays PEPC cDNA, under the control of 35S promoter, in Arabidopsis thaliana resulted in ~7-10 fold higher protein abundance and ~7-10 fold increase in PEPC activity in the transgenic lines than that in the vector control. We suggest that overexpression of PEPC played an anaplerotic role to increase the supply of 4-carbon carboxylic acids, which provided carbon skeletons for increased amino acid and protein synthesis. Higher protein content must have been responsible for increased metabolic processes including chlorophyll biosynthesis, photosynthesis, and respiration. Consequently, the PEPC-overexpressed transgenic plants had higher chlorophyll content, enhanced electron transport rate (ETR), lower non-photochemical quenching (NPQ) of chlorophyll a fluorescence, and a higher performance index (PI) than the vector control. Consistent with these observations, the rate of CO2 assimilation, the starch content, and the dry weight of PEPC-overexpressed plants increased by 14-18%, 10-18%, and 6.5-16%, respectively. Significantly, transgenics were tolerant to salt stress as they had increased ability to synthesize amino acids, including the osmolyte proline. NaCl (150mM)-treated transgenic plants had higher variable to maximum Chl a fluorescence (F v/F m) ratio, higher PI, higher ETR, and lower NPQ than the salt-treated vector controls. These results suggest that expression of C4 photosynthesis enzyme(s) in a C3 plant can improve its photosynthetic capacity with enhanced tolerance to salinity stress.

Overexpression of phosphoenolpyruvate carboxylase from Jatropha curcas increases fatty acid accumulation in Nicotiana tabacum

Jatropha curcas L. is an excellent biofuel crop, which displays a high efficiency of carbon absorption, and seed oil of Jatropha can be efficiently processed to produce high-quality biodiesel. Plant phosphoenolpyruvate carboxylases (PEPCs) play important roles not only in initial fixation of atmospheric CO2 in C4 and Crassulacean acid metabolism (CAM) plants, but also in fatty acid biosynthesis in seeds of oil plants by regulating carbon partitioning. Here, we identified JcPEPC1 from J. curcas L. by homology cloning, and alignment analysis of protein sequence revealed JcPEPC1 was a plant C3-type PEPC, and shared high similarity to PEPC of castor oil plant Ricinus communis. We implemented detailed functional characterization of JcPEPC1 by expression analysis and transgenic tobacco. JcPEPC1 gene expressed in the leaves and seeds of J. curcas L., and remarkable increase of expression level was also detected at seed oil-accumulating stages. We overexpressed JcPEPC1 in tobacco, and showed the enzymatic activity of PEPC in transgenic plants was notably higher than wild type. Gas chromatography (GC) analysis elucidated the composition and total content of fatty acids were also altered. This study indicated JcPEPC1 played a fundamental role in fatty acid biosynthesis in Jatropha seeds. Our results proposed enhanced PEPC activity of Jatropha could improve biosynthesis of fatty acid, which implied critical functions in primary metabolism of non-photosynthetic PEPC.

Nutrient Dependence of RNase E Essentiality in Escherichia coli

Escherichia coli cells normally require RNase E activity to form colonies (colony-forming ability [CFA]). The CFA-defective phenotype of cells lacking RNase E is partly reversed by overexpression of the related endoribonuclease RNase G or by mutation of the gene encoding the RNA helicase DeaD. We found that the carbon source utilization by rne deaD doubly mutant bacteria differs from that of rne(+) cells and from that of cells mutated in deaD alone and that the loss of rne function in these bacteria limits conversion of the glycolytic pathway product phosphoenolpyruvate to the tricarboxylic acid (TCA) cycle intermediate oxaloacetic acid. We show that the mechanism underlying this effect is reduced production of the enzyme phosphoenolpyruvate carboxylase (PPC) and that adventitious overexpression of PPC, which facilitates phosphoenolpyruvate utilization and connects the glycolytic pathway with the TCA cycle, restored CFA to rne deaD mutant bacteria cultured on carbon sources that otherwise were unable to sustain growth. We further show that bacteria producing full-length RNase E, which allows formation of degradosomes, have nutritional requirements different from those of cells supplied with only the N-terminal catalytic region of RNase E and that mitigation of RNase E deficiency by overexpression of a related RNase, RNase G, is also affected by carbon source. Our results reveal previously unsuspected effects of RNase E deficiency and degradosome formation on nutrient utilization by E. coli cells.

High Light Tolerance is Enhanced by Overexpressed PEPC in Rice Under Drought Stress

Overexpression of phosphoenolpyruvate carboxylase (PEPC) gene in transgenic rice (Oryza sativa L.) may alleviate inhibition on photosynthesis under drought stress condition. In the present research, photosynthetic light curve, chlorophyll α fluorescence parameters, pigment content, and reactive oxygen metabolism were studied in 2 PEPC transgenic rice lines under drought stress at flowering stage. The results showed that under drought stress, especially severely drought stress, net photosynthetic rate decreased dramatically under high photosynthetic active radiation (PAR) in untransformed wild type rice, while maintained unchanging in the PEPC transgenic lines under high PAR higher than 1200 μmol m−2 s−1. The photochemistry activities (Fv/Fm, ϕPSII, and qP) decreased slightly under drought stress in both PEPC transgenic lines. These results indicated that PEPC enhanced the photoinhibition tolerance of rice under drought stress. The increased zeaxanthin content in leaves of the PEPC transgenic rice lines dispersed more light energy as heat under drought stress, thus decreased the producing rate in photosystem II. At the same time, the activities of superoxide dismutase, peroxidase, and catalase were higher in the PEPC transgenic rice plants than the untransformed wild type under drought stress. These enzymes could effectively diminish the reactive oxygen species and reduce the membrane lipid peroxidation.

Constitutive and dark‐induced expression of Solanum tuberosum phosphoenolpyruvate carboxylase enhances stomatal opening and photosynthetic performance of Arabidopsis thaliana

The effect of constitutive and dark-induced expression of Solanum tuberosum phosphoenolpyruvate carboxylase (PEPC) on the opening state of stomata and photosynthetic performance in Arabidopsis thaliana plants was studied. Transcript accumulation analyses of the A. thaliana dark-induced (Din10 and Din6) and the Pisum sativum asparagine synthetase 2 promoters (Asn2) in transiently transformed tobacco leaves showed that Din10 promoter induced more DsRed accumulation in the dark compared to the other din genes. Overexpression of PEPC under the control of the constitutive enhanced CaMV 35S (p35SS) and dark-induced Din10 promoter in stably transformed A. thaliana plants increased the number of opened stomata in dark adapted leaves. Gas exchange measurements using A. thaliana plants transgenic for p35SS-PEPC and Din10-PEPC revealed a marked increase in stomatal conductance, transpiration, and dark respiration rates measured in the dark compared to wild-type plants. Moreover, measurement of CO(2) assimilation rates at different external CO(2) concentrations (C(a) ) and different light intensities shows an increase in the CO(2) assimilation rates in transgenic Arabidopsis lines compared to wild-type plants. This is considered as first step towards transferring the aspects of Crassulacean acid metabolism-like photosynthetic mechanism into C3 plants.

Alleviation of Drought Stress Inhibition on Photosynthesis by Overexpression of PEPC in Rice

Introducing enzymes involved in photosynthesis of C 4 plants into rice ( Oryza sativa L.) is supposed to enhance the photosynthesis and crop productivity. However, only a few researches showed that the photosynthesis and crop productivity have been improved by introducing phosphoenolpyruvate carboxylase (PEPC) gene into rice. In the present study, the photosynthetic rates ( P n) in 42 rice lines overexpressing PEPC gene were investigated. The average P n value of transgenic lines was almost the same as that of the wild type (control) in paddy field, but was much higher than that of the control in upland field. Only a few transgenic lines showed higher P n in paddy field and most of them showed higher P n in upland field. Similar results were found in the water controlled experiment. Two transgenic lines with different relative activities of PEPC (10 and 25 folds) were selected to study their photosynthesis under different water potentials (0, −20, and −40 kPa). In both lines, the P n values were similar to that in the wild type under normal condition (0 kPa) and higher under drought conditions (−20 kPa and −40 kPa). In both experiments, the transgenic lines had higher P n under drought conditions, with a slower decreasing rate than the wild type. Therefore, the present results suggested that the overexpressed PEPC could not improve the photosynthetic rate of transgenic rice plants. But the photosynthetic rate of transgenic rices declined slowly under drought condition. It is supposed that PEPC might be involved in drought resistance to decrease the inhibition of drought stress on photosynthesis in rice.

Carbon metabolism in transgenic rice plants that express phospho enolpyruvate carboxylase and/or phospho enolpyruvate carboxykinase

Transgenic rice plants expressing phospho enolpyruvate carboxykinase (PCK) in the chloroplasts showed C4-like carbon flow [S. Suzuki, N. Murai, J.N. Burnell, M. Arai, Changes in photosynthetic carbon flow in transgenic rice plants that express C4-type phospho enolpyruvate carboxykinase from Urochloa panicoides, Plant Physiol. 124 (2000) 163–172]. Here we report on the carbon metabolism of newly generated transgenic rice plants expressing either phospho enolpyruvate carboxylase (PEPC) or both PCK and PEPC. The incorporation of 14CO 2 into 4C compounds in PEPC transgenic rice was two- to three-fold that in control plants, and the incorporation in PEPC/PCK-transgenic rice was three-fold that in PEPC transgenic rice, but the enhanced level in PEPC/PCK-transgenic rice was almost the same as that in PCK-transgenic rice. Higher rates of incorporation of 14C into photoassimilates (sucrose and 3-PGA plus sugar phosphates) from l-[4- 14C]malate were observed in PEPC, PCK and PEPC/PCK-transgenic rice, but the incorporation rates in PEPC transgenic rice were reduced to those found in control plants by pretreatment with NaN 3. Levels of PEP, malate and aspartate in PEPC/PCK-transgenic rice were similar to those in PCK-transgenic rice but different in PEPC transgenic rice. PEPC/PCK-transgenic rice showed a low chlorophyll concentration and swollen thylakoid membranes although PCK and PEPC transgenic rice showed normal phenotypes. These results suggest that the overexpression of PEPC enhances the anaplerotic pathway rather than the initial carbon fixation of the C4-like photosynthetic pathway, and that elevated PEPC activity in combination with PCK activity contributes little to C4-like carbon flow.

Overexpression of phosphoenolpyruvate carboxylase from Corynebacterium glutamicum lowers the CO2 compensation point ([IMAGE]*) and enhances dark and light respiration in transgenic potato

Overexpression of phosphoenolpyruvate carboxylase from Corynebacterium glutamicum lowers the CO2 compensation point ([IMAGE]*) and enhances dark and light respiration in transgenic potato

Overexpression of phosphoenolpyruvate carboxylase from Corynebacterium glutamicum lowers the CO2 compensation point ( *) and enhances dark and light respiration in transgenic potato

The effect of decreased or increased phosphoenolpyruvate carboxylase (PEPC) activity on the CO2 compensation point, respiration in the light or dark as well as the partitioning of carbon into starch, soluble sugars, organic acids, and amino acids was investigated using transgenic potato plants. Engineered PEPC activity ranged from 0.5-fold wild-type level in antisense plants to 5-fold wild-type levels in lines overexpressing the cppc gene of Corynebacterium glutamicum encoding for a PEPC not modulated by protein phosphorylation. The CO2 compensation point determined according to Brooks and Farquhar (1985) was lower in PEPC overexpressors (32 µl l−1 CO2) compared to control potato lines (38 µl l−1 CO2), but was increased in antisense PEPC plants (42 µl l−1 CO2). 3-fold overexpression of PEPC gave a minimum CO2 compensation point of 32 µl l−1 CO2. Increased PEPC activity resulted in enhanced respiration in the light and dark. Altered PEPC activity had no effect on the pattern of 14CO2 incorporation into leaf discs in the light. 14C pulse-chase experiments in the dark, demonstrated that substantially more total label was lost in the leaf discs from PEPC overexpressors. Metabolite levels were determined in 21 PEPC overexpressing lines after 8 h in the light. A 5-fold increase in PEPC over the wild-type increased malate (61%), starch (75%) and significantly increased sucrose contents (150%). Total amino acid contents were only marginally increased. From gas exchange characteristics and labelling experiments it was concluded that PEP carboxylation, followed by an increased rate of respiratory CO2 release, might work as a HCO−3/CO2 pump. This might result in elevated CO2/O2 ratios in the mesophyll, concomitant with a more favoured carboxylation/oxygenation ratio of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco).

Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli.

Fermentative production of succinic acid from glucose by Escherichia coli was significantly increased by overexpression of phosphoenolpyruvate carboxylase. In contrast, overexpression of phosphoenolpyruvate carboxykinase had no effect. Under optimized conditions, induction of the carboxylase resulted in a 3.5-fold increase in the concentration of succinic acid, making succinic acid the major fermentation product by weight.