Seed-specific expression of a bacterial phosphoenolpyruvate carboxylase in Vicia narbonensis increases protein content and improves carbon economy

An ambitious aim in plant breeding and biotechnology is to increase the protein content of crop seeds used for food and feed. Using an approach to manipulate assimilate partitioning, we succeeded in elevating the protein content in legume seeds up to 50%. Transgenic bean plants were generated which express a Corynebacterium glutamicum phosphoenolpyruvate carboxylase (PEPC) in a seed-specific manner. The bacterial enzyme was not feedback inhibited by malate. Transgenic seeds showed a higher [14C]-CO2 uptake and about a threefold increased incorporation of labelled carbon into proteins. Changed metabolite profiles of maturing cotyledons indicated a shift of metabolic fluxes from sugars/starch into organic acids and free amino acids. These changes were consistent with an increased carbon flow through the anaplerotic pathway catalysed by PEPC. Consequently, transgenic seeds accumulated up to 20% more protein per gram seed dry weight. Additionally, seed dry weight was higher by 20%-30%. We conclude that PEPC in seeds is a promising target for molecular plant breeding.     

An engineered phosphoenolpyruvate carboxylase redirects carbon and nitrogen flow in transgenic potato plants

Phosphoenolpyruvate carboxylase (PEPC) plays a central role in the anaplerotic provision of carbon skeletons for amino acid biosynthesis in leaves of C3 plants. Furthermore, in both C4 and CAM plants photosynthetic isoforms are pivotal for the fixation of atmospheric CO2. Potato PEPC was mutated either by modifications of the N-terminal phosphorylation site or by an exchange of an internal cDNA segment for the homologous sequence of PEPC from the C4 plant Flaveria trinervia. Both modifications resulted in enzymes with lowered sensitivity to malate inhibition and an increased affinity for PEP. These effects were enhanced by a combination of both mutated sequences and pulse labelling with 14CO2 in vivo revealed clearly increased fixation into malate for this genotype. Activity levels correlated well with protein levels of the mutated PEPC. Constitutive overexpression of PEPC carrying both N-terminal and internal modifications strongly diminished plant growth and tuber yield. Metabolite analysis showed that carbon flow was re-directed from soluble sugars and starch to organic acids (malate) and amino acids, which increased four-fold compared with the wild type. The effects on leaf metabolism indicate that the engineered enzyme provides an optimised starting point for the installation of a C4-like photosynthetic pathway in C3 plants.     

Manipulating PEPC levels in plants

This review examines the current understanding of the structural, functional and regulatory properties of C4 and C3 forms of higher plant phosphoenolpyruvate carboxylase. The emphasis is on the interactive metabolic and post-translational controls acting on the enzyme in the physiological context of C4 photosynthesis and the anaplerotic pathway. A brief overview is given concerning the recent developments of PEPC-based genetic engineering of C3 plants with the aim of improving photosynthetic performance in normal and limiting environmental conditions. So far, in spite of achieving a considerable increase in PEPC levels, more work needs to be done with respect to the correct dosage and location before that goal is reached. Some unpublished results on the transformation of maize with a sorghum C4 PEPC cDNA are also presented. They show that it is possible to increase photosynthetic PEPC levels in this C4 plant and that the modification in enzyme content has a pleiotropic physiological impact and, notably, an improved water use efficiency when water is limited.     

A field-grown transgenic tomato line expressing higher levels of polyamines reveals legume cover crop mulch-specific perturbations in fruit phenotype at the levels of metabolite profiles, gene expression, and agronomic characteristics

Genetic modification of crop plants to introduce desirable traits such as nutritional enhancement, disease and pest resistance, and enhanced crop productivity is increasingly seen as a promising technology for sustainable agriculture and boosting food production in the world. Independently, cultural practices that utilize alternative agriculture strategies including organic cultivation subscribe to sustainable agriculture by limiting chemical usage and reduced tillage. How the two together affect fruit metabolism or plant growth in the field or whether they are compatible has not yet been tested. Fruit-specific yeast S-adenosylmethionine decarboxylase (ySAMdc) line 579HO, and a control line 556AZ were grown in leguminous hairy vetch (Vicia villosa Roth) (HV) mulch and conventional black polyethylene (BP) mulch, and their fruit analysed. Significant genotypexmulch-dependent interactions on fruit phenotype were exemplified by differential profiles of 20 fruit metabolites such as amino acids, sugars, and organic acids. Expression patterns of the ySAMdc transgene, and tomato SAMdc, E8, PEPC, and ICDHc genes were compared between the two lines as a function of growth on either BP or HV mulch. HV mulch significantly stimulated the accumulation of asparagine, glutamate, glutamine, choline, and citrate concomitant with a decrease in glucose in the 556AZ fruits during ripening as compared to BP. It enables a metabolic system in tomato somewhat akin to the one in higher polyamine-accumulating transgenic fruit that have higher phytonutrient content. Finally, synergism was found between HV mulch and transgenic tomato in up-regulating N:C indicator genes PEPC and ICDHc in the fruit.     

磷酸烯醇式丙酮酸羧化酶和磷酸烯醇式丙酮酸羧激酶介导的草酰乙酸回补途径对谷氨酸棒杆菌V1氨基酸代谢的影响

【目的】谷氨酸棒杆菌是工业生产氨基酸的主要菌株,以缬氨酸高产菌株谷氨酸棒杆菌V1为研究对象,探讨磷酸烯醇式丙酮酸羧化酶(PEPC)和磷酸烯醇式丙酮酸羧激酶(PCK)介导的草酰乙酸回补途径对菌株生理特性以及主要氨基酸代谢流量的影响。【方法】通过基因工程手段,在谷氨酸棒杆菌V1中过表达pepc(编码PEPC)和pck(编码PCK),比较重组菌与出发菌关键酶活性、发酵特性以及主要氨基酸积累量变化。【结果】构建两株重组菌V1-pepc(强化草酰乙酸回补途径)和V1-pck(弱化草酰乙酸回补途径),重组菌生长均较出发菌延缓,总生物量、葡萄糖和硫酸铵消耗基本不变;过表达pck,PCK活性提高22.8%,丙氨酸、缬氨酸、谷氨酸、精氨酸积累量分别提高了11.8%、17.2%、27.8%和19.5%;过表达pepc,PEPC活性提高27.5%,同时PC活性降低12.9%,天冬氨酸族和谷氨酸族氨基酸的整体流量变化不大,丙氨酸族氨基酸的整体流量降低了14.7%。【结论】丙氨酸族氨基酸受此回补途径影响较大,天冬氨酸族氨基酸受此影响较小。     

Simultaneous Overexpression of Citrate Synthase and Phosphoenolpyruvate Carboxylase in Leaves Augments Citrate Exclusion and Al Resistance in Transgenic Tobacco

Phosphoenolpyruvate carboxylase (PEPC) and citrate synthase (CS) are two key enzymes in organic acid synthesis metabolism. In the present study, a cytoplasmic form of CS from tobacco and a mutant (with reduced sensitivity to organic acid inhibition) PEPC from Synechococcus vulcanus were overexpressed simultaneously using a light-inducible promoter in tobacco leaves. The analysis for enzyme activity showed that CS and PEPC enzyme activities were increased by 235% to 257% and 218% to 236% in the selected cs and pepc (double-gene) overexpression lines, respectively, compared with those in the wild-type plants (WT). The measurement for the relative root elongation rate of the tobacco plants exposed to 30???M aluminum (Al) indicated that Al tolerance in the double-gene overexpression lines was stronger than that of the transgenic cs or pepc lines and WT plants. The ¹³C-NMR analysis with NaH¹³CO₃ showed that overexpression of CS and PEPC in the transgenic tobacco successfully constructed a new citrate synthesis pathway. Under the conditions with Al stress, the amount of citrate secreted from the double-transgenic tobacco roots was the largest among the tested plants. When grown on sandy soil supplied with a nutritional solution containing 500???M Al, the growth of the double-transgenic tobacco was better than that of the transgenic cs or pepc tobacco and WT, and their root biomass was the highest among the tested plants. These results demonstrated that construction of a new citrate synthesis pathway by simultaneous overexpression of CS and PEPC in the cytoplasm of transgenic plant leaves could enhance Al resistance in plants.     

Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

Phosphoenolpyruvate carboxylase is an ubiquitous cytosolic enzyme that catalyzes the ß-carboxylation of phosphoenolpyruvate (PEP) and is encoded by multigene family in plants. It plays an important role in carbon economy of plants by assimilating CO₂ into organic acids for subsequent C₄ or CAM photosynthesis or to perform several anaplerotic roles in non-photosynthetic tissues. In this study, a cDNA clone encoding for PEPC polypeptide possessing signature motifs characteristic to ZmC₄PEPC was isolated from Pennisetum glaucum (PgPEPC). Deduced amino acid sequence revealed its predicted secondary structure consisting of forty alpha helices and eight beta strands is well conserved among other PEPC homologs irrespective of variation in their primary amino acid sequences. Predicted PgPEPC quartenary structure is a tetramer consisting of a dimer of dimers, which is globally akin to maize PEPC crystal structure with respect to major chain folding wherein catalytically important amino acid residues of active site geometry are conserved. Recombinant PgPEPC protein expressed in E. coli and purified to homogeneity, possessed in vitro ß-carboxylation activity that is determined using a coupled reaction converting PEP into malate. Tetramer is the most active form, however, it exists in various oligomeric forms depending upon the protein concentration, pH, ionic strength of the media and presence of its substrate or effecters. Recombinant PgPEPC protein confers enhanced growth advantage to E. coli under harsh growth conditions in comparison to their respective controls; suggesting that PgPEPC plays a significant role in stress adaptation.     

Trehalose 6–phosphate coordinates organic and amino acid metabolism with carbon availability

Trehalose 6–phosphate (Tre6P) is an essential signal metabolite in plants, linking growth and development to carbon metabolism. The sucrose–Tre6P nexus model postulates that Tre6P acts as both a signal and negative feedback regulator of sucrose levels. To test this model, short‐term metabolic responses to induced increases in Tre6P levels were investigated in Arabidopsis thaliana plants expressing the Escherichia coli Tre6P synthase gene (otsA) under the control of an ethanol‐inducible promoter. Increased Tre6P levels led to a transient decrease in sucrose content, post‐translational activation of nitrate reductase and phosphoenolpyruvate carboxylase, and increased levels of organic and amino acids. Radio‐isotope (¹⁴CO₂) and stable isotope (¹³CO₂) labelling experiments showed no change in the rates of photoassimilate export in plants with elevated Tre6P, but increased labelling of organic acids. We conclude that high Tre6P levels decrease sucrose levels by stimulating nitrate assimilation and anaplerotic synthesis of organic acids, thereby diverting photoassimilates away from sucrose to generate carbon skeletons and fixed nitrogen for amino acid synthesis. These results are consistent with the sucrose–Tre6P nexus model, and implicate Tre6P in coordinating carbon and nitrogen metabolism in plants.     

Organic acid accumulation may inhibit N2 fixation in phosphorus-stressed lupin nodules.

Nodulated lupins (Lupinus angustifolius cv. Wonga) were hydroponically grown under conditions of low phosphate (LP) or adequate phosphate (HP) to assess the effect of phosphoenolpyruvate carboxylase (PEPC)-derived organic acids on nitrogen assimilation in LP nodules. LP conditions are linked to altered organic acid metabolism, by the engagement of PEP metabolism via PEPC. In LP nodules, the enhanced organic acid synthesis may reduce the available organic carbon for nitrogen assimilation. The diversion of carbon between the organic acid- and amino acid pools was assessed through key nodular enzymes and (14)CO(2) metabolism. Under LP conditions, increased rates of organic acid synthesis via PEPC and malate dehydrogenase (MDH), coincided with reduced nitrogen assimilation via aspartate aminotransferase (AAT), aspartate synthetase (AS) and glutamine synthetase (GS)/glutamate synthase (GOGAT) activities. There was a preferential metabolism of nodular (14)CO(2) into organic acids and particularly into malate. High malate levels were associated with reduced N(2) fixation and synthesis of amino acids. These results indicate that phosphorus deficiency can enhance malate synthesis in nodules, but that excessive malate accumulation may inhibit N(2) fixation and nitrogen assimilation.     

植物磷酸烯醇式丙酮酸羧化酶的功能及其在基因工程中的应用

植物磷酸烯醇式丙酮酸羧化酶(Phosphoenolpyruvate carboxylase,PEPC,EC 4.1.1.31)是广泛存在的一种细胞质酶,催化磷酸烯醇式丙酮酸(PEP)和HCO3-生成草酰乙酸(OAA),后者可转化生成三羧酸循环的多种中间产物.PEPC在植物细胞中参与植物的光合碳同化等重要代谢途径,并且在不同组织中具有多种生理功能.PEPC同时也参与调控植物种子的营养物质合成与代谢过程,控制糖类物质流向脂肪酸合成或蛋白质合成途径.以下介绍了植物PEPC的种类、蛋白质结构特点及其在植物组织中的调控方式,并重点论述了PEPC在生物基因工程中的应用方面的进展,随着对其功能机制和应用研究的深入,将有助于植物PEPC在高产优质农作物育种、能源植物和工业微生物等的开发利用等方面得到更好的发展与应用.     

Cloning and nucleotide sequence of the phosphoenolpyruvate carboxylase-coding gene of Corynebacterium glutamicum ATCC13032

As a first step in determining the importance of the anaplerotic function of phosphoenolpyruvate carboxylase (PEPC) in amino acid biosynthesis, the ppc gene coding for PEPC of Corynebacterium glutamicum ATCC13032 has been cloned by complementation of an Escherichia coli ppc mutant strain. PEPC activity encoded by the cloned gene is not affected by acetyl-CoA under conditions where the E. coli enzyme is strongly activated, whereas acetyl-CoA is able to relieve inhibition by L-aspartate used singly or in combination with alpha-ketoglutarate. Amplification of the ppc gene in a C. glutamicum lysine-excreting strain resulted in increased PEPC-specific activity and lysine productivity. The nucleotide sequence of a DNA fragment of 4885 bp encompassing the ppc gene has been determined. At the amino acid level, PEPC from C. glutamicum presents overall a high degree of similarity with corresponding enzymes from three different organisms. The location of some strictly conserved regions may have important implications for PEPC activity and allostery.     

The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled enzyme located at the core of plant C-metabolism that catalyses the irreversible β-carboxylation of PEP to form oxaloacetate and Pi. The critical role of PEPC in assimilating atmospheric CO(2) during C(4) and Crassulacean acid metabolism photosynthesis has been studied extensively. PEPC also fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and nitrogen assimilation. An impressive array of strategies has evolved to co-ordinate in vivo PEPC activity with cellular demands for C(4)-C(6) carboxylic acids. To achieve its diverse roles and complex regulation, PEPC belongs to a small multigene family encoding several closely related PTPCs (plant-type PEPCs), along with a distantly related BTPC (bacterial-type PEPC). PTPC genes encode ~110-kDa polypeptides containing conserved serine-phosphorylation and lysine-mono-ubiquitination sites, and typically exist as homotetrameric Class-1 PEPCs. In contrast, BTPC genes encode larger ~117-kDa polypeptides owing to a unique intrinsically disordered domain that mediates BTPC's tight interaction with co-expressed PTPC subunits. This association results in the formation of unusual ~900-kDa Class-2 PEPC hetero-octameric complexes that are desensitized to allosteric effectors. BTPC is a catalytic and regulatory subunit of Class-2 PEPC that is subject to multi-site regulatory phosphorylation in vivo. The interaction between divergent PEPC polypeptides within Class-2 PEPCs adds another layer of complexity to the evolution, physiological functions and metabolic control of this essential CO(2)-fixing plant enzyme. The present review summarizes exciting developments concerning the functions, post-translational controls and subcellular location of plant PTPC and BTPC isoenzymes.     

The use of RNA interference for the metabolic engineering of plants (Review)

The metabolic engineering of plants is aimed at the realization of new biochemical reactions by transgenic cells. These reactions are determined by enzymes encoded by foreign or self-modified genes. Plants are considered to be the most interesting objects for metabolic engineering. Although they are characterized by the same pathways for the synthesis of basic biological compounds, plants differ by the astonishing diversity of their products: sugars, aromatic compounds, fatty acids, steroid compounds, and other biologically active substances. RNA interference aimed at modifying metabolic pathways is a powerful tool that allows for the obtainment of plants with new valuable properties. The present review discusses the main tendencies for research development directed toward the obtainment of transgenic plants with altered metabolism.     

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 form 0.5-fold wild-type level in antisense plants to 5-fold wild-type levels in lines overexpressing the ccpc 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 l l^-1 CO2) compared to control potato lines (38 l l^-1 CO2), but was increased in antisense PEPC plants (42 l^-1 CO2). 3-fold overexpression of PEPC gave a minimum CO2 compensation point of 32 l 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 9150%). Total amino acid contents were only marginally increased. From gas exchange characteristics and labeling experiments it was concluded that PEP carboxylation, followed by an increased rate of respiratory CO2 release, might work as a HCO3^-/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).     

一种中间代谢途径代谢通量的定量分析方法

以¹³C标记的碳源,用二维核磁共振技术(¹H-¹³C,HMQC)测定代谢中产生的氨基酸标记模式,研究对中间代谢途径胞内代谢通量进行定量分析的方法.通过开发软件包,改进同位素分布的数学模型,提出了反应映射矩阵(RMM)等概念.由简化算法,提高程序的执行效率,建立了定量分析胞内代谢通量的平台.代谢模型涉及了糖酵解途径、磷酸戊糖途径、三羧酸循环、几种回补反应、发酵途径和氨基酸合成途径.     

Phosphorylation of phosphoenolpyruvate carboxylase is not essential for high photosynthetic rates in the C4 species Flaveria bidentis

Phosphoenolpyruvate carboxylase (PEPC; EC4.1.1.31) plays a key role during C(4) photosynthesis. The enzyme is activated by metabolites such as glucose-6-phosphate and inhibited by malate. This metabolite sensitivity is modulated by the reversible phosphorylation of a conserved serine residue near the N terminus in response to light. The phosphorylation of PEPC is modulated by a protein kinase specific to PEPC (PEPC-PK). To explore the role PEPC-PK plays in the regulation of C(4) photosynthetic CO(2) fixation, we have transformed Flaveria bidentis (a C(4) dicot) with antisense or RNA interference constructs targeted at the mRNA of this PEPC-PK. We generated several independent transgenic lines where PEPC is not phosphorylated in the light, demonstrating that this PEPC-PK is essential for the phosphorylation of PEPC in vivo. Malate sensitivity of PEPC extracted from these transgenic lines in the light was similar to the malate sensitivity of PEPC extracted from darkened wild-type leaves but greater than the malate sensitivity observed in PEPC extracted from wild-type leaves in the light, confirming the link between PEPC phosphorylation and the degree of malate inhibition. There were, however, no differences in the CO(2) and light response of CO(2) assimilation rates between wild-type plants and transgenic plants with low PEPC phosphorylation, showing that phosphorylation of PEPC in the light is not essential for efficient C(4) photosynthesis for plants grown under standard glasshouse conditions. This raises the intriguing question of what role this complexly regulated reversible phosphorylation of PEPC plays in C(4) photosynthesis.     

Nitrogen fixation and carbon metabolism in legume nodules

A large amount of energy is utilized by legume nodules for the fixation of nitrogen and assimilation of fixed nitrogen (ammonia) into organic compounds. The source of energy is provided in the form of photosynthates by the host plant. Phosphoenol pyruvate carboxylase (PEPC) enzyme, which is responsible for carbon dioxide fixation in C4 and crassulacean acid metabolism plants, has also been found to play an important role in carbon metabolism in legume root nodule. PEPC-mediated CO2 fixation in nodules results in the synthesis of C4 dicarboxylic acids, viz. aspartate, malate, fumarate etc. which can be transported into bacteroids with the intervention of dicarboxylate transporter (DCT) protein. PEPC has been purified from the root nodules of few legume species. Information on the relationship between nitrogen fixation and carbon metabolism through PEPC in leguminous plants is scanty and incoherent. This review summarizes the various aspects of carbon and nitrogen metabolism in legume root nodules.     

Systems metabolic engineering of Corynebacterium glutamicum for high-level production of 1,3-propanediol from glucose and xylose

Corynebacterium glutamicum is a versatile chassis which has been widely used to produce various amino acids and organic acids. In this study, we report the development of an efficient C. glutamicum strain to produce 1,3-propanediol (1,3-PDO) from glucose and xylose by systems metabolic engineering approaches, including (1) construction and optimization of two different glycerol synthesis modules; (2) combining glycerol and 1,3-PDO synthesis modules; (3) reducing 3-hydroxypropionate accumulation by clarifying a mechanism involving 1,3-PDO re-consumption; (4) reducing the accumulation of toxic 3-hydroxypropionaldehyde by pathway engineering; (5) engineering NADPH generation pathway and anaplerotic pathway. The final engineered strain can efficiently produce 1,3-PDO from glucose with a titer of 110.4 g/L, a yield of 0.42 g/g glucose, and a productivity of 2.30 g/L/h in fed-batch fermentation. By further introducing an optimized xylose metabolism module, the engineered strain can simultaneously utilize glucose and xylose to produce 1,3-PDO with a titer of 98.2 g/L and a yield of 0.38 g/g sugars. This result demonstrates that C. glutamicum is a potential chassis for the industrial production of 1,3-PDO from abundant lignocellulosic feedstocks.