Transforming wheat vacuolar invertase into a high affinity sucrose:sucrose 1‐fructosyltransferase

Schroeven, Lindsey; Lammens, Willem; Laere, André Van; Ende, Wim Van den

doi:10.1111/j.1469-8137.2008.02603.x

Cited by 57 publications

(67 citation statements)

References 57 publications

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“…Each sequence shared more than 99% identity with its T. officinale orthologue and more than 79% identity with the corresponding proteins from chicory (Ci1‐SST AFB83198 and Ci1‐FFT AAD00558) and Jerusalem artichoke (Ht1‐SST CAA08812 and Ht1‐FFT CAA08811). Multiple sequence alignments using MUSCLE revealed the presence of three GH32 family‐specific conserved regions including the three catalytically active amino acids shown in bold: x‐x‐x‐ D ‐P‐D/N‐G; R D P; and E C (Altenbach and Ritsema, 2007; Altenbach et al ., 2005; Edgar, 2004; Schroeven et al ., 2008) (Figure S1). Furthermore, the fructosyltransferase‐specific motif x‐A/G‐Y/F was found in Tb1‐SST, Tk1‐SST, Tb1‐FFT and Tk1‐FFT (Altenbach et al ., 2005; Lasseur et al ., 2009).…”

Section: Resultsmentioning

confidence: 99%

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

Stolze¹,

Wanke²,

Deenen³

et al. 2017

Plant Biotechnology Journal

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SummaryNatural rubber (NR) is an important raw material for a large number of industrial products. The primary source of NR is the rubber tree Hevea brasiliensis, but increased worldwide demand means that alternative sustainable sources are urgently required. The Russian dandelion (Taraxacum koksaghyz Rodin) is such an alternative because large amounts of NR are produced in its root system. However, rubber biosynthesis must be improved to develop T. koksaghyz into a commercially feasible crop. In addition to NR, T. koksaghyz also produces large amounts of the reserve carbohydrate inulin, which is stored in parenchymal root cell vacuoles near the phloem, adjacent to apoplastically separated laticifers. In contrast to NR, which accumulates throughout the year even during dormancy, inulin is synthesized during the summer and is degraded from the autumn onwards when root tissues undergo a sink‐to‐source transition. We carried out a comprehensive analysis of inulin and NR metabolism in T. koksaghyz and its close relative T. brevicorniculatum and functionally characterized the key enzyme fructan 1‐exohydrolase (1‐FEH), which catalyses the degradation of inulin to fructose and sucrose. The constitutive overexpression of Tk1‐FEH almost doubled the rubber content in the roots of two dandelion species without any trade‐offs in terms of plant fitness. To our knowledge, this is the first study showing that energy supplied by the reserve carbohydrate inulin can be used to promote the synthesis of NR in dandelions, providing a basis for the breeding of rubber‐enriched varieties for industrial rubber production.

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Section: Resultsmentioning

confidence: 99%

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

Stolze¹,

Wanke²,

Deenen³

et al. 2017

Plant Biotechnology Journal

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“…However, most of these amino acids represent semiconserved substitutions (Fig. 1) (Reddy and Maley, 1996;Schroeven et al, 2008), and only the amino acids within a 15-Ǻ distance from the catalytic triad center were taken into account (Fig. 2).…”

Section: Designing Mutants Based On Multiple Sequence Alignments and mentioning

confidence: 99%

“…Moreover, a molecular modeling approach realized in silico (using Arabidopsis [Arabidopsis thaliana] cell wall invertase 1 [AtcwINV1] as a template; Verhaest et al, 2006; Protein Data Bank identifier 2AC1) allowed us to further reduce the number of promising candidates. The active site of all GH32 enzymes is characterized by three highly conserved amino acids in the WMNDPNG, RDP, and EC motifs (Reddy and Maley, 1996;Schroeven et al, 2008), and only the amino acids within a 15-Ǻ distance from the catalytic triad center were taken into account (Fig. 2).…”

Section: Designing Mutants Based On Multiple Sequence Alignments and mentioning

confidence: 99%

“…The very high identity at the sequence level between Lp6G-FFT/1-FFT and Lp1-SST inspired us to study in depth the molecular differences between these functionally different enzymes. Despite their high identity, these enzymes differ greatly in substrate specificity and product formation, the two most crucial differences being that (1) Lp1-SST uses Suc as a donor substrate, while Suc is a very bad donor substrate for the recombinant Lp6G-FFT/1-FFT and (2) Lp1-SST can only create a b(2-1) linkage between two fructosyl residues, while Lp6G-FFT/1-FFT can create both a b(2-1) linkage between two fructosyl residues and a b(2-6) linkage between one fructosyl residue and one glucosyl residue.Although substantial progress has been made to transform a VI into a FT (Ritsema et al, 2006;Schroeven et al, 2008) and a CWI into a FEH , the transformation of one type of FT activity into another type of FTactivity has not been reported before. Here, we describe, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/ 1-FFT from perennial ryegrass: Lp6G-FFT/1-FFT) into an S-type enzyme (1-SST) by site-directed mutagenesis and compare the kinetic properties of the best mutant with the wild-type 1-SST from perennial ryegrass (Lp1-SST).…”

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confidence: 99%

“…Although substantial progress has been made to transform a VI into a FT (Ritsema et al, 2006;Schroeven et al, 2008) and a CWI into a FEH (Le , the transformation of one type of FT activity into another type of FTactivity has not been reported before. Here, we describe, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/ 1-FFT from perennial ryegrass: Lp6G-FFT/1-FFT) into an S-type enzyme (1-SST) by site-directed mutagenesis and compare the kinetic properties of the best mutant with the wild-type 1-SST from perennial ryegrass (Lp1-SST).…”

mentioning

confidence: 99%

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Transforming a Fructan:Fructan 6G-Fructosyltransferase from Perennial Ryegrass into a Sucrose:Sucrose 1-Fructosyltransferase

et al. 2008

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Fructosyltransferases (FTs) synthesize fructans, fructose polymers accumulating in economically important cool-season grasses and cereals. FTs might be crucial for plant survival under stress conditions in species in which fructans represent the major form of reserve carbohydrate, such as perennial ryegrass (Lolium perenne). Two FT types can be distinguished: those using sucrose (S-type enzymes: sucrose:sucrose 1-fructosyltransferase , sucrose:fructan 6-fructosyltransferase) and those using fructans (F-type enzymes: fructan:fructan 1-fructosyltransferase , fructan:fructan 6G-fructosyltransferase [6G-FFT]) as preferential donor substrate. Here, we report, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/1-FFT) into an S-type enzyme (1-SST) using perennial ryegrass 6G-FFT/1-FFT (Lp6G-FFT/1-FFT) and 1-SST (Lp1-SST) as model enzymes. This transformation was accomplished by mutating three amino acids (N340D, W343R, and S415N) in the vicinity of the active site of Lp6G-FFT/1-FFT. In addition, effects of each amino acid mutation alone or in combination have been studied. Our results strongly suggest that the amino acid at position 343 (tryptophan or arginine) can greatly determine the donor substrate characteristics by influencing the position of the amino acid at position 340. Moreover, the presence of arginine-343 negatively affects the formation of neofructan-type linkages. The results are compared with recent findings on donor substrate selectivity within the group of plant cell wall invertases and fructan exohydrolases. Taken together, these insights contribute to our knowledge of structure/function relationships within plant family 32 glycosyl hydrolases and open the way to the production of tailor-made fructans on a larger scale.Fructans are Fru polymers that can be considered as an extension of Suc, accumulating above a certain threshold Suc concentration (Cairns and Pollock, 1988;Guerrand et al., 1996;Maleux and Van den Ende, 2007). Fructans occur in many dicot and monocot plant species mainly belonging to Asteraceae, Liliaceae, and Poaceae (Hendry, 1993;Prud'homme et al., 2007;Shiomi et al., 2007;Van den Ende and Van Laere, 2007;Yoshida et al., 2007). Nowadays, research is mainly focused on economically important cereals (wheat [Triticum aestivum] and barley [Hordeum vulgare]) and forage grasses (mainly Lolium species). Depending on the linkage type between the fructosyl residues and the position of the Glc residue (Lewis, 1993), several fructan types can be distinguished. Fructans with a terminal Glc residue include the b(2-1)-type fructans (inulin, principally occurring in dicots) and the linear b(2-6) (levan)-or branched-type fructans (graminan) with both b(2-6) and b(2-1) linkages (as occurring in bacteria and cereals, respectively). Fructans with an internal Glc residue include the neoinulin and neolevan types (occurring in monocots such as Allium, Asparagus, and Lolium).Apart from their function as a vacuolar storage carbohydrate, fructans may help to protect plants ag...

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Sucrose Metabolism

Lunn¹

2016

Encyclopedia of Life Sciences

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Sucrose is one of the main products of photosynthesis in plants, and the most common form of carbohydrate transported from source to sink organs. It also functions as a storage reserve, compatible solute and signal metabolite in plants. Sucrose is synthesised via the phosphorylated intermediate sucrose‐6′‐phosphate, by sucrose‐phosphate synthase (SPS) and sucrose‐phosphatase (SPP). In sink organs, sucrose is broken down by invertase or sucrose synthase to provide carbon and energy for growth and accumulation of storage reserves, such as starch, oil and fructans. Sucrose and trehalose are the only common nonreducing disaccharides found in nature, and their metabolism is inextricably linked in plants. Trehalose‐6‐phosphate, the intermediate of trehalose synthesis, is a signal of sucrose availability in plant cells, regulating photoassimilate partitioning in leaves and the utilisation of sucrose in sink organs. Key Concepts Sucrose is one of the main products of photosynthesis and the most common transport sugar in plants. Sucrose is a nonreducing disaccharide and is synthesised in the cytosol via the phosphorylated intermediate, sucrose‐6′‐phosphate. In leaves, the rate of sucrose synthesis is tightly coordinated with the rates of photosynthetic carbon dioxide fixation and starch synthesis in the chloroplasts. Sucrose is transported from leaves via the phloem, to provide the rest of the plant with carbon and energy for growth and storage product synthesis. Sucrose is unloaded from the phloem in sink organs. It can be hydrolysed by cell wall invertases and imported into the cells as hexose sugars, or taken up intact and metabolised by intracellular invertases or sucrose synthase. Fructans are soluble polymers of fructose found in about 15% of all flowering plants. Dicotyledonous plants generally make inulin‐type fructans, whereas monocotyledonous plants also make levan, inulin neoseries, levan neoseries and mixed levan (graminan)‐type fructans. Fructans are synthesised from sucrose via trisaccharide intermediates – 1‐kestose, 6‐kestose or 6G‐kestose – by various fructosyltransferases in the vacuole. Trehalose is a nonreducing disaccharide found in many bacteria, archaea, invertebrates and fungi, and is often used as a stress protectant, compatible solute, storage reserve or transport sugar. Trehalose metabolism is ubiquitous in plants, and essential for their normal growth and development, but most flowering plants accumulate only trace amounts of trehalose. Trehalose‐6‐phosphate, the intermediate of trehalose synthesis, is a signal of sucrose availability in plants, regulating photoassimilate partitioning in leaves and the utilisation of sucrose in sink organs.

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Transforming wheat vacuolar invertase into a high affinity sucrose:sucrose 1‐fructosyltransferase

Cited by 57 publications

References 57 publications

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

Transforming a Fructan:Fructan 6G-Fructosyltransferase from Perennial Ryegrass into a Sucrose:Sucrose 1-Fructosyltransferase

Sucrose Metabolism

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