Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins

Abstract

A new approach in the field of plant gums is described which presents a new solution to the production of hydroxyproline(Hyp)-rich glycoproteins (HRGPs), repetitive proline-rich proteins (RPRPs) and arabinogalactan-proteins (AGPs). The expression of synthetic genes designed from repetitive peptide sequences of such glycoproteins, including the peptide sequences of gum arabic glycoprotein (GAGP), is taught in host cells, including plant host cells.

Description

[0001] The present invention relates generally to the field of plant gums and other hydroxyproline-rich glycoproteins, and in particular, to the expression of synthetic genes designed from repetitive peptide sequences.[0002] Gummosis is a common wound response that results in the exudation of a gum sealant at the site of cracks in bark. A. M. Stephen et al., "Exudate Gums", Methods Plant Biochem. (1990). Generally the exudate is a composite of polysaccharides and glycoproteins structurally related to cell wall components such as galactans [G. O. Aspinall, "Plant Gums", The Carbohydrates 2B:522536 (1970)] and hydroxyproline-rich glycoproteins [Anderson and McDougall, "The chemical characterization of the gum exudates from eight Australian Acacia species of the series Phyllodineae." Food Hydrocolloids, 2: 329 (1988)].[0003] Gum arabic is probably the best characterized of these exudates (although it has been largely refractory to chemical analysis). It is a natural plant exudate secre...

AI Technical Summary

Benefits of technology

[0102] The Extensins occupy an intermediate position in the glycosylation continuum, containing about 50% carbohydrate which occurs mainly as Hyp-arabinosides (1-4 Ara residues), but not as Hyp-arabinogalactan polysaccharide. Extensins contain the repetitive, highly arabinosylated, diagnostic Ser-Hyp.sub.4 (SEQ ID NO:3) glycopeptide module. The precise function of this module is unknown, but earlier work indicates that these motifs of arabinosylated Hyp help stabilize the extended polyproline-II helix of the extensins. Monogalactose also occurs on the Ser residues.

[0107] In one embodiment, the present invention contemplates a substantially purified polypeptide comprising at least a portion of the amino acid consensus sequence Ser-Hyp-Hyp-Hyp-[Hyp / Thr]-Leu-Ser-Hyp-Ser-H-yp-Thr-Hyp-Thr-Hyp-Hyp-Hyp-Gly-Pro-His (SEQ ID NO:1 and SEQ ID NO:2) or variants thereof. While an understanding of the natural mechanism of glycosylation is not required for the successful operation of the present invention, it is believed that this GAGP 19-amino acid consensus repeat (which contains both contiguous Hyp and non-contiguous Hyp repeats) is glycosylated in native GAGP with both Hyp-arabinosides and Hyp-polysaccharide in molar ratios. It is further believed that the high molecular weight protein component of gum arabic (i.e. GAGP) is responsible for the remarkable (and advantageous) emulsifying and stabilizing activity exploited by the food and soft drink industries.

[0139] Tomato cultures are the ideal recipients for repetitive HRGP modules to be hydroxylated and glycosylated: Tomato is readily transformed. The cultures produce cell surface HRGPs in high yields easily eluted from the cell surface of intact cells and they possess the required posttranslational enzymes unique to plants--HRGP prolyl hydroxylases, hydroxyproline O-glycosyltransferases and other specific glycosyltransferases for building complex polysaccharide side chains. Furthermore, tomato genetics, and tomato leaf disc transformation / plantlet regeneration are well worked out.

[0143] Other particle bombardment methods are also available for the introduction of heterologous nucleic acid sequences into plant cells. Generally, these methods involve depositing the nucleic acid sequence of interest upon the surface of small, dense particles of a material such as gold, platinum, or tungsten. The coated particles are themselves then coated onto either a rigid surface, such as a metal plate, or onto a carrier sheet made of a fragile material such as mylar. The coated sheet is then accelerated toward the target biological tissue. The use of the flat sheet generates a uniform spread of accelerated particles which maximizes the number of cells receiving particles under uniform conditions, resulting in the introduction of the nucleic acid sample into the target tissue.

[0145] One of skill in the art knows that the efficiency of transformation by Agrobacterium may be enhanced by using a number of methods known in the art. For example, the inclusion of a natural wound response molecule such as acetosyringone (AS) to the Agrobacterium culture has been shown to enhance transformation efficiency with Agrobacterium tumefaciens [Shahla et al. (1987) Plant Molec. Biol. 8:291-298]. Alternatively, transformation efficiency may be enhanced by wounding the target tissue to be transformed. Wounding of plant tissue may be achieved, for example, by punching, maceration, bombardment with microprojectiles, etc. [see, e.g., Bidney et al. (1992) Plant Molec. Biol. 18:301-313].

[0161] In one preferred embodiment, the consensus sequence and portions thereof is selected from Ser-Hyp-Hyp-Hyp-A-Leu-Ser-Hyp-Ser-Hyp-Thr-Hyp-Th-r-Hyp-Hyp-B-Gly-Pro-His (SEQ ID NO:179), where A is selected from Hyp, Thr and Ser, and B is selected from Hyp and Leu (Table 6). Remarkably, fifteen amino acid residues of this sequence are "quasi-palindromic," i.e., the side chain sequence is almost the same whether read from the N-terminus or C-terminus. Without limiting the invention to a particular theory or mechanism, it is the inventors' consideration that such peptide symmetry, which occurs frequently in extensins and AGPs, may enhance molecular packing, recognition, and self-assembly. Indeed, palindromic symmetry rigidified by contiguous Hyp motifs in the motifs: Ser-Hyp-Hyp-Hyp-(Hyp) and Thr-Hyp-Hyp-(Hyp), may impart self-ordering properties in GAGP and other HRGPs. Thus, it is the inventor's consideration that GAGP properties are related to the polysaccharide substituents. In particular, the repeating glycopeptide symmetry of two central polysaccharides flanked by Hyp arabinosides may enhance gum arabic's remarkable properties which include: an anomalously low viscosity [Churms et al. (1983) Carbohydrate Research 123:267], the ability to act as a flavor emulsifier and stabilizer, and GAGP's biological role as a component of a plastic sealant.

Problems solved by technology

It is a tedious process involving piercing and stripping the bark of the trees, then returning later to gather the dried tear drop shaped, spherical balls that form in response to mechanical wounding.

However, this production depends on the environmental and political stability of the region producing the gum.

Again in 1985, drought brought about shortages of the gum, resulting in a 600% price increase.

Second, additives have been investigated to supplement inferior gum arabic.

The effort to find other gums in other regions of the world has met with some limited success.

However, this approach has limitations.

Unfortunately, conditions have not been found which lead to the expression of gum arabic in culture. A. Mollard and J -P. Joseleau, "Acacia senegal cells cultured in suspension secrete a hydroxyproline-deficient arabinogalactan-protein" Plant Physiol. Biochem. 32:703 (1994).

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