GusPlus Positive Selection

Various projects at CAMBIA have attempted to develop screening and positive selection systems for plant transgenesis. This concept, first proposed by CAMBIA a number of years ago, would address some of the problems associated with currently used selectable marker genes:

  • In certain jurisdictions, intellectual property rights restrict access to most practical negative selection methods.
  • Public acceptance questions use of certain selectable marker genes, notably genes encoding resistance to antibiotics.
  • Some regulatory agencies discourage use of such systems in plants to be released into the field.
  • Selection systems based on genes conferring resistance to antibiotics or herbicides (“negative selection”) also stress living transgenic cells, in part by causing the release of phenolic substances from non-transformed, dying plant cells that may affect growth of transformed cells. This effect, which can hurt regeneration, limits their value in challenging tissue culture systems.

The goal of CAMBIA’s work in this area, not yet fully realised, has been to develop a positive-selection system based on a glucose-releasing procompound that circumvents these limitations. In plants, the disaccharide sucrose releases glucose when hydrolyzed by the enzyme invertase. The glucose released can then act an energy substrate to drive plant growth. In an attempt to “mimic” this principle, we chose the disaccharide cellobiouronic acid (CbA) as the substrate for our selection system. CbA is composed of glucuronic acid linked ß(1-4) to glucose. As CbA was not commercially available when this work began, CAMBIA had to develop a new method to prepare the sugar. US Patent US 6,268,493 describes this work and can be licensed royalty-free under open source principles for both research and commercial uses; potential commercial suppliers are of interest. Because of its structure, we expect CbA to be hydrolyzed by any of a variety of different ß-glucuronidase (GUS) enzymes. Plant cells lacking GUS activity are unable to grow on CbA as the sole carbon source. Transformation with a gene encoding GUS, however, would allow them to access the glucose “immobilized” within CbA and use it as an energy source for growth.

positive_selection

Additional advantages of the CbA/GUS selection system being developed are that CbA can be produced from gellan gum, a commonly used food additive in ice creams and other products, and that GUS has already been comprehensively tested for safety in products approved by many national regulatory agencies for human consumption. The ß-glucuronidase (GUS) enzyme that currently works best for this application was isolated from Thermotoga and together with the gene that encodes it is described in published patent applications WO 00/055333 and US 2003229921, and in U.S. Patent No. 7,087,420.

The license CAMBIA offers covers the gene and others similar to it, the protein, and a variety of uses of the protein, which may have industrial applications beyond positive selection. This enzyme is of interest for other applications because it is highly thermostable, and it can be licensed royalty-free under open source principles. We expect that continuing work by anyone who wishes to collaborate within the open source licensee community will optimize the enzyme characteristics for cleavage of CbA in the spaces around the growing cells. CAMBIA has done some codon manipulation toward getting it to work in plants, still not entirely successful.

For positive selection, due to dominating patents (see www.lens.org) there may not be freedom to operate in all jurisdictions even with the workable substrate and gene variants, but in many countries you would be free to research and even commercialise this concept, and we definitely welcome collaboration. We have been developing a technology landscape around positive selection, on which we also welcome collaboration.