Fungi

Summary

“Fungal” aspects

Fungi constitute one of the life kingdoms. Fungi are eukaryotic (eu=true; karyon=nucleus) organisms with a cell wall like plants, but they do not have chlorophyll. Fungi are not able to ingest their food like animals do, nor can they manufacture their own food the way plants do. Instead, fungi feed by absorption of nutrients from the surrounding environment. They accomplish this by growing through and within the substrate on which they are feeding.

Fungi are divided into two big groups: yeasts and moulds. Yeasts are solitary rounded forms that reproduce by making more rounded forms through mechanisms such as budding or fission. Moulds, on the other hand, have bodies composed of thread-like long cells called hyphae. Thus, moulds are also known as filamentous fungi. The filamentous cells are connected end-to-end and grow in a branching fashion forming a network called mycelium. The mycelium that grows over and within a substrate that is used as a source of nourishment is called vegetative mycelium. In the life cycle, the vegetative mycelium may give rise to a large organized reproductive structure called fruit body, which bears the reproductive cells or spores and is produced solely for the release of spores.

In taxonomic terms, moulds are present in all five divisions of Eumycota (Eu=true; mycota=fungus): Mastigomycotina (e.g. Phytophtora , Achlya), Zygomycotina (e.g. Rhizopus, Mucur), Ascomycotina (e.g. some species of Aspergillus, Neurospora ), Basidiomycotina (e.g. Agaricus, Pleurotus) and Deuteromycotina (e.g. Fusarium, Trichoderma).

Filamentous fungi or moulds are vital for the maintenance of ecosystems. By breaking down dead organic material, they continue the cycle of nutrients through ecosystems. Some of them act as plant pathogens causing severe crop losses from disease and post-harvest food spoilage. In the reagent industry and medicine areas, filamentous fungi are the source of commercial enzymes, organic acids, and numerous drugs such as antibiotics (e.g. penicillin, cefalosporin). Among filamentous fungi are highly appreciated edible fungi such asAgaricus bisporus, the popular cultivated mushroom; Pleurotus spp., the “oyster mushroom”, Tuber spp., “truffles”, and Morchella spp., “Morels”, among others. Thus, in many areas, the industrial production of genetically engineered fungi has tremendous potential.

IP aspects

The selected patents and patent applications presented in this section are directed to transformation of moulds or filamentous fungi with Agrobacterium.

There are two institutions that have filed patent applications related to Agrobacterium-mediated transformation of filamentous fungi: Unilever N.V., in The Netherlands, has a granted United States patent and several applications around the world, and The Penn State Research Foundation, in the United States, has a PCT and a United States patent application.

Unilever‘s invention is directed to a transformed mould with A. tumefaciens having a vector containing an expressible gene between T-DNA borders. Although the Penn State ‘s invention also refers to the transformation of a mould, which the inventors called filamentous fungi, they limit the invention to a particular tissue to be transformed: the fruit body tissue of a filamentous fungi. Unilever does not claim the transformation of any tissue in particular, and their claims are therefore broader in that respect. In addition, some of the claims filed in the Penn State’s applications are also directed to the transformation of a particular filamentous fungus: Agaricus bisporus , the cultivated mushroom.

In conclusion

There is an overlap between the inventions as both refer to transformation of any filamentous fungi or mould with Agrobacterium. However, the invention disclosed by Penn State Research Foundation is more defined as it encompasses a particular fungal tissue to be transformed.
It remains to be seen whether the claims as filed in the applications by Penn State Research Foundation are granted as filed. Unilever‘s United States patent is fairly broad, however, and may pose freedom to operate problems if the species A. tumefaciens is used for transforming any mould.

Granted patent and patent application filed by Unilever Patent Holdings B.V.

Unilever discloses in its applications a process for producing a transformed mould with A. tumefaciens. In the method, an A. tumefaciens vector containing at least one expressible gene is introduced into the mould.

Moulds are defined by the inventors to include fungi from all five subdivisions of the division Eumycota. The examples in the disclosures include fungi from the genera Aspergillus, Fusarium, Trichoderma, Neurospora and Colletotrichum. The first three genera contain species important in large scale fermentation and production of homologous and heterologous proteins. Other species within these genera are fungal pathogens and fungi that serve as important model organisms for basic research.

Specific Patent Information

Patent Number

Title, Independent Claims and Summary of Claims

Assignee

US 6255115

Earliest priority – 7 April 1997
Filed – 7 October 1999
Granted – 3 July 2001
Expected expiry – 7 October 2019

Title – Agrobacterium-mediated transformation of moulds, in particular those belonging to the genus Aspergillus

Claim 1A process for producing a transformed mould, comprising: A) inserting a DNA fragment containing at least one expressible gene to be introduced into a mould into a vector of Agrobacterium tumefaciens between the T-DNA borders present in that vector;
B) introducing the vector containing the DNA fragment between the T-DNA borders into an Agrobacterium tumefaciens strain containing a vir region in its DNA;
C) inducing vir genes to release T-DNA containing said DNA fragment from said Agrobacterium tumefaciens, and incubating the Agrobacterium tumefaciens strain with the mould to be transformed; and
D) selecting the transformed mould from the untransformed mould depending on the characteristics of the introduced DNA or its expression product, and optionally culturing the transformed mould.

The United States patent claims

a method for producing a transgenic mould by inserting into it at least an expressible gene carried in an A. tumefaciens vector. A. tumefaciens containing the vector and the mould are co-cultivated and the transformed mould is selected.

Note that although the title of the patent refers to the genus Aspergillus, the independent claims are not limited to this genus or any other genera of filamentous fungi.

Unilever Patent
Holdings B.V.

EP 973917 B1

Earliest priority – 7 April 1997
Filed – 24 March 1998
Granted – 3 March 2004
Expected expiry – 23 March 2018

Title – Agrobacterium-mediated transformation of moulds, in particular those belonging to the genus Aspergillus

Claim 1A process for producing a transformed mould, characterized in that:

1) a DNA fragment containing at least one expressible gene to be introduced into a mould is first cloned into a vector of Agrobacterium tumefaciens between the T-DNA borders present in that vector, wherein T-DNA borders are 24 basepair imperfect direct repeats flanking the T-DNA;
2) the vector containing the DNA fragment between the T-DNA borders is introduced into an Agrobacterium tumefaciens strain containing a vir region in its DNA;
3) release of T-DNA containing said DNA fragment from said Agrobacterium tumefaciens by addition of a vir-inducing compound, and the Agrobacterium tumefaciens strain is incubated with the mould to be transformed; and
4) the transformed mould is selected from the untransformed mould depending on the characteristics of the introduced DNA or its expression product, and optionally the transformed mould is cultured.

The claims as filed in the granted EP patent are directed to the same subject matter as the granted claims of the United States patent.

Designated contracting States at the time of grant are: Switzerland, Germany, Spain, France, United Kingdom, Italy, Liechtenstein.

* As the independent claims of the United States patent and the European patent application are worded slightly different, the claims are presented independently. However, the claims have practically the same scope.

EP 973917 A1

Earliest priority – 7 April 1997
Filed – 24 March 1998
Granted as EP 973917 B1 (see above)

Title – Agrobacterium-mediated transformation of moulds, in particular those belonging to the genus Aspergillus

Claim 1 (granted with minor changes)A process for producing a transformed mould, characterized in that:

1) a DNA fragment containing at least one expressible gene to be introduced into a mould is first cloned into a vector of Agrobacterium tumefaciens between the T-DNA borders present in that vector;
2) the vector containing the DNA fragment between the T-DNA borders is introduced into an Agrobacterium tumefaciens strain containing a vir region in its DNA;
3) release of T-DNA containing said DNA fragment from said Agrobacterium tumefaciens by addition of a vir-inducing compound, and the Agrobacterium tumefaciens strain is incubated with the mould to be transformed; and
4) the transformed mould is selected from the untransformed mould depending on the characteristics of the introduced DNA or its expression product, and optionally the transformed mould is cultured.

Remarks

Related applications also filed in Brazil (BR 9807941 A), Indonesia (ID 22929), and South Africa (ZA 9802905 A).
National phase entry of WO 1998/45455 in Australia (AU 74283/98) has lapsed on 23 December 1999.
National phase entry of WO 1998/45455 in Canada (CA 2286307) and Japan (JP 2001/518786 T2) are still pending.
National phase entry of WO 1998/45455 in China (CN 1151264) has been granted on 26 May 2004.
Related patent family of WO 1999/32641 ‘A process for site-directed integration of multiple copies of a gene in a mould’ describes a method of site-specific transformation of mould using a rare restriction endonuclease system. The first independent claim does notrecite transformation using Agrobacterium.

Note: Patent information on this page was last updated on 9 February 2006.

Patent applications filed by the Penn State Research Foundation

The present PCT and United States patent applications relate to the transformation of the fruit body tissue of a filamentous fungus with Agrobacterium. The genetically modified fungi can serve as biofermentators for the mass production of commercial products. As examples of filamentous fungi, the applicant mentions fungi belonging to phyla Ascomycota and Basidiomycota such as Coprinus, Agaricus, Morchella , and Coriolus, among others. In particular, the invention contemplates the transformation of the cultivated mushroom Agaricus bisporus, which accounts for 38% of the world production of cultivated mushrooms.

Specific Patent Information

Patent Number

Specific Patent Information

Assignee

WO 2002/00896 A2

Earliest priority – 28 June 2000
Filed – 28 June 2001
OPI – 3 January 2002

Title – Methods and compositions for highly effective transformation of filamentous fungi

Claim 1A method of transforming filamentous fungi comprising:

introducing to a fruit body tissue cell of said fungi a polynucleotide construct said construct comprising sequences for the expression of a structural gene or for the inhibition of an endogenous gene, the presence of which is desired in said fungi cell.

Claim 16A filamentous fungi transformation method comprising:

A) obtaining an Agrobacterium vector, said vector comprising a polynucleotide sequence the presence of which is desired in a recipient filamentous fungi cell; and
B) introducing said vector to fruit body tissue cells of said fungi in the presence of an active vir Agrobacterium region and a vir inducing compound.

Claim 26A method for transforming a fungi cell comprising:

A) obtaining an Agrobacterium-derived Ti plasmid, said plasmid comprising a polynucleotide sequence the expression of which is desired in a host fungal cell;
B) introducing said plasmid to said cell in the presence of vir active genes and a vir inducing agent.

Claim 30A method for transformation of Agaricus bisporus cells comprising:

A) obtaining an Agrobacterium vector, said vector comprising a polynucleotide sequence the presence of which is desired in a recipient filamentous fungi cell; and
B) introducing said vector to fruit body tissue cells of said Agaricus bisporus cells in the presence of an active vir Agrobacterium region and a vir inducing compound.

The claims as filed in the PCT application recite:

a method for transforming any filamentous fungus by introducing into the fruit body tissue a construct for the expression of a structural gene or for the inhibition of an endogenous gene; and
a method for transforming the fruit body tissue of any filamentous fungus and Agaricus bisporus in particular with an Agrobacteriumvector in the presence of a vir acting region and a vir inducing compound.

Although these claims are fairly broad as they refer to the transformation of any filamentous fungus without specifying a method in one case and specifying Agrobacterium in another claim, an apparent limitation lies in the type of fungal tissue to be transformed. The invention recites only transformation of fruit body tissue. It remains to be seen whether granted claims will have the same breadth as the filed claims.

Penn State Research Foundation

US 6964866

Earliest priority – 28 June 2000
Filed – 28 June 2001
Granted – 15 November 2005
Expected expiry – 1 September 2022 (patent term adjustment: 431 days)

Title – Methods and compositions for highly efficient transformation of filamentous fungi

Claim 1
A method of transforming Agaricus bisporus comprising:(a) introducing to fruit body tissue cells of said Agaricus bisporus a polynucleotide construct desired in said Agaricus bisporus, said construct comprising a promoter sequence capable of regulating expression in Agaricus bisporus cells, wherein said introducing is byAgrobacterium-mediated transformation by co-cultivation of vir induced Agrobacterium with Agaricus fruit body tissue cells; and
(b) thereafter regenerating a transgenic Agaricus bisporus from said fruit body tissue cells.

Claim 7
An Agaricus bisporus transformation method comprising:(a) obtaining an Agrobacterium vector, said vector comprising a polynucleotide sequence the presence of which is desired in a recipientAgaricus bisporus cell, said polynucleotide sequence operatively linked to a promoter sequence capable of regulating expression in Agaricus bisporus cells;
(b) introducing said vector to fruit body tissue cells of said Agaricus bisporus in the presence of an active vir Agrobacterium region and a vir inducing compound by co-cultivation of vir induced Agrobacterium with Agaricus fruit body tissue cells; and
(c) regenerating a transgenic Agaricus bisporus from said fruit body cells.

Claim 14
A method for transformation of Agaricus bisporus fruit body tissue cells comprising:(a) obtaining an Agrobacterium vector, said vector comprising a polynucleotide sequence the presence of which is desired in a recipientAgaricus bisporus fruit body tissue cell, said polynucleotide sequence operatively linked to a promoter sequence capable of regulating expression in Agaricus bisporus cells and introducing said vector to said Agaricus bisporus fruit body tissue cells in the presence of an active vir Agrobacterium region and a vir inducing compound by co-cultivation of vir induced Agrobacterium with Agaricus fruit body tissue cells.

Granted United States patent US 6964866 recites an Agrobacterium-mediated transformation method of Agaricus bisporus, where fruit body tissue cells of A. bisporus are introduced to a vir-induced Agrobacterium.

US 2005/70007 A1

Earliest priority – 28 June 2000
Filed – 30 September 2004
Application pending

Title – Methods and compositions for highly efficient transformation of filamentous fungi

Claim 1
A method of transforming filamentous fungi comprising:

introducing to a fruit body tissue cells of said fungi a polynucleotide construct said construct comprising sequences for the expression of a structural gene or for inhibition of an endogenous gene, the presence of which is desired in said fungi cell.

Claim 8
A filamentous fungi transformation method comprising:(a) obtaining an Agrobacterium vector, said vector comprising a polynucleotide sequence the presence of which is desired in a recipient filamentous fungi cell, and
(b) introducing said vector to fruit body tissue cells of said fungi in the presence of an active vir Agrobacterium region and a vir inducing compound.

Claim 10
A method for transforming a fungi cell comprising:(a) obtaining an Agrobacterium derived ti-plasmid, said plasmid comprising a polynucleotide sequence the expression of which is desired in a host fungal cell,
(b) introducing said plasmid to said cell in the presence of vir active genes and a vir inducing agent.

United States patent application US 2005/070007 is a divisional of now granted US 6964866 (see above).

Independent claim 1 in this application recites a method of transforming any filamentous fungus by using fruit tissue body cells that is not limited to Agrobacterium-mediated transformation.