News Column

Patent Issued for Method of Modifying the Carbohydrate Content of a Plant

June 4, 2014



By a News Reporter-Staff News Editor at Biotech Week -- A patent by the inventors Boussiengui-Boussiengui, Gino (Libreville, GA); Kossmann, Jens (Somerset West, ZA), filed on September 17, 2010, was published online on May 20, 2014, according to news reporting originating from Alexandria, Virginia, by NewsRx correspondents (see also Stellenbosch University).

Patent number 8729338 is assigned to Stellenbosch University (Stellenbosch, ZA).

The following quote was obtained by the news editors from the background information supplied by the inventors: "Sucrose, harvested primarily from sugarcane, is both a major human food as well as an important feedstock for fuel ethanol production. The commercial production of sugar may therefore benefit by either increasing the sugar content of the crop or increasing cane yield. Since increasing the sugar content of sugarcane results in increased sugar yields with a relatively small increase in associated production costs, gains in sugar content are viewed as economically more beneficial than corresponding increases in cane yield. This means that increased sugar content has become an important objective of sugarcane breeding programs and transgenic research using biotechnology-based approaches.

"Many biotechnology-based approaches for increasing the reduced carbon or nitrogen content of crops are directed towards increasing the concentrations of direct precursors of the target product (Sonnewald et al., 1997; Stark et al., 1992), These approaches tend to focus on increasing the concentration of the relevant metabolites, however have never resulted in significant increases of the target metabolite in a storage organ.

"A need therefore exists for an alternative method of modifying the carbohydrate content of crops such as sugar cane, and for crops such as sugar cane with a relatively higher sugar content."

In addition to the background information obtained for this patent, NewsRx journalists also obtained the inventors' summary information for this patent: "According to the invention, there is provided a method of increasing the content of at least one carbohydrate in one or more plant cells, the method including inhibiting UMP synthase (UMPS) activity in the one or more plant cells.

"In some embodiments, the method of inhibiting UMPS activity will include the step of inserting into one or more plant cells at least one gene silencing cassette. Expressing the gene silencing cassette(s) thereby results in decreased UMP synthase activity and increased content of at least one carbohydrate (e.g. sucrose). Decreased UMPS activity may be brought about by various mechanisms (see e.g. discussion in the detailed description below regarding methods for reducing UMPS expression).

"In some embodiments, the one or more gene silencing cassettes will be inserted into a population of cells (e.g. a callus).

"In some embodiments, a transgenic plant is regenerated from one or more plant cells produced as described above. Plant tissue and plant parts (for example, seeds, storage organs etc.) may be harvested from the plant.

"In some embodiments, plant tissue having an increased content of at least one carbohydrate is produced from one or more plant cells transformed as described above. This may optionally comprise regenerating a transgenic plant from one or more transformed plant cells. The term 'tissue' is used herein in a broad sense and includes a reference to an aggregate of cells. The cells of the tissue may be differentiated or undifferentiated. In some embodiments the cells may have a similar structure and/or function. In some embodiments the cells may be specialized to perform a particular function. In one embodiment, the tissue is a callus.

"In some embodiments, one or more plant cells are transformed with one or more gene silencing cassette(s) which include nucleic acid operably linked to one or more transcription elements for transcribing the nucleic acid wherein transcription of the nucleic acid decreases activity of UMP synthase.

"Optionally the nucleic acid comprises (and optionally consists of) one or more of the following: (i) the antisense sequence of a UMPS ORF (e.g. the nucleotide sequence of SEQ ID NO 1), or part thereof (e.g. at least 10, 15, 20, 25, 30, 40, 50, 70, 100, 150, 200, 300, 400, 500, 600, 700 nucleotides of the sequence), (ii) a nucleotide sequence at least 80%, 85%, 90%, 95%, or 99% similar, to the antisense sequence of a UMPS ORF (e.g. SEQ ID NO 1), or to part thereof, (iii) the sense sequence of a UMPS ORF (e.g. the nucleotide sequence of SEQ ID NO 2), or part thereof, (iv) a nucleotide sequence at least 80%, 85%, 90%, 95%, or 99% similar, to the sense sequence of a UMPS ORF (e.g. SEQ ID NO 2), or to part thereof, (v) (a) (i) or (ii) as above, and (b) (iii) or (iv) as above, optionally with a spliceable intron sequence between (a) and (b), and wherein the spliceable intron sequence is preferably at least 70 or 74 bp in length, preferably at least 100 bp, 200 bp, 300 bp, 400 bp, 450 bp, or 500 bp in length, and more preferably at least 74 bp in length.

"In at least some embodiments, the UMPS ORF corresponds to the UMPS ORF of the transformed plant cell. Thus, for instance, where the plant cell is a sugarcane plant cell the UMPS ORF is the ORF of sugarcane and thus, for example, in accordance with (i) above, the nucleic acid comprises the antisense sequence of a sugarcane UMPS ORF, or part thereof.

"In some embodiments, two or more gene silencing cassettes may be inserted into one or more plant cells, e.g. a first gene silencing cassette with nucleic acid according to (i) or (ii) above, and a second gene silencing cassette with nucleic acid according to (iii) or (iv) above. Thus, embodiments are envisaged wherein sense and antisense molecules are expressed from single or separate gene silencing cassettes. Such molecules may be used in double-stranded RNA interference whereby a sense and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule of a UMPS gene are expressed in the same plant cell, resulting in inhibition of the expression of the corresponding endogenous mRNA encoding UMPS.

"In some embodiments, the one or more transcription elements include a promoter, optionally a monocotyledonous promoter (e.g. a UBI promoter).

"In some embodiments, the at least one carbohydrate is selected from the group consisting of sucrose, starch, glucose, and fructose. In some embodiments, the method results in an increased content of both starch and sucrose and optionally one or more further carbohydrates (e.g. glucose).

"In some embodiments, the plant is a sugar-storing plant, optionally selected from the group including sugarcane, sweet sorghum, and sugar beet. In some embodiments the plant is of the genus Saccharum, optionally sugarcane.

"Plant cells, plant tissue, plants and plant parts (e.g. seeds, storage organs, sugar cane) obtained or obtainable by the method described above are included within the scope of the invention.

"According to the invention there is further provided a vector comprising one or more gene silencing cassettes for decreasing UMPS activity in a plant cell.

"In some embodiments the vector is a RNAi vector.

"In some embodiments, the gene silencing cassette(s) include nucleic acid operably linked to one or more transcription elements for transcribing the nucleic acid wherein transcription of the nucleic acid decreases activity of UMPS.

"Optionally the nucleic acid comprises (and optionally consists of) one or more of the following: (i) the antisense sequence of a UMPS ORF (e.g. the nucleotide sequence of SEQ ID NO 1), or part thereof (e.g. at least 10, 15, 20, 25, 30, 40, 50, 70, 100, 150, 200, 300, 400, 500, 600, 700 nucleotides of the sequence), (ii) a nucleotide sequence at least 80%, 85%, 90%, 95%, or 99% similar, to the antisense sequence of a UMPS ORF (e.g. SEQ ID NO 1), or to part thereof, (iii) the sense sequence of a UMPS ORF (e.g. the nucleotide sequence of SEQ ID NO 2), or part thereof, (iv) a nucleotide sequence at least 80%, 85%, 90%, 95%, or 99% similar, to the sense sequence of a UMPS ORF (e.g. SEQ ID NO 2), or to part thereof, (v) (a) (i) or (ii) as above, and (b) (iii) or (iv) as above, optionally with a spliceable intron sequence between (a) and (b), and wherein the spliceable intron sequence is preferably at least 70 or 74 bp in length, preferably at least 100 bp, 200 bp, 300 bp, 400 bp, 450 bp, or 500 bp in length, and more preferably at least 74 bp in length.

"In some embodiments, the one or more transcription elements include a promoter, optionally a monocotyledonous promoter (e.g. a UBI promoter).

"In some embodiments, the at least one carbohydrate is selected from the group consisting of sucrose, starch, glucose, and fructose. In some embodiments, the method results in an increased content of both starch and sucrose and optionally one or more further carbohydrates (e.g. glucose).

"In some embodiments, the plant is a sugar-storing plant, optionally selected from the group including sugarcane, sweet sorghum, and sugar beet. In some embodiments the plant is of the genus Saccharum, optionally sugarcane.

"According to the invention there is also provided a transformed plant cell which includes a vector comprising one or more gene silencing cassettes for decreasing the activity of UMPS in a plant cell.

"In some embodiments the vector is a RNAi vector.

"In some embodiments, the gene silencing cassette(s) include nucleic acid operably linked to one or more transcription elements for transcribing the nucleic acid wherein transcription of the nucleic acid decreases activity of UMPS.

"Optionally the nucleic acid comprises (and optionally consists of) one or more of the following: (i) the antisense sequence of a UMPS ORF (e.g. the nucleotide sequence of SEQ ID NO 1), or part thereof (e.g. at least 10, 15, 20, 25, 30, 40, 50, 70, 100, 150, 200, 300, 400, 500, 600, 700 nucleotides of the sequence), (ii) a nucleotide sequence at least 80%, 85%, 90%, 95%, or 99% similar, to the antisense sequence of a UMPS ORF (e.g. SEQ ID NO 1), or to part thereof, (iii) the sense sequence of a UMPS ORF (e.g. the nucleotide sequence of SEQ ID NO 2), or part thereof, (iv) a nucleotide sequence at least 80%, 85%, 90%, 95%, or 99% similar, to the sense sequence of a UMPS ORF (e.g. SEQ ID NO 2), or to part thereof, (v) (a) (i) or (ii) as above, and (b) (iii) or (iv) as above, optionally with a spliceable intron sequence between (a) and (b), and wherein the spliceable intron sequence is preferably at least 70 or 74 bp in length, preferably at least 100 bp, 200 bp, 300 bp, 400 bp, 450 bp, or 500 bp in length, and more preferably at least 74 bp in length.

"In some embodiments, the one or more transcription elements include a promoter, optionally a monocotyledonous promoter (e.g. a UBI promoter).

"In some embodiments, the at least one carbohydrate is selected from the group consisting of sucrose, starch, glucose, and fructose. In some embodiments, the method results in an increased content of both starch and sucrose and optionally one or more further carbohydrates (e.g. glucose).

"In some embodiments, the plant is a sugar-storing plant, optionally selected from the group including sugarcane, sweet sorghum, and sugar beet. In some embodiments the plant is of the genus Saccharum, optionally sugarcane.

"According to the invention there is further provided a transgenic plant, plant part or plant tissue containing or derived from a transformed plant cell which includes a vector comprising one or more gene silencing cassettes for decreasing the activity of UMPS in a plant cell.

"In some embodiments the vector is a RNAi vector.

"In some embodiments, the gene silencing cassette(s) include nucleic acid operably linked to one or more transcription elements for transcribing the nucleic acid wherein transcription of the nucleic acid decreases activity of UMPS.

"Optionally the nucleic acid comprises (and optionally consists of) one or more of the following: (i) the antisense sequence of a UMPS ORF (e.g. the nucleotide sequence of SEQ ID NO 1), or part thereof (e.g. at least 10, 15, 20, 25, 30, 40, 50, 70, 100, 150, 200, 300, 400, 500, 600, 700 nucleotides of the sequence), (ii) a nucleotide sequence at least 80%, 85%, 90%, 95%, or 99% similar, to the antisense sequence of a UMPS ORF (e.g. SEQ ID NO 1), or to part thereof, (iii) the sense sequence of a UMPS ORF (e.g. the nucleotide sequence of SEQ ID NO 2), or part thereof, (iv) a nucleotide sequence at least 80%, 85%, 90%, 95%, or 99% similar, to the sense sequence of a UMPS ORF (e.g. SEQ ID NO 2), or to part thereof, (v) (a) (i) or (ii) as above, and (b) (iii) or (iv) as above, optionally with a spliceable intron sequence between (a) and (b), and wherein the spliceable intron sequence is preferably at least 70 or 74 bp in length, preferably at least 100 bp, 200 bp, 300 bp, 400 bp, 450 bp, or 500 bp in length, and more preferably at least 74 bp in length.

"In some embodiments, the one or more transcription elements include a promoter, optionally a monocotyledonous promoter (e.g. a UBI promoter).

"In some embodiments, the at least one carbohydrate is selected from the group consisting of sucrose, starch, glucose, and fructose. In some embodiments, the method results in an increased content of both starch and sucrose and optionally one or more further carbohydrates (e.g. glucose).

"In some embodiments, the plant is a sugar-storing plant, optionally selected from the group including sugarcane, sweet sorghum, and sugar beet. In some embodiments the plant is of the genus Saccharum, optionally sugarcane.

"In some embodiments, the plant is of the genus Saccharum, optionally sugarcane, and the transgenic plant part a callus.

"The term 'carbohydrate' as used herein refers to energy-producing organic compounds such as starches and sugars.

"The term 'UMP Synthase' as used herein refers to an enzyme capable of catalyzing the final two steps of de novo pyrimidine biosynthesis and having both orotate phosphoribosyl transferase and orotidylate decarboxylase activity."

URL and more information on this patent, see: Boussiengui-Boussiengui, Gino; Kossmann, Jens. Method of Modifying the Carbohydrate Content of a Plant. U.S. Patent Number 8729338, filed September 17, 2010, and published online on May 20, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=83&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=4142&f=G&l=50&co1=AND&d=PTXT&s1=20140520.PD.&OS=ISD/20140520&RS=ISD/20140520

Keywords for this news article include: Synthase, Sugar Cane, Agriculture, Enzymes and Coenzymes, Stellenbosch University.

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