Positive selection using glucuronide

An isolated nucleic acid molecule comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO:14) or a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase.

An isolated nucleic acid molecule that encodes one of the amino acid sequences of SEQ ID NOs.: 19-21 (19, 20, 21), or a variant thereof wherein the variant has at least 75% amino acid identity to one of SEQ ID NOs.: 19-21 and which encodes a functional β-glucuronidase.

An expression vector comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, wherein the β-glucuronidase is encoded by a nucleic acid molecule comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO: 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase.

An expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, wherein the microbial β-glucuronidase comprises one of the amino acid sequences of SEQ ID NOs.: 19-21 (19. 20, 21), or variant thereof, wherein the variant has at least 75% amino acid identity to one of SEQ ID NOs.: 19-21, and which encodes a functional β-glucuronidase.

A method for monitoring expression of a gene of interest or a portion thereof in a host cell, comprising: (a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid molecule comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO: 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO: 14) and which encodes functional β-glucuronidase and a nucleic acid molecule encoding a product of the gene of interest; wherein the β-glucuronidase and the gene of interest are co-expressed; (b) detecting the presence of the microbial β-glucuronidase, thereby monitoring expression of the gene of interest.

A method for transforming a host cell with a gene of interest or portion thereof, comprising: (a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid sequence comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO: 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase, and a nucleic acid sequence encoding a product of the gene of interest, such that the vector construct integrates into the genome of the host cell; wherein the β-glucuronidase and the gene of interest are co-expressed; (b) detecting the presence of the microbial β-glucuronidase, thereby establishing that the host cell is transformed.

A method for positive selection for a transformed cell, comprising: (a) introducing into a host cell a vector construct, the vector construct comprising a nucleic acid sequence comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO: 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase; (b) exposing the host cell to a sample comprising a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that an aglycone is released, wherein the aglycone is required for growth of the host cell; wherein a host cell that expresses the β-glucuronidase grows, thereby positively selecting a transformed cell.

Claim 17

An isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 22, or a variant thereof wherein the variant has at least 90% amino acid identity to SEQ ID NO: 22 and which encodes a functional β-glucuronidase.

An expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, wherein the microbial β-glucuronidase comprises the amino acid sequence of SEQ ID NO: 22, or variant thereof, wherein the variant has at least 90% amino acid identity to SEQ ID NO: 22, and which encodes a functional β-glucuronidase.

An isolated nucleic acid molecule consisting essentially of a nucleotide sequence that encodes a microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase.

An isolated nucleic acid molecule encoding a thermostable β-glucuronidase, wherein the β-glucuronidase has a half-life of at least 10 min at 65° C.

An isolated nucleic acid molecule encoding a microbial β-glucuronidase, wherein the β-glucuronidase converts at least 50 nmoles of p-nitrophenyl-glucuronide to p-nitrophenyl per minute per μg of protein at 37° C.

An isolated nucleic acid molecule encoding a microbial β-glucuronidase, wherein the β-glucuronidase retains at least 80% of its activity in 10 mM glucuronic acid.

An isolated nucleic acid molecule encoding a fusion protein of a microbial β-glucuronidase or an enzymatically active portion thereof and a second protein.

An expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase.

An isolated form of recombinant microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase.

A method for monitoring expression of a gene of interest or a portion thereof in a host cell, comprising:

    (a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid molecule according to claim 1 and a nucleic acid molecule encoding a product of the gene of interest or a portion thereof;    

    (b) detecting the presence of the microbial glucuronidase, thereby monitoring expression of the gene of interest.

A method for transforming a host cell with a gene of interest or portion thereof, comprising:

    (a) introducing into the host cell a vector construct, the vector construct comprising

        a nucleic acid sequence encoding a microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase, and

        a nucleic acid sequence encoding a product of the gene of interest or a portion thereof, such that the vector construct integrates into the genome of the host cell;

    (b) detecting the presence of the microbial β-glucuronidase, thereby establishing that the host cell is transformed.

A method for positive selection for a transformed cell, comprising:

(a) introducing into a host cell a vector construct, the vector construct comprising-nucleic acid sequence encoding a microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli p-glucuronidase;

(b) exposing the host cell to the sample comprising a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that the compound is released, wherein the compound is required for cell growth.

A method of producing a transgenic plant that expresses a microbial β-glucuronidase, comprising:

    (a) introducing an expression vector comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase, into an embryogenic plant cell; and

    (b) producing a plant from the embryogenic plant cell, wherein the plant expresses the β-glucuronidase.

A method for positive selection for a transformed cell, comprising:

    (a) introducing into a host cell a vector construct, the vector construct comprising nucleic acid sequence encoding a microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase;

    (b) exposing the host cell to the sample comprising a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that the compound is released, wherein the compound is required for cell growth.

A transgenic plant cell comprising an expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase.

A transgenic plant comprising an expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase.

A transgenic aquatic animal cell comprising an expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter.

A transgenic aquatic animal comprising an expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter.

A method for identifying a microorganism that secretes β-glucuronidase, comprising:

    (a) culturing the microorganism in a medium containing a substrate for β-glucuronidase, wherein the cleaved substrate is detectable, and wherein the microorganism is an isolate of a naturally occurring microorganism or a transgenic microorganism; and
    (b) detecting the cleaved substrate in the medium; therefrom identifying an organism that secretes β-glucuronidase.

A method for providing an effector compound to a cell in a transgenic plant, comprising:

    (a) growing a transgenic plant that comprises an expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter and a nucleic acid sequence comprising a gene encoding a cell surface receptor for an effector compound.
    (b) exposing the transgenic plant to a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that the effector compound is released.

Claim 1

An isolated nucleic acid molecule that encodes a microbial β-glucuronidase, comprising nucleotides 1-1689 of Figures 4I-J (SEQ ID NO.14) or a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of Figure 4I-J (SEQ ID NO.14) and which encodes a functional β-glucuronidase.

Claim 16

An isolated form of recombinant microbial β-glucuronidase, wherein the microbial β-glucuronidase comprises the amino acid sequence presented in Figure 3B panel E (SEQ ID NO: 6) or a variant thereof having at least 75% amino acid sequence identity thereto.

Claim 17
A method for monitoring expression of a gene of interest or a portion thereof in a host cell, comprising:

(a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid molecule according to claims 1 or 2 and a nucleic acid sequence encoding a product of the gene of interest or a portion thereof;
(b) detecting the presence of the microbial β-glucuronidase, thereby monitoring expression of the gene of interest.

Claim 18
A method for transforming with a host cell a gene of interest or a portion thereof, comprising:

(a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid sequence according to claims 1 or 2 and a nucleic acid sequence encoding a product of the gene of interest or a portion thereof such that the vector construct integrates into the genome of the host cell; and
(b) detecting the presence of the microbial β-glucuronidase, thereby establishing that the host cell is transformed.

Claim 19
A method for positive selection for a transformed cell, comprising:

(a) introducing into a host cell a vector construct, the vector construct comprising nucleic acid sequence according to claims 1 or 2; and
(b) exposing the host cell to the sample comprising a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that the compound is released, wherein the compound is required for cell growth.

Claim 22
A method of producing a transgenic plant that expresses a microbial β-glucuronidase, comprising:
(a) introducing into an embryogenic plant cell an expression vector comprising a nucleic acid sequence according to claims 1 or 2 in operative linkage with a heterologous promoter; and
(b) producing a plant from the embryogenic plant cell, wherein the plant expresses the β-glucuronidase.

Claim 28
A method for providing an effector compound to a cell of a transgenic plant, comprising:
(a) growing a transgenic plant that comprises an expression vector, comprising nucleic acid sequence encoding a microbial β-glucuronidase according to claims 1 or 2 in operative linkage with a heterologous promoter and a nucleic acid sequence comprising a gene encoding a cell surface receptor for an effector compound; and
(b) exposing the transgenic plant to a glucuronide, wherein the glucuronide in cleaved by the β-glucuronidase, such that the effector compound is released.

A method of preparing cellobiuronic acid comprising: exposing gellan gum to partially hydrolyzing conditions to produce a hydrolysate comprising anionic saccharides, the anionic saccharides comprising at least 50 wt. % cellobiuronic acid; and isolating the cellobiuronic acid by anion exchange chromatography.

A method of hydrolyzing gellan gum comprising (a) contacting gellan gum with a hydrolyzing agent selected from the group consisting of acid, base and hydrolytic enzyme, under conditions that provide a hydrolysate comprising anionic saccharides, the anionic saccharides comprising at least 50 wt. % cellobiuronic acid, where the cellobiuronic acid is present in the hydrolysate at a concentration of at least 5 wt. % based on the total weight of gellan gum, and wherein the gellan gum has not previously been subjected to oxidizing conditions; and (b) isolating an anionic fraction comprising cellobiuronic acid from a neutral fraction comprising one or more monosaccharides using anion exchange chromatography.

A method of hydrolyzing gellan gum comprising contacting gellan gum with an aqueous composition having a pH between 2 and 7, under conditions effective to partially hydrolyze the gellan to form a hydrolysate comprising anionic saccharides, the anionic saccharides comprising at least 50 wt. % cellobiuronic acid, isolating the cellobiuronic acid from neutral saccharides by anion exchange chromatography, and separating the cellobiuronic acid from water.

A method of preparing cellobiuronic acid comprising:
    exposing gellan to partially hydrolyzing conditions to produce a hydrolysate comprising saccharides, the saccharides comprising cellobiuronic acid; and
    isolating the cellobiuronic acid.

A method of hydrolyzing gellan comprising contacting gellan with a hydrolyzing agent selected from the group consisting of acid, base and hydrolytic enzyme, under conditions that provide a hydrolysate comprising disaccharides and monosaccharides, wherein the disaccharid comprises cellobiuronic acid, and the cellobiuronic acid is present in the hydrolysate at a concentration of at least 5 wt. % based on the total weight of gellan, and wherein the gellan has not previously been subjected to oxidizing conditions.

A method of hydrolyzing gellan comprising contacting gellan with an aqueous composition having a pH between 2 and 7, under conditions effective to partially hydrolyze the gellan to cellobiuronic acid and separate the cellobiuronic acid from water.