Positive selection using glucuronide

CAMBIA holds patents in the United States and Australia that have claims on, among others, methods for selecting transformed cells based on the metabolism of various glucuronides that, when cleaved, could result in promotion of the growth of transformed cells.

Technology overview

GUS gene has been widely used as reporter gene for plant transformation since 1987. The enzyme coded by the GUS gene is β-glucuronidase, which hydrolyzes a wide variety of glucuronides. Therefore, the application of GUS gene can be extended to be used as a positively selectable marker. The utility of β-glucuronidase as selective marker relies on the fact that cells cannot grow on a β-glucuronide carbon source such as a glucuronide disaccharide unless β-glucuronidase is provided to cleave the β-glucuronide bond. The most useful example of such a disaccharide is cellobiuronic acid, which comprises β-glucuronic acid in [1-4] linkage to glucose. Only cells expressing β-glucuronidase can grow on a carbon source consisting only of cellobiuronic acid.

The positive selection system based on glucuronide metabolism is not limited to plant cells and can be applied to any host cells that do not have endogenous β-glucuronidase activity.

Patent information concerning selection using glucuronide

Patent/Application Number

Title, Independent Claims and Summary

Assignee
US 6641996

  • Earliest priority – 09 Sep 1997
  • Filed – 17 Mar 1999
  • Granted – 04 Nov 2003
  • Expected expiry – 08 Sep 2018

Title – Microbial β-glucuronidase genes, gene products and uses thereof

Claim 1
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.
Claim 2
An isolated nucleic acid molecule that encodes one of the amino acid sequences of SEQ ID NOs.: 19-21 (192021), 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.
Claim 3
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.
Claim 11
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 (192021), 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.
Claim 13
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.
Claim 14
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.
Claim 15
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.

Claim 18
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.

CAMBIA

US 2003/229921 A1

  • Earliest priority – 09 Sep 1997
  • Filed – 12 Feb 2003
  • Granted – Pending
  • Expected expiry – N/A
Title – Microbial β-glucuronidase genes, gene products and uses thereof

Claim 1
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.
Claim 7
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.
Claim 9
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.
Claim 10
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.
Claim 11
An isolated nucleic acid molecule encoding a fusion protein of a microbial β-glucuronidase or an enzymatically active portion thereof and a second protein.
Claim 13
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.
Claim 32
An isolated form of recombinant microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase.
Claim 38
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.
Claim 39
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 comprisinga 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.

Claim 40
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.
Claim 43
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.
Claim 45
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.
Claim 46
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.
Claim 47
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.
Claim 48
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.
Claim 50
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.
Claim 51
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.
Claim 54
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.

This application is a Continuation of US 6641996 and the patent will soon be granted. The claim of interest is claim 40.

AU 775238 B2

  • Earliest priority – 17 Mar 1999
  • Filed – 16 Mar 2000
  • Granted – 22 Jul 2004
  • Expected expiry – 16 Mar 2020

Title – Microbial β-glucuronidase genes, gene products and uses thereof

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.

Another related patent with claims to use of a β-glucuronidase in positive selection has issued in Australia (760275).

Remarks

Related patent applications were also filed in Europe (EP 1175495 A1), New Zealand (NZ 503020), Japan (JP 2001515724) and Canada (CA 2303423).  A related patent (application published as WO 2000/055333) has been issued in New Zealand as Patent Number 1485.  A related patent (application published as WO 1999/13085) has been issued in the US (7087420) and is pending in Australia, Canada, Europe and further applications are pending in the United States as application numbers 10/120, 145 and 10/364, 649 (both recently allowed and soon to issue) in Brazil, Canada, Europe and Israel.

A patent on producing a substrate for glucuronidase positive selection issued in the United States as Patent Number 6268493 is shown in the table below.

Patent information for cellobiuronic acid preparation

Patent/Application Number

Title, Independent Claims and Summary

Assignee
US 6268493

  • Earliest priority – 07 Aug 1998
  • Filed – 07 Aug 1998
  • Granted – 31 Jul 2001
  • Expected expiry – 07 Aug 2018
Title – Preparation of cellobiuronic acid from polysaccharide

Claim 1
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.
Claim 17
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.
Claim 26
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.

CAMBIA

AU 764652 B2

  • Earliest priority – 07 Aug 1998
  • Filed – 06 Aug 1999
  • Granted – 28 Aug 2003
  • Expected expiry – 06 Aug 2019
Title – Preparation of cellobiuronic acid from polysaccharide

Claim 1A 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.
Claim 18A 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.
Claim 27
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.

Remarks

The related PCT application was filed as WO 2000/08039.