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Alcohol-regulated promoters

Scientific aspects

The promoter system in its natural environment

In a bacterial positive-mode operon or positive operon the genes are off by default and are turned on by activators. The interaction between a regulatory protein and a regulatory region on the DNA to which it binds turns transcription on.

The ethanol utilization pathway of the filamentous fungus Aspergillus nidulans is a well-characterized positive operon system. It controls the cellular response to ethanol and other related chemicals. The first enzyme in the pathway of ethanol utilization is alcohol dehydrogenase I (Adh-I) encoded by the alcA gene. The transcriptional activator protein AlcRbinds target sequences within the alcA gene promoter in the presence of ethanol, ethyl methyl ketone or other alcohols/ketones. These compounds act as inducers of the gene expression.

A promoter system transferred into plants

One system transferred into plants provides the alcR gene under the control of a strong constitutive promtoer such as CaMV 35S, and a modfied alcA promoter linked to a gene of interest or target gene. The modified or chimeric alcA promoter (referred to as the target promoter) comprises the regulatory sequence of the alcA promoter which is bound by AlcR and a core promoter region (a TATA box and a transcription start site) of a plant-expressible gene promoter. In the presence of an inducer (e.g., an alcohol or ketone), the transactivator protein AlcR binds to the specific sequences of the modified alcA promoter and the target gene is expressed.

Among the advantages of the system are:

  • the lack of an equivalent alcR gene in plants, which rules out interference with plant endogenous Adh activity,
  • the chemical inducers are relatively simple molecules with low toxicity, and
  • under normal growth conditions of a plant, the levels of natural inducers, (e.g., ethanol) that are produced when plants are waterlogged or under artificial anoxia are very low and do not induce alcA expression.

The alcohol-inducible system can be used for:

  • production of high levels of recombinant proteins, especially those that interfere with growth or biomass production,
  • providing an inducible male sterility system, and
  • providing inducible expression of resistance genes.

Nevertheless, there are still some difficulties in using the system for agricultural purposes. Specifically, the means to introduce the inducer, e.g., by spraying or drenching, and its volatility, impact the effectiveness of the system for field work.

IP issues

Syngenta has patents related to alcohol-inducible expression systems for plants.  Its patents were granted in United States and Europe.

Approximate scope of protection

The only patent grant is in Europe. The patent claims:

  • a chemically-inducible plant gene expression cassette that contains:
    • a promoter linked to an AlcR encoding gene, and
    • an inducible promoter linked to a gene of interest.
  • a method for controlling plant gene expression by using the mentioned expression cassette, and
  • a chimeric chemically-inducible promoter containing:
    • regulatory sequences of the alcA gene promoter, and
    • a transcription initiation sequence from a plant-expressible gene promoter.

Note that the inducible promoter of the expression cassette is not restricted to any particular promoter and may be other than alcA (claim 1). The promoter is activatable by the regulatory protein (AlcR protein) in the presence of an effective inducer. The inducible promoter may be derived from aldehyde dehydrogenase gene or other alcohol dehydrogenase genes that are involved in the ethanol utilization pathway.

The transcription initiation sequence of the chimeric chemically-inducible promoter refers to the CAAT box, TATA box and surrounding sequences that define the transcription start point for the structural gene.

Specific Patent Information

Patent Number Title, Summary of Claims and Independent Claims Assignee
EP 637339 B1

  • Earliest priority -13 April 1992
  • Filed – 13 April 1993
  • Granted – 31 October 2001
  • Expected expiry – 13 April 2013
 Title – DNA constructs and plants incorporating them

Claim 1
A chemically-inducible plant gene expression cassette comprising
a first promoter operatively linked to the alcR regulator sequence obtainable from Aspergillus nidulans which encodes the AlcR regulator protein, and
an inducible promoter operatively linked to a target gene, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer whereby application of the inducer causes expression of the target gene.
Claim 11
A method for controlling plant gene expression comprising
transforming a plant cell with a chemically-inducible plant gene expression cassette which has
a first promoter operatively linked to the alcR regulator sequence obtainable from Aspergillus nidulans which encodes the AlcR regulator protein, and
an inducible promoter operatively linked to a target gene, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer whereby application of the inducer causes expression of the target gene.
Claim 12
A chimeric promoter comprising
an upstream region containing a promoter regulatory sequence obtainable from the alcA gene promoter of Aspergillus nidulans and
a downstream region containing a transcription initiation sequence, characterised in that said upstream and downstream regions are heterologous, the promoter is chemically inducible and the transcription initiation sequence is obtainable from the core promoter region of a promoter which is active in plant cells.

The claims are generally to:

  • A chemically-inducible expression cassette comprising a first promoter linked to the alcR sequence and an inducible promoter linked to a target gene. The AlcR protein activates the inducible promoter in the presence of an inducer and causes the expression of the gene.
  • A chimeric promoter comprising an upstream region with a chemically-inducible alcA gene promoter and a downstream region with a transcription initiation sequence from a core plant-expressible promoter region, in which the two regions are heterologous.
  • A method for controlling plant gene expression by transforming a plant with an expression cassette as described.

Syngenta Ltd.
US 6605754

  • Earliest priority -13 April 1992
  • Filed – 13 March 1998
  • Granted 12 August 2003
  • Expected expiry – 12 March 2018
 Title – DNA constructs and plants incorporating them

Claim 1
A plant cell which contains stably integrated into its genome a gene expression cassette, said gene expression cassette comprising
a first promoter operatively linked to a sequence comprising an alcR coding sequence from Aspergillus nidulans and which encodes an ALCR regulator protein, and
an inducible promoter from an ALCR-activatable gene, which gene is the alcA gene from Aspergillus nidulans, operatively linked to a target gene, said inducible promoter being activated by the ALCR regulator protein in the presence of an alcohol and/or ketone inducer, so that application of a sufficient amount of a suitable inducer causes expression of the target gene.
Claim 7
A method for controlling plant gene expression comprising
transforming a plant cell with a chemically-inducible gene expression cassette which has
a first promoter operatively linked to a sequence comprising an alcR coding sequence from Aspergillus nidulans, and which encodes an ALCR regulator protein, and
an inducible promoter from an ALCR-activatable gene, which gene is the alcA gene from Aspergillus nidulans, operatively linked to a target gene, said inducible promoter being activated by the ALCR regulator protein in the presence of an alcohol and/or ketone inducer, so that application of a sufficient amount of a suitable inducer causes expression of the target gene.
Claim 8
A plant cell containing a chimeric promoter operatively linked to a target gene, said chimeric promoter comprising
an upstream region containing a promoter from an ALCR-activatable alcA gene from Aspergillus nidulans and     a downstream region containing a transcription initiation sequence, wherein the upstream region and the downstream region are heterologous and the chimeric promoter is inducible by an alcohol and/or ketone, so that application of a sufficient amount of a suitable inducer, in the presence of the ALCR regulator protein encoded by the alcR gene from Aspergillus nidulans, causes expression of the target gene.

The claims of this US patent is genearlly directed to:

  • Plant cells containing a chemically-inducible plant gene expression cassette or chimeric promoter operatively linked to a target gene as described in EP 637339 B1.
  • A method for controlling plant gene expression by transforming a plant cell with the chemically-inducible gene expression cassette as described so that gene expression is controlled by an alcohol and/or ketone inducer.

Remarks

 The application in Australia (AU 39019/93 A1) has lapsed.

Note: Patent information was last updated on 11 May 2006. Search terms: “promoter” in abstract and “Syngenta” in application. Patent database: PatentLens in combination with INPADOC.