# Patent

## Title
Artificial and mutated nucleotide sequences

## Abstract

The present invention relates to artificial nucleotide sequences, including specific mutations therein.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
Artificial and synthetic DNA sequences have gained extensive use with development of the field of synthetic biology in the past decade. The present invention relates to use of artificial nucleotide sequences in the field of bioluminescence, which is emission of light by living organisms. Bioluminescence of bacterial organisms is mediated by the bacterial LUX operon. The LUX operon encodes for the bacterial luciferase, the light emitting enzyme, as well as enzymes responsible for synthesis of luciferins, substrates required for the light emission reaction. The operon contains genes named C-D-A-B-E(-G), where Lux A and Lux B code for the components of the luciferase and Lux C, D and E code for a fatty acid reductase complex producing an aldehyde necessary for the reaction. LuxG codes for an enzyme thought to participate in the turnover of the second luciferin, the flavin mononucleotide.
2. Description of Related Art
In biotechnology, genes of the LUX operon have a wide range of applications. For instance, the LUX operon is utilized as a reporter in a variety of bacterial and plant biosensors. Bacterial cells of naturally non-glowing species such as E. coli have been engineered to contain the LUX operon inducible by pre-determined classes of chemicals. These cells start glowing in the presence of these specific compounds, reporting on the composition or toxicity of the sample. Plants engineered with a fully functional LUX operon have been contemplated for use as phytosensors, monitoring the conditions of the plant and the environment. Furthermore, ornamental plants have been engineered to contain a LUX operon to produce novel and unique types of glowing ornamental plants. The U.S. ornamentals market was sized at approximately $21B in the early 2000's, and the entire worldwide market for ornamental plants has been estimated to be over $100B.
The ornamental plant market is driven by innovation, where outdated varieties are inevitably replaced by new types of plants and flowers. New colors of roses and carnations, and new shapes and colors of petunias, find their way to the marketplace every year. Generation of new and esthetically pleasing varieties is known to be the key force driving the floriculture industry and stimulating its growth.
However, one of the major limitations of the applicability of LUX operon-based technologies, particularly in plants, is low levels of light emission in plants expressing LUX genes. Therefore means to engineer the LUX operon to enhance and augment plant light emission are needed.
The present invention addresses this problem, and provides several means of enhancing light emission, instrumental in producing new, exciting varieties of highly autoluminescent ornamental plants, as well as additional plant products, such as more effective autoluminescent plant phytosensors.
SUMMARY OF THE INVENTION
The present invention discloses novel artificial DNA sequences, i.e., SEQ ID NOs:1-8 and 11-12, shown in the section entitled “Nucleotide and Amino Acid Sequences of the Invention”, variously encoding for LUX and other polypeptides, useful in enhancing autoluminescence.
In another aspect, the present invention discloses specific mutations in the LuxC and LuxE genes that are highly effective in enhancing light emission in an organism, such as a bacterium or plant, containing these genes in a mutated LUX operon.
More specifically, in its various aspects, the present invention provides:
[1] A nucleic acid construct, comprising the nucleotide sequences shown in SEQ ID NOs: 1-5, operably linked for expression.
[2] A nucleic acid construct, comprising the nucleotide sequences shown in SEQ ID NOs: 1-6, operably linked for expression.
[3] The nucleic acid construct of [1] or [2], further comprising, operably linked for expression, the nucleotide sequence shown in SEQ ID NO:7.
[4] The nucleic acid construct of any one of [1]-[3], further comprising, operably linked for expression, the nucleotide sequence shown in SEQ ID NO:8.
[5] The nucleic acid construct of any one of [1]-[4], which is an expression cassette.
[6] An expression vector, comprising the expression cassette of [5].
[7] A living cell, containing any one or more of the nucleotide sequences shown in SEQ ID NOs:1-8 or 11-12.
[8] The living cell of [7], containing the nucleotide sequences shown in SEQ ID NOs:1-5, operably linked for expression.
[9] The living cell of [7], containing the nucleotide sequences shown in SEQ ID NOs: 1-6, operably linked for expression.
[10] The living cell of [8], further comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8, operably linked for expression.
[11] The living con of [9], further comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8, operably linked for expression.
[12] The living cell of any one of [7]-[11], which is selected from the group consisting of a bacterial cell and a plant cell.
[13] The living cell of [12], which is autoluminescent.
[14] A plant, a cell of which contains said nucleic acid construct, expression cassette, expression vector, or nucleotide sequences of any one of [1]-[6].
[15] The plant of [14], wherein said nucleic acid construct, expression cassette, expression vector, or nucleotide sequences are located in a plastid.
[16] The plant of [15], wherein the plastid is a chloroplast.
[17] The plant of any one of [14]-[16], wherein said nucleotide sequences are expressed.
[18] The plant of [17], which is autoluminescent.
[19] Progeny of the plant of [18].
[20] The progeny of [19], which are produced sexually or asexually.
[21] The progeny of [20], which are produced asexually from cuttings.
[22] A part of said plant or progeny of any one of [14]-[21].
[23] The part of said plant or progeny of [22], which is selected from the group consisting of a protoplast, a cell, a tissue, an organ, a cutting, and an explant.
[24] The part of said plant or progeny of [22], which is selected from the group consisting of an inflorescence, a flower, a sepal, a petal, a pistil, a stigma, a style, an ovary, an ovule, an embryo, a receptacle, a seed, a fruit, a stamen, a filament, an anther, a male or female gametophyte, a pollen grain, a meristem, a terminal bud, an axillary bud, a leaf, a stem, a root, a tuberous root, a rhizome, a tuber, a stolon, a corm, a bulb, an offset, a cell of said plant in culture, a tissue of said plant in culture, an organ of said plant in culture, and a callus.
[25] A method of producing an autoluminescent plant, comprising asexually propagating a cutting of said plant or progeny of any one of [14]-[21].
Further scope of the applicability of the present invention will become apparent from the detailed description and drawings provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
The above and other aspects, features, and advantages of the present invention will be better understood from the following detailed description taken in conjunction with the accompanying drawings, all of which are given by way of illustration only, and are not limitative of the present invention, in which:
FIG. 1 shows an example of enhanced light output of transgenic autoluminescent tobacco containing a combination of artificial LUX sequences SEQ ID NOs:1, 2, 11, 4, 12, and 6 (right panel) vs. autoluminescent tobacco plants containing known wild-type LUX sequences (left panel). Light emission is detected using a ChemiDoc XRS Molecular Imager; inverse images are shown.
FIG. 2 shows the further effect of mutations in structural LUX genes in enhancing light emission of the LUX operon in E. coli. (A) Change of Ala to Gly in LuxC amino acid sequence position 389 (SEQ ID NO:3) enhances light emission; (B) Change of Gln to Glu in LuxE amino acid position 167 (SEQ ID NO:5) used in combination with SEQ ID NO:3 further enhances light emission compared to the use of SEQ ID NO:3 alone. E. coli cultures carrying the mutations imaged using ChemiDoc XRS Molecular Imager. Left panel: culture plates in light; right panel: photographic exposure of the cultures; 100 sec exposure for (A), 10 sec exposure for (B).



NUCLEOTIDE AND AMINO ACID SEQUENCES
SEQ ID NO:1: artificial Lux A nucleotide sequence;
SEQ ID NO:2: artificial Lux B nucleotide sequence;
SEQ ID NO:3: artificial Lux C nucleotide sequence, incorporating Ala Gly mutation at amino acid position 389;
SEQ ID NO:4: artificial Lux D nucleotide sequence;
SEQ ID NO:5: artificial Lux E nucleotide sequence, incorporating Gln Glu mutation at amino acid position 167;
SEQ ID NO:6: artificial Lux G nucleotide sequence;
SEQ ID NO:7: artificial E. coli Fre nucleotide sequence;
SEQ ID NO:8: artificial V. fischeri Yellow Fluorescent Protein nucleotide sequence;
SEQ ID NO:9: amino acid sequence of wild-type Photobacterium leiognathi LuxC protein; 
SEQ ID NO:10: amino acid sequence of wild-type Photobacterium leiognathi LuxE protein;
SEQ ID NO:11: artificial Lux C nucleotide sequence without Ala→Gly mutation at amino acid position 389. Compare to SEQ ID NO:3;
SEQ ID NO:12: artificial Lux E nucleotide sequence without Gln→Glu mutation at amino acid position 167. Compare to SEQ ID NO:5.
Although not listed above, the present invention also encompasses the amino acid sequences of the proteins encoded by the nucleotide sequences listed. Such amino acid sequences can be deduced by, for example, conventional bioinformatics methods, including the use of publicly available and proprietary computer programs designed for this purpose.
DETAILED DESCRIPTION OF THE INVENTION
The following detailed description of the invention is provided to aid those skilled in the art in practicing the present invention. Even so, the following detailed description should not be construed to unduly limit the present invention, as modifications and variations in the embodiments herein discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.
The contents of each of the references cited herein is herein incorporated by reference in its entirety.
Methods and techniques for generating transgenic, transplastomic, and otherwise genetically modified plants are well known in the art.
The use of native LUX genes to produce autoluminescent plants has been previously described in the art. Patent applications by Krichevsky, i.e., WO 2009/017821 and WO 2011/106001, disclose the use of naturally occurring LUX genes in the form of an operon in plastids, and U.S. Pat. No. 7,663,022 by Hudkins prophetically contemplates nuclear expression of LUX genes from separate vectors.
However, none of these references either discloses or suggests the artificial LUX, E. coli Fre, or V. fischeri Yellow Fluorescent Protein (YFP) sequences SEQ ID NOs:1-8 and 11-12 of the present invention. Further, the art does not teach or suggest the mutations in LuxC and LuxE genes disclosed herein.
In one embodiment, LUX operon genes are used in variety of biotechnology applications, which can further benefit from enhancement of light output generated by the LUX operon. For example, the problem of enhancing the light output of the autoluminescent plants disclosed in the above-noted PCT applications and producing brighter glowing plants, which are appealing and attractive to the consumer, is solved by the artificial DNA sequences of the present invention. Expression of these sequences, or combinations thereof, results in autoluminescent plants that are several fold brighter than plants expressing wild-type LUX genes.
Examples of certain preferred combinations of the artificial sequences disclosed herein include, but are not limited to: SEQ ID NOs:1-5 in combination; SEQ ID NOs:1-6 in combination; or further, combination of SEQ ID NOs:1-5 in combination or SEQ ID NOs:1-6 in combination, further in combination with SEQ ID NO:7; and further, such foregoing combinations, further in combination with SEQ ID NO:8. In each of these cases, the nucleotide sequences are operably linked for expression.
One skilled in the art will recognize that the individual sequences disclosed herein can be used in combination, as indicated above, in any order, and are independent of one another.
As used herein, the phrase “operably linked for expression” and the like encompasses nucleic acid sequences linked in the 5′ to 3′ direction in such a way as to facilitate expression of an included nucleotide coding sequence.
The following examples are meant to be illustrative, and not limiting, of the practice or products of the present invention.
Example 1
Enhanced Light Emission by a Combination of SEQ ID NOs:1, 2, 11, 4, 12, and 6
An example of light emission enhancement in transgenic tobacco by a combination of artificial sequences of the present invention, employing SEQ ID NOs: 1, 2, 11, 4, 12, and 6 operably linked for expression, compared to that produced by a combination of wild-type LUX operon genes C-D-A-B-E(-G) operably linked for expession, is shown in FIG. 1. The methods employed to produce these plants are disclosed in the inventor's PCT International Publications WO 2009/017821 and WO 2011/106001.
Artificial sequences SEQ ID NOs:7 and 8, encoding FRE and YFP proteins, respectively, are designed to further improve light output and change the emitted light color, respectively, of the autoluminescent plants encompassed by the present invention.
Example 2
Light Emission Enhancement by LUX Structural Gene Mutants
A number of mutations in the regulatory genes governing expression of the entire LUX operon in bacteria are known in the art. However, mutations in the LUX structural genes, and particularly in LuxC and LuxE, enhancing or otherwise modulating light emission generated by the LUX operon have not been previously identified.
Disclosed herein are two novel mutations in the structural LUX genes C (encoding an Ala→Gly mutation at amino acid position 389) and E (encoding a Gln→Glu mutation at amino acid position 167), which greatly enhance light emission of the LUX operon.
The known sequence of the Photobacterium leiognathi LUX operon (GeneBank #M63594) discloses the following sequences of the wild-type LuxC and LuxE genes:
LuxC (Gene Bank M63594) (SEQ ID NO:9)








MIKKIPMIIGGVVQNTSGYGMRELTLNNNKVNIPIITQSDVEAIQSLNIENKLTINQIVNFLYTVGQKW



 


KSETYSRRLTYIRDLIKFLGYSQEMAKLEANWISMILCSKSALYDIVENDLSSRHIIDEWIPQGECYVK


 


ALPKGKSVHLLAGNVPLSGVTSILRAILTKNECIIKTSSADPFTATALVNSFIDVDAEHPITRSISVMY


 


WSHSEDLAIPKQIMSCADVVIAWGGDDAIKWATEHAPSHADILKFGPKKSISIVDNPTDIKAAAIGVAH


 


DICFYDQQACFSTQDIYYIGDSIDIFFDELAQQLNKYKDILPKGERNFDEKAAFSLTERECLFAKYKVQ


 


KGESQSWLLTQSPAGSFGNQPLSRSAYIHQVNDISEVIPFVHKAVTQTVAIAPWESSFKYRDILAEHGA


 


ERIIEAGMNNIFRVGGAHDGMRPLQRLVNYISHERPSTYTTKDVSVKIEQTRYLEEDKFLVFVP






LuxE (Gene Bank M63594) (SEQ ID NO:10)









MSTLLNIDATEIKVSTEIDDIIFTSSPLTLLFEDQEKIQKELILESFHYHYNHNKDYKYYCNIQGVDEN



 


IQSIDDIPVFPTSMFKYSRLHTADESNIENWFTSSGTKGVKSHIARDRQSIERLLGSVNYGMKYLGEFH


 


EHQLELVNMGPDRFSASNVWFKYVMSLVQLLYPTTFTVENDEIDFEQTILALKAIQRKGKGICLIGPPY


 


FIYLLCHYMKEHNIEFNAGAHMFIITGGGWKTKQKEALNRQDFNQLLMETFSLFHESQIRDIFNQVELN


 


TCFFEDSLQRKHVPPWVYARALDPVTLTPVEDGQEGLMSYMDASSTSYPTFIVTDDIGIVRHLKEPDPF


 


QGTTVEIVRRLNTREQKGCSLSMATSLK






As disclosed herein, substitution of Ala (position 389) in the amino acid sequence of LuxC (SEQ ID NO:9; emphasized in the sequence in 14 point type, bold, and underlined) with, for example Gly, strongly enhances light emission generated by the operon. Moreover, substitution of Gln (position 167) in the amino acid sequence of LuxE (SEQ ID NO:10; emphasized in the sequence in 14 point type, bold, and underlined) with, for example Glu, further increases light emission of E. coli harboring the mutated operons (FIG. 2).
Methods for nucleic acid mutagenesis are known in the art (e.g., Chen et al. (1997) “High efficiency of site-directed mutagenesis mediated by a single PCR product.” Nuc. Acid Res, 25(3): 682-4). Methods for transforming E. coli are well known in the art.
In the particular example shown in FIG. 2, the mutant LuxC gene (SEQ ID NO:3) is first introduced into the operon (SEQ ID NOs: 1-4, 12, and 6) introduced into the bacteria (Panel A), followed by introduction of the mutant LuxE gene (SEQ ID NO:5) into the operon (SEQ ID NOs:1-6) introduced into the bacteria (Panel B). One skilled in the art will appreciate that these mutations can be introduced in any order, and are independent of one another. Each mutation can be used for light emission enhancement separately, in combination with one another, or as indicated above, each can be used separately, or together, in combination with any other mutations or sequences. This includes, for example, the combinations listed above.
This example demonstrates the luminescence-enhancing effect of LuxC Ala (389) substitution to Gly and LuxE Gln (167) substitution to Glu in E. coli. 
Specifically, FIG. 2 shows the further effect of mutations in these structural LUX genes in enhancing light emission of the LUX operon in E. coli. Panel (A) shows that change of Ala to Gly in LuxC amino acid sequence position 389 (SEQ ID NO:3) enhances bacterial light emission; Panel (B) shows that change of Gln to Glu in LuxE amino acid position 167 (SEQ ID NO:5), used in combination with SEQ ID NO:3, further enhances light emission compared to the use of SEQ ID NO:3 alone.
In view of these results, it is fully expected that use of artificial nucleotide sequences encoding LUXC comprising the Ala→Gly mutation, e.g., SEQ ID NO:3, and LUXE, comprising the Gln→Glu mutation, e.g., SEQ ID NO:5, either alone, or together, in combination with the other artificial LUX operon sequences disclosed herein, will produce a similar light-enhancing effect in plants.
As noted above, preferred combinations of the artificial sequences disclosed herein include, but are not limited to: SEQ ID NOs:1-5 in combination; SEQ ID NOs:1-6 in combination; or further, combination of SEQ ID NOs:1-5 in combination or SEQ ID NOs:1-6 in combination, further in combination with SEQ ID NO:7; and further, such foregoing combinations, further in combination with SEQ ID NO:8. In each of these cases, the nucleotide sequences are operably linked for expression.
One skilled in the art can appreciate that substitution of LuxC with other amino acids in position (389) and LuxE in position (167) can result in different modifications of light emission, which can be further used to modulate LUX operon luminescence. Furthermore, depending on the type of bacteria from which the LUX operon is derived, positions of these critical amino acids can be shifted within close proximity to the described residues of P. leiognathi, or located in sequences with high homology to sequences surrounding position (389) in LuxC and position (167) in LuxE.
The artificial DNA sequences of the present invention incorporate the above described LuxC and LuxE mutations (SEQ ID NOs:3 and 5, respectively), designed to further enhance light output of the LUX operon. The utility and applicability of the current invention includes, for example, generating bright autoluminescent plants. Besides applications in ornamental plants, where bright plants are attractive to consumers, the present sequences have utility in producing highly effective plant biosensors emitting light in response to various types of stress or other conditions when operons containing these sequences are under the control of appropriate promoters, e.g., stress-inducible promoters, and are thus useful in agriculture for crop or environmental monitoring.
Plants encompassed by the present invention include both monocots and dicots, ornamentals as well as crop plants. Non-limiting examples include ornamental plants such as petunias, poinsettias, and roses, as well as crop plants such as corn and oil producing palms.
Also encompassed by the present invention are parts of such plants including, for example, a protoplast, a cell, a tissue, an organ, a cutting, and an explant. Such parts further include an inflorescence, a flower, a sepal, a petal, a pistil, a stigma, a style, an ovary, an ovule, an embryo, a receptacle, a seed, a fruit, a stamen, a filament, an anther, a male or female gametophyte, a pollen grain, a meristem, a terminal bud, an axillary bud, a leaf, a stem, a root, a tuberous root, a rhizome, a tuber, a stolon, a corm, a bulb, an offset, a cell of said plant in culture, a tissue of said plant in culture, an organ of said plant in culture, and a callus. The present invention also encompasses progeny, whether produced sexually or asexually, of transgsenic plants of the invention containing sequences disclosed herein.
In regard to methods of propagating autoluminescent plants encompassed by the present invention, methods of propagation and reproduction of such plants are well known in the art, and include both sexual and asexual techniques.
Asexual reproduction is the propagation of a plant to multiply the plant without the use of seeds to assure an exact genetic copy of the plant being reproduced.
Any known method of asexual reproduction which renders a true genetic copy of the plant may be employed in the present invention. Acceptable modes of asexual reproduction include, but are not limited to, rooting cuttings; grafting; explants; budding; apomictic seeds; bulbs; division; slips; layering; rhizomes; runners; corms; tissue culture; nucellar embryos; and any other conventional method of asexual propagation. The present invention encompasses all such methods of propagation and reproduction of plants encompassed by the present invention.
In additional examples, the presently disclosed DNA sequences are further useful in generating more efficient plant research systems, where their autoluminescent properties can be used as a reporter system for gene expression and other scientific assays.
The invention being thus described, it will be recognized that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

## Claims

1. A nucleic acid construct, comprising the nucleotide sequences shown in SEQ ID NOs: 1-5, operably linked for expression.

2. A nucleic acid construct, comprising the nucleotide sequences shown in SEQ ID NOs:1-6, operably linked for expression.

3. The nucleic acid construct of claim 1, further comprising, operably linked for expression, the nucleotide sequence shown in SEQ ID NO:7.

4. The nucleic acid construct of claim 2, further comprising, operably linked for expression, the nucleotide sequence shown in SEQ ID NO:7.

5. The nucleic acid construct of claim 1, further comprising, operably linked for expression, the nucleotide sequence shown in SEQ ID NO:8.

6. The nucleic acid construct of claim 2, further comprising, operably linked for expression, the nucleotide sequence shown in SEQ ID NO:8.

7. The nucleic acid construct of claim 3, further comprising, operably linked for expression, the nucleotide sequence shown in SEQ ID NO:8.

8. The nucleic acid construct of claim 4, further comprising, operably linked for expression, the nucleotide sequence shown in SEQ ID NO:8.

9. The nucleic acid construct of claim 1, which is an expression cassette.

10. The nucleic acid construct of claim 2, which is an expression cassette.

11. The nucleic acid construct of claim 3, which is an expression cassette.

12. The nucleic acid construct of claim 4, which is an expression cassette.

13. The nucleic acid construct of claim 5, which is an expression cassette.

14. The nucleic acid construct of claim 6, which is an expression cassette.

15. The nucleic acid construct of claim 7, which is an expression cassette.

16. The nucleic acid construct of claim 8, which is an expression cassette.

17. A living cell, containing the nucleotide sequences shown in SEQ ID NOs:1-5, operably linked for expression.

18. The living cell of claim 17, comprising the nucleotide sequences shown in SEQ ID NOs:1-6, operably linked for expression.

19. The living cell of claim 17, further comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8, operably linked for expression.

20. The living cell of claim 18, further comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8, operably linked for expression.