WO2021219844A1

WO2021219844A1 - Metabolic engineering of plants enriched in l-dopa

Info

Publication number: WO2021219844A1
Application number: PCT/EP2021/061385
Authority: WO
Inventors: Cathie MARTIN; Eugenio Butelli; Dario BREITEL
Original assignee: John Innes Centre
Priority date: 2020-05-01
Filing date: 2021-04-30
Publication date: 2021-11-04
Also published as: GB202006463D0

Abstract

The present invention relates to a method of producing L-DOPA in plants, and in particular, in non-betalain-producing plants. The invention also relates to plants obtained by the method, as well as the fruits thereof, and pharmaceutical compositions comprising L-DOPA obtained from the plants of the invention.

Description

Metabolic engineering of plants enriched in L-DOPA

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

L-DOPA, also known as Levodopa or L-3,4-dihydroxyphenylalanine, has been the gold standard therapy for Parkinson’s Disease (PD), since its establishment as a drug in 1967. It is one of the essential medicines, as declared by the World Health Organisation (WHO Model List, Essential Medicines, 19th edition, April 2015). The market value of L-DOPA was 101 billion dollars and 250 tons per year, in 2005. The most common source of L- DOPA is chemical synthesis but biological and natural sources are also available and have been reported to offer some advantages over chemical sources. For example, natural sources of L-DOPA have been reported to have different ‘pharmacokinetics’ (the effect is quicker and can last longer after administration) and also reduced ‘dyskinesia’ (involuntary muscle movements - a common side effect seen with chemical L-DOPA). In addition, natural L-DOPA may be more bioavailable after ingestion. Only a few plants have been reported to contain measurable quantities of L-DOPA, mainly in seeds, with the most studied and best known being the velvet bean, Mucuna pruriens, which can contain up to 10% w/w L-DOPA in its seeds. However, the level of L-DOPA in velvet beans is highly variable, and furthermore, large-scale production of these obscure beans is not practical. To date, no metabolic engineering of a non-betalain synthesising plant to accumulate L-DOPA has been reported in the scientific literature.

Although L-DOPA draws a lot of attention as a drug, its role in plants has not been extensively investigated. It was suggested to have repellent properties in preventing seeds from being attacked, or defensive roles in velvet bean. It can also serve as an allelochemical to prevent neighbouring plants from growing nearby, once excreted from the roots. The toxicity of L-DOPA results in inhibition of root growth and has been attributed to the fact that L-DOPA is a precursor for melanin and causes damage while it is being polymerised. The toxic effects of L-DOPA can be reversed by decreasing the activity of Polyphenol Oxidase (PPO), that promotes its oxidation, or by application of ascorbic acid. In high concentrations, L-DOPA has been shown to have antioxidant properties, as well. L-DOPA is an essential precursor for synthesis of betalain pigments and for some specialised alkaloids, such as epinephrine and codeine.

L-DOPA is synthesised through the hydroxylation of L-tyrosine by a tyrosinase, which may be a polyphenol oxidase or a cytochrome P450 (CYP450). Recently, progress in understanding the biosynthesis of betalains was made when a group of CYP450 proteins was identified in beetroot (Beta vulgaris) that catalyse the conversion of tyrosine to L- DOPA, in the first step of the betalain biosynthetic pathway. While some of these CYP450 enzymes, such as the beetroot CYP76AD1, have a dual activity of tyrosine hydroxylation and oxidation of L-DOPA to cyclodopa, a very closely related protein, CYP76AD6 catalyses only tyrosine hydroxylation.

There is a need for biological sources of L-DOPA, particularly in countries where access to commercial (chemically synthesised) pharmaceuticals is limited and/or too expensive. To date, no naturally occurring or engineered plants exist that can produce the level of L-DOPA required for use as a pharmaceutical. The present invention addresses this need.

SUMMARY OF THE INVENTION

L-DOPA, also known as Levodopa or L-3,4-dihydroxyphenylalanine, is a non-standard amino acid, and the gold standard drug for the treatment for Parkinson’s Disease. Recently, a gene encoding the enzyme that is responsible for its synthesis, as a precursor of the coloured pigment group betalains, was identified in beetroot, BvCYP76AD6. We have engineered tomato fruit enriched in L-DOPA through overexpression of BvCYP76AD6 in a fruit specific manner. Analysis of the transgenic fruit revealed the feasibility of accumulating L-DOPA in a non-naturally betalain producing plant. Fruit accumulating L-DOPA also showed major effects on the fruit metabolome. Some of these changes include elevation of amino acids levels, changes in the levels of intermediates of the Tricarboxylic Acid Cycle (TCA) and glycolysis pathways and reductions in the levels of phenolic compounds and nitrogen-containing specialised metabolites. Furthermore, we were able to increase the L-DOPA levels further by elevating the expression of the metabolic master regulator, MYB12, specifically in tomato fruit, together with BvCYP76AD6. We have elucidated new roles for L-DOPA in plants, because it impacted fruit quality parameters including antioxidant capacity and firmness. The L-DOPA levels achieved in tomato fruit were comparable to the levels in other non-seed organs of L-DOPA- accumulating plants, offering an opportunity to develop further biological sources of L-DOPA by widening the repertoire of L-DOPA containing plants. These tomato fruit could be used as an alternative source of this important pharmaceutical.

In a first aspect of the invention, there is provided a method of producing L-DOPA, the method comprising expressing or increasing the expression of a gene encoding a cytochrome p450 enzyme and a gene encoding a Myb12 transcription factor in a plant.

In a preferred embodiment, cytochrome p450 and Myb12 are expressed in fruit.

In a further embodiment, the method comprises introducing and expressing in a plant a nucleic acid construct comprising a nucleic acid sequence encoding a cytochrome p450 enzyme and a nucleic acid construct comprising a nucleic acid sequence encoding a Myb12 transcription factor. In one embodiment, the method comprises introducing a first nucleic acid construct comprising a nucleic acid sequence encoding a cytochrome p450 enzyme into a first plant and introducing and expressing a second nucleic acid construct comprising a nucleic acid sequence encoding a Myb12 transcription factor into a second plant. Preferably, the method further comprises combining the first and the second plant and selecting progeny that express both a cytochrome p450 enzyme and a Myb12 transcription factor.

In an alternative embodiment, the method comprises introducing and expressing a nucleic acid construct expressing a nucleic acid sequence encoding a cytochrome p450 enzyme and a nucleic acid sequence encoding a Myb12 transcription factor.

In a preferred embodiment, each nucleic acid sequence is operably linked to a regulatory sequence, which may be the same or a different regulatory sequence. Preferably, the regulatory sequence is a fruit-specific promoter, preferably the E8 promoter. Even more preferably the E8 promoter comprises a nucleic acid sequence as defined in SEQ ID NO: 5 or a functional variant or homolog thereof. In another embodiment, the method comprises introducing at least one mutation into the plant genome, wherein said mutation is the insertion of at least one or more additional copy of a nucleic acid sequence encoding a cytochrome p450 enzyme and at least one or more additional copy of a nucleic acid sequence encoding a Myb12 transcription factor, such that said sequences are operably linked to a regulatory sequence. Preferably, the mutation is introduced by genome editing, preferably ZFNs, TALENs or CRISPR.

In another embodiment, the method further comprises reducing or abolishing the expression of one or more genes involved in the production of polyphenols from phenylalanine. More preferably, the one or more genes is selected from chalcone synthase (CHS), hydroxycinnamoyl transferase (HQT). In one embodiment, the method comprises introducing at least one mutation into the nucleic acid sequence encoding one or more genes involved in the production of polyphenols from phenylalanine. Preferably the mutation is introduced using targeted genome modification, preferably ZFNs, TALENS or CRISPR or wherein the mutation is introduced using mutagenesis, preferably TILLING or T-DNA insertion. In another embodiment, the method comprises using RNA interference to reduce or abolish the expression of one or more genes involved in the production of polyphenols from phenylalanine.

In a further embodiment, the method further comprises obtaining L-DOPA from the fruit of the plant.

In another aspect of the invention, there is provided a genetically altered plant, part thereof or plant cell, wherein the plant, part thereof or plant cell expresses or has an increased level of expression of a cytochrome p450 enzyme and a Myb12 transcription factor.

In one embodiment, the genetically altered plant expresses at least one nucleic acid construct comprising a nucleic acid sequence encoding a cytochrome p450 enzyme and a Myb12 transcription factor. In a further embodiment, the plant expresses a first nucleic acid construct comprising a nucleic acid sequence encoding a cytochrome p450 enzyme and a second nucleic acid construct comprising a nucleic acid sequence encoding a Myb12 transcription factor. Preferably, the nucleic acid construct(s) are stably integrated into the genome of the plant. In an alternative embodiment, the genetically altered plant has at least one mutation in its genome, wherein the mutation leads to the accumulation or increased production of L-DOPA in the plant. Preferably, the mutation is the insertion of at least one or more additional copy of a nucleic acid encoding a cytochrome p450 enzyme and the insertion of at least one or more additional copy of a nucleic acid encoding a Myb12 transcription factor or homolog or functional variant thereof under the control of a regulatory sequence.

In one embodiment, the genetically altered plant part is a seed. Accordingly, in a further aspect, there is provided a seed derived from the plant described above, wherein the seed comprise a nucleic acid sequence encoding a cytochrome p450 enzyme and a nucleic acid sequence encoding a Myb12 transcription factor. Preferably, the nucleic acid sequences are stably integrated into the genome.

In another aspect of the invention, there is provided a method of making a genetically altered plant that produces L-DOPA or has an increased level of production of L-DOPA, the method comprising introducing and expressing at least one nucleic acid construct comprising a nucleic acid sequence encoding a cytochrome p450 enzyme and a Myb12 transcription factor in the plant.

In an alternative aspect of the invention, there is provided a method of making a genetically altered plant that produces L-DOPA or has an increased level of production of L-DOPA, the method comprising introducing a mutation, into the plant wherein the mutation leads to the accumulation or increased production of L-DOPA in the plant. In a preferred embodiment, the mutation is introduced using targeted genome editing, preferably CRISPR.

In one embodiment, the mutation is the insertion of at least one or more additional copy of a nucleic acid encoding a cytochrome p450 enzyme and the insertion of at least one or more additional copy of a nucleic acid encoding a Myb12 transcription factor or homolog or functional variant thereof, wherein both cytochrome p450 enzyme and Myb12 are under the control of one or more regulatory sequence(s).

In another aspect of the invention there is provided a nucleic acid construct comprising a nucleic acid sequence encoding a cytochrome p450 enzyme and a nucleic acid sequence encoding a Myb12 transcription factor. Preferably, the nucleic acid sequence is operably linked to a regulatory sequence, wherein preferably, the regulatory sequence is a tissue-specific promoter. More preferably, the tissue-specific promoter is the E8 promoter.

In another aspect of the invention there is provided a vector comprising the nucleic acid construct and a host cell comprising the nucleic acid construct or vector described above. In another aspect of the invention there is provided the use of the nucleic acid construct or vector described above to produce L-DOPA or increase the production of L-DOPA in a plant.

In a further aspect of the invention, there is provided L-DOPA obtained or obtainable by the methods described above.

In another aspect, there is provided a method of making a pharmaceutical composition, the method comprising combining L-DOPA obtained or obtainable by the method described above with a pharmaceutically acceptable carrier.

In a further aspect, there is provided a pharmaceutical composition obtained or obtainable by the method described above.

In another aspect, there is provided a method of treating Parkinson’s disease, the method comprising administering L-DOPA obtained or obtainable by the method described above or the pharmaceutical composition described above to a patient in need thereof.

In one embodiment, the plant is selected from tomato, tomato ( Solanum lycopersicum), potato (Solanum tuberosum), eggplant (Solanum melongena), banana (Musa sp.), soybean (Glycine max) and oil seed plants such as Camelina sp. and Brassica rapa.

In another aspect of the invention there is also provided a plant or plant progeny obtained or obtainable by any of the methods described above. In another aspect, there is provided pollen, propagule, progeny, or part of the plant derived from the plant described above wherein said pollen, propagule, progeny, or part comprise a nucleic acid sequence encoding a cytochrome p450 enzyme and a nucleic acid sequence encoding a Myb12 transcription factor.

DESCRIPTION OF THE FIGURES

The invention is further described in the following non-limiting figures:

Figure 1 shows the gene expression analysis and L-DOPA accumulation in fruit from T 1 plants. Fruit from three lines of CYP76AD6 transgenic plants were harvested seven days post breaker and analysed for CYP76AD6 expression, and L-DOPA accumulation in fresh tissue and in dry tissue. As expected, the BvCYP76AD6 transgene was expressed only in the transgenic lines, CYP786AD6#1-#3. These fruit accumulated L-DOPA at a significantly higher level than wt fruit, which contained only traces. Significant changes, compared to wt, in a student’s t-test, ^a- P-value≤0.0001; ^b-P-value≤0.005; ^c- P- value≤0.05.

Figure 2 shows a comparison of the activity of CYP76AD1 and CYP76AD6 in yeast. Yeast harbouring expression cassettes of BvCYP76AD1 or BvCYP76AD6 were analysed for L-DOPA accumulation levels, compared to a control cassette expressing the GUS gene. (A) L-DOPA was released into the growth medium, although a large amount of the metabolite remained inside the yeast cells. (B) Although both BvCYP76AD6 and BvCYP76AD1 could drive the synthesis of L-DOPA in yeast, BvCYP76AD6 was more efficient.

Figure 3 shows the changes in the profile of primary metabolites in L-DOPA-enriched tomatoes. GC-MS analysis of fruit expressing CYP76AD6 harvested seven days post breaker was carried out to identify any changes in the profile of primary metabolites. Fruit with high levels of L-DOPA showed significantly higher levels of (A) standard and (B) non-standard amino acids. (C) Tyrosine levels were reduced in the fruit enriched in L- DOPA, but values were not statistically significantly lower than those of the control. (D) Intermediates in glycolysis, the TCA cycle and the Calvin cycle, showed altered levels. (E) L-DOPA- enriched fruit had higher levels of the polyamine ornithine, and (F) reduced levels of total tocopherols. Student’s t-test significance: (*)P-value≤0.05; (**)P- value<0.01 ;(***)P-value≤0.001. Figure 4 shows the changes in the profiles of secondary metabolites in L-DOPA- enriched tomatoes. LC-MS analysis of L-DOPA- enriched tomato fruit, harvested seven days post breaker, was carried out to identify changes in the profiles of secondary metabolites. Fruit with high levels of L-DOPA showed reduced levels of (A) polyphenols and (B) nitrogen-containing compounds (alkaloids). Student’s t-test significance- (*)P- value≤0.05; (**)P-value≤0.01 ;(***)P-value≤0 .001 .

Figure 5 shows the analysis of L-DOPA levels in tomato fruit expressing CYP76AD6. (A) Tyrosine levels in Br+7 fruit, from MYB12- overexpressing plants were analysed by GC-MS and found to be higher than in the wt fruit. (B-D) Fruit from CYP76AD6- expressing plants and their crosses with MYB12 plants, were harvested seven days post breaker and analysed for (B)transgene expression, (C) L-DOPA accumulation in fresh tissue and (D) in dry tissue. (A) The BvCYP76AD6 transgene was expressed only in the transgenic lines CYP786AD6#1-#2 and their crosses with MYB12. MYB12 was expressed only in the MYB12 line and in its crosses to CYP76AD6. (B,C) L-DOPA accumulated in the CYP76AD6 expressing tomato lines. The L-DOPA levels increased when MYB12 overexpression was also introduced to the plants. Student’s t-test significance: P-value≤0.05.

Figure 6 shows the fruit shelf life analyses in L-DOPA-enriched tomatoes. Parameters correlated with fruit shelf life were studied in L-DOPA accumulating tomatoes and in fruit from crosses with MYB12. Fruit with higher contents of L-DOPA exhibited (A) higher antioxidant capacity, and (B) kept firm for longer after harvest. The firmness of the fruit was correlated with low expression levels of (D) PECTATE LYASE and (E) POLYGALACTURONASE. (C) No significant differences were observed in the water loss rate between the different genotypes. (F) L-DOPA accumulating fruit exhibited smaller lesions than wt following inoculation with Botrytis cinereal. Student’s t-test significance- P-value≤0.05.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics, which are within the skill of the art. Such techniques are explained fully in the literature.

As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. They can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene" or “gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.

The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

The term "promoter" typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved in the binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue- specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or -10 box transcriptional regulatory sequences. The aspects of the invention involve recombination DNA technology and exclude embodiments that are based solely on generating plants by traditional breeding methods.

For the purposes of the invention, a “genetically altered” or “mutant” plant is a plant that has been genetically altered compared to the naturally occurring wild type (WT) plant. In one embodiment, a mutant plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant using a mutagenesis method, such as the mutagenesis methods described herein. In one embodiment, the mutagenesis method is targeted genome modification or genome editing. In one embodiment, the plant genome has been altered compared to wild type sequences using a mutagenesis method. In one example, mutations can be used to insert a cytochrome p450 enzyme and a Myb12 transcription factor gene sequence to increase the levels of L-DOPA. In this example, the gene sequences are operably linked to an endogenous promoter. Therefore, in this example, the production of L-DOPA in plants is conferred by the presence of an altered plant genome and is not conferred by the presence of transgenes expressed in the plant. In other words, the genetically altered plant can be described as transgene-free.

Nonetheless, in an alternative embodiment, the genetically altered plant is a transgenic plant. For the purposes of the invention, "transgenic", “transgene” or "recombinant" means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either

(a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or

(b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or

(c) a) and b) are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. A plant according to all aspects of the invention described herein may be a monocot or a dicot plant. In a preferred embodiment, the plant is a non-betalain producing plant. In one embodiment, the plant is selected from tomato ( Solanum lycopersicum), potato (Solanum tuberosum), eggplant (Solanum melongena), banana (Musa sp.), soybean (Glycine max) and oil seed plants such as Camelina sp.and Brassica rapa. In a preferred embodiment, the plant is tomato ( Solanum lycopersicum).

The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, wherein each of the aforementioned produce L- DOPA, for example, by one of the methods described herein. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned produce L-DOPA, for example, by one of the methods described herein.

The invention also extends to harvestable parts of a plant of the invention as described herein, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. The aspects of the invention also extend to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins. The invention also relates to a product derived from a plant as described herein or from a part thereof, more preferably a pharmaceutical product.

In a most preferred embodiment, the plant part or harvestable product is the fruit. Therefore, in a further aspect of the invention, there is provided fruit produced from a genetically altered plant as described herein.

In an alternative embodiment, the plant part is pollen, a propagule or progeny of the genetically altered plant described herein. Accordingly, in a further aspect of the invention there is provided pollen, a propagule or progeny produced from a genetically altered plant as described herein.

A control plant as used herein according to all of the aspects of the invention is a plant which has not been modified according to the methods of the invention. Accordingly, in one embodiment, the control plant does not produce L-DOPA, as described above. In an alternative embodiment, the plant has not been genetically modified, as described above. In one embodiment, the control plant is a wild type plant. The control plant is typically of the same plant species, preferably having the same genetic background as the modified plant.

In a first aspect of the invention, there is provided a method of producing L-DOPA, the method comprising expressing or increasing the expression of a cytochrome p450 enzyme and a Myb12 transcription factor in a plant. In an alternative aspect of the invention, there is provided a method of producing L-DOPA in a plant, the method comprising expressing or increasing the expression of a cytochrome p450 enzyme and a Myb12 transcription factor in a plant.

As used herein, the term “expressing” means that a cytochrome p450 enzyme and a Myb12 transcription factor are expressed in a plant that does not already express these genes. The term “increasing the expression” means that the cytochrome p450 enzyme and Myb12 transcription factor are already expressed in the plant, and the level of expression is increased over wild-type or control levels. The level of increase may be at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more compared to the level in the wild-type or control plant. Methods for determining expression of the cytochrome p450 enzyme or the Myb12 transcription factor would be well-known to the skilled person using standard techniques in the art.

L-DOPA (Levodopa or L-3,4-dihydroxyphenylalanine) is a an amino acid precursor of dopamine. Levels of L-DOPA in a plant can be measured by any technique known to the skilled person. One example is described in the materials and methods section below. As shown in Figure, 5 the method of the present invention leads to the significant production of L-DOPA. In one embodiment, the method results in the production of between 5 and 30mg/100g fresh weight of fruit, preferably between 10 and 20mg/100g fresh fruit weight, even more preferably between 12 and 15mg/100 fresh fruit weight and most preferably around 14mg/100g fresh weight of fruit. Alternatively, in another embodiment, the method results in the production of between 0.005 and 0.030% of the fresh fruit weight, more preferably between 0.010 and 0.020% of the fresh fruit weight and even more preferably around 0.015% of the fresh fruit weight.

In one embodiment, the cytochrome p450 enzyme is CYP76AD6. In a further preferred embodiment, the CYP76AD6 is from Beetroot ( Beta vulgaris) and comprises or consists of an amino acid sequence as defined in SEQ ID NO: 1 or a functional variant or homologue thereof. In a further preferred embodiment, CYP76AD6 comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 2 or a functional variant or homologue thereof.

In another embodiment, Myb12 comprises or consists of an amino acid sequence as defined in SEQ ID NO: 3 or a functional variant or homologue thereof. In a further preferred embodiment, Myb12 comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 4 or a functional variant or homologue thereof.

In one embodiment, the method comprises introducing and expressing at least one nucleic acid construct where the nucleic acid construct comprises a nucleic acid sequence encoding a cytochrome p450 enzyme and a nucleic acid sequence encoding a Myb12 transcription factor, as defined herein.

In one embodiment, the method comprises introducing and expressing a first nucleic acid construct comprising a nucleic acid sequence encoding a cytochrome p450 enzyme and introducing and expressing a second nucleic acid construct comprising a nucleic acid sequence encoding a Myb12 transcription factor. The first and second nucleic acid constructs may be expressed in the same or different plants. If expressed in different plants, the first and second plants may be combined subsequently, for example through crossing and progeny selected that express both the first and second constructs. In some embodiments, the step of crossing the plants does not form part of the invention.

In an alternative embodiment, the method comprises introducing and expressing a nucleic acid construct expressing a nucleic acid sequence encoding a cytochrome p450 enzyme and a nucleic acid sequence encoding a Myb12 transcription factor, as defined herein.

In a preferred embodiment, the nucleic acid sequences (encoding a cytochrome p450 enzyme or Myb12) are operably linked to a regulatory sequence. Accordingly, in one embodiment, the nucleic acid sequences are expressed using a regulatory sequence that drives overexpression in specific cells or tissues. Overexpression according to the invention means that the transgene is expressed or is expressed at a level that is higher than the expression of any endogenous cytochrome p450 enzyme or Myb12 gene whose expression is driven by its endogenous counterpart. In one embodiment, the nucleic acid and regulatory sequence are from the same plant family. In another embodiment, the nucleic acid and regulatory sequence are from a different plant family, genus or species - for example, Beetroot CYP76AD6 or Arabidopsis Myb12 is expressed in a plant that is not Beetroot or Arabidopsis respectively.

In one embodiment, where the nucleic acid construct expresses both a nucleic acid sequence encoding a cytochrome p450 enzyme and a nucleic acid sequence encoding Myb12, each nucleic acid sequence may be operably linked to the same or separate regulatory sequences. In a preferred embodiment, the nucleic acid sequences are operably linked to the same regulatory sequences. In one embodiment, to allow two proteins to be expressed from a single construct, the construct may further comprise ribosomal skipping sequences, which may be added to the 5’ and/or 3’ ends of the individual nucleic acid sequence (i.e. cytochrome p450 enzyme and Myb12). Suitable ribosomal skipping sequences will be well-known to the skilled person, and include 2A- like peptides.

A "plant promoter" comprises regulatory elements that mediate the expression of a coding sequence segment in plant cells. The promoters upstream of the nucleotide sequences useful in the nucleic acid constructs described herein can also be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3'-regulatory region such as terminators or other 3' regulatory regions which are located away from the ORF. It is furthermore possible that the activity of the promoter is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms.

In a preferred embodiment, the promoter is a tissue-specific promoter. Tissue specific promoters are transcriptional control elements that are active only in particular cells or tissues at specific times during plant development. In one example, the tissue-specific promoter is a fruit specific promoter. An example of a fruit-specific promoter is the E8 promoter. In one embodiment, the E8 promoter comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 5 or a functional variant thereof. In another embodiment, particularly where the plant is potato, the promoter is a tuber-specific promoter. The term "operably linked" as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.

In one embodiment, the progeny plant is stably transformed with the nucleic acid construct or constructs described herein and comprises the exogenous polynucleotides, which are heritably maintained in the plant cell. The method may further include steps to verify that the construct is stably integrated. The method may also comprise the additional step of collecting seeds from the selected progeny plant.

In an alternative embodiment, the method comprises introducing at least one mutation into the plant genome, wherein said mutation is the insertion of at least one or more additional copy of a nucleic acid sequence encoding a cytochrome p450 enzyme and a Myb12 transcription factor, such that said sequences are operably linked to a regulatory sequence. For example, the mutation may comprise the insertion of at least one or more additional copy of a nucleic acid encoding an CYP76AD6 enzyme as defined in SEQ ID NO: 1 or a functional variant or homolog thereof, and the insertion of at least one or more additional copy of a nucleic acid encoding a Myb12 transcription factor, as defined in SEQ ID NO: 3 or a functional variant or homolog thereof, so that both sequences are operably linked to a regulatory sequence.

The term “functional variant of a nucleic acid sequence” as used herein with reference to any of the sequences described herein refers to a variant gene or amino acid sequence or part of the gene or amino acid sequence that retains the biological function of the full non-variant sequence. A functional variant also comprises a variant of the gene of interest that has sequence alterations that do not affect function, for example in non- conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active. Alterations in a nucleic acid sequence which result in the production of a different amino acid at a given site that do not affect the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

In one embodiment, a functional variant has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,

46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,

61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,

76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,

91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the non-variant nucleic acid or amino acid sequence.

The term homolog, as used herein, also designates a CYP76AD6 or Myb12 gene orthologue from other plant species. A homolog may have, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,

52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,

67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,

97%, 98%, or at least 99% overall sequence identity to the amino acid represented by any of SEQ ID NO: 1 or 3 or to the nucleic acid sequences as shown by SEQ ID NOs: 2 or 4. Functional variants of CYP76AD6 and Myb12 homologs are also within the scope of the invention.

In one embodiment, the CYP76AD6 is homolog is selected from one of the following families, Amaranthaceae, Aizoaceae, Phytollacoideae, Nyctaginaceae, Rivinoideae and Portulacineae. As such, in one example, a CYP76AD6 homolog is defined in SEQ ID NO: 6 or 8 or a variant thereof. In a further embodiment, the CYP76AD6 homolog comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 7 or 9 or a variant thereof. In a further embodiment, the Myb12 polypeptide of the invention may comprise one or more of the following conserved motifs.

SG7-1 motif: GRTxRSxMK (SEQ ID NO: 10);

SG7-2 motif: [W/x][L/x]LS (SEQ ID NO: 11)

Where X represents any amino acid, and wherein SG7 indicated MYB subgroup 7.

Accordingly, in one embodiment, the method comprises expressing or increasing the expression of a cytochrome p450 enzyme and a Myb12 transcription factor, wherein the cytochrome p450 enzyme comprises or consists of one of the following sequences: a. a nucleic acid sequence encoding a cytochrome p450 polypeptide as defined in SEQ ID NO: 1 or homolog or a functional variant thereof; or b. a nucleic acid sequence as defined in SEQ ID NO: 2 or a homolog or functional variant thereof; or c. a nucleic acid sequence capable of binding under stringent hybridisation conditions as defined herein to one of the sequences in (a) or (b); and wherein the Myb12 transcription factor comprises or consists of one of the following sequences: d. a nucleic acid sequence encoding a Myb12 polypeptide as defined in SEQ ID NO: 3 or homolog or a functional variant thereof; or e. a nucleic acid sequence as defined in SEQ ID NO: 4 or a homolog or functional variant thereof; or f. a nucleic acid sequence encoding a cytochrome p450 enzyme polypeptide, wherein the polypeptide comprises at least one motif as defined in SEQ ID NO: 10 or 11 or a variant thereof, wherein the variant has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to SEQ ID NO: 10 or 11 or g. a nucleic acid sequence capable of binding under stringent hybridisation conditions as defined herein to one of the sequences in (e), (f) or (g);

By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing).

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In one example, stringent conditions may be crossing and washing of the membrane in a DNA or RNA crossing experiment at 65°C using a solution of 0.1 x SSPE (or 0.1 x SSC), 0.1% SDS.

The skilled person would understand that suitable homologues can be identified by sequence comparisons and identifications of conserved domains. There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function. Homologous positions can thus be determined by performing sequence alignments once the homologous sequence has been identified. For example, a cytochrome p450 enzyme and a Myb12 transcription factor homolog can be identified using a BLAST search of the plant genome of interest using Beta vulgaris cytochrome p450 enzyme or Arabidopsis Myb12 transcription factor as a query.

Thus, the nucleotide sequences of the invention and described herein can also be used to isolate corresponding sequences from other organisms, particularly other plants, for example non-betalain plants In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences described herein. Topology of the sequences and the characteristic domain structure can also be considered when identifying and isolating homologs. Sequences may be isolated based on their sequence identity to the entire sequence or to fragments thereof. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e. , genomic or cDNA libraries) from a chosen plant. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labelled with a detectable group, or any other detectable marker. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al. , (1989) Molecular Cloning: A Library Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

In an alternative embodiment of the invention, the method comprises introducing at least one mutation into the plant genome, wherein said mutation is the insertion of at least one or more additional copy of a nucleic acid sequence encoding a cytochrome p450 enzyme and/or a Myb12 transcription factor, such that said sequences are operably linked to a (endogenous) regulatory sequence. Preferably the regulatory sequence is an E8 promoter sequence.

Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events. In one embodiment, the mutation is introduced using ZFNs, TALENs or CRISPR/Cas9.

In a preferred embodiment, the targeted genome editing technique is CRISPR. The use of this technology in genome editing is well described in the art, for example in US 8,697,359 and references cited herein. In short, CRISPR is a microbial nuclease system involved in defence against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage (sgRNA). Three types (l-lll) of CRISPR systems have been identified across a wide range of bacterial hosts. One key feature of each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers). The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre- crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.

One major advantage of the CRISPR-Cas9 system, as compared to conventional gene targeting and other programmable endonucleases is the ease of multiplexing, where multiple positions or sites on genes can be mutated simultaneously simply by using multiple sgRNAs each targeting a different site. In addition, where two sgRNAs are used flanking a genomic region, the intervening section can be deleted or inverted (Wiles et al., 2015).

Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms. Codon optimized versions of Cas9, which is originally from the bacterium Streptococcus pyogenes, can also be used to increase efficiency. Cas9 orthologues may also be used, such as Staphylococcus aureus (SaCas9) or Streptococcus thermophiles (StCas9).

The single guide RNA (sgRNA) is the second component of the CRISPR/Cas system that forms a complex with the Cas9 nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities. The canonical length of the guide sequence is 20 bp. In plants, sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3. Alternatively, Cpf1 , which is another Cas protein, can be used as the endonuclease. Cpf1 differs from Cas9 in several ways: Cpf1 requires a T-rich PAM sequence (TTTV) for target recognition, Cpf1 does not require a tracrRNA, and as such only crRNA is required unlike Cas9 and the Cpf1 -cleavage site is located distal and downstream to the PAM sequence in the protospacer sequence (Li et al. , 2017). Furthermore, after identification of the PAM motif, Cpf1 introduces a sticky-end-like DNA double-stranded break with several nucleotides of overhang. As such, the CRISPR/CPf1 system consists of a Cpf1 enzyme and a crRNA.

Cas9 and Cpf1 expression plasmids for use in the methods of the invention can be constructed as described in the art. Cas9 or Cpf1 and the one or more sgRNA molecule may be delivered as separate or as a single construct. Where separate constructs are used for the delivery of the CRISPR enzyme (i.e. Cas9 or Cpf1) and the sgRNA molecule(s), the promoters used to drive expression of the CRISPR enzyme/sgRNA molecule may be the same or different. In one embodiment, RNA polymerase (Pol) II- dependent promoters can be used to drive expression of the CRISPR enzyme. In another embodiment, Pol Ill-dependent promoters, such as U6 or U3, can be used to drive expression of the sgRNA.

The genome editing constructs may be introduced into a plant cell using any suitable method known to the skilled person. In an alternative embodiment, any of the nucleic acid constructs described herein may be first transcribed to form a preassembled Cas9- sgRNA ribonucleoprotein and then delivered to at least one plant cell using any of the above described methods, such as lipofection, electroporation, biolistic bombardment or microinjection.

Specific protocols for using the above-described CRISPR constructs would be well known to the skilled person. As one example, a suitable protocol is described in Ma & Liu (“CRISPR/Cas-based multiplex genome editing in monocot and dicot plants”) incorporated herein by reference.

In another embodiment, of the invention, the method further comprises reducing or abolishing the expression of one or more genes involved in the production of polyphenols from phenylalanine. In a preferred embodiment, the one or more genes is selected from chalcone synthase (CHS), hydroxycinnamoyl transferase (HQT). In one example, HQT comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 12 or a homolog or functional variant thereof. In a further example, CHS is CHS2 and comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 13 or a homolog or functional variant thereof. In one embodiment, the expression of one or more genes may be reduced using RNA interference, mutagenesis or targeted genome editing (such as CRISPR, which is described above). In one example, sgRNA constructs, which can be designed using known techniques in the art, can be used to introduce a targeted mutation into the endogenous gene or promoter of a gene involved in the production of polyphenols, as described above, such that the mutation reduces or abolishes the expression of that gene. In one example, sgRNA constructs may be designed that target a sequence in HOT such as SEQ ID NO: 15 or 16. In another example, sgRNA constructs may be designed that target a sequence in HOT such as SEQ ID NO: 17 or 18.

In another embodiment, conventional mutagenesis techniques, such as T-DNA insertional mutagenesis or any known physical or chemical mutagen can be used disrupt the genes or promoters described above. In a further example, the expression of one or more of the above genes can be reduced at the level of transcription or translation using gene silencing methods known to the skilled person, such as, but not limited to, the use of small interfering nucleic acids (siNAs) against one or more of the genes involved in the production of polyphenols from phenylalanine. For example, the siRNA may include, short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), antagomirs and short hairpin RNA (shRNA) capable of mediating RNA interference. In one example, the siRNA construct comprises a sequence as defined in SEQ ID NO: 14 or a variant thereof. Such an RNAi construct (which is a fragment of 547 bp; two copies inserted in opposite orientation) may be used in vector pFRN and the 35S promoter replaced with tomato E8 promoter.

The term “reducing” means a decrease in the levels of one or more genes involved in the production of polyphenols from phenylalanine by up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% when compared to the level in a wild-type or control plant. The term “abolish” expression means that no expression of these genes is detectable. Methods for determining levels of gene expression would be well known to the skilled person. Finally, in a further embodiment, the methods described herein include the step of obtaining L-DOPA from the plant. L-DOPA can be obtained from the plant by any suitable method known to the skilled person. For example, production of purified L-DOPA from a plant could be achieved by homogenisation in water-methanol mixtures with added ascorbate to limit oxidation. L-DOPA can be separated from the mixture by any suitable technique, such as column chromatography, thin layer chromatography or the like [56] This would allow local, cheap, low-tech production of this important pharmaceutical for PD patients who currently lack access as a result of their location and/or its cost.

In another aspect of the invention, there is provided a method for improving one or more properties of a fruit, the method comprising expressing or increasing the expression of a cytochrome p450 enzyme and a Myb12 transcription factor in a plant, as described above, wherein the one or more properties is selected from quality, antioxidant levels, fruit firmness and resistance to biotic stress. In one example, resistance to biotic stress may be resistance to a fungal pathogen such as Botrytis cinerea (as shown in Figure 6F).

In another aspect of the invention, there is provided a nucleic acid construct comprising a nucleic acid sequence encoding a cytochrome p450 enzyme as defined above and a nucleic acid sequence encoding Myb12, as defined above. The nucleic acid sequences may be operably linked to the same or separate regulatory sequences. In a preferred embodiment, the nucleic acid sequences are operably linked to the same regulatory sequence. In one embodiment, to allow two proteins to be expressed from a single construct, the construct may further comprise ribosomal skipping sequences, which may be added to the 5’ and/or 3’ ends of the individual nucleic acid sequence (i.e. cytochrome p450 enzyme and Myb12). Suitable ribosomal skipping sequences will be well-known to the skilled person, and include 2A-like peptides.

In another aspect, the invention relates to an isolated host cell transformed with the nucleic acid construct or vector as described above. The host cell may be a bacterial cell, such as Agrobacterium tumefaciens, or an isolated plant cell. The invention also relates to a culture medium or kit comprising a culture medium and an isolated host cell as described below. Thus, in a further aspect, the invention relates to a transgenic plant expressing the nucleic acid construct as described herein. Also described herein is a transgenic plant obtained or obtainable by the above-described methods.

In another aspect, the invention relates to the use of a nucleic acid construct as described herein to produce L-DOPA.

In another aspect of the invention, there is provided a genetically altered plant, part thereof or plant cell, characterised in that the plant produces L-DOPA, wherein the plant is a non-betalain synthesising plant. In one embodiment, the plant, part thereof or plant cell expresses or has an increased level of expression of a cytochrome p450 enzyme and a Myb12 transcription factor.

In one embodiment, the plant or plant cell expresses at least one nucleic acid construct comprising nucleic acid sequence encoding a cytochrome p450 enzyme and a Myb12 transcription factor, as described above. In one embodiment, the construct is stably incorporated into the genome.

In an alternative embodiment, the plant may be produced by introducing a mutation into the plant genome by any of the above-described methods. In one embodiment, the mutation is the insertion of at least one additional copy of a nucleic acid encoding a cytochrome p450 enzyme and the insertion of at least one or more additional copy of a nucleic acid encoding a Myb12 transcription factor or homolog or functional variant thereof under the control of a regulatory sequence, as described above.

In another aspect of the invention, there is provided a method of making a genetically altered plant accumulating L-DOPA or having an increased level of production of L- DOPA, the method comprising introducing and expressing at least one nucleic acid construct comprising a nucleic acid sequence encoding a cytochrome p450 enzyme and a Myb12 transcription factor, as described above.

The terms "introduction", “transfection” or "transformation" as referred to herein encompass the transfer of an exogenous polynucleotide or construct (such as a nucleic acid construct or a genome editing construct as described herein) into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.

Transformation of plants is now a routine technique in many species. Any of several transformation methods known to the skilled person may be used to introduce one or more genome editing constructs of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation.

Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant (microinjection), gene guns (or biolistic particle delivery systems (bioloistics)) as described in the examples, lipofection, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, ultrasound-mediated gene transfection, optical or laser transfection, transfection using silicon carbide fibers, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Transgenic plants can also be produced via Agrobacterium tumefaciens mediated transformation, including but not limited to using the floral dip/ Agrobacterium vacuum infiltration method as described in Clough & Bent (1998) and incorporated herein by reference.

Optionally, to select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above- described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. As described in the examples, a suitable marker can be kanamycin or the nptll gene (aminoglycoside phosphotransferase) Alternatively, the transformed plants are screened for the presence of a selectable marker, such as, but not limited to, GFP, GUS (b-glucuronidase). Other examples would be readily known to the skilled person. Alternatively, no selection is performed, and the seeds obtained in the above-described manner are planted and grown and L-DOPA levels measured at an appropriate time using standard techniques in the art. This alternative, which avoids the introduction of transgenes, is preferable to produce transgene-free plants.

Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using PCR to detect the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, integration and expression levels of the newly introduced DNA may be monitored using Southern, Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.

The method may further comprise selecting one or more mutated plants, preferably for further propagation. The selected plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).

In a further related aspect of the invention, there is also provided a method of obtaining a genetically modified plant as described herein, the method comprising a. selecting a part of the plant; b. transfecting at least one cell of the part of the plant of paragraph (a) with at least one nucleic acid construct as described herein, using the transfection or transformation techniques described above; c. regenerating at least one plant derived from the transfected cell or cells; d. selecting one or more plants obtained according to paragraph (c) that show production or increased production of L-DOPA.

In another aspect of the invention, there is provided a method of making a genetically altered plant accumulating L-DOPA or having an increased level of production of L- DOPA, the method comprising introducing a mutation, wherein the mutation leads to the increased production of L-DOPA in the plant. In one embodiment, the mutation is introduced using targeted genome editing, preferably CRISPR. In a further embodiment, the mutation is the insertion of at least one or more additional copies of a nucleic acid encoding a cytochrome p450 enzyme and the insertion of at least one or more additional copies of a nucleic acid encoding a Myb12 transcription factor or homolog or functional variant thereof under the control of a regulatory sequence.

In a further embodiment, the method also comprises the step of screening the genetically modified plant for the introduction of one or more additional copies of a cytochrome p450 enzyme and a Myb12 transcription factor nucleic acid, as described herein. In one embodiment, the method comprises obtaining a DNA sample from a transformed plant and carrying out DNA amplification to detect one of the mutations described above. In a further embodiment, the methods comprise generating stable T2 plants preferably homozygous for the mutation.

A genetically altered plant of the present invention may also be obtained by transference of any of the sequences of the invention by crossing, e.g., using pollen of the genetically altered plant described herein to pollinate a wild-type or control plant, or pollinating the gynoecia of plants described herein with other pollen that does not contain at least one of the above-described mutations. The methods for obtaining the plant of the invention are not exclusively limited to those described in this paragraph; for example, genetic transformation of germ cells from the ear of wheat could be carried out as mentioned, but without having to regenerate a plant afterward.

In a further aspect of the invention there is provided a plant obtained or obtainable by any of the above-described methods. Also included in the scope of the invention is the progeny obtained from the plants. In a particular aspect of the invention, there is provided fruit obtained or obtainable by any of the methods described herein. In a further aspect of the invention there is provided a method for producing L-DOPA, said method comprising a. producing a plant wherein the expression of a cytochrome p450 enzyme and a Myb12 transcription factor as described herein, is increased; b. obtaining fruit from said plant; c. obtaining L-DOPA from the fruit.

In another aspect of the invention, there is provided L-DOPA obtained or obtainable by any of the methods described herein.

In a further aspect of the invention, there is provided a method of making a pharmaceutical composition, the method comprising combining L-DOPA obtained or obtainable by any of the methods described herein with a pharmaceutically acceptable carrier, comprising excipients and other components, which facilitate processing of the active compounds into preparations suitable for pharmaceutical administration. Also included in the scope of the invention is a pharmaceutical composition obtained or obtainable by the above method.

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers known in the art in dosages suitable for oral administration. Such carriers enable the compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like suitable for ingestion by the subject.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds if desired to obtain tablets or dragee cores. Suitable excipients include carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilising agents may be added, such as cross linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof. In another aspect of the invention, there is provided a method of treating Parkinson’s disease, the method comprising administering L-DOPA obtained or obtainable by any of the methods described above or by administering a pharmaceutical composition described above to a patient in need thereof. In a further aspect of the invention, there is provided L-DOPA obtained or obtainable by any of the methods described above or the pharmaceutical composition described above for use in the treatment of Parkinson’s disease. Finally, in a further aspect of the invention, there is provided the use of L-DOPA obtained or obtainable by any of the methods described above or the use of the pharmaceutical composition described above in the manufacture of a medicament for the treatment of Parkinson’s disease.

In a further aspect of the invention, there is provided a health/dietary supplement or nutraceutical comprising L-DOPA obtained or obtainable by the methods described above. The supplement or nutraceutical may be used to improve cognitive functions, improve sleep, motivation or as an aphrodisiac. In one example the supplement or nutraceutical may comprise the fruit obtained by a plant of the invention.

While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. The foregoing application, and all documents and sequence accession numbers cited therein or during their prosecution ("application cited documents") and all documents cited or referenced in the application cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

The invention is now described in the following non-limiting example.

EXAMPLES

L-DOPA is naturally produced in plants, and in certain circumstances is administered from natural plant sources. We wanted to explore the feasibility of engineering its synthesis in plants that do not normally accumulate it, and to offer opportunities for new production systems for L-DOPA in plants. Tomato is a crop model with fleshy fruit and a complete well-characterised genome, comprehensive gene expression platforms, extensive metabolite analysis data, useful genetic resources and a wide range of analytical tools and protocols. These tools and resources, together with tomato being one of the most commonly-consumed crops in the world as well as its high nutritional value in the human diet, have made tomato a good model for metabolic engineering. Here, L-DOPA accumulating tomatoes were generated, in a fruit-specific manner, to avoid yield penalties or possible toxicity effects of L-DOPA on plant development. Additionally, tomato fruit are relatively rich in ascorbate, which could prevent the oxidation of L-DOPA and the generation of melanin, which might, in turn, cause further oxidative stress. We have shown the effect of ectopic L-DOPA production in this crop on metabolite and fruit quality properties, and the feasibility of synthesis of L-DOPA for development of new biological sources.

Example I: CYP76AD6 vs CYP76AD1 activity in yeast Recently, two genes were found to catalyse the hydroxylation of tyrosine to L-DOPA in beetroot, CYP76AD1 and CYP76AD6. To decide which gene should be used to generate L-DOPA-enriched tomato fruit, we took advantage of the established BY4742 yeast system (Fig.1). First, we checked whether L-DOPA accumulated in yeast cells or was released into the medium. Yeast transformed with CYP76AD6 and GUS, under the GAL1 inducible promoter were induced for 72 hours. The L-DOPA levels were quantified separately in the medium and in the precipitated yeast cells (Fig. 1A). Though there was a significant difference between the fractions, the medium contained twice the amount of L-DOPA as the yeast fraction. Therefore, we decided to measure the L-DOPA levels in cells and medium together, to determine total L-DOPA production.

To compare the ability of CYP76AD1 and CYP76AD6 to produce L-DOPA, yeast cells expressing CYP76AD1 or CYP76AD6 were generated and analysed (Fig. 1B). Constructs harbouring GUS expression cassettes were used as controls. L-DOPA levels were monitored up to 48 hours post induction and normalised to OD600=1. L-DOPA accumulation in yeast strains expressing the CYP76AD1 was much lower than the levels accumulating in strains expressing CYP76AD6. CYP76AD1 reached saturation after eight hours, and still produced about five times lower levels of L-DOPA than CYP76AD6- expressing yeast at the same time. After 48 hours, the L-DOPA levels in the CYP76AD6- expressing yeast were 14 times higher than in the CYP76AD1-ex pressing line.

These yeast expression assays suggested that CYP76AD6 would promote the accumulation of L-DOPA in plants better than CYP76AD1, and therefore it was chosen for engineering L-DOPA accumulation in tomatoes.

Example II: Generation of L-DOPA- engineered tomato plants

Since L-DOPA has been shown to have toxic effects and might inhibit growth leading to yield losses, we made use of the fruit specific E8 promoter of tomato, to drive the overexpression of CYP76AD6 gene. The CYP76AD6 gene from beetroot was cloned into an expression cassette containing the E8 promoter and 35S terminator from CaMV. Three stably transformed tomato lines were established, containing the CYP76AD6 expression cassette (CYP76AD6-#1, CYP76AD6-#2, CYP76AD6-#3). Fruit from these plants were analysed seven days post breaker (Br+7) for CYP76AD6 gene expression levels, by quantitative real-time PCR (qRT-PCR), with samples from wild type (wt) fruit as controls (Figure 2). All the three CYP76AD6 transformed lines expressed the CYP76AD6 transgene, however line #3 showed lower expression of CYP76AD6 than line #1 and line #2. We completed this analysis by quantifying the L-DOPA levels in these fruit (Figure 2). Fruit from CYP76AD6-#3, in line with its low expression of CYP76AD6, contained only 0.002% L-DOPA (2.38mg per 100g fruit fresh weight), while lines #1 and #2 contained 0.01% each (CYP76AD6-#1 had 10.43mg and CYP76AD6-#2 had 8.48mg per 100g fruit). Wt fruit showed only traces of L-DOPA, that were 76 times lower than in fruit from line #3, 333 times lower than in fruit of line #1 , and 270 times lower than in fruit of line #2 (on average). Since biological sources of L-DOPA have been reported, mainly in seeds which, unlike tomatoes, have low water content, we freeze dried the fruit tissue to estimate L-DOPA levels on the basis of dry weight. Once water had been eliminated from the tissue (91-95%) the L-DOPA content was 0.04% in CYP76AD6-#3, 0.18% in CYP76AD6-#1 and 0.13% in CYP76AD6-#2 of the total dry weight of the fruit. Wt L- DOPA levels in this analysis were 5.5 X10^-5% of the fruit dry weight, which was below the limit for accurate quantification by our system.

These results showed that the heterologous expression of BvCYP76AD6 for the synthesis of L-DOPA was not only feasible, but these plants were also capable of storing L-DOPA, in a sink organ (fruit). This motivated us to investigate the effect of accumulating L-DOPA in vivo, further. We carried out more detailed analyses on lines #1 and #2, which had the highest levels of L-DOPA.

Example III: Changes in the metabolic profile in L-DOPA containing fruit

L-DOPA is a secondary metabolite that is synthesised from tyrosine, a primary amino acid, the synthesis of which is tightly regulated. We explored the effect of L-DOPA synthesis and accumulation on the metabolic profile of tomato fruit. To do this, we carried out untargeted metabolite analysis by GC-MS (Fig. 3) and LC-MS (Fig. 4) on fruit, 7 days post-breaker from lines CYP76AD6-#1 and CYP76AD6-#2.

Changes in primary metabolites were identified primarily by GC-MS analysis (Fig. 3). We observed higher levels of amino acids in L-DOPA-accumulating fruit than in wt fruit, including significantly higher levels of alanine, asparagine, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, and valine (Fig. 3A). In addition, nonstandard amino acids and other downstream metabolites derived from standard amino acids increased in L-DOPA fruit (Fig. 3B). These metabolites included beta alanine, pipecolic acid, serine-O-acetyl and pyroglutamic acid, which has been suggested to function as a storage form of glutamate. Tyrosine levels were reduced compared to wt fruit, which was expected since tyrosine is consumed in the biosynthesis of L-DOPA. However, these reductions in tyrosine levels were not statistically significant (Fig. 3C), emphasising the likely tight regulation of tyrosine levels. Other compounds annotated as intermediates in glycolysis, the TCA cycle and the Calvin cycle also showed altered levels. The L-DOPA accumulating fruit had lower levels of glucose-6-phosphate, fumaric acid and succinic acid, and higher levels of glycerol-3- phosphate and malic acid (Fig. 3D). Interestingly, the levels of the polyamine ornithine, were 10 times higher in the L-DOPA fruit than in wt fruit (Fig. 3E). Tocopherol levels were reduced by 50% in the transgenic fruit. Tocopherols require tyrosine as an intermediate in their biosynthesis, suggesting that available tyrosine was redirected for the synthesis of L-DOPA, instead of other tyrosine-dependant metabolites in the CYP76AD6 fruit (Fig. 3F).

Further investigation of the metabolome in the L-DOPA-accumulating fruit, primarily for secondary metabolites, was carried out by LC-MS (Fig. 4). Unlike the changes that were observed in primary metabolites and their derivatives by GC-MS, all the specialised metabolites that exhibited different levels, were reduced compared to wt (Fig. 4). Two groups of specialised metabolites were reduced significantly, polyphenols (Fig. 4A) and nitrogen containing compounds (Fig. 3B). Among the reduced polyphenols were quercetin 3-0-rutinoside-7-0-glucoside, caffeic acid-hexose, naringenin chalcone, naringenin-dihexose, homovanillic acid-O-hexoside, and other phenolics and flavones that were not identified, specifically. All these polyphenols are derived from phenylalanine via general phenylpropanoid biosynthesis, suggesting that tyrosine consumption for L-DOPA production feeds back to repress general phenylpropanoid metabolism. Since phenylalanine levels increased in CYP76AD6 fruit, this suggests that this feedback control operates on the activity of PAL (phenylalanine ammonia lyase) as reported for L-DOPA treatment of soyabean. Among the nitrogen containing compounds, conjugates of glycoalkaloids (including hydroxytomatine) and other N-containing compounds, showed reduced levels in the fruit accumulating L-DOPA. Our analysis suggested that the consumption of tyrosine to form L-DOPA caused an increase in primary metabolism to form higher levels of most amino acids, while reducing the synthesis of specialised metabolites that compete for aromatic amino acids.

Example IV: Overexpression of CYP76AD6 in the background of MYB12 results in further accumulation of L-DOPA

Tomato fruit from plants overexpressing Arabidopsis MYB12 (MYB12), were harvested at 7 days post breaker, and analysed by GC-MS. Tyrosine levels were -50% higher than in wt fruit, confirming the previous findings (Fig.5A). Therefore, to increase the L-DOPA accumulation in the fruit further, two independent lines (CYP76AD6-#1 , CYP76AD6-#2) were crossed with tomatoes overexpressing the MYB12 gene from Arabidopsis (MYB12) specifically in fruit. Fruit harvested from F1 plants, 7 days post breaker (Br+7), were analysed for gene expression, together with wild type (wt) and MYB12 fruit as controls, and fruit harvested from the T2 plants of the CYP76AD6 lines (Fig. 5B). The two original L-DOPA accumulating lines expressed the CYP76AD6 transgene, as expected, and the expression levels of the CYP76AD6 and MYB12 transgenes, were not significantly different between the crosses (CYP76AD6#1XMYB12 and CYP76AD6# 2XMYB12 lines) and their CYP76AD6 and MYB12 parents.

Next, we analysed the L-DOPA levels in this set of fruit (Fig. 5C). The lines crossed with MYB12 showed increased L-DOPA accumulation, to 0.015% of the fruit fresh weight (14.9 mg/100g fruit), which was 30-45% more than in the CYP76AD6 parental line. In addition, in freeze-dried fruit, the CYP76AD6XMYB12 crossed fruit contained on average 0.27% L-DOPA on the basis of dry weight (Fig. 5D). We believe these results were due to the increase in flux of carbon from primary metabolism to its precursor, tyrosine, caused by enhanced MYB12 expression in fruit.

Example V: Accumulation of L-DOPA enhance fruit quality properties

L-DOPA has been reported to have antioxidant properties, and therefore we tested the L-DOPA accumulating tomatoes for their antioxidant capacities using the Trolox Equivalent Antioxidant Capacity assay (TEAC; Fig. 6A). Increases in antioxidant capacities in MYB12 tomato fruit relative to wt fruit have been reported. Indeed, fruit from both CYP76AD6-#1 and #2 lines, had higher antioxidant capacity than wt fruit, and similar antioxidant capacities to MYB12 fruit. Fruit from CYP76AD6 lines crossed with MYB12, exhibited higher antioxidant levels than either of the parental lines individually, confirming the positive association between L-DOPA levels and antioxidant capacity.

Higher levels of antioxidants and improved antioxidant capacities have been shown to be positively associated with improved shelf life in many soft fruits. To test whether the high antioxidant capacity of tomato fruit enriched in L-DOPA impacted shelf-life as well, we recorded the changes in fruit firmness and water loss in detached fruit (Fig. 6B,C), from fourteen days post- breaker to five weeks later. The rate of water loss was slightly faster in the L-DOPA-accumulating fruit than in wt fruit, although these differences were not statistically significant. In contrast, starting from the point of harvest, the difference in shelf-life was dramatically different. Fruit from the wt plants were already soft at fourteen days post breaker, completing softening within a week, with the lowest values of firmness recorded. Upon harvest, MYB12 and the L-DOPA fruit were firmer than wt fruit. The MYB12 fruit were slightly less firm than the CYP76AD6-expressing fruit. The differences in firmness between the genotypes were more obvious one week post-harvest; MYB12 fruit underwent faster softening, reaching their softest points, earlier than the L-DOPA- accumulating lines. Fruit expressing CYP76AD6 showed complete softening three to five weeks post-harvest. Interestingly, both CYP76AD6XMYB12 crosses showed no significant difference in firmness compared to their respective L-DOPA-accumulating parental lines, suggesting that there were no additive effects of MYB12 overexpression on the effects of L-DOPA accumulation, on fruit shelf-life. Softening of tomato fruit during ripening is positively correlated with the expression levels of two genes, PECTATE LYASE (PL) and POL YGALAC TURONA SE2a (PG). We analysed the expression of these softening-associated genes by qRT-PCR in the transgenic fruits, seven days post breaker (Fig. 6D,E). Expression of both, PL and PG, was reduced in all fruit accumulating L-DOPA as well as in fruit additionally expressing MYB12, in line with the reduced softening rates observed in these fruits, compared to wt. The expression levels of these two markers of over-ripening were not significantly different in the L-DOPA fruit from those in MYB12 fruit. Since the L-DOPA fruit showed delayed softening compared to the MYB12 fruit, it may be that L-DOPA contributes to fruit firmness in additional ways to Myb12.

A second feature important to shelf life of tomato is the response of fruit to infection by pathogens. We have analysed the effect of L-DOPA accumulation on the response of tomato fruit to the necrotrophic fungus, Botrytis cinerea. Fruit at 14 to 21 days post- breaker, were wounded and inoculated with the pathogen and lesions were examined three days post inoculation (dpi). While fungal inoculation of wt fruit caused the development of mycelium, fruit accumulating L-DOPA showed no signs of mycelium and had smaller lesions (Fig. 6F). These results strengthened further the association between antioxidant capacity, fruit firmness upon ripening and susceptibility to pathogens, as previously reported in tomato fruit and other crops.

Summary

L-DOPA is a non-standard amino acid, and its importance in the medical field is very well established in the treatment of Parkinson’s disease (PD). L-DOPA is generally not present at high levels in plants because it is consumed by different biosynthetic pathways, synthesising betalains, morphine, melanin, and other specialised metabolitess. A restricted number of plants accumulate L-DOPA, most notably the velvet bean, Mucuna puriens, in its seeds. Natural sources of L-DOPA can be used for the treatment of PD in cases where the patient suffers from adverse effects of chemically synthesised L-DOPA, such as nausea, vomiting and behavioural complications. Indeed, the most widely studied natural source of L-DOPA is velvet bean ( Mucuna pruriens) which is also used as a herbal drug. However, this resource is far from ideal, and problems arise from harvest through processing to its final applications. The plant is covered with urticating hairs that contain mucunain, that can cause irritation and allergic reactions in field workers that harvest the crop, and the beans themselves contain high levels of tryptamines, that can cause hallucinations in PD patients.

As such, there is a need to increase the repertoire of plants accumulating L-DOPA, and to address this need, we introduced the enzyme responsible for converting tyrosine to L- DOPA in the betalain biosynthetic pathway into a non-producing plant, tomato, and produced L-DOPA-enriched tomato fruit. Tomato was the plant of choice, since it has been engineered to accumulate several other secondary metabolites, which can reach high levels particularly when limiting production specifically to fruit, using the fruit-specific E8 promoter. We have further identified further methods to enhance L-DOPA levels, such as overexpressing the fruit specific MYB12 transcription factor. Furthermore, since tomato is widely cultivated, this crop can be used for scale-up and as such, offers a standardised and controlled natural source for L-DOPA. The engineered tomato fruit accumulated up to 0.15% L-DOPA as a proportion of the fruit dry weight (Fig. 5C). This almost doubled to 0.27% when MYB12 was ectopically expressed in fruit to increase flux to tyrosine (Fig. 5A,C). These L-DOPA levels are similar to those accumulating in other non-seed organs of L-DOPA producing plants. A common dose for L-DOPA treatment is less than 500mg/day. This dose could be achieved by about 200 grams of dry matter (~2 kg fresh fruit) from our engineered tomato fruit. Furthermore, different resources of L-DOPA are widely accepted as a traditional drug for other purposes, such as male infertility and as an aphrodisiac, where lower doses are consumed.

Interestingly, the ectopic introduction of L-DOPA in tomato, revealed several accompanying effects, related to L-DOPA accumulation in plants. Firstly, the shelf life properties of the fruit were improved, and this effect correlated with higher antioxidant capacities and reduced expression of genes involved in cell wall degradation, resulting in improved fruit firmness post-harvest, and reduced susceptibility to B. cinerea (Fig. 6). Synthesis of L-DOPA also had a major effect on the plant metabolome, including increased levels of both standard and non-standard amino acids in fruit (Fig. 3). Surprisingly, the levels of tyrosine itself were not significantly reduced, despite tyrosine being the precursor for L-DOPA, suggesting that metabolic regulation may be channelled to maintain tyrosine levels. This supports the reported tight regulation of tyrosine biosynthesis through feedback inhibition. Tyrosine levels may be maintained by increases in the levels of intermediates in glycolysis and the TCA cycle. Reduced tocopherol levels indicated that metabolic flux had been shifted towards L-DOPA from other tyrosine- utilising pathways. This interpretation was confirmed by LC-MS analysis, which showed reduced levels of other nitrogen-containing and phenolic compounds in fruit accumulating L-DOPA (Fig. 4).

We have demonstrated that the use of CYP76AD6 and Myb12 expressing tomatoes as a source of L-DOPA is feasible, and they could be considered further as a source of L- DOPA for treatment of Parkinson’s Disease (PD) in places where access to commercial pharmaceuticals is limited and/or relatively expensive. At estimated levels of 150 mg L- DOPA per kg tomatoes, the L-DOPA tomatoes yield much lower than microbial fermentation systems or immobilised tyrosinase bioproduction systems. However, scale-up production of tomatoes is low-tech and high yields can be achieved without major investment. In addition, we have shown that L-DOPA stored in the harvested tomatoes without major oxidation to melanin, provided the fruit are not wounded. Production of purified L-DOPA from these tomatoes could be achieved by homogenisation in water-methanol mixtures with added ascorbate to limit oxidation, followed by thin layer chromatography. This would allow local, cheap, low-tech production of this important pharmaceutical for PD patients who currently lack access as a result of their location and/or its cost.

In addition, natural sources of L-DOPA, such as velvet been (which contains between 0.5-9% L-DOPA in its seeds), have been reported to show significantly better effects than commercially synthesised L-DOPA in the treatment of PD. The plant matrix plays a major role in increasing the therapeutic effects of Mucuna extracts and the antioxidant activity of extracts of velvet bean seeds can further prevent the progress of oxidative stress. An increased antioxidant capacity resulting from accumulation of L-DOPA was also observed in CVP76AD6-expressing tomato fruit (Fig. 4), suggesting that natural extracts of these tomatoes (such as tomato water) could offer a substitute for velvet bean extracts used as an aphrodisiac or to treat male infertility.

Materials and Methods Yeast transformation

BvCYP76AD6 and BvCYP76AD1 were cloned in pAG423GAL-ccdB and pYES-DEST52 as previously described, and transformed into BY4742 yeast strain using the polyethylene glycol/ lithium acetate (PEG/LiAc) method. Vectors transformed with BETA- GLUCORONIDASE ( GUS ) instead of the CYP76AD6 genes, were used as controls. Yeast were grown in synthetic defined (SD) medium overnight, containing 2% glucose and lacking amino acids as necessary for selection. The yeast were pelleted and resuspended to OD₆oo=1 in SD medium with 2mM ascorbate, 1% Raffinose and 2% galactose and lacking the relevant amino acids. Five hundred microliter culture was sampled as detailed in the main text, freeze-dried and analysed for L-DOPA levels, using 1ml of extraction buffer.

Generation of CYP76AD6 tomato plants pBI N-E8-BVCYP76AD6 was cloned using pDONR207-BvCYP76AD6 and pJIT160-E8. Agrobacterium-mediated transformation to Money Maker tomato variety was carried out as previously described. Transgenic plants were confirmed by kanamycin resistance and PCR amplification using gene and promoter specific primers.

Obtaining tomato samples Fruit were harvested seven days post breaker, in at least three biological repeats, placenta and seeds removed, and frozen in liquid nitrogen. Samples were stored in - 80°C until ground with liquid nitrogen and analysed.

Gene expression analysis

RNA was extracted from fruit with Tri Reagent, according to manufacturer’s protocol (Sigma), following lithium acetate precipitation. DNasel-treated RNA samples (Sigma), were reverse transcribed using High Capacity cDNA reverse transcription kit (Applied Biosystems). Gene expression levels were analysed using SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma) and BioRad CFX real-time PCR instrument. TIP41 was used as endogenous control.

L-DOPA measurements

Fruit was extracted for 30 minutes shaking at room temperature in extraction buffer (80% MeOH, 0.1% Formic acid, 50ug/ml labelled L-DOPA as internal standard (Sigma). This was followed by five minutes sonication, centrifugation in 4°C and filtration of the supernatant through a 0.22um filter. Extracts were diluted 1000 times in 0.1% formic acid. Samples were kept at 4°C in the dark until injected. Standard curve was generated for 0 to 50ug/ml L-DOPA (Sigma). Samples were analysed using Waters Acquity LC combined with a Xevo TQS mass spectrometer and Accucore-150-Amide-HILIC 2.6u 100*2.1mm column. L-DOPA was identified by 152.06 fragment and normalised to the 154.33 mass of the labelled L-DOPA.

Firmness and water loss analysis

Fruit were harvested 14 days post breaker and stored at 16°C, in the dark, in a nylon bag. The fruit were weighed and scored for firmness (1 to 5 scale; 5-hardest, 1-softest) every week, for five weeks.

Antioxidant capacity analysis, GC-MS and LC-MS for metabolite analyses

Ground samples were freeze-dried overnight and extracted (30mg/ml) in extraction buffer (ribitol 1.5mg/l in 80% methanol). Samples were shaken at room temperature for 30 minutes, followed by 10 minutes sonication and 10 minutes centrifugation in 4°C. Five microliters were used for Trolox equivalent antioxidant capacity assays (TEAC) as previously described. GC-MS and LC-MS analyses were carried out as previously detailed [59, 60], using 120mI and 400mI evaporated extract volumes, respectively. Botrytis cinerea wound inoculation

Botrytis cinerea wound inoculations and scoring in tomato fruit were carried out as previously detailed in [61] REFERENCES

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62. Zhu, O. and G. Chen, The extracting condition of melanin from the banana skin. Food Res Dev, 2009. 10: p. 171-173. SEQUENCE LISTING

SEQ ID NO: 1 Beta vulgaris CYP76AD6 amino acid sequence

MDNATLAVILSILFVFYHIFKSFFTNSSSRRLPPGPKPVPIFGNIFDLGEKPHRSFANLSK

IHGPLISLKLGSVTTIVVSSASVAEEMFLKNDQALANRTIPDSVRAGDHDKLSMSWLPV

SQKWRNMRKISAVQLLSNQKLDASQPLRQAKVKQLLSYVQVCSEKMQPVDIGRAAFT

TSLNLLSNTFFSIELASHESSASQEFKQLMWNIMEEIGRPNYADFFPILGYIDPFGIRRR

LAGYFDKLIDVFQDIIRERQKLRSSNSSGAKQTNDILDTLLKLHEDNELSMPEINHLLVDI

FDAGTDTTASTLEWAMAELVKNPEMMTKVQIEIEQALGKDCLDIQESDISKLPYLQAIIK

ETLRLHPPTVFLLPRKADNDVELYGYVVPKNAQVLVNLWAIGRDPKVWKNPEVFSPE

RFLDCNIDYKGRDFELLPFGAGRRICPGLTLAYRMLNLMLATLLQNYNWKLEDGINPK

DLDMDEKFGITLQKVKPLQVIPVPRN

SEQ ID NO: 2 Beta vulgaris CYP76AD6 coding sequence

AT GG AT AACGC AACACTTGCT GT G ATCCTTTCCATTTT GTTT GT GTTTT ACCACATT TT CAAATCCTTTTT CACCAATT CTT CAT CTCGT AGGCTTCCTCCT GGTCCCAAACCC GT GCC AATTTTT GGCAACATTTTCG AT CTTGGCGAAAAGCCT CATCG AT CTTTT GC CAAT CT AT CT AAAATT CACGGCCCTTT GATT AGCCT AAAGTT AGG AAGT GT AAC AA CT ATT GTT GTTTCCTCGGCCT CTGT GGCCG AGG AAAT GTTCCTT AAAAAT G ACCAA GCACTT GOT AACCGAACCATTCCT GACTCGGTT AGGGCTGGT GACCACGACAAAT T ATCCAT GTCGTGGTTGCCT GTTTCCCAAAAATGG AG AAAT AT GAG AAAAAT CTCC GOT GT CCAATT ACTCTCCAACCAAAAACTT GAT GOT AGTCAACCT CTT AG ACAAGC T AAGGT G AAACAACTTTT AT CAT ACGT ACAAGTTT GTTCCGAAAAAATGCAACCCG TCGAT ATTGGACGGGCCGCATTT ACAACGTCACTT AATTT ATT ATCAAACACATTTT T CT CAATCGAATT AGCAAGTCAT G AAT CTAGT GCTTCCCAAG AGTTT AAACAACT C AT GTGGAAT ATT ATGGAGGAAATTGGAAGGCCT AATT AT GCT GATTTTTTCCCT ATT CTTGGTT ACATT G ATCCCTTT GGTAT AAG ACGTCGTTT GGCTGGTT ACTTT GAT AA ACT CATT GAT GTTTTCCAAGACATT ATTCGT G AAAGACAAAAGCTTCG AT CTT CT AA TT CTTCCGGCGCAAAACAAACAAAT GACATT CTT GAT ACT CTT CTT AAACTCCAT GA AGAT AAT G AGTT G AGT ATGCCT GAAATT AAT CACCTT CTCGTGG AT AT CTTT G ACG CCGGAACAGACACAACAGCAAGCACATTAGAATGGGCGATGGCCGAACTTGTGA AAAACCCGG AAAT GAT G ACT AAAGTT CAAATT GAAATCG AACAAGCT CTTGGAAAA G ATTGCTT AGACAT ACAAGAATCCGACAT CT CAAAACT ACCTT ATTT AC AAGCCATT AT AAAAGAAACGTT ACGTTT ACACCCTCCT ACT GT GTTTTTGCTGCCTCGAAAGGC AGACAAT GACGT AGAGTT AT ATGGCT ACGTT GT ACCAAAGAATGCTCAAGTCCTT G TCAATCTTTGGGCAATTGGTCGTGATCCAAAGGTATGGAAAAATCCGGAAGTATTT TCTCCT G AAAGGTTTTT AGATT GC AAT ATCG ATT AT AAAGGACG AG ATTTCGAACTT TTACCCTTTGGTGCTGGTAGAAGGATATGCCCTGGACTTACTTTGGCATATAGAAT GTT GAACTT GAT GTTGGCTACT CTT CTT CAAAACT ACAATTGGAAACTT G AAG AT G GT ATCAATCCT AAGGATTT AGACAT GGAT GAGAAATTTGGGATT ACATTGCAAAAG GTT AAACCT CTT CAAGTT ATTCCAGTTCCCAGAAACT

SEQ ID NO: 3: Arabidopsis thaliana Myb12 amino acid sequence

MGRAPCCEKVGIKRGRWTAEEDQILSNYIQSNGEGSWRSLPKNAGLKRCGKSCRLR

WINYLRSDLKRGNITPEEEELVVKLHSTLGNRWSLIAGHLPGRTDNEIKNYWNSHLSR

KLHNFIRKPSISQDVSAVIMTNASSAPPPPQAKRRLGRTSRSAMKPKIHRTKTRKTKKT

SAPPEPNADVAGADKEALMVESSGAEAELGRPCDYYGDDCNKNLMSINGDNGVLTF

DDDIIDLLLDESDPGHLYTNTTCGGDGELHNIRDSEGARGFSDTWNQGNLDCLLQSC

PSVESFLNYDHQVNDASTDEFIDWDCVWQEGSDNNLWHEKENPDSMVSWLLDGDD

EATIGNSNCENFGEPLDHDDESALVAWLLS

SEQ ID NO: 4 Arabidopsis thaliana Myb12 coding sequence ATGGGAAGAGCGCCATGTTGCGAGAAGGTCGGTATCAAGAGAGGGCGGTGGAC GGCGGAGGAGGACCAGATTCTCTCCAACTACATTCAATCCAACGGTGAAGGTTC TTGGAGATCTCTCCCCAAAAAT GCCGGATT AAAAAGGT GTGGAAAGAGCT GT AGA TT G AG ATGG AT AAACT AT CT AAG AT CAGACCT CAAGCGTGGAAACAT AACTCCAGA AGAAGAAGAACTCGTT GTT AAATTGCATTCCACTTTGGGAAACAGGTGGT CACT AA TCGCGGGTCATCTACCAGGGAGAACAGACAACGAAATAAAAAATTATTGGAACTC T CAT CT CAGCCGT AAACTCCACAACTT CATT AGG AAGCCATCCAT CT CT C AAGACG TCTCCGCCGTAATCATGACGAACGCTTCTTCAGCGCCACCGCCGCCGCAGGCAA AACGCAGACTTGGGAGAACGAGTAGGTCCGCTATGAAACCAAAAATCCACAGAAC AAAAACTCGT AAAACGAAG AAAACGTCT GCACCACCGG AGCCT AACGCCG AT GTA GCT GGGGCT GAT AAAGAAGCATT AAT GGTGGAGTCAAGT GGAGCCGAGGCT GAG CT AGG ACGACCAT GT GACT ACT ATGG AG AT GATT GT AACAAAAAT CT CAT G AGC AT T AATGGCGAT AATGGAGTTTT AACGTTT GAT GAT GAT ATCATCGATCTTTT GTTGGA CGAGTCAGATCCTGGCCACTTGTACACAAACACAACGTGCGGTGGTGATGGGGA GTTGCAT AACAT AAGAGACTCT GAAGGAGCCAGAGGGTTCTCGGAT ACTTGGAAC CAAGGGAAT CTCG ACT GTCTT CTT CAGTCTT GTCCATCGGTGGAGTCGTTTCTCAA CT ACGACCACCAAGTT AACGAT GCGTCGACGGAT GAGTTT ATCGATTGGGATT GT GTTT GGCAAGAAGGT AGT GAT AAT AATCTTT GGCAT GAGAAAGAGAATCCCGACT CAATGGTCTCGT GGCTTTT AGACGGT GAT GAT GAGGCCACGATCGGGAAT AGT AA TT GT GAGAACTTTGGAGAACCGTT AGATCAT GACGACGAAAGCGCTTTGGTCGCT TGGCTTCTGTCATG

SEQ ID NO: 5 Solanum lycopersicum E8 promoter sequence

CATCCCT AAT GAT ATT GTT CACGT AATT AAGTTTT GTGGAAGT G AGAG AGTCCAAT TTT GAT AAG AAAAG AGT C AG AAAACGT AAT ATTTT AAAAGTCT AAAT CTTT CT AC AA AT AAGAGCAAATTT ATTT ATTTTTT AATCCAAT AAAT ATT AATGGAGGACAAATTCAA TT CACTT GGTTGT AAAAT AAACTT AAACCAAT AACCAAAG AACT AAT AAATCCT G AA GT GGAATT ATT AAGGAT AAAT GT ACAT AGACAAT GAAGAAAT AAT AGGTTCGAT GA ATT AAT AAT AATT AAGG AT GTT ACAAT CAT CAT GTGCCAAGT AT AT ACACAAT ATT C T AT GGG ATTT AT AATTTCGTT ACTT CACTT AACTTTTGCGT AAAT AAAACGAATT AT C T GAT ATTTT AT AAT AAAACAGTT AATT AAGAACC AT CATTTTT AACAACAT AG AT AT A TT ATTT CT AAT AGTTT AAT GAT ACTTTT AAAT CTTTT AAATTTT AT GTTT CTTTT AG AA AAT AAAAATT CAAAAAATT AAAT AT ATTT ACAAAAACT ACAAT C AAACACAACTT CAT AT ATT AAAAGCAAAAT AT ATTTT G AAAATTT CAAGT GTCCT AACAAAT AAGACAAG A GGAAAAT GT ACGAT GAGAGACAT AAAGAGAACT AAT AATT GAGGAGTCCT AT AAT A T AT AAT AAAGTTT ATT AGT AAACTT AATT ATT AAGG ACTCCT AAAAT AT AT GAT AGGA GAAAAT GAATGGT GAGAGAT ATTGGAAAACTT AAT AATT AAGGATTTT AAAAT AT AT GGT AAAAGAT AGGCAAAGT ATCCATT ATCCCCTTTT AACTT GAAGTCT ACT AGGCG CAT GT GAAAGTT G ATTTTTT GTCACGT CAT AT AGCT AT AAT GT AAAAAAAG AAAGT A AAATTTTT AATTTTTTTT AAT AT AT GACAT ATTTT AAACGAAAT AT AGGACAAAAT GT AAAT GAAT AGT AAAGG AAACAAAG ATT AAT ACTT ACTTT GT AAG AATTT AAG AT AAA TTT AAAATTT AAT AG AT CAACTTT ACGTCT AG AAAG ACCC AT AT CT AGAAGG AATTT CACGAAATCGGCCCTT ATT CAAAAAT AACTTTT AAAT AAT G AATTTT AAATTTT AAG A AAT AAT ATCCAAT GAAT AAAT GACAT GT AGCATTTT ACCT AAAT ATTT CAACT ATTTT AATCCAAT ATT AATTT GTTTT ATTCCCAACAAT AG AAAGTCTT GTGCAG AC ATTT AAT CT G ACTTTT CCAGT ACT AAAT ATT AATTTT CT G AAG ATTTTCGGGTTT AGTCCACAA GTTTT AGT GAGAAGTTTTGCTCAAAATTTT AGGT GAGAAGGTTT GAT ATTT ATCTTT TGTT AAATT AATTT ATCTAGGT G ACT ATT ATTT ATTT AAGT AG AAATT CAT AT CATT A CTTTTGCCAACTT GT AGTCAT AAT AGGAGT AGGT GT AT AT GAT GAAGGAAT AAACA AGTCCAGT GG AGT GATT AAAAT AAAAT AT AATTT AGGT GT ACAT C AAAT AAAAACCT T AAAGTTT AGAAAGGCACCGAAT AATTTTGCAT AGAAGAT ATT AGT AAATTT AT AAA AAT AAAAGAAAT GT AGTT GTCAAGTT GTCTT CTTTTTTTT GG AT AAAAAT AGC AGTT GGCTTAT GTCATT CTTTT ACAACCTCCAT GCC ACTT GTCCAATT GTT G AC AC TT AACT AAT C AGTTT GATT CAT GTAT G AAT ACT AAAT AATTTTTT AGG ACT G ACT CAA AT ATTTTT AT ATT AT CAT AGT AAT ATTT ATCT AATTTTT AGG ACC ACTT ATT ACT AAAT AAT AAATT AACT ACT ACT AT ATT ATT GTT GT G AAGCAACAACGTTTT GGTTGTTATG AT GAAACGT ACACT AT AT CAGT AT GAAAAATT CAAAACGATT AGT AT AAATT AT ATT G AAAATTT GAT ATTTTT CT ATT CTT AAT CAGACGT ATTGGGTTT CAT ATTTT AAAAAG GGACT AAACTT AGAAG AGAAGTTT GTTT G AAACT ACTTTT GTCT CTTT CTT GTTCCC ATTT CT CT CTT AGATTT CAAAAAGT G AACT ACTTT ATCTCTTT CTTT GTT CAC ATTTT ATTTT ATT CT ATT AT AAAT ATGGCATCCT CAT ATT GGG ATTTTT AGAAATT ATTCT AA T CATT CAC AGTGCAAAAG AAG

SEQ ID NO: 6 Beta vulgaris CYP76AD5 amino acid sequence

MDNTTLALILSSLFVCFQLIRSFINHAKKSNKLPPGPKRMPIFGNIFDLGEKPHRSFANL

AKIHGPLVSLQLGSVTTVVVSSADVAKEMFLKNDQALANRTIPDSVRAGDHDKLSMS

WLPVSAKWRNLRKISAVQLLSTQRLDASQAHRQSKVQQLLEYVHDCSKKGQPVDIGR

AAFTTSLNLLSNTFFSVELASHESSASQEFKQLMWNIMEEIGRPNYADFFPILGYLDPF

GIRRRLAGYFDQLIAVFQDIIGERQKIRSANLSGGKQTTNDILDTLLNLYDEKELSMGEI

NHLLVDIFDAGTDTTASTLEWAMAELVKNPDMMVKVQDEIEQAIGKGCSMVQESDISK

LPYLQAIIKETLRLHPPTVFLLPRKADADVELYGYVVPKNAQVLVNLWAIGRDPKVWKN

PEVFSPERFLESNIDYKGRDFELLPFGAGRRICPGLTLAYRMLNLMMANFLHSYDWKL

EDGMHPKDLDMDEKFGITLQKVKPLQVIPVPRK

SEC ID NO: 7 Beta vulgaris CYP76AD5 nucleic acid sequence 1 atggataaca ctacacttgc attgatactt tcttctttat ttgtatgttt tcaacttatt 61 cgatctttca ttaaccatgc taaaaaatcc aacaaacttc caccagggcc aaaaagaatg 121 ccgatttttg gcaatatttt tgatcttggt gaaaaacctc atcgctcatt tgcgaatctt 181 gctaagattc acggcccttt ggtgagccta cagttaggaa gtgttacaac tgttgtagta 241 tcatcagcag atgtggctaa agaaatgttc cttaaaaatg atcaagcact tgctaacaga 301 actatccctg attcagttag agcaggtgat catgataagc tgtctatgtc atggttgcct 361 gtatcggcta aatggcggaa cctaagaaaa atctccgctg tgcaattgct ttcgacgcaa 421 cgacttgatg ctagtcaagc gcatagacaa tccaaggtgc aacaacttct tgaatatgtg 481 catgattgtt ctaaaaaagg acaacctgtt gacattggaa gggcagcatt tactacttca 541 ctcaatttat tatcaaacac atttttctca gttgaattag ctagccatga atctagtgct 601 tcacaagagt ttaagcaact catgtggaac attatggagg aaattggtag gcctaattat 661 gctgatttct tccccattct tggctacctt gatccttttg gcataaggcg tcgtttggct 721 ggttactttg atcaacttat tgctgttttt caagacatta ttggtgaaag gcaaaagatt 781 cgatctgcta atctttctgg tgggaaacaa acaacaaatg acattcttga cactcttctc 841 aacctctatg atgagaaaga gttgagtatg ggtgaaatca atcatctcct agtggatatc 901 tttgacgctg gaacagacac tacagctagc acattggaat gggcaatggc agagctagtt 961 aaaaatccgg atatgatggt caaagttcaa gacgaaatcg agcaagcgat tggaaaaggt 1021 tgttcaatgg ttcaagaatc cgatatctca aaactcccat acttgcaagc tattatcaag 1081 gaaacattgc gtctacatcc tccaactgta tttctcttac ctcgaaaggc agacgctgac 1141 gtggagttat atggttatgt tgtacccaaa aatgcacaag ttctagtcaa tctatgggca 1201 attggtcgtg atccaaaggt atggaaaaat ccagaagtat tttctcctga aaggttttta 1261 gagagtaata ttgattacaa gggacgagat tttgagcttt taccatttgg tgctggaaga 1321 aggatatgtc ctggactcac tctagcttat agaatgttaa atttgatgat ggccaatttt 1381 cttcattcct atgattggaa gcttgaagat ggtatgcatc caaaagattt ggacatggat 1441 gagaaatttg gtataacttt gcagaaggtt aagcctctcc aagttattcc cgtacctagg 1501 aaataa

SEQ ID NO: 8 Mirabilis jalapa cytochrome P450 CYP76AD15 amino acid sequence

MENTMLGVILATIFLTFHIMKMLFSPSKVKLPPGPRPLPIIGNILELGDKPHRSFANLAKI

HGPLVTLKLGSVTTIVVSSSEVAKEMFLKNDQPLANRTIPDSVRAGNHDKLSMSWLPV

SPKWRNLRKISAVQLLSTQRLDASQAHRQAKIKQLIEYVKKCSKIGQYVDIGQVAFTTS

LNLLSNTFFSKELASFDSDNAQEFKQLMWCIMEEIGRPNYADYFPILGYVDPFGARRR

LSRYFDQLIEVFQVIIRERLTHDNNIVGNNNDVLATLLDLYKQNELTMDEINHLLVDIFDA

GTDTTASTLEWAMSELIKNPHIMAKAQEEVRRATMSHGGATVAEIQESDINNLPYIQSII

KETLRLHPPTVFLLPRKADVDVQLFGYVVPKNAQVLVNLWAIGRDPNVWPDPEVFSP

ERFMDCEIDVKGRDFELLPFGAGRRICPGLSLAYRMLNLMLANMVHSFDWKLPGVEN

GSGSEMDSLDMDEKFGIALQKTKP

SEQ ID NO: 9 Mirabilis jalapa cytochrome P450 CYP76AD15 nucleic acid sequence 1 atggaaaaca caatgttagg tgttatccta gcaaccattt tcctcacttt tcacataatg 61 aagatgttat ttagtccttc caaggttaaa ctacccccgg gtccgagacc attgccaatt 121 attggtaata ttctcgagct tggggataaa ccacatcgtt cttttgcaaa ccttgcgaaa 181 attcacggtc ctttagttac tttgaaactc gggagtgtaa ccactattgt ggtttcctct 241 tctgaagttg ctaaagaaat gtttttgaaa aatgaccaac ctttggcaaa tcgtaccata 301 cctgactcag taagagcagg taaccatgac aaactatcaa tgtcgtggtt gcctgtatca 361 cccaaatggc gaaatcttag aaagatttca gccgtccaat tgctctcaac tcaacgactt 421 gatgcaagtc aagctcatag acaagctaaa atcaaacaac ttattgagta cgtaaaaaaa 481 tgcagtaaaa tcggccaata cgtcgatatt ggccaagttg cattcactac atcacttaat 541 ttactatcaa acacattctt ttcaaaagaa ctagcatcat ttgattcaga taatgcacaa 601 gagttcaaac aactaatgtg gtgcattatg gaagaaattg gtaggcctaa ttatgccgat 661 tattttccta tcttgggtta tgtcgatcca ttcggtgcta gacgtcgact ttctcgttac 721 ttcgatcaac taattgaagt atttcaagtg attattcgtg agagacttac acatgataat 781 aatattgtgg gtaataacaa tgatgtttta gctacgttgc tcgatcttta taaacaaaac 841 gagttaacta tggatgaaat caaccattta ctagtggaca tttttgatgc tggtacggat 901 acaacagcaa gtacactaga atgggcaatg tcagagctca taaaaaatcc acacataatg 961 gccaaagctc aagaggaggt ccggcgagcc accatgtctc acggcggagc tacggtggcg 1021 gaaatacaag aatcggatat caataatctt ccatacatac aatctattat taaagaaaca 1081 cttcgtttac acccaccaac tgtgttttta cttcctagaa aagctgacgt ggatgtccaa 1141 ttattcggct atgtggtccc caaaaatgct caagtcctag tcaatttatg ggccattggt 1201 cgtgacccaa atgtgtggcc cgacccggaa gtttttagtc ccgaaagatt tatggattgt 1261 gagattgatg tcaagggtcg tgattttgag ctattgcctt tcggggcggg tcgtcggatt 1321 tgtccgggat tgtctttggc ttatcggatg cttaatttga tgttggctaa tatggtacat 1381 tcttttgatt ggaaattacc cggtgttgaa aatggatccg ggtcggaaat ggatagtttg 1441 gatatggatg agaaatttgg catcgcttta caaaagacta aacca

SEQ ID NO: 12: HQT nucleic acid sequence

(target regions for CRISPR are underlined in bold). Coding sequences are underlined.

ATTACTCCTCCATCTTCCTTATCTCTTTAGCTCTTTCTCCCTTCACATTTCACAAATAATAATATTCCAAAAATACATATTTTTATTA TTTTTAAAAACACCCTTCCCTCTCCTTGACCTAATTTTTTTTTTTACCTCTTTTGGAAATAAATGGGAAGTGAAAAAATGATGAAAAT TAATATCAAAGAATCAACACTAGTGAAACCATCAAAACCAACACCAACAAAGAGAATTTGGAGTTCTAATTTGGATTTAATTGTTGGA AGAATTCATCTTTTGACTGTTTATTTTTATAAACCAAATGGATCTTCAAATTTTTTTGATAATAAAGTTATTAAAGAAGCATTAAGTA ATGTTTTACTTTCATTTTATCCAATGGCTGGAAGATTAGGTAC-GGATGAACAAGGTAGAATTGAAGTTAATTGTAATGGTGAAGGTGT _ _ _

GTTGAAACCTCTGGAGATATCTCAACTTTCCCACTAGTTATATTTCAGGTAAAAAAGAATTTTTACATTATCAGTATTACGATATTTT ATTTATATATTTATATATATATATAGATAGTATAAAAATTTTCTTATTTATATTTGTTATATGTGTTGATAATATAAAGATTCTTTCT TAGTGTTTATTTACCTATATGTTATTTTATTAGAGGATTGGTTTTTTTTTGTCTTCATTATATATATATGTTCCATGTTATTATACTA GTGATTCTTTTAAGGTAAAATAATTTTTATGCTATTATATTTGTATGTGTTGATAGTAGTACAAAGATCTTTTTATGTGGTTGGTATT TTTTTTATTTTACATGTTTATGATTGTTAGGTTATTAATTTTATTTTTTAGCTACTTGTAGAAGGGATGGATGTTGTATGATATAATT TATCTGAACTAAATAGCTTGAATTTGAATCTCGAATAATAAGGTAATTTTTTTTGTTTATAAATAAGAATTTTCAATTGATTTCTAAT TGATTAAATCAATTGAGGTGTAGTAGAAAATTGAGTTTAATCTAATTTAAAATCTATCACTATTTGTAGTTATATTATTTTTTGAAAT AAAAATAAAATGATCAAGGGGAGAAAATGCATTTCATATAAAAATCAAGAATAGTATATTTTACTTAAAATGAATAATCAATAATTTA ATTTTAAAGCAAGTCGGTTTATCTTTTTTTTAAAAAAATCAAAATCTAAAGGGGTAGAAAAAATGACATGTGCCTAATTTTTAAAAAA AGAATAATTGGTATTATTGTTCTTGTTCCTTGTCTTTTCTAATAAGAAAATTTAGATATTTTCTACTAACCAAACAAAGAAAAAAAGT TATTTATTTTAACATCAAGATAGGGGCAAATAACAAAAATGATTAAGAATGAAAATTGCATAATTAATGTCTCTAATTAAGTGTTTAG TAGAATTTTAATAGAAGAGAACTTAGTAAAGTATTTAAGAATAGCATACTTTTTAAGTAATATTTTCTAGGTGACCTATATGATTACT ACTATAGTATAGACTAATTAAAATAACAAGCTAGGTGACAAGTTAAGTCACATTATTTTCTAAAAATAAAATAAAAATCACCCACTTG AATTACAAATTCAAAATTTAATAAAAAGTATAGTTTTTTCTTCCATATTTTATTTACATTTTGTATAAATATGTGGTACAAGGTATTT TGTAATATTTATTTTTTCCTTAGACTCCTTCATATACCATTCAAAATTTAAATTTAACGAATTCGATCTTAAAATTTTAAGACATAGT ATTTTGAGAAATTACCAAAAATAAAAAAATATTACTATAATTCAATTGAAGAGTATTTTATATTCTGAGTTGCTAGATCTAATAATAG TAATATTTAATTCTTGACAGCTATCAATTTATCAAGATGGTTCTAGTCTTACAAAAACCTACCAAACTAGTACTTTATTCATTCATAA ACTAGTTGTTATATTATTTTTTTTTCGAATTTAGTTGACTCAATTTTTAAAAAATATAATCTAATTATATTTAAAATTATATTGTCAC ATCAAACTGGAGTATAAATATCCTAATTATTTTCACATTAATTTAGGAGAACAAGAGAAATGATTTGATAGTTAGATACGGTAATTAA TATAGGAAGAGAAAAAATAAATTCTAGAATGAATAATTTTTCAAAATATTAAAAGAAGGAGAAATTTAACAAACCATAAATTAGTGCT TATCTTACTCTTTTAACAATTGCTTTACCTATTTTTATTTTTATTTTTTGGCTTAAAAGAACAAAAAACATTATTTGATTAAGTATAT GATGAAATAATCTAAGTTACATCATTGTTTTAGGAATGAAATAATATTCATAAATTCTTACAACAACTTGTGTAGTGGGTAGGCCATA CTTGTTTTGAAAAAACCAAATACTTTTTTTCTGAAATTGTGCATGTGAATGCATTAATAATAAATAGATAAAATTTGAAGTTGATCAA GTCACACTTCCCTAGCATATTGGTCAATGAAGTATTTGTTTATTTCCTTCTAGATAGTGCAAGTGAATTTGTAAGTGATCTCTTCCAA

CTTTGATGTCTATATAGTTTTTATTTAATATACTGATAGTGTAAAAAATATTTATAGATTACTCGTTTCAAGTGTGGCGGAGTCGCTC TT6GTGGTGGASTATTCCACACGTTATCCSATGGTCTCTCATCCATCCACTTCATCAACACSTSGTCGGACATCGCCCGTSGCCTCTC

CGTCGCAGTCCCGCCGTTCATCGATCGGACGCTCCTCCGTGCAAGGGACCCACCGACATCTTCTTTCGAGCACGTTGAGTACCATCCT

CCACCTACCCTAARCTCATCGAAAAATCGCGAGTCCAGTACCACGACCATGTTGAAATTCTCGAGTGAACAACTCGGGCTTCTTAAGT

CCAAGTCCAAAAATGAGGGTAGCACCTATGAAATCCTCGCAGCCCATATTTGGCGATGCACGTGCAAGGCACGTGGATTGCCAGAGGA

TCAATTGACCAAATTACACGTGGCCACCGACGGAAGGTCAAGGCTTTGCCCTCCCTTGCCACCGGGTTACCTAGGAAACGTCGTGTTC

ACGGCAACCCCAATAGCTAAATCATGCGAACTTCAATCAGAGCCGTTGACAAATTCCGTCAAGAGAATTCACAACGAGTTGATCAAAA

TGGACGACAATTACCTAAGATCAGCACTGGATTACCTCGAATTACAACCTGATTTATCAACCCTAATTCGGGGCCCGGCTTACTTTGC

TAGCCCTAACCTCAATATTAATAGTTGGACTAGGTTGCCTGTCCATGAGTGTGATTTTGGATGGGGTAGGCCAATTCATATGGGACCA

GCTTGCATTTTATATGAAGGGACAATTTATATTATACCAAGTCCAAATTCTAAAGATAGGAACTTGCGTTTGGCTGTTTGTCTAGATG

CTGGTCACATGTCACTATTTGAAAAATATTTATATGAATTATGA

SEC ID NO: 13: CHS2 nucleic acid sequence.

(target regions for CRISPR are underlined). Coding sequences are undrelined.

ATGGTCACCGTTGAGGAGGTTCGAAGGGCGCAACGTGCAAAGGGACCAGCTACTAT CATGGCCATAGGCACGGCGACTCCTTCGAACT GTGTT GAT CAAAGCACTTATCCTGATTATTATTTTCGAAT CACTAATAGTGAACATATGACTGAGCTTAAGGAGAAATTTAAGCGCAT GT GT AAGAAAT AT AC C CAT T T T T T GAAAT TAT T GT T TAT CAT CTAAT CT TACACAT T T T TAGT GT GGAT TAT AT AT T TAAATAAGATA TAATTACATATACCAAAACTATGTTTTGGCTATTGATAATTAAATGGTATTCCTAATTAATTGTAT CACACATGATATACTTTACTTG AT AC T AAC TAAT TAT AT AT C AACAT ACAACAAAGAT GAAT T T TAT TAGGT T T T C T C GAT AGAT T GAT AAAT AT TCTTTTTTT GAT TAT TATTTTTCGTTTAACGCAGGTGATAAATCGATGATTAATAAGAGATATATGCATTTAACTGAAGAAATTTTAAAAGAAAACCCAAATA TTTGTGAATACATGGCTCCTTCTTTGGATGCTAGACAAGATATAGTGGTGGTTGAAGTGCCAAAACTTGGCAAAGAAGCAGCCCAAAA GGCCATTAAAGAATGGGGTCAGCCCAAGTCCAAGAT CACCCATGTGGTCTTTTGCACCACTAGTGGGGTGGACATGCCTGGGGCCGAC TACCAACTCACCAAGCTTCTTGGGCTTCGACCTTCGGTTAAGCGGCTCATGATGTAT CAACAAGGTTGCTTTGCTGGTGGGACCGTTA TCCGACTGGCAAAGGACTTAGCTGAGAACAACAAGGGTGCTCGAGTTCTTGTTGTTTGCTCAGAGAT CACTGCAGTTACTTTTCGTGG TCCAAGTGATACTCATTTGGATAGTATGGTTGGACAAGCCCTTTTTGGTGATGGGGCAGCCGCAATGATTATAGGTTCAGATCCATTA CCAGAAGTTGAAAGGCCTTTGTTTGAACTCGTCTCTGCAGCCCAAACTCTACTCCCTGATAGCGAAGGTGCTATTGATGGTCACCTTC GCGAAGTTGGGCTAACAT TT CACT TGCTCAAAGATGTTCCT GGAT T GAT CTCGAAAAACATTGAAAAGAGTT TAAT TGAAGCATT CCA ACCGTTAGGCATTTCTGATTGGAATTCCATCTTTTGGATCGCGCACCCTGGTGGGCCGGCGATTCTAGAT CAAGTTGAACTAAAACTG AGCCTAAAGCCCGAAAAACTTCGGGCTACTAGGCAAGTTTTAAGTGACTATGGAAATATGTCTAGTGCTTGTGTTCTATTTATTTTAG ATGAAATGAGAAAGGCCTCATCCAAAGAAGGGCTTAGTACCACAGGTGAAGGCCTTGATTGGGGTGTACTTTTTGGATTTGGGCCTGG

GCT TACT GTTGAGACTGTTGTGCTCCATAGTGTGTCTACT TAG

SEQ ID NO: 14: CHs1-RNAi

TCCAGCT ACG AT CTT AGCCATTGGAACAT CT ACGCCTT CT AACT GTGTT GAT CAGA GT ACTT ATCCT GATT ATT ATTTTCGT AT CACT AACAGT G AGCACAAG ACT G AGCT G AAAGAG AAATTT AAGCGCAT GTGT GAT AAAT CAAT GATT AAG AAG AG AT AT AT GCA CTT AACCGAAGAAATCTT GAAAGAGAACCCT AACAT GT GTGCAT ACAT GGCACCTT CCCTT GAT GCAAGGCAAGACAT AGTT GTT GTT GAAGTGCCT AAACTTGGAAAAAGA GGCACCCAAAAGGCCATCAAAGAATGGGGCCAGCCCAAATCCAAGATTACCCATT TGGTCTTTT GT ACCACT AGTGGT GTGGACAT GCCCGCGT GT GACT ACCAACTCGC TAAGCTCCTACCCGTTCGCCCATCAGTCAAGCGACTCATGATGTACCAACAAGGT TGCTTTGCCGGGGGAACAGT ACTTCGGCT AGCCAAGGACTTGGCT GAGAACAAC AAGGGTGCT AGAGTCCTT GTT GTTTGCTCT GAGATCACT GCAGTT ACG SEQ ID NO: 15 HQT CRISPR target sequence CCTT AT CT CTTT AGCT CTTT CT C

SEQ ID NO: 16 HQT CRISPR target sequence G AAGTT AATT GT AATGGT G AAGG

SEQ ID NO: 17 CHS2 CRISPR target sequence CCGTT ATCCGACT GGCAAAGGAC SEQ ID NO: 18 CHS2 CRISPR target sequence GATGGTCACCTTCGCGAAGTTGG

Claims

CLAIMS:

1. A method of producing L-DOPA, the method comprising expressing or increasing the expression of a gene encoding a cytochrome p450 enzyme and the expression of a gene encoding a Myb12 transcription factor in a plant, wherein preferably the cytochrome p450 enzyme is CYP76AD6.

2. The method of claim 1, wherein the cytochrome p450 enzyme and Myb12 transcription factor are expressed in fruit.

3. The method of claim 1 or 2, wherein the gene for CYP76AD6 encodes an amino acid sequence as defined SEQ ID NO: 1 or a functional variant or homolog thereof, and wherein the gene for Myb12 encodes an amino acid sequence as defined in SEQ ID NO: 3 or a functional variant or homolog thereof.

4. The method of any preceding claim, wherein the method comprises introducing and expressing in a plant at least one nucleic acid construct comprising at least one nucleic acid sequence encoding the cytochrome p450 enzyme and the Myb12 transcription factor.

5. The method of claim 4, wherein the nucleic acid sequence is operably linked to a regulatory sequence, preferably wherein the regulatory sequence is a fruit- specific promoter, preferably the E8 promoter, wherein preferably, the E8 promoter comprises a nucleic acid sequence as defined in SEQ ID NO: 5 or a functional variant or homolog thereof.

6. The method of claim 1 to 3, wherein the method comprises introducing at least one mutation into the plant genome, wherein said mutation is the insertion of at least one or more additional copy of a nucleic acid sequence encoding a cytochrome p450 enzyme and at least one or more additional copy of a nucleic acid sequence encoding a Myb12 transcription factor, such that said sequences are operably linked to a regulatory sequence.

7. The method of any preceding claim, where the method further comprises reducing or abolishing the expression of one or more genes involved in the production of polyphenols from phenylalanine, wherein preferably, the one or more genes is selected from chalcone synthase (CHS) and hydroxycinnamoyl transferase (HQT).

8. The method of claim 7, wherein the method comprises using RNA interference to reduce or abolish the expression of one or more genes involved in the production of polyphenols from phenylalanine.

9. The method of any preceding claim, where the plant is selected from tomato, tomato ( Solanum lycopersicum), potato (Solanum tuberosum), eggplant (Solanum melongena), banana (Musa sp.), soybean (Glycine max) and oil seed plants such as Camelina sp. and Brassica rapa.

10. The method of any preceding claim, where the method further comprises obtaining L-DOPA from the fruit of the plant.

11. A genetically altered plant, part thereof or plant cell, wherein the plant, part thereof or plant cell expresses or has an increased level of expression of a cytochrome p450 enzyme and a Myb12 transcription factor.

12. The genetically altered plant of claim 11 , wherein the plant expresses at least one nucleic acid construct comprising at least one nucleic acid sequence encoding a cytochrome p450 enzyme and a Myb12 transcription factor.

13. The genetically altered plant of claim 26, wherein the plant expresses a first nucleic acid construct comprising a nucleic acid sequence encoding a cytochrome p450 enzyme and a second nucleic acid construct comprising a nucleic acid sequence encoding a Myb12 transcription factor, wherein the nucleic acid constructs are stably incorporated into the genome.

14. The genetically altered plant of claim 12 or 13, wherein the nucleic acid sequence encodes a cytochrome p450 enzyme as defined in SEQ ID NO: 1 or a functional variant or homologue thereof, and wherein the nucleic acid sequence encodes a Myb12 transcription factor as defined in SEQ ID NO: 3 or a functional variant or homologue thereof.

15. The genetically altered plant of claim 14, wherein the plant has at least one mutation in its genome, wherein the mutation leads to the accumulation or increased production of L-DOPA in the plant.

16. The genetically altered plant of claim 15, wherein the mutation is the insertion of at least one or more additional copy of a nucleic acid encoding a cytochrome p450 enzyme and the insertion of at least one or more additional copy of a nucleic acid encoding a Myb12 transcription factor or homolog or functional variant thereof under the control of a regulatory sequence.

17. The genetically altered plant of any of claims 11 to 16, wherein the plant is selected from tomato ( Solanum lycopersicum), potato (Solanum tuberosum), eggplant (Solanum melongena), banana (Musa sp.), soybean (Glycine max) and oil seed plants such as Camelina sp.and Brassica rapa.

18. A nucleic acid construct comprising a nucleic acid sequence encoding a cytochrome p450 enzyme and a nucleic acid sequence encoding a Myb12 transcription factor, wherein the nucleic acid sequence is operably linked to a regulatory sequence, wherein preferably, the regulatory sequence is a tissue- specific promoter.

19. A host cell comprising the nucleic acid construct of claim 18.

20. The use of the nucleic acid construct of claim 18 to produce L-DOPA or increase the production of L-DOPA in a plant.

21. L-DOPA obtained or obtainable by the method of any of claims 1 to 10.

22. A method of making a pharmaceutical composition, the method comprising combining L-DOPA obtained or obtainable by the method of any of claims 1 to 10 with a pharmaceutically acceptable carrier.

23. A pharmaceutical composition obtained or obtainable by the method of claim 22.

24. A method of treating Parkinson’s disease, the method comprising administering L-DOPA obtained or obtainable by the method of any of claims 1 to 10 or the pharmaceutical composition of claim 23 to a patient in need thereof.

25. A seed derived from the plant as claimed in any of claims 11 to 17, wherein the seed comprise a nucleic acid sequence encoding a cytochrome p450 enzyme and a nucleic acid sequence encoding a Myb12 transcription factor.