Transcription array

Abstract

The disclosure relates to transcription cassettes, expression vectors and microbial cells adapted for the expression of genes regulated by the RBA transcription factor.

Description

[0001] TRANSCRIPTION ARRAY

[0002] Field of the Disclosure

[0003] The disclosure relates to a transcription expression array obtained from Papaver somniferum that is mutated in a gene encoding a transcription factor (REGULATOR OF BENZYLISOQUINOLINE ALKALOIDS (RBA) that is a global regulator of genes involved in the production of poppy benzylisoquinoline (BIA) alkaloids and related genes; the disclosure includes transcription cassettes including nucleic acid molecules encoding said regulated genes, expression vectors and microbial cells adapted for expression of said nucleic acid molecules encoding said BIA regulated genes.

[0004] Background to the Disclosure

[0005] The opium poppy, Papaver somniferum is an important source of a variety of BIAs. Due to their narcotic and analgesic properties, some BIAs, and their derivatives, are desired for use in therapy. P. somniferum is a source of clinically useful alkaloids such as morphine, codeine, thebaine, noscapine and papaverine. BIAs are extracted from latex harvested from the green seed pods of opium poppy or from the poppy straw which is the dried mature plant. The pathway to produce alkaloids and the various genes involved in the pathway are known and are disclosed in US15 / 182,761 and US15 / 304,455 the contents of which are hereby incorporated by reference in their entirety. Morphinan alkaloids such as codeine and morphine are known to derive from the intermediate (R)- reticuline. (R)-reticuline is thought to be formed by its enantiomer (S)-reticuline in a two- step isomerization process. (R)-reticuline is then further transformed to thebaine. Thebaine is transformed either to oripavine to morphinone and morphine or via an alternative route to codeinone and codeine which is then subsequently transformed to morphine.

[0006] Fast Neutron Mutagenesis (FNM) was carried out on seed of a morphine & noscapine producing P. sominferum cultivar. The mutagenized M1 seed was sown and the M1 generation self-pollinated to generate M2 seed. The M2 generation was then screened for any unusual metabolite profiles compared to the non-mutagenized parental variety. The screen identified two independent mutants that no longer accumulate any BIAs and no longer express most BIA biosynthesis genes. Molecular characterisation of the mutants at the DNA level revealed that each mutant carries a unique deletion spanning a shared region encoding a transcription factor. Because of the loss of BIA synthesis gene expression and lack of BIA accumulation in the mutants, the transcription factor was named REGULATOR OF BENZYLISOQUINOLINE ALKALOIDS (RBA) and the mutants retrospectively labelled rba-1 and rba-2 mutants. WO2022 / 249068 discloses these rba mutants and their phenotype, the content of which is incorporated by reference in its entirety.

[0007] This disclosure relates to an expression array of genes prepared from rba mutant Papaver somniferum plants which are down regulated in the absence of RBA expression because of the deletion of the RBA gene. We disclose expression cassettes and vectors for the expression of genes that are RBA regulated and microbial cells and plant cells adapted for expression of said genes.

[0008] STATEMENTS OF INVENTION

[0009] In its broadest aspect there are provided expression cassettes, expression vectors and microbial cells adapted for the expression of genes regulated by the RBA transcription factor as disclosed in table 1.

[0010] According to an aspect of the invention there is provided a transcription cassette comprising a nucleic acid molecule that encodes a polypeptide with benzylisoquinoline (BIA) alkaloid transporter activity or is associated with redox regulation wherein said nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence as represented by the sequence selected from the group: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27; ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence selected from the group: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27 wherein said nucleic acid molecule encodes a polypeptide with benzylisoquinoline (BIA) alkaloid transporter activity; iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28 and v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) above and which has benzylisoquinoline (BIA) alkaloid transporter activity or is associated with redox regulation

[0011] According to a further aspect of the invention there is provided a transcription cassette comprising a nucleic acid molecule that encodes a polypeptide with transcription or translation associated activity wherein said nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence as represented by the sequence selected from the group: SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 45,47, 51, 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71, 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111 , 113, 115, 117, 119 and 121 ; ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence selected from the group: SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 45, 47, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111 , 113, 115, 117, 119 and 121 wherein said nucleic acid molecule encodes a polypeptide with transcription or translation associated activity; iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 and 122; v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) above and which has transcription factor or translation associated activity.

[0012] According to an aspect of the invention there is provided a transcription cassette comprising a nucleic acid molecule that encodes a polypeptide with benzylisoquinoline (BIA) alkaloid activity wherein said nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group: i) a nucleotide sequence as represented by the sequence selected from the group SEQ ID NO 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181 , 183, 185, 187, 189, 191, 193, 195, 197, 199, 201 , 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283 and 49; ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence selected from the group: SEQ ID NO: 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,

[0013] 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,

[0014] 193, 195, 197, 199, 201 , 203, 205, 207, 209, 211, 213, 215, 217, 219,

[0015] 221 , 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,

[0016] 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275,

[0017] 277, 279, 281 , 283 and 49; wherein said nucleic acid molecule encodes a polypeptide with benzylisoquinoline (BIA) alkaloid activity; iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group SEQ ID NO: 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,

[0018] 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,

[0019] 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,

[0020] 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 240, 242, 244,

[0021] 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,

[0022] 274, 276, 278, 280, 282, 284 and 50; and v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) above and which has benzylisoquinoline (BIA) alkaloid activity.

[0023] Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The Tmis the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:

[0024] Very High Stringency (allows sequences that share at least 90% identity to hybridize)

[0025] Hybridization: 5x SSC at 65°C for 16 hours

[0026] Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

[0027] Wash twice: 0.5x SSC at 65°C for 20 minutes each

[0028] High Stringency (allows sequences that share at least 80% identity to hybridize)

[0029] Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

[0030] Wash twice: 2x SSC at RT for 5-20 minutes each

[0031] Wash twice: 1x SSC at 55°C-70°C for 30 minutes each

[0032] Low Stringency (allows seguences that share at least 50% identity to hybridize)

[0033] Hybridization: 6x SSC at RT to 55°C for 16-20 hours

[0034] Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

[0035] In a preferred embodiment of the invention said nucleic acid molecule is at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over the full-length nucleotide sequence selected from the group: i) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27; ii) SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 45, 47, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119 and 121; iii) SEQ ID NO:123, 125, 127, 129, 131, 133, 135, 137, 139, 141 , 143, 145, 147, 149,

[0036] 151 , 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,

[0037] 185, 187, 189, 191, 193, 195, 197, 199, 201 , 203, 205, 207, 209, 211, 213, 215, 217,

[0038] 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,

[0039] 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283 and 49.

[0040] A modified polypeptide as herein disclosed may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations that may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics. The following non-limiting list of amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or enhance the same biological function and activity as the reference polypeptide from which it varies.

[0041] In a preferred embodiment of the invention, said polypeptide comprising a modified amino acid sequence has at least 50% identity, even more preferably at least 55% identity, still more preferably at least 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 95% identity, and at least 99% identity with most or the full-length amino acid sequence illustrated herein.

[0042] According to a further aspect of the invention there is provided an expression vector comprising a transcription cassette according to the invention.

[0043] In a preferred embodiment of the invention said expression vector includes a nucleotide sequence comprising a promoter that confers constitutive, regulatable, or inducible expression on said nucleic acid molecule encoding said polypeptide of said transcription cassette according to the invention wherein said promoter is operably linked to the nucleic acid molecule encoding said polypeptide.

[0044] Preferably said expression vector comprises a heterologous promoter that confers constitutive, regulatable, or inducible expression on said nucleic acid molecule encoding said polypeptide of said transcription cassette in a microbial host cell.

[0045] Preferably the nucleic acid molecule in the expression vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, (e.g., bacterial, yeast cell), or plant cell. The vector may be a bi-functional expression vector which functions in multiple hosts.

[0046] By "promoter" is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental, or other promoters for expression in plant cells comprised in microbial cells or plants depending on design. Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells. "Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter. In an aspect, the promoter is a tissue specific promoter, an inducible promoter, or a developmentally regulated promoter.

[0047] Of interest in the present context are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success in plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148. Suitable vectors may include plant viral-derived vectors (see e.g., EP194809). If desired, selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes such as resistance to herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate).

[0048] According to a further aspect of the invention there is provided a cell transformed with a transcription cassette or expression vector according to the invention.

[0049] In a preferred embodiment of the invention said cell is a transformed microbial cell.

[0050] In a preferred embodiment of the invention said microbial cell is a bacterial cell, a fungal cell or a yeast cell, for example a Saccharomyces cerevisiae cell.

[0051] The provision of microbial expression systems, for example modified microbial cell-lines e.g. fungal or bacterial, to produce plant metabolic intermediates, for example benzylisoquinoline (BIA) alkaloids is disclosed.

[0052] WO20 18 / 029282, the content of which is incorporated by reference in its entirety, discloses the engineering of microbial host cells that lack alcohol dehydrogenase or aldehyde reductase and optionally one or more second alcohol dehydrogenases or aldehyde reductases and its use in the production of benzylisoquinoline alkaloids or benzylisoquinoline alkaloid precursors, for example (S)-reticuline or (S)-norcoclaurine, by the engineered microbial cells. WO2018 / 211331, the content of which is incorporated by reference in its entirety, discloses a recombinant host cell that expresses one or more genes encoding a cytochrome P450 enzyme capable of N-demethylating and / or O-demethylating reticuline and / or derivatives thereof wherein reticuline and derivatives thereof can be (S)-reticuline, 1 ,2 dehydroreticuline, (R)-reticuline, salutaridine, salutaridinol, thebaine, oripavine, neopinone, codeinone, codeine, morphinone, morphine, hydrocodone, 14- hydroxycodeinone, 7-O-acetyl-salutaridinol or oxycodone.

[0053] WO20 18 / 229305, the content of which is incorporated by reference in its entirety, discloses norcoclaurine synthases with increased activity in host cells such as yeast mutated norcoclaurine synthase amino acid sequences originating from Coptis japonica having an increased catalysation, when compared to the wild type synthase, of the condensation of 4-HPAA and dopamine to (S)-norcoclaurine and / or 3,4-DHPAA and dopamine to (S)-norlaudanosoline.

[0054] WO20 19 / 243624, the content of which is incorporated by reference in its entirety, provides a recombinant Saccharomyces cerevisiae host cell capable of producing one or more benzylisoquinoline alkaloids, comprising a recombinant gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine and having reduced expression of one or more endogenous transporter genes or one or more endogenous transcription factor genes that regulates expression of the one or more endogenous transporter genes and including one or more endogenous genes encoding one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor or one or more transcription factor genes that regulate expression of the one or more endogenous genes; and / or one or more endogenous genes encoding one or more NCS-compatible substrate pathway polypeptides or one or more transcription factor genes that regulate expression of the one or more endogenous genes.

[0055] W02020 / 078837, the content of which is incorporated by reference in its entirety, discloses a microbial-based recombinant expression in Saccharomyces cerevisiae of a number of different transporter proteins to determine a positive influence on the yield of any of several opioids and the in vivo bioconversion of thebaine and / or oripavine to relevant downstream opioid biosynthesis compounds and intermediates.

[0056] W02020 / 144371, the content of which is incorporated by reference in its entirety, discloses a recombinant microbial host cell comprising a biosynthetic metabolic pathway capable of producing one or more target compounds for example, L-dopa, dopamine, (S)-norcoclaurine and derivatives thereof. The pathway comprising one or more heterologous L-tyrosine hydroxylases converting L-Tyrosine into L-dopa capable of increasing the cell production of the target compound(s).

[0057] WO2021 / 069714, the content of which is incorporated by reference in its entirety, discloses a genetically modified host cell, for example a yeast cell, efficient at producing highly pure benzylisoquinoline alkaloids as well as demethylating benzylisoquinoline alkaloids thebaine and / or oripavine in host cells into the corresponding northebaine and / or nororipavine.

[0058] W02024 / 100063 , the content of which is incorporated by reference in its entirety, discloses recombinant host cells , for example yeast cells wherein the recombinant host cell ' s transport system is modified to upregulate the endogenous host cell transporters involved in the efflux desirable BIAs and / or BIA derivatives and / or functional addition of heterologous transporters to enhance efflux of desirable BIAs and / or BIA derivatives .

[0059] In an alternative embodiment of the invention said cell is a transformed plant cell, for example a Papaver spp plant cell such as Papaver somniferum.

[0060] If microbial cells are used as organisms in the process according to the invention they are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism. As a rule, microorganisms are grown in a liquid medium comprising a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0°C and 100°C, preferably between 10°C and 60°C, while gassing in oxygen.

[0061] The pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not. The cultures can be grown batchwise, semi-batchwise or continuously. Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously. The methylated opiate alkaloids produced can be isolated from the organisms as described above by processes known to the skilled worker, for example by extraction, distillation, crystallization, if appropriate precipitation with salt, and / or chromatography. To this end, the organisms can advantageously be disrupted beforehand. In this process, the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8.

[0062] The culture medium to be used must suitably meet the requirements of the strains in question. Descriptions of culture media for various microorganisms can be found in the textbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981).

[0063] As described above, these media which can be employed in accordance with the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0064] Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. The addition of mixtures of a variety of carbon sources may also be advantageous. Other possible carbon sources are oils and fats such as, for example, soya oil, sunflower oil, peanut oil and / or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and / or linoleic acid, alcohols and / or polyalcohols such as, for example, glycerol, methanol and / or ethanol, and / or organic acids such as, for example, acetic acid and / or lactic acid.

[0065] Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture.

[0066] Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0067] Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur- containing fine chemicals, in particular of methionine. Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.

[0068] Chelating agents may be added to the medium to keep the metal ions in solution. Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.

[0069] The fermentation media used according to the invention for culturing microorganisms usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium. The exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

[0070] All media components are sterilized, either by heat (20 min at 1.5 bar and 121 °C) or by filter sterilization. The components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.

[0071] The culture temperature is normally between 15°C and 45°C, preferably at from 25°C to 40°C and may be kept constant or may be altered during the experiment. The pH of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters. To maintain the stability of plasmids it is possible to add to the medium suitable substances having a selective effect, for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture. The temperature of the culture is normally 20°C to 45°C and preferably 25°C to 40°C. The culture is continued until formation of the desired product is at a maximum. This aim is normally achieved within 10 to 160 hours. The fermentation broth can then be processed further. The biomass may, according to requirement, be removed completely or partially from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth. It is advantageous to process the biomass after its separation.

[0072] However, the fermentation broth can also be thickened or concentrated without separating the cells, using known methods such as, for example, with the aid of a rotary evaporator, thin-film evaporator, falling-film evaporator, by reverse osmosis or by nanofiltration. Finally, this concentrated fermentation broth can be processed to obtain the BIA products or intermediates present therein.

[0073] According to an alternative aspect of the invention there is provided a plant cell transfected with a transcription cassette or expression vector according to the invention.

[0074] According to a further aspect of the invention there is provided a plant comprising a plant cell according to the invention.

[0075] In a preferred embodiment of the invention said plant cell or said plant is of the genus Papaver spp preferably Papaver somniferum.

[0076] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers, or steps. “Consisting essentially” means having the essential integers but including integers which do not materially affect the function of the essential integers.

[0077] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. Where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0078] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. An embodiment of the invention will now be described by example only and with reference to the following figure:

[0079] Figure 1: Functional characterization of the STORR fusion protein by heterologous expression in S. cereviseae. (A) HPLC-MS analysis of the in-vitro conversion of dehydroreticuline to (R)-reticuline. Crude soluble (sol) or microsomal (ms) preparations harboring the empty pESC-TRP vector (empty vector), vector containing the oxidoreductase module (oxired), an opium poppy cytochrome P450 reductase (CPR) redox partner (CPR), CPR + CYP82Y2 (CPR + CPY82Y2) or CPR + CYP82Y2- oxidoreductase fusion (CPR+CYP82Y2-oxired) were assayed (R. S. Allen et al., Nat. Biotechnol. 22, 1559-1566 (2004)).

[0024] , The solid lines of the HPLC-MS chromatograms show the normalized total ion count at m / z 328 corresponding to 1,2-dehydroreticuline (substrate) whereas the dotted lines show the normalized total ion count at m / z 330 corresponding to reticuline (product). The inset panel shows the chiral analysis of reticuline: The grey trace is for an (S)- and (R)-reticuline standard, the purple and red traces correspond to reticuline derived by activity of the oxidoreductase and CYP82Y2- oxidoreductase fusion, respectively. (B) HPLC-MS analysis of the conversion of (S)- reticuline into dehydroreticuline and (R)-reticuline. Crude microsomal preparations obtained from S. cerevisiae harboring expression vector pESC-TRP containing CPR only (black), CPR + CYP82Y2 module (blue) or CPR + CYP82Y2-oxidoreductase fusion (red) were assayed. The solid lines of the HPLC-MS chromatograms show the normalized total ion count at m / z 328 corresponding to 1,2-dehydroreticuline, dotted lines show the normalized total ion count at m / z 330 corresponding to reticuline. The inset panel shows the chiral analysis of reticuline, with the same abbreviations as for the main panel.

[0080] Figure 2: Heterologous expression of the methyltransferase PSMT1 in yeast. PSMT1 was cloned into yeast vector pESC-TRP and transformed into Saccharomyces cerevisiae G175. Transformed cultures were grown on media containing glucose, washed and resuspended in 5mL induction medium in shake-flasks containing galactose with raffinose added as a carbon source. Six hours after induction, 15pM scoulerine was added to a subset of cultures. After 3 days, cultures were harvested by centrifugation and a 2pL aliquot of supernatant analysed by UPLC-MS. Off-set base peak chromatograms for the empty vector control and a representative assay are shown. Tetrahydrocolumbamine appeared in all four transformants and was not detectable in any controls (empty vector + substrate, wild-type ± substrate).

[0081] Figure 3. STORR codon optimised sequences MATERIALS, METHODS & EXAMPLES

[0082] Plant growth for RNA sampling from stems

[0083] HN4 wild type and rba-1 and rba-2 mutant plants were grown in a substrate consisting of 50% sand and 50% Terragreen (Oil-Dri Ltd, Wisbech, UK). The plants were watered daily with Vitalink Hydro MAX SW Grow A & B hydroponic nutrient solution (total Nitrogen 4.8%, Ammoniacal Nitrogen (NH4) 0.4%, Nitrate Nitrogen (NO3) 4.4%, Potassium oxide K2O 6.7%, Phosphorous pentoxide (MgO) 2.3%, Calcium Oxide (CaO) 4.2%, Magnesium oxide (MgO) 1.4%, Sulphur trioxide (SO3) 2.6%, Boron (B) 0.008%, Cobalt (Co) 0.001%, Copper (Cu) -chelated by EDTA 0.0016%, Iron (Fe) - chelated by EDDH0A 0.065%, Manganese (Mn) -chelated by EDTA 0.023%, Zinc (Zn) - chelated by EDTA 0.008%, Molybdenum (Mo) 0.001%, Nickel (Ni) 0.001%. The soft water used to make up the nutrient solution did not require any adjustments of the pH.

[0084] RNA isolation for RNA sequencing

[0085] Upper stem samples (defined as the 2 cm stem section immediately underneath the flower head) were collected from rba-1 and rba-2 mutant plants as well as HN4 wild type 1-2 days after anthesis. Three biological replicates were collected per material. The samples were flash frozen in liquid nitrogen and stored at -80°C until extraction. Grinding was performed in jars chilled in liquid nitrogen on the Qiagen TissueLyser as follows: 15 seconds at 20Hz, followed by re-chilling in liquid nitrogen, then a further 15 seconds at 20Hz. 100mg of ground material was used per RNA extraction. RNA was isolated from the powder using a CTAB-based extraction method (Chang et al. (1993) Plant Mol. Biol. Rep.11: 113-116) with small modifications: (i) three sequential extractions with chloroform:isoamylalcohol (24:1) were performed and (ii) the RNA was precipitated overnight with lithium chloride at -20°C. Following extraction, the samples were treated with DNAse I using Ambion’s DNA-free kit according to the manufacturers’ protocol.

[0086] Library preparation and RNA sequencing

[0087] RNA sample quality control, mRNA library preparation (poly A enrichment) and 150 base paired-end Illumina sequencing were carried out by Novogene (Cambridge, UK) according to their inhouse protocols and procedures. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. Libraries were pooled before running them on Illumina's Novaseq 6000 sequencing platform.

[0088] RNA sequencing analysis:

[0089] Three biological replicates for each sample were sequenced. The RNA-seq reads were mapped to the reference transcript dataset of the 55,316 annotated proteins coding genes of the updated HN1 reference genome (Yang et al. (2021) Nat. Comm. 12, Article number 6030), using BWA mapping software with default parameters. Mapped reads were counted and retrieved using SAMTOOLS software package and the expression matrix were normalised to TPM (Transcripts per Million mapped reads) values for subsequent comparative analyses.

[0090] Genes were classified as RBA-regulated if they showed a minimum of average expression value of at least 5 TPM in HN4 stems and about 10-fold reduction of expression in stems of both rba mutants.

[0091] STORR Recombinant Microbial Expression

[0092] Yeast transformation

[0093] Saccharomyces cerevisiae G175 (Sorger D. et al. (2004) J. Biol. Chem. 279: 31190-6) was transformed with the respective vectors as well as the respective empty vector controls using the lithium acetate / single-stranded carrier DNA / PEG method (Gietz. and Schiestl (2007) Nature Protocols 2: 35-37). Transformants were selected on synthetic medium plates containing glucose (0.67% (w / v) yeast nitrogen base without amino acids, 2% (w / v) glucose, 0.13% (w / v) amino acid drop out mix not containing tryptophan, 2% (w / v) Bacto Agar) after a 3-day incubation at 30°C.

[0094] Heterologous expression in yeast

[0095] The transformed yeast strains were cultivated in Synthetic Defined (SD) medium composed of 2 % carbon source, 0.5 % ammonium sulfate, 1.7 g L-1 yeast nitrogen base (without amino acids and ammonium sulfate) and 1.92 g L'1or tryptophan drop-out supplement (Sigma-Aldrich, Gillingham, UK). Cultures grown using glucose as a carbon source were used to inoculate 200 ml cultures with raffinose as a carbon source to an GD600 nm of 0.10 in 1 L conical flasks. These were grown at 30 °C, 250 rpm, and at an GD600 nm of 1.8 to 2.2, 20 mL of 20 % galactose was added to induce expression on the plasmid-borne transgenes. After a further 16-20 hours of cultivation, the yeast cells were harvested by centrifugation at 2,000 g for 10 min. The yeast cells were then washed once with 50 mL of water, then suspended in 2 mL of extraction buffer containing 50 mM Bis-tris propane (pH 8.0), 1.2 M sorbitol, 100 mM NaCI, 1 mM EDTA, 1 mM DTT and 1 / 250 dilution of protease inhibitor cocktail (Sigma-Aldrich P8215). The resuspension was then used to prepare crude cell lysates and / or microsomes.

[0096] Preparation of yeast crude cell lysates and soluble and microsomal fractions

[0097] Soluble and microsomal preparations were prepared as described by King A. et al. (Planta 226, 381-94, 2007). The protocol uses beating with glass beads for breaking yeast cell walls to obtain crude cell lysate. To obtain soluble and microsomal fractions, crude lysate preparation was followed by ultracentrifugation. Alternatively, the crude lysate was used directly in enzymatic assays.

[0098] Heterologous protein expression in Saccharomyces cerevisiae

[0099] Genscript created synthetic DNA sequences were created for PsCPR [Genbank accession AAC05021 , (Rosco A. et al. (1997) Arch. Biochem. Biophys. 348, 369-77), the full length STORR (CYP82Y2-Oxidoreductatse (“Oxired”)) protein, the N-terminal CYP82Y2 module and C-terminal oxidoreductase (Oxidoreductase) module., that were codon optimised for expression in Saccharomyces cerevisiae using standard methods.

[0100] The codon optimized genes (SEQ ID NO 285-288, Figure 3) were then amplified by PCR using the following primers which incorporate a restrictions site and 5’-AAAA-3’ Kozak sequences preceding the start codon:

[0101] Primer name Target Sequence

[0102] CPR XhoI FCodon°Ptimlzed5 ’ -AAAAGGATCCAAAAATGGGTTCAAACAACTTAGCCAACTC-3 ’ cytochrome P450

[0103] CPR BamHI R 5 ’ -AAAACTCGAGTT ACC A AAC ATCTCTC AAAT ATCTTTCTTC-3 ’ reductase (CPR)

[0104] CYP82X- Codon optimized 5’-

[0105] Oxidoreductase_NotI_F full-length AAAAGCGGCCGCAAAAATGGAATTACAATACATCTCCTACTTTC-

[0106] CYP82Y2- 3’

[0107] CYP82X- Oxidoreductasefusion 5 ’ -AAAATTAATTAATTATGCTTCGTCATCCCATAATTC-3 ’

[0108] Oxidoreductase_PacI_R protein

[0109] Codon optimized

[0110] CYP82X Notl F 5’-

[0111] N-terminal

[0112] AAAAGCGGCCGCAAAAATGGAATTACAATACATTTCTTACTTTC-

[0113] CYP82Y2 module

[0114] 3’ CYP82X_PacI_R 5 ’ -AAAATTAATTAATCTCTTTCAGATGCAGCAC-3 ’

[0115] Codon optimized

[0116] Oxidoreductase_NotI_F C-terminal 5 ’ -AAAAGCGGCCGCAAAAATGGAATCCTCTGGTGTCCCTG-3 ’

[0117] Oxidoreductase_PacI_R Oxidoreductase 5 ’ -AAAATTAATTAATTATGCTTCGTCATCCCACAATTC-3 ’ module

[0118] Expression vector pESC-TRP: :oxired was created by digesting the corresponding PCR product with Notl and Pad and inserting this behind the GAL10 promoter of pESC-TRP. pESC-TRP::CPR was created by digesting the corresponding PCR product with BamHI and Xhol and inserting this behind the GAL1 promoter of pESC-TRP. This was then used to created pESC-TRP: :CPR::CYP82Y2 and pESC-TRP::CPR::CYP82Y2- Oxidoreductase. These plasmids were then transformed in S. cerevisiae G175 (Sorger D. et al. (2004) J. Biol. Chem. 279, 31190-6) using lithium acetate protocol (Geitz R. D. and Woods R. A. (2002) Methods in Enzymology 350, 87-96). Yeasts were cultivated in Synthetic Defined (SD) medium composed of 2 % carbon source, 0.5 % ammonium sulfate, 1.7 g L'1yeast nitrogen base (without amino acids and ammonium sulfate) and 1.92 g L'1or tryptophan drop-out supplement (Sigma-Aldrich, Gillingham, UK). Cultures grown using glucose as a carbon source were used to inoculate 200 ml cultures with raffinose as a carbon source to an OD 600 nm of 0.10 in 1 L conical flasks. These were grown at 30 °C, 250 rpm, and at and OD 600 nm of 1.8 to 2.2, 20 mL of 20 % galactose was added to induce expression on the plasmid-borne transgenes. After a further 16 hours of cultivation, the yeast cells were harvested by centrifugation at 2,000 g for 10 min. The yeast cells were then washed once with 50 mL of water, then suspended in 2 mL of extraction buffer containing 50 mM Bis-tris propane (pH 8.0), 1.2 M sorbitol, 100 mM NaCI, 1 mM EDTA, 1 mM DTT and 1 / 250 dilution of protease inhibitor cocktail (Sigma-Aldrich P8215). Soluble and microsomal preparations were then prepared as described previously (King A. et al. (2007) Planta 226, 381-94).

[0119] STORR Enzyme Assays

[0120] General reaction set up was carried out as previously described (Lenz and Zenk (1995) FEBS 233:132-139). Substrates (S)-reticuline, 1,2-dehydroreticuline and coclaurine were purchased from TRC Chemicals (Toronto, Canada) and codeinone was supplied by MacFarlan-Smith (Edinburgh, UK). Assay reactions contained 300 mM NADPH, 5 pg oxidoreductase A or B preparation, 100 mM buffer covering a range of pH values (potassium phosphate pH 6-8, glycine-NaOH pH 9) and 75 pM substrate. Reactions were incubated for 2 h at 37°C and immediately frozen at -80°C. Reactions were dried down to powder in speedvac Gene Vac EZ-2 plus (Ipswich, UK) at 40°C and resuspended in 100 L 1:1 Hexane: Ethanol (v / v), 0.1% Diethylamine (v / v).

[0121] Heterologous expression in yeast demonstrates that the STORR fusion protein is capable of catalyzing the two-step conversion of (S)- to (R)-reticuline with each of is modules performing an individual step in the sequential reaction.

[0122] For direct functional characterization the STORR fusion and the separate modules were expressed in Saccharomyces cerevisiae and enzyme assays performed on soluble extracts and microsomal preparations (Figure 1). 1,2-dehydroreticuline is converted to

[0123] (R)-reticuline with 100% conversion efficiency by both the STORR fusion and the oxidoreductase module but not by the CYP82Y2 module plus redox partner (Figure 1A) In contrast, the CYP82Y2 module plus redox partner catalyzed near- 100% conversion of

[0124] (S)-reticuline to 1,2-dehydroreticuline demonstrating that it acts as a 1,2- dehydroreticuline synthase (Figure 1B). Microsomal preparations harbouring the entire STORR fusion converted about 20% of the added (S)-reticuline to (R)-reticuline, confirming the bifunctional role of the protein in performing sequential reactions in the epimerization of reticuline (Figure 1 B).

[0125] P450-redox systems where the P450 enzyme is covalently linked to redox partner reductase components are well known in both prokaryotes and lower eukaryotes (Guengerich F.P. and Munro A.W. (2013) J. Biol. Chem. 288, 17065-73). The discovery of the STORR fusion demonstrates that covalent linkage of a P450 to a reductase that is involved in a sequential reaction rather than as a redox partner exists in nature. Other forms of bifunctional P450 fusions with oxygenase / peroxidase, hydrolase and dioxygenase modules have been reported to occur in ascomycetes and all of these also appear to catalyze sequential reactions (Brodhun F. et al. (2009) J. Biol. Chem. 284, 11792-805; Hansen B.G. et al. (2012) Appl. Environ. Microbiol. 78, 4908-13; Hoffmann I. et al. (2014) J. of Lipid Res. 55, 2113-23). STORR represents the first example of a P450 fusion protein from higher eukaryotes. A possible explanation as to why such fusion proteins evolve is that they facilitate efficient channeling of highly unstable, reactive or potentially toxic intermediates. Evidence for efficient substrate channeling in the case of the STORR fusion comes from the observation that microsomal fractions harboring the fusion protein directly convert (S)- to (R)-reticuline with no detectable accumulation of 1 ,2-dehydroreticuline (Figure 1 B).

[0126] PSMT1 Recombinant Microbial Expression Generation of the plasmid construct for heterologous expression of PSMT1 gene product in Saccharomyces cerevisiae

[0127] Cloning and protein production in S. cerevisiae were performed using the pESC Yeast Epitope Tagging System (Agilent Technologies, USA). The full-length coding sequence of PSMT1 (SEQ ID NO 157) was amplified from cDNA extracted from HN1 variety using primers which introduced BamHI (forward primer) and Xho\ sites (reverse primer). The resulting PCR product was directionally cloned using the BamHI and Xho\ sites of the pESC-TRP vector (Agilent Technologies, USA). The obtained pESC-TRP:PSMT1 construct was transformed and propagated into E. coli strain DH5a with fidelity of the insert confirmed subsequently by DNA sequencing.

[0128] Yeast transformation and PSMT1 protein production pESC-TRP: :PSMT1 was transformed into Saccharomyces cerevisiae strain G175 using the lithium acetate method following the protocol described by the manufacturer (Agilent Technologies, USA). Briefly, G175 strain was grown overnight in 50 mL YAPD broth (0.0075% (w / v) L-adenine hemisulfate salt, 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose and diluted to an ODeoo = 0.25 in fresh YPAD broth and incubated at 30°C until ODeoo = 1.0. Cells were harvested and resuspended in 10 mL LTE buffer (100 mM LiOAc, 10 mM Tris-HCI pH 7.5, 1 mM EDTA), spun at 3,000 rpm for 5 min and resuspended in 0.5 mL LTE buffer. 50 pL of this cell suspension was then used for each transformation, carried out by mixing 1 pg of pESC-TRP: PSMT1 , 300 pL transformation mix (40% (w / v) PEG 3350, 100 mM LiOAc, 10 mM Tris-HCI pH 7.5, 1 mM EDTA) followed by a 30 min incubation at 30°C and a 15 min incubation at 42°C. Transformants were selected on synthetic medium plates containing glucose (0.67% (w / v) yeast nitrogen base without amino acids, 2% (w / v) glucose, 0.13% (w / v) amino acid drop out mix not containing tryptophan, 2% (w / v) Bacto Agar) after a 3-day incubation at 30°C. Colonies were then streaked out on fresh selective medium and tested for the presence of insert. One yeast colony was resuspended in 50pL 10 mM Tris-CI pH 7.5, 1 mM EDTA, Lyticase 100-50 U / pL and incubated for 30 min at 37°C and then 10 min at 95°C. The suspension was clarified by spinning the products for 5 min at 13,000 rpm on a benchtop microfuge. PSMT1 insert was checked by PCR using vector primers Gall FOR 5’ATT TTC GGT TTG TAT TAC TTC-3’ and Gall Rev 5’-GTT CTT AAT ACT AAC ATA ACT-3’ using the cycling conditions described by the manufacturer.

[0129] Recombinant protein production was carried out following described methods (Hawkins and Smolke (2008) Nat. Chem. Biol. 4, 564) with the following modifications. Selected transformants carrying the PSMT1 insert were inoculated in 10 mL synthetic broth containing glucose and grown overnight at 30°C in shake flasks. Cells were harvested by spinning down cultures at 3,000 rpm on a benchtop centrifuge for 5 min and washed twice in sterile 0.9% (w / v) NaCI. Cell pellets were resuspended in 5 mL synthetic broth containing raffinose and galactose (0.67% (w / v) yeast nitrogen base without amino acids, 2% (w / v) galactose, 1.5% (w / v) raffinose, 0.13% (w / v) amino acid drop out mix not containing tryptophan) and incubated at 30°C for 6 h at 200 rpm to induce the expression of recombinant PSMT1. Sterile scoulerine HCI (APIN Chemicals Ltd, Oxon, UK) prepared in 100 mM Acetate buffer pH4.5 was added to the cultures to a final concentration of 15 pM. After 3 days, cultures were harvested by centrifugation and a 2pL aliquot of supernatant analysed by UPLC-MS.

[0130] We expressed the PSMT1 gene product in Saccharomyces cerevisiae. PSMT1 converts scoulerine to tetrahydrocolumbamine at high efficiency (Figure 2). We therefore conclude that PSMT1 is responsible for the first committed step in the pathway to noscapine synthesis.

[0131] Table 1 Expression of RBA regulated genes

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

Claims

CLAIMS1. A transcription cassette comprising a nucleic acid molecule that encodes a polypeptide with benzylisoquinoline (BIA) alkaloid transporter activity or associated with redox regulation wherein said nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group: i) a nucleotide sequence as represented by the sequence selected from the group consisting of in SEQ ID NO: 1 , 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27; ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25 and 27 wherein said nucleic acid molecule encodes a polypeptide with benzylisoquinoline (BIA) alkaloid transporter activity or is associated with redox regulation; iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28 and v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) above and which has benzylisoquinoline (BIA) alkaloid transporter activity or is associated with redox regulation.

2. A transcription cassette comprising a nucleic acid molecule that encodes a polypeptide with transcription or translation associated activity wherein said nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group: i) a nucleotide sequence as represented by the sequence selected from the group: SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 45, 47, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111 , 113, 115, 117, 119 and 121 ; ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence selected from the group: SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 45, 47, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111 , 113, 115, 117, 119 and 121 wherein said nucleic acid molecule encodes a polypeptide with transcription or translation associated activity; iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 and 122; v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) above and which has transcription factor or translation associated activity.

3. A transcription cassette comprising a nucleic acid molecule that encodes a polypeptide with benzylisoquinoline (BIA) alkaloid activity wherein said nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence as represented by the sequence selected from the group: SEQ ID NO123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171,173, 175, 177, 179, 181 , 183, 185, 187, 189, 191, 193, 195, 197, 199,201 , 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227,229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255,257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283 and 49 ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence selected from the group: SEQ ID NO: 123, 125, 127, 129, 131 , 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201 , 203, 205, 207, 209, 211, 213, 215, 217, 219,221 , 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275,277, 279, 281 , 283 and 49; wherein said nucleic acid molecule encodes a polypeptide with benzylisoquinoline (BIA) alkaloid activity;iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group: SEQ ID NO: 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 240, 242, 244,246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,274, 276, 278, 280, 282, 284 and 50; and v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) above and which has benzylisoquinoline (BIA) alkaloid activity.

4. The transcription cassette according to any one of claims 1 to 3 wherein said nucleic acid molecule is at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over the full-length nucleotide sequence selected from the group: i) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27; and / or ii) SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 45, 47, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119 and 121; and / or iii) SEQ ID NO: 123, 125, 127, 129, 131 , 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201 , 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241 , 243, 245, 247, 249,251 , 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281 , 283 and49.

5. The transcription cassette according to any one of claims 1 to 3 wherein said polypeptide comprising a modified amino acid sequence has at least 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 95% identity, and at least 99% identity with most or the full- length amino acid sequence illustrated herein.

6. An expression vector comprising a transcription cassette according to any of claims 1 to 5.

7. The expression vector according to claim 6 wherein said expression vector includes a nucleotide sequence comprising a promoter that confers constitutive, regulatable, or inducible expression on said nucleic acid molecule encoding said polypeptide of the transcription cassette according to any one of claims 1 to 5 wherein said promoter is operably linked to the nucleic acid molecule encoding said polypeptide.

8. The expression vector according to claims 6 or 7 wherein said promoter is a heterologous promoter that confers constitutive, regulatable, or inducible expression on said nucleic acid molecule encoding said polypeptide of said transcription cassette according to any one of claims 1 to 5 in a microbial or plant host cell.

9. The expression vector according to any one of claims 6 or 7 wherein the nucleic acid molecule of said transcription cassette according to any one of claims 1 to 5 is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial or plant cell.

10. The expression vector according to claim 9 wherein the microbial cell is a bacterial cell or yeast cell.

11. A cell transformed with a transcription cassette according to any one of claims 1 to 5 or expression vector according to any one of claims 6 to 10.

12. The cell according to claim 11 wherein said cell is a microbial cell.

13. The cell according to claim 12 wherein said microbial cell is a bacterial cell, a fungal cell or a yeast cell.

14. The cell according to claim 13 wherein said cell is a plant cell,15. The cell according to claim 14 wherein said plant cell is of the genus Papaver.

16. The cell according to claim 15 wherein said plant cell is Papaver somniferum cell.

17. A plant comprising a plant cell according to any one of claims 14 to 16.

18. The plant according to claim 17 wherein said plant is of the genusPapaver spp.

19. The plant according to claim 18 wherein said plant is Papaver somniferum.

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