Aurone-producing transgenic plants

Abstract

The present disclosure relates to a bryophyte cell that is transformed or genetically modified to express aureusidin synthase and chalcone-4'-glycosyltransferase. The aureusidin synthase and chalcone-4'-glycosyltransferase may be expressed as a single biscistronic cassette separated by a P2A peptide or separately through insertion of two unique gene cassettes into said bryophyte cell. The expression may be driven by an exogenous promoter. The transformed bryophyte cell may be exposed to UV radiation to enhance production of exogenous flavonoids.

Description

AURONE-PRODUCING TRANSGENIC PLANTSTECHNICAL FIELD:

[0001] This present disclosure relates to a transgenic plant exhibiting enhanced production of aurones and derivatives of aurones. This present disclosure further relates to enhancing the production of aurones in genetically modified bryophyte cells.BACKGROUND:

[0002] Aurones are tricyclic flavonoids comprising a benzofuranone core and a phenyl group linked by a carbon-carbon exocylic bond. Aurones are considered to be a minor group of flavonoids due to their relatively low abundance and sporadic distribution across the plant kingdom (Bohm 1988). As a rare class of plant phenolics, aurones were first identified in midtwentieth century and named for the characteristic bright gold colour that they confer to floral tissues. The first identified aurone, Leptosidin 6-O-glucoside, was reported in the flowers of Coreopsis grandiflora L. in 1943 (Geissman & Heaton), followed by aureusidin 6-O-glucoside in Antirrhinum majus L. in 1950 (Seikel and Geissman). Since then, more than 100 aurone-related flavonoids of different structures have been reported, including those occurring in: (i) the eudicot families Anacardiaceae, Asteraceae, Gesneriaceae, Faba-ceae, Oxalidaceae, Plumbaginaceae, Rubiaceae, Rhamnaceae, Rosaceae, Cactaceae, Moraceae or Plantaginaceae (Anderson and Jordhiem 2010; Boucherle et al. 2017); and (ii) the monocot family Cyperaceae (Boucherle et al. 2017: Davis et al. 2020). Structurally, aurones are defined by a distinctive 6:5 benzofuranone core and 2-aryl substitution at the C2 position (Mazziotti et al., 2022). These pigments predominantly accumulate in flowers, where they generate yellow hues and patterning that attract pollinators by contrasting with anthocyanins and serving as nectar guides (Davies et al., 2020). Aurones mostly accumulate in floral tissue where they play an essential role in attracting pollinators (Davies et al. 2006; Penny et al. 1983). Beyond floral tissues, aurones may also be found in roots, tree heartwood, and nectar where they are believed to act as antimicrobial and anti-feedants (Boucherle et al. (2017).

[0003] The sporadic occurrence of aurones across the plant kingdom and elucidation of several distinctive aurone biosynthetic pathways suggest multiple unique events of convergent evolution resulting in aurone biosynthesis (Davies et al. 2020; Nakayama et al. 2022). To date, members from two distinct enzyme classes have been reported to facilitate aurone biosynthesis:peroxidases and polyphenol oxidases (PPOs). PPOs are a class of enzymes that catalyze the oxidation of phenolic substrates. PPOs are classified as either tyrosinases (TYRs) or catechol oxidases (CO) (Panis et al. 2020). PPOs are primarily localized within the plastids of intact plant cells. Upon cellular disruption resulting from herbivory or microbial infection, PPOs interact with phenolic substrates previously sequestered in the vacuole, resulting in the formation of toxic o- quinones and tissue browning which serve to protect the plant from infection and herbivory (Zhang et al. 2023). In addition, PPOs involved in aurone biosynthesis may perform additional specialized roles in plants by contributing to floral pigmentation rather than assuming a general role in defense (Nakayama et al. 2022; Boucherle et al. 2017).

[0004] Studies conducted in the 1970s on crystalline horseradish peroxidase, a type III peroxidase which catalyzes the formation of a phenoxy radical from a phenolic hydrogen doner using H2O2 as a hydrogen acceptor, demonstrated conversion of isoliquiritigenin to the aurone hispidol in vitro (Wilson & Wong 1976). This finding led to the proposal that peroxidases enzymes are involved in production of hispidol in legumes such as soybean (Glycine max), garbanzo (C / cer arietinum), and common bean (Phaseolus vulgaris) (Wilson & Wong 1976). This proposal was later supported by the characterization of two class III peroxidases (MtPRXI and MtPRX2), which were found to convert isoliquiritigenin to the aurone hispidol, in alfalfa, Medicago truncatula (Farag et al. 2009). According to the proposed mechanism for producing hispidol, MtPRXI and MtPRX2 catalyze the initial formation of an isoliquiritigenin phenoxy radical with subsequent conversion to hispidol occurring non enzymatically (Nakayama et al. 2022; Farag et al. 2009).

[0005] The first fully characterized aurone synthase, aureusidin synthase (AmAUSI), is a PPO derived from snap dragon (Antirrhinum majus) which catalyzes the oxygen-dependent production of aureusidin 6-O-glucoside from tetrahydroxychalcone 4'-O-glucoside (Nakayama et al., 2000; Sato et al., 2001 ; Strack and Schliemann 2001 ; Nakayama 2002). Notably, AmAUSI displays narrow substrate specificity for tetrahydroxychalcone 4'-O-glucoside and does not accept tyrosinase the standard substrate for PPOs. Such exhibited specificity suggests that the AmAUSI is a CO (Nakayama et al. 2000; Nakayama et al. 2022). Two members of the PPO gene family found in the Asterales species Coreopsis grandiflora, CgAS1 and CgAS2, were found to catalyze the formation of 4-deoxyaurones from 6'-deoxychalcones, and more specifically the production of butein and isoliquirtigenin to hispidol (Molitar et al. 2015). Similar to AmAUSI , both CgAS1 and CgAS2 exhibit hydroxylase activity towards its natural substrate isoliquiritigenin but do not accept classical tyrosinase substrates; as a result, CgAS1 and CgAS2 are considered Cos (Kampatsikas& Rompel 2021). In the liverwort M. polymorpha, overexpression of the transcription factor MpMYB14 led to the accumulation of the red-pigment auronidins and the intermediate aureusidin (Berland etal. 2019: Furadate etal. 2023). Moreover, cell-free extracts from these lines produced aureusidin in a time-dependent manner when incubated with naringenin chaicone, indicating a similar pathway to that in snapdragon, involving a PPO (Furadate et al. 2023). Among the six highly expressed PPOs in MpMYB14OE lines, only Mp2g05850.1 converted naringenin chaicone to aureusidin when expressed in yeast (Furadate et al. 2023). However, phylogenetic analysis of the PPO family in M. polymorpha demonstrates that MpAS1 appears to have evolved independently from the PPO family in higher plants (Furadate et al. 2023).

[0006] Over the past two decades, aurones have garnered significant interest for their capacity to function as antiviral, anti-cancer and anti-inflammatory agents (Mazziotti et al. 2022). For example, aurones have demonstrated broad-spectrum antibacterial activity, proving effective against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Proteus vulgaris, and Mycobacterium tuberculosis (Olleik et al. 2019; Campanico et al. 2019; Sutton et al. 2017). It has also been reported that aurones can inhibit RdRp: a key enzyme responsible for the viral replication of hepatitis C (Waheed et al. 2012; Megeullati et al. 2014). Sulfuretin, an aurone found in Coreopsis grandiflora, has been shown to counteract the activities of the pro-inflammatory molecules nitric oxide (NO) and prostaglandin E (PGE2) (Moon et al. 2015). Moreover, aurones have garnered significant interest for their ability to inhibit tumour proliferation. For example, hamiltrone, an aurone found in the shrub Uvaria hamiltonii, was found to modulate the DNA strand-scission activity of rapidly proliferating tumour cells (Huang, 1998). However, aurones naturally occur in some plant tissues only and only accumulate at low levels in such plant tissues, thus making extraction and purification difficult and inefficient at an industrial scale (Boucherle et al. 2017). Moreover, synthetic approaches to aurone biosynthesis require complex precursors and harsh heavy metal catalysts.

[0007] Another major obstacle in the production of aurones is the rapid conversion of chaicone precursors to inert flavanones through either the enzymatic action of chaicone isomerase (CHI) or spontaneous ring closure (Austin & Noel 2003; Nakayama et al. 2022). Aurone producing plants have evolved several mechanisms to overcome the challenge of ring closure all of which include compartmentalization of the aurone synthase biosynthetic step away from the cytosol. In snap dragon, naringenin chaicone is glycosylated to naringenin-6-O-glyscoside by the 4’ glycosyl transferase Am4’GT (Ono et al. 2006) and transported to the vacuole where AmAllSI is localised(Ono et al. 2006). The addition of a glycosyl group has been proposed to not only hamper the activity of CHI (Fujino et al. 2018) but also serve as a signal for translocation into the vacuole (Ono et al. 2006; Nakayama 2022). Moreover, spontaneous ring closure of naringein-4-O- glycoside occurs more slowly in the acidic vacuole permitting AmAllSI to oxidate the chaicone to aureusidin-6-O-glucoside (Austin & Noel 2003; Nakayama 2022). In Coreopsis, the 6’- deoxychalcone isoliquiritigenin undergoes ring closure at a rate that is 30 fold slower than that of naringenin chaicone (Jez & Noel 2002). Moreover, CHSs in the Asterales family are unable to accept 6’-deoxychalcones as substrates. Therefore, it has been proposed that in C. grandiflora, isoliquitigenin is produced in the cytoplasm and transported to the plastid where CgALISI is localized, converted to sulfuretin, transported back into the cytoplasm, glycosylated to produce sulfuretin-6-O-glucoside and sequestered in the vacuole (Molitar et al. 2015). In alfalfa, the peroxidases, MtPRX1&2 are proposed to localize in the vacuole and they convert cytosolically produced isoliquitigenin-6-O-glucoside to hispidol (Farag et al. 2009). In M. polymorpha, it has been proposed that the synthesis of the aurone back bone occurs in the more acidic apoplastic space where MpAS1 is localized and at a pH of 5.5 where it is most active (Furadate et al. 2023). Accumulation of aureusidin 6-O-glucuronide (Markham & Porter 1978) and aureusidin 4-O- neohesperidoside in M. polymorpha (Furadate et al. 2023) indicates that, similar to snap dragon, MpAS1 accepts chaicone glycosides as a substrate.

[0008] Despite the promising therapeutic potential of aurones, and the potential to synthesize derivatives thereof, commercialization of aurones continues to face significant manufacturing challenges. Naturally occurring aurones only accumulate at very low levels in plant tissues, thereby making industrial-scale harvesting and extraction unfeasible (Boucherle et al. 2017). Moreover, previously reported synthetic approaches to rebuild the 2-functionalized benzofuran scaffold central to aurones require multi-step chemistry, complex precursors and harsh heavy metal catalysts. Presently, there is a lack of cost-effective and environmentally friendly means of producing aurones (Mazziotti et al. 2022). High volume design-build-learn cycles may overcome some of these challenges, but current high-throughput methods for analyzing complex metabolomic data remain too slow and costly (Li et al. 2022).

[0009] Notwithstanding their low abundance, there has been scientific interest in developing techniques that would enhance the production of aurones through genetically modified plants. For example, United States publication number 2013 / 0305408 teaches the production of aurones in leafy vegetables through genetically modifying such leafy vegetables, though it does not teachthe same in non-vascular species. Production of heterologous glycosylated proteins in transformed bryophytes has also been taught in the patent literature such as international patent application number PCT / EP2006 / 006831.

[0010] Recently, the use of genetically modified microbial cultures grown in bioreactors has been proposed as an alternative for the industrial scale production of plant-derived small molecules. While microbial bioreactor systems are excellent at producing small molecules, phenolic compounds such as aurones can be toxic to the microbes in such bioreactor systems. In addition, reconstitution of flavonoid pathways in microbial hosts is challenging, as plant enzymes display lower efficiency, mislocalize, and produce undesirable side products when expressed in plant systems (Li et al., 2022).

[0011] In addition, flavonoids are believed to have evolved over 400 million years ago to protect early land plants and assist in facilitating such plants’ transition out of water by protecting the photosynthesizing tissue within such plants from the harsh LIV radiation (Davies et al. 2020). As a result, flavonoids are found exclusively in vascular plants and some bryophytes, including mosses (Davies et al. 2020). Flavonoids have since evolved into one of the most diverse class of secondary metabolites produced by plants; to date, over 9000 unique structural variants have been identified (Williams and Grayer 2004) and such variants are classified based on their aglycone structure. P. patens is known to synthesize several flavonoids and express 19 chaicone synthase (CHS) genes, four of which are UV-responsive (Wolfe et al., 2010). Interestingly, P. patens lacks type II CHI and instead expresses noncatalytic type IV CHI proteins, suggesting flavanone production may proceed via nonenzymatic isomerization (Morita et al., 2014). To date, no aurone has been identified or detected in P. patens.SUMMARY:

[0012] The present disclosure relates to one or more teachings directed to modifying a bryophyte cell to produce flavonoids that are not normally expressed or produced in such bryophyte cell.

[0013] According to a part of the disclosure, there is a genetically modified bryophyte cell comprising a gene cassette encoding: (a) any one of: (i) a biological sequence set forth in SEQ ID NO: 1 and a biological sequence set forth in SEQ ID NO: 3; (ii) a biological sequence set forth in SEQ ID NO: 5 and a biological sequence set forth in SEQ ID NO: 7; (iii) a biological sequence set forth in SEQ ID NO: 10; (iv) a biological sequence set forth in SEQ ID NO: 12; and (v) abiological sequence set forth in SEQ ID NO: 14; or (b) any biological sequence having at least 80% sequence identity to any of the foregoing biological sequences identified in (a)(i) to (a)(v).

[0014] The biological sequence set forth in SEQ ID NO: 1 and the biological sequence set forth in SEQ ID NO: 3 may be linked by a biological sequence set forth in SEQ ID NO: 2. The biological sequence set forth in SEQ ID NO: 5 and the biological sequence set forth in SEQ ID NO: 7 may be linked by a biological sequence set forth in SEQ ID NO: 6.

[0015] The cell may further comprise a promoter for promoting the combined sequence within the cell. The promoter may be a maize ubiquitin promoter.

[0016] According to another part of the disclosure, there is a method of enhancing a production of naringenin in a bryophyte cell, the method comprising introducing into a vacuole of the cell and integrating into a genome of the cell a biological sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 8, or any biological sequence having at least 80% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 8, via homologous recombination using flanking sequences from P. patens genome locus 108.

[0017] According to another part of the disclosure, there is a method of enhancing a production of aureusidin in a bryophyte cell, the method comprising introducing into the cell and integrating into a genome of the cell the biological sequence set forth in SEQ ID NO: 10, or any biological sequence having at least 80% sequence identity to SEQ ID NO: 10, via homologous recombination using flanking sequences from P. patens genome locus 108.

[0018] According to another part of the disclosure, there is a method of enhancing a production of luteolin in a bryophyte cell, the method comprising introducing into the cell and integrating into a genome of the cell either the biological sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 14, or any biological sequence having at least 80% sequence identity to SEQ ID NO: 12 or SEQ ID NO: 14, via homologous recombination using flanking sequences from P. patens genome locus 108.

[0019] In some instances, the bryophyte cell may be exposed to LIV radiation to enhance production of total flavonoids. Examples of such flavonoids may include, but may not be limited to, aureusidin and auresudin-O-6-glycoside. In some instances, the bryophyte cell may be further modified to express additional flavonoid modifying enzymes. Examples of such flavonoidmodifying enzymes may include, but are not necessarily limited to, chaicone synthases, chaicone isomerases, flavonoid glycosyl transferases, flavonoid hydroxylases, flavone synthase I, flavone synthase II, isoflavone synthase, flavanol synthase, isoflavone hydroxylases, stilbene synthases, chaicone reductases, enzymes behaving like chaicone synthase, dihydroflavonol 4-reductase, isoflavone reductase, and flavonoid O-methyltransferases. In some instances, the bryophyte cell may be further modified to not express endogenous flavonoid biosynthetic enzymes. This may be done through complete deletion of the gene from the genome, truncation via insertion of a premature stop codon, or RNAi suppression of expression.

[0020] This summary does not necessarily describe the entire scope of all aspects of the disclosure. Other aspects, features and advantages will be apparent to those of ordinary skill in the art upon review of the following description of specific embodiments.BRIEF DESCRIPTION OF THE FIGURES

[0021] In the accompanying drawings:

[0022] FIGURE 1 depicts a compilation of LC-MS extracted ion chromatograms (EIC) acquired in negative ion mode at m / z 285.042 ± 0.01 , showing the EIC of a purified aureusidin standard, the EIC of a soluble phenolic extract from wild-type P. patens protonema grown on solid media, and the EIC of a soluble phenolic extract from wild-type P. patens protonema grown on solid media spiked with purified aureusidin.

[0023] FIGURE 2 depicts a compilation of LC-MS total ion chromatograms (TIC) of AmAUS-2A- 4'GT expressing P. patens lines (Total Ion Current). TICs of soluble phenolic extracts from two independent AmAUS-2A-4 'GT expressing protonemal lines compared to two wild-type replicates. Expression of the transgene alters the overall phenolic profile, with the appearance of multiple novel peaks. In particular, new signals emerging at ~7.5-8.0 min are consistent with the accumulation of compounds with masses near that of naringenin, suggesting the formation of naringenin adducts or derivatives not detected in wild type.

[0024] FIGURE 3 depicts a compilation of LC-MS extracted ion chromatograms (EIC) analyzing AmAUS-2A-4'GT expressing P. patens lines. EICs at m / z 271.06 ± 0.01 , corresponding to the aglycone naringenin, are shown for: (i) a purified naringenin standard; (ii) wild-type extracts; and (iii) transgenic moss expressing AmAUS-2A-4'GT protonema grown on solid media. Transgeniclines exhibit a marked increase in the peak at ~10 min, consistent with aglycone naringenin accumulation, as well as the emergence of multiple additional peaks containing the 271 ion, suggesting accumulation of naringenin derivatives or conjugates not observed in wild-type tissue.

[0025] FIGURE 4 shows a LC / MS-based quantification of aurone pathway metabolites in wildtype and AmAUS-2A-4’GT moss lines. The bar graph showing extracted ion chromatogram (EIC) peak areas of auruesidin, luteolin, and naringenin from wild-type (WT) and AmAUS-2A-4’GT- expressing Physcomitrium patens protonema grown on solid media.

[0026] FIGURE 5 shows total Flavonoid Content in Phenolic Extracts from P. patens. Bar graph showing total flavonoid content in phenolic extracts from wild-type Physcomitrium patens and transgenic lines expressing AmAUS-2A-4'GT or CgAS1. Flavonoid levels were quantified using the colorimetric aluminum chloride assay, with naringenin as the calibration standard. Results are expressed as micrograms of naringenin equivalents (pg NE) per milligram of dried extract.

[0027] FIGURE 6 shows MAFFT alignment of predicted polyphenol oxidase (PPG) family protein sequences from Physcomitrium patens with previously characterized aurone synthases from Antirrhinum majus (AmAUSI) and Coreopsis grandiflora (CgAS1). Conserved residues characteristic of plant catechol oxidases are annotated.

[0028] FIGURE 7 depicts LC-MS extracted ion chromatograms (EIC) of samples at m / z 287.0 ± 0.1. LC-MS analysis was performed to detect aureusidin in wild-type and transgenic P. patens lines protonema grown in liquid media. The EIC trace at m / z 287, corresponding to the deprotonated molecular ion of aureusidin, is shown for: (top to bottom) wild-type protonema (replicate 1), wild-type protonema (replicate 2), CgAS 7-expressing protonema (replicate 1), CgASI-expressing protonema (replicate 2), and an aureusidin standard. A peak matching the retention time and m / z of the standard is clearly observed in CgAS 7-expressing lines but is absent or present only at trace levels in wild type, indicating successful production of aureusidin in the engineered moss.

[0029] Figure 8 depicts LC / MS-based quantification of aurone pathway metabolites in wild type (WT) and CgAS1 moss lines. The bar graph showing extracted ion chromatogram (EIC) peak areas of aureusidin, luteolin, and naringenin from wild-type (WT) and CgAS 7-expressing Physcomitrium patens protonema grown on solid media. Metabolite levels were determined by LC / MS analysis in negative ion mode and normalized to the fresh weight of moss tissue at thetime of extraction. EIC peak areas were integrated based on retention time and mass accuracy, using elution times of authenticated standards. Expression of CgAS1 led to substantial increase in aureusidin and luteolin compared to WT. Data represent mean ± standard deviation (SD) from n = 2 biological replicates.

[0030] Figure 9 depicts a proposed aurone biosynthesis pathway and subcellular localization in P. patens.

[0031] Figure 10 depicts neighbor joining tree of polyphenol oxidases (PPO) family in P. patens, along with previously characterized aurone synthases AmAUS1 |ABR57233.1, CgAS|AHN09736.1 & MpAS1|Mapoly0021s004. Tree was built from MAFFT aligned amino acid sequences using the JTT model of protein substitution and 1000 bootstrap replicates.

[0032] Figure 11 depicts a linearized construct used for transformation of moss protoplasts containing a codon optimized PpAS1 (SEQ. ID NO: 12) sequence driven by the maize ubiquitin promoter and containing a hygromycin resistance cassette driven by a 35S constitutive promoter (SEQ. No 13). Construct is flanked by sequences from the P. patens 108 locus to mediate homologous recombination.

[0033] Figure 12 depicts a linearized construct used for transformation of moss protoplasts containing a codon optimized PpAS2 (SEQ. ID NO: 14) sequence driven by the maize ubiquitin promoter and containing a hygromycin resistance cassette driven by a 35S constitutive promoter PpAS2 (SEQ. ID NO: 15). Construct is flanked by sequences from the P. patens 108 locus to mediate homologous recombination.

[0034] Figure 13 depicts LC / MS-based quantification of aurone pathway metabolites in WT, PpAS1 PpAS2 moss lines. The bar graph shows extracted ion chromatogram (EIC) peak areas of aureusidin, luteolin, and naringenin from wild-type (WT) and PpAS1, PpAS2 expressing Physcomitrium patens protonema grown on solid media. Metabolite levels were determined by LC / MS analysis in negative ion mode and normalized to the fresh weight of moss tissue at the time of extraction. EIC peak areas were integrated based on retention time and mass accuracy, using elution times of authenticated standards. Expression of the PpASIand PpAS2 led to a substantial increase in luteolin compared to WT. Data represent mean ± standard deviation (SD) from n = 2 biological replicates.

[0035] FIGURE 14 depicts a linearized construct used for transformation of moss protoplasts containing the aureusidin synthase-2A-4’glycosyltransferase (AUS-2A-4GT, SEQ ID NO: 8) sequence driven by the maize ubiquitin promoter and containing a hygromycin resistance cassette driven by a 35S constitutive promoter. The construct is flanked by sequences from the P. patens 108 locus to mediate homology recombination.

[0036] FIGURE 15 depicts SEQ ID NO. 9 moss overexpression vector pTH-UBI-GATE with the aureusidin synthase- 2A-4’glycosyltransferase (AUS-2A-4GT, SEQ ID NO: 8) inserted between attB sites. The pTH-UBI-GATE vector has been modified to contain the DNA sequences originating from the Pp108 locus which flank a non-disruptive locus in the P. patens genome, as moss can be genetically engineered by homologous recombination.

[0037] FIGURE 16 depicts LC-MS analysis of soluble phenolic extracts from protonema tissue of P. patens moss lines (AUS-2A-4GT) expressing AUS-2A-4GT, SEQ ID NO: 4 and exhibiting significant differences in overall metabolic profile, wherein: (i) Figure 16(a) shows total ion current; and (ii) Figure 16(b) is an ion chromatograph for m / z= 271.06 (+ / - 0.01), clear accumulation of naringenin and, ostensibly naringenin chaicone.DETAILED DESCRIPTION:

[0038] Directional terms such as “top,” “bottom,” “upwards,” “downwards,” “vertically,” and “laterally” are used in the following description for the purpose of providing relative reference only, and are not intended to suggest any limitations on how any article is to be positioned during use, or to be mounted in an assembly or relative to an environment. The use of the word “a” or “an” when used herein in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.” Any element expressed in the singular form also encompasses its plural form. Any element expressed in the plural form also encompasses its singular form. The term “plurality” as used herein means more than one, for example, two or more, three or more, four or more, and the like.

[0039] As used herein and unless otherwise specified, the term “about”, when used to describe a recited value, means within 10% of the recited value.

[0040] As used herein and unless otherwise specified, the term “AS” refers to aureusidin synthase.

[0041] As used herein and unless otherwise specified, the terms “comprising”, “having”, “including”, “containing”, and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, un-recited elements and / or method steps. For example, "A includes 1, 2, and 3" means that A includes but is not limited to 1, 2, and 3.

[0042] As used herein and unless otherwise specified, the term “consisting essentially of” when used herein in connection with a composition, use, or method, denotes that additional elements, method steps or both additional elements and method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method, or use functions.

[0043] As used herein and unless otherwise specified, the term “consisting of” when used herein in connection with a composition, use, or method, excludes the presence of additional elements and / or method steps.

[0044] As used herein and unless otherwise specified, the term “ectopic endogenous promoter” refers to a promoter originating from P. patens that is introduced in front of a nucleic acid sequence and is operably associated therewith.

[0045] As used herein and unless otherwise specified, the term “exogenous promoter” refers to a promoter originating from a different species that is introduced in front of a nucleic acid sequence and is operably associated therewith. For example, an exogenous promoter may be one that has been placed in front of a selected nucleic acid component as herein defined and does not consist of the natural or native promoter usually associated with the nucleic acid component of interest as found in wild type circumstances.

[0046] As used herein and unless otherwise specified, the term "heterologous" has a meaning as understood by a person skilled in the art in cell biology or biochemistry.

[0047] Unless otherwise specified, the use of either of the terms "homology" and "homologous" herein does not imply any necessary evolutionary relationship between compared sequences.

[0048] As used herein and unless otherwise specified, the phrase "such as" is intended to be open-ended.

[0049] The present disclosure relates to a bryophyte cell that has been transformed or geneticallymodified to produce flavonoids (e.g., aurones such as but not limited to aureusidin) that are not normally expressed or produced in said bryophyte cell. In an example, bryophyte cells described herein can include those that have been previously transformed with heterologous genes (and translations thereof) that encode enzymes involved in the biosynthesis of flavonoids, plant secondary metabolites, or both. Bryophyte cells described herein may also include those that have been transformed severally (for example, simultaneously or over time) with nucleotide sequences coding for at least a primary protein sequence of interest such as, but not limited to, a plant derived enzyme involved in the biosynthesis or modification of phenolic secondary metabolites. Examples of such enzymes include, but are not limited to, chaicone synthases, chaicone isomerases, flavonoid glycosyl transferases, flavonoid hydroxylases, flavone synthase I, flavone synthase II, isoflavone synthase, flavanol synthase, isoflavone hydroxylases, stilbene synthases, chaicone reductases, enzymes that behave like chaicone synthase, dihydroflavonol 4-reductase, isoflavone reductase, and flavonoid O-methyltransferases. The bryophyte cell can be one from a bryophyte selected from the group consisting of species from the genera Physcomitrella, Funaria, Sphagnum, and Ceratodon. Preferably, the bryophyte cell is selected from P. patens.

[0050] The present disclosure further relates to a bryophyte cell that has been transformed with vectors or constructs. Within the bryophyte cell, the one or more introduced nucleotide sequences (or translations thereof) may be incorporated within the chromosome. Accordingly, a bryophyte cell may have incorporated therein one or more heterologous nucleotide sequences (or translations thereof) into its genome under operative control of regulatory sequences for control of expression. The coding sequence may be operably linked to one or more regulatory sequences which may be heterologous or foreign to the nucleic acid sequences disclosed herein.

[0051] The present disclosure further relates to a method of making a transformed bryophyte cell, such as a P. patens cell: (i) by introducing one or more nucleotide sequences disclosed herein (or translations thereof) into a bryophyte cell; or (ii) by introducing a suitable vector comprising the one or more nucleotide sequences disclosed herein (or translations thereof) into a bryophyte cell, thereby initiating recombination between the vector and the bryophyte cell genome for introducing said one or more sequences into the bryophyte cell genome.

[0052] The transgene can be on an extra-genomic vector or otherwise incorporated into the genome. In some instances, a heterologous gene may replace an endogenous equivalent gene;in some instances, the inserted sequence may be additional to the endogenous gene or other sequence. Advantageously, a heterologous gene can be expressed under the control of a promoter of choice. A sequence within a bryophyte or other host cell may be identifiably heterologous, exogenous or foreign.Detection of aureusidin in wild-type P. Patens

[0053] To evaluate P. patens as a host for aureusidin production, soluble phenolics from wildtype protonema were analyzed by LC-MS. Figure 1 shows LC-MS extracted ion chromatograms (EIC) acquired in negative ion mode at m / z 285.042 ± 0.01 , showing a purified aureusidin standard (see top EIC in Figure 1), soluble phenolic extract from wild-type P. patens protonema grown on solid media (see middle EIC in Figure 1), and the same extract spiked with purified aureusidin (see bottom EIC in Figure 1). A peak with matching retention time and m / z to the standard is present in wild-type samples, indicating the presence of trace levels of aureusidin. A compound co-eluting with an authentic aureusidin standard was detected in trace amounts, matching both retention time (7.5 min) and mass spectrum (285.042) (see Figure 1). In addition, supplementation with purified aureusidin standard resulted in an increase in the observed peak intensity further supporting the conclusion that trace amounts of aureusidin are present in wild type P. patens protonema.

[0054] While P. patens is known to produce other flavonoids, such as luteolin, quercetin, kaempferol and apigenin (Wolfe et al., 2010), aurones have not been previously documented in P. patens. Aureusidin’s presence in P. patens, albeit in trace amounts, is therefore surprising given that, as a model organism, its genome has been extensively studied in combination with metabolomic studies (Resning et al. 2020; Higara et al. 2020; Erxleben et al 2012; Resemann et al. 2022).

[0055] This finding suggests a previously unrecognized capacity for aurone biosynthesis in P. patens, However, the trace amounts of endogenous presence of aureusidin suggests that without targeted analytical efforts, this metabolite would likely remain undetected. This fact underscores the challenges inherent in identifying low-abundance compounds within the chemically complex matrices of plant metabolomes, and this disclosure relates to enhancing the production of aurones in genetically modified bryophyte cells.Enhancing the biosynthesis of aureusidin in P. patens: Examples of transformedbryophyte cells

[0056] In an embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express aureusidin synthase (SEQ ID NO: 1) and chalcone- 4'-O-glycosyltransferase (SEQ ID NO: 3) as a single biscistronic cassette separated by a P2A peptide (SEQ ID NO: 2). In other words, the single biscistronic cassette comprises aureusidin synthase-2A-chalcone-4'-O-glycosyltransferase (SEQ ID NO: 4) or any biological sequence having at least 80% sequence identity thereto. Said expression can be driven by an exogenous or an ectopic endogenous promoter. Said promoter can confer constitutive expression of such single gene.

[0057] In another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 1 (or any biological sequence having at least 80% sequence identity thereto) and SEQ ID NO: 3 (or any biological sequence having at least 80% sequence identity thereto) separately through insertion of two unique gene cassettes into said bryophyte cell. Said expression can be driven by separate exogenous or ectopic endogenous promoters. Each promoter can confer constitutive expression of its respective gene.

[0058] In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 1 (or any biological sequence having at least 80% sequence identity thereto) and SEQ ID NO: 3 (or any biological sequence having at least 80% sequence identity thereto) as a single biscistronic cassette separated by a P2A peptide (SEQ ID NO: 2). In other words, the single biscistronic cassette comprises aureusidin synthase-2A-chalcone-4’-O-glycosyltransferase (SEQ ID NO: 4), or any biological sequence having at least 80% sequence identity thereto. Said expression can be driven by an exogenous or an ectopic endogenous promoter. Said promoter can confer inducible expression of such single gene.

[0059] In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 1 (or any biological sequence having at least 80% sequence identity thereto) and SEQ ID NO: 3 (or any biological sequence having at least 80% sequence identity thereto) separately through insertion of two unique gene cassettes into said bryophyte cell. Said expression can be driven by separate exogenous or anectopic endogenous promoters. Each promoter can confer inducible expression of its respective gene.

[0060] In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 5 (or any biological sequence having at least 80% sequence identity thereto) and SEQ ID NO: 7 (or any biological sequence having at least 80% sequence identity thereto) as a single biscistronic cassette separated by SEQ ID NO: 6.

[0061] In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 8 or any biological sequence having at least 80% sequence identity thereto. In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 10 or any biological sequence having at least 80% sequence identity thereto. In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 12 or any biological sequence having at least 80% sequence identity thereto. In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 14 or any biological sequence having at least 80% sequence identity thereto. In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 16 or any biological sequence having at least 80% sequence identity thereto. In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 17 or any biological sequence having at least 80% sequence identity thereto. In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 18 or any biological sequence having at least 80% sequence identity thereto. In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 32 or any biological sequence having at least 80% sequence identity thereto. In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 33 or any biological sequence having at least 80% sequence identity thereto. In yet another embodiment of a transformed bryophyte cell, there is a bryophyte cell that is transformed or genetically modified to express SEQ ID NO: 34 or any biological sequencehaving at least 80% sequence identity thereto.

[0062] A bryophyte promoter, in particular a P. patens promoter, can be any DNA sequence capable of binding a host DNA-dependent RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g. structural gene) into mRNA. Such promoter has a transcription initiation region that is located proximal to the 5' end of the coding sequence and that typically includes an RNA polymerase binding site and a transcription initiation site. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the AUG start codon on the mRNA. Examples of promoters include the rice P-actin1 promoter, the cauliflower mosaic virus (CaMV)35S promoter, the Agrobacterium tumefaciens nopaline synthase (nos) promoter, the rice actin 1 gene (ActT) promoter, a soybean heat-shock promoter and an artificial TOP10 promoter (Holtorf et al. 2002), the 2x CaMV 35S promoter and the CaMV long promoter (Kay et al. 1987), the human cytomegalo virus (CMV) promoter and the simian virus (SV) 40 promoter (Horstmann et al. 2004), and the maize UBQ promoter and variants thereof (Streatfield et al. 2004).

[0063] In some instances, the bryophyte promoter also has a second domain called an upstream activator sequence (UAS) which, if present, is usually distal to the structural gene. Whereas constitutive expression of a gene occurs in the absence of a UAS, the UAS permits regulated (inducible) expression of a gene. Moreover, bryophyte promoter sequences (naturally occurring or synthetic) encoding enzymes in bryophyte metabolic pathways can provide particularly useful promoter sequences (see, for example, Zeidler et al. (1996) Plant. Mol . Biol. 30, 199-205). Furthermore, a bryophyte promoter can include naturally occurring promoters of non-bryophyte origin (see, for example, Zeidler et al. (1999) J. Plant Physiol. 154, 641-650; Cohen et al., Proc. Natl. Acad. Sci. USA, 77: 1078, 1980; Henikoff et al., Nature, 283: 835, 1981 ; Hollenberg et al., Curr. Topics Microbiol. Immunol., 96: 119, 198).

[0064] The person skilled in the art or science to whom this disclosure is addressed will appreciate that each nucleotide sequence coding for, for example, aureusidin synthase-2A- chalcone-4'-glycosyltransferase (SEQ ID NO: 4), aureusidin synthase (SEQ ID NO: 1), chalcone- 4'-glycosyltransferase (SEQ ID NO: 3), or any other suitable biological sequence disclosed herein, will be under regulatory control of its own exogenous or an extopic endogenous promoter and terminator. In particular, when introducing selected aureusidin synthase and chalcone-4'-glycosyltransferase nucleic acid sequences, or any other biological sequence disclosed herein, into a bryophyte cell, certain considerations known to the person skilled in the art or science would be taken into account. For example, the nucleic acid(s) to be inserted into the bryophyte should be assembled within a cassette. The construct should contain effective regulatory elements which in turn will drive transcription. In addition, a method of transporting the construct into the bryophyte cell must be available.

[0065] In some embodiments, the bryophyte cell is exposed to LIV radiation to enhance production of exogenous flavonoids. Examples of such flavonoids include, but are not limited to, aureusidin and auresudin-O-6-glycoside. In some embodiments, the bryophyte cell is further modified to express additional flavonoid modifying enzymes. Examples of such flavonoid modifying enzymes include, but are not limited to, chaicone synthases, chaicone isomerases, flavonoid glycosyl transferases, flavonoid hydroxylases, flavone synthase I, flavone synthase II, isoflavone synthase, flavanol synthase, isoflavone hydroxylases, stilbene synthases, chaicone reductases, enzymes behaving like chaicone synthase, dihydroflavonol 4-reductase, isoflavone reductase, and flavonoid O-methyltransferases. In some embodiments, the bryophyte cell is further modified to not express endogenous flavonoid biosynthetic enzymes. This can be done through complete deletion of the gene from the genome, truncation via insertion of a premature stop codon, or RNAi suppression of expression.

[0066] Expression constructs can be integrated into the bryophyte genome with an integrating vector. An integrating vector may be directed to a specific locus in moss by selecting the appropriate homologous sequence for inclusion in the vector. An integrating vector may also be directed to a random location in the genome through Agrobacterium mediated transformation. Methods of introducing exogenous DNA into bryophyte cells are documented in the scientific literature (see for example, Schaefer D. G. "Principles and protocols for the moss Physcomitrella patens," (May 2001) Institute of Ecology, Laboratory of Plant Cell Genetics, University of Lausanne Didier; Reutter K. and Reski R., Plant Tissue Culture and Biotechnology September 1996, Vol.2, No.3; Zeidler M et al., (1996), Plant Molecular Biology 30:199-205). Suitable vectors can be chosen or constructed as appropriate (see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press; Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992). Examples of vector backbones suitable for targeted integration into the P. patens genome via homologous recombination include pLGVneoll03 (Hain et al. 1985), pBI426 (Bommineni et al.1994), pGL-108, pGL-240 and pHP-213 (Shaeffer & Zyrd 1997). Examples of vector backbones suitable for non-targeted integration into the P. patens genome via Agrobacterium mediated transformation include the plasmid BIBAC (Hamilton et al. 1997), pMSP (Lee et al. 2007) and PZP-RCS2 (Goderis et al. 2002).

[0067] In another embodiment, there are polypeptides comprising amino acid sequences with SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34 function as defined herein and as obtainable using sequence information as provided herein. In some embodiments, an allele, variant, derivative, mutant derivative, mutant or homologue of the specific sequence may show little overall homology. For example, homology may be about 20%, or about 25%, or about 30%, or about 35%, or about 40% or about 45%, with the specific sequence. However, in functionally significant domains or regions, the amino acid homology may be higher. Functionally significant domains or regions of different polypeptides may be combined for expression from encoding nucleic acid as a fusion protein.

[0068] According to an embodiment, to enhance the biosynthesis of aureusidin in P. patens, two distinct transgenic strategies were engineered. Each strategy targeted different subcellular compartments of the putative endogenous aurone biosynthetic pathway: the vacuolar compartment and the plastidial compartment.Vacuolar Compartment

[0069] To assess whether the endogenous pathway occurs via a vacuolar compartmentalization strategy in P. patens, a bicistronic gene cassette encoding aureusidin synthase (AmAllSI , SEQ ID NO: 1) and chaicone 4'-O-glycosyltransferase (4GT, SEQ ID NO: 3) was constructed. To optimize expression in moss, both coding sequences were codon-optimized for expression in P. patens and linked via a self-cleaving P2A peptide (SEQ ID NO: 2). The entire cassette was placed under the control of the maize ubiquitin promoter and integrated into the moss genome via homologous recombination using flanking sequences from P. patens genome locus 108 to facilitate targeted homologous recombination (SEQ ID NO: 4). Soluble phenolic compounds were extracted from protonemal tissue of wild-type P. patens and transgenic lines expressing AmAUS1-P2A-4'GT analyzed by LC-MS. Compared to wild-type controls, AmAUS-2A-4'GT expressing lines exhibited substantial alterations in soluble phenolic profiles. Referring to Figure2, total ion chromatograms (TIC) revealed a broad shift in the soluble phenolic profile of ArnAUSI- 2A-4'GT expressing lines.

[0070] Referring to Figure 3, extracted ion chromatograms (EIC) at m / z 271.06 ± 0.01 , a prominent peak with the same elution time of purified naringenin standard was observed in the transgenic lines, with higher intensity than in wild-type extracts. For reference, Figure 3 shows the extracted ion chromatograms (EIC) at m / z 271.06 ± 0.01 , corresponding to the aglycone naringenin, are shown for: (i) a purified naringenin standard (see top EIC); (ii) wild-type extracts (see middle EIC); and (iii) transgenic moss expressing AmAUS-2A-4'GT protonema grown on solid media (see bottom EIC). Transgenic lines exhibit a marked increase in the peak at ~10 min, consistent with aglycone naringenin accumulation, as well as the emergence of multiple additional peaks containing the 271 ion, suggesting accumulation of naringenin derivatives or conjugates not observed in wild-type tissue.

[0071] Referring to Figure 4, quantification of aglycone naringenin in wild-type P. patens vs P. patens comprising AmAUS1-2A-4'GT expressing lines revealed an increase of about 100x. Metabolite levels were determined by LC / MS analysis in negative ion mode and normalized to the fresh weight of moss tissue at the time of extraction. EIC peak areas were integrated based on retention time and mass accuracy, using elution times of authenticated standards. Expression of the AmAUS-2A-4'GT bicistronic construct led to a substantial increase in luteolin and naringenin levels compared to WT. Data represent mean ± standard deviation (SD) from n = 2 biological replicates. Further quantification of the flavonoids aureusidin and luteolin showed a modest increase in aureusidin relative to wild type, along with significant accumulation of luteolin, suggesting a redistribution of metabolic flux within the flavonoid pathway. This is further supported by an increase of approximately 2x in total flavonoid content, as shown in Figure 5. However, despite co-expression of Am4'GT with the vacuole-localized AmAllSI , only a modest increase in aureusidin levels was observed, and this increase did not significantly exceed the overall rise in total flavonoid content in AmAUS-2A-4'GT expressing moss lines (see Figure 5). This pattern suggests the possibility of an alternative compartmentalization of the endogenous aureusidin pathway in P. patens.

[0072] Referring to Figures 4 and 5, and in addition to the major naringenin peak, transgenic lines exhibited several additional peaks with identical m / z values but distinct retention times, suggesting the presence of naringenin-like derivatives. These derivatives may undergo in-sourcefragmentation during LC-MS analysis, resulting in signals matching the m / z of the naringenin aglycone standard despite differing elution profiles. It is also possible that some of the naringenin signal observed at ~10 minutes (see Figure 3) originates from the spontaneous cyclization of naringenin chaicone during sample extraction and LC-MS processing (Austin & Noel, 2003). Naringenin chaicones are chemically unstable and prone to non-enzymatic ring closure under aqueous conditions, forming flavanones such as naringenin without enzymatic catalysis (Austin & Noel, 2003; Adnan et al., 2020). Compounding this, the m / z of naringenin and its chaicone precursor are indistinguishable by MS alone. As a result, naringenin chalcone-4’-O-glycoside, the expected product of Am4'GT activity, may undergo deglycosylation and spontaneous cyclization during extraction, generating naringenin-like signal. Moreover, given the identical m / z of naringenin and its chaicone, it is possible that a glycosylated form of naringenin chaicone also accounts for some of the additional peaks observed at m / z 271. However, the absence of a verified standard for naringenin chalcone-4’-O-glycoside prevents definitive identification of these peaks. Taken together, these chemical and analytical constraints complicate efforts to distinguish true naringenin accumulation from artefactual products arising during sample preparation and analysis.Plasticidal Compartment

[0073] To investigate whether the endogenous aureusidin biosynthetic pathway in P. patens is plastid-localized, the aurone synthase gene from Coreopsis grandiflora (Molitar etal. 2016; Kaintz etal. 2014), which encodes a plastid-targeted aurone synthase (SEQ ID NO: 10), was expressed. Referring to Figure 6, the coding sequence was codon-optimized for expression in moss and assembled under the control of the maize ubiquitin promoter, then integrated into the P. patens genome via homologous recombination using flanking sequences from genome locus 108 to ensure targeted insertion. The CgAS1 construct (SEQ ID NO: 10) was introduced using PEG- mediated protoplast transformation, and stable transgenic lines were selected and validated. Referring to Figures 7 and 8, expression of CgAS1 in P. patens led to a marked increase in aureusidin accumulation, approximately 18-fold compared to wild-type levels.

[0074] In C. grandiflora, CgAS1 converts the more stable chaicone isoliquiritigenin to sulfuretin within the plastid, after which the product is glycosylated and transported to the vacuole (Molitor etal., 2015). Interestingly, CgAS1 appears capable of utilizing naringenin chaicone as a substrate, leading to the production of aureusidin in P. patens. This suggests that aurone synthases exhibitflexibility in accepting structurally related substrates. In P. patens, it is likely that naringenin chaicone, potentially in a 4’0-glycosylated form, acts as a suitable substrate for CgAS1 , enabling its conversion into aureusidin. This functional promiscuity underscores the potential to program aurone biosynthesis in moss by leveraging the substrate tolerance of CgAS1 . Co-expression of enzymes that generate alternative chaicone precursors, such as butein or isoliquiritigenin, could further diversify the aurone profile produced in P. patens, broadening the range of bioactive compounds accessible through this system.

[0075] It is believed that plastid-targeted expression of CgAS1 not only enhances aureusidin biosynthesis but also influences endogenous flavonoid metabolism, leading to luteolin accumulation. Referring to Figure 8, analysis of endogenous flavonoids revealed that P. patens lines expressing plastid-targeted CgAS1 accumulated about 18x more aureusidin and 27-fold more luteolin than wild-type controls. This substantial increase, along with the detectable production of aureusidin in engineered lines, suggests that the endogenous biosynthetic machinery required for aureusidin formation is present and active within the plastid and over overexpression of the plastid localized CgAS1 activate this latent pathway, elevating aureusidin levels well beyond the trace amounts observed in wild type. Notably, the concurrent increase in luteolin, a 3',4'-hydroxylated flavone, is unexpected, as flavone biosynthesis is generally considered to occur in the cytosol or along the endoplasmic reticulum (Zhou et al. 2020;Winkel et al., 2019).

[0076] It is believed that aurone biosynthesis in P. patens may occur natively within the plastid and that targeting pathway enzymes to this compartment enhances metabolic efficiency. Referring to Figure 9, it is believed that, in P. patens, naringenin chaicone may be glycosylated at the 6-position and subsequently targeted to the plastid, rather than the vacuole, for aurone biosynthesis. In moss expressing AUS-2A-4GT, naringenin chaicone glucoside accumulates. While aureusidin localizes to the vacuole, the substrate may be transported into the plastid via an unknown mechanism. Overexpression of plastid-targeted CgAS1 from C. grandiflora results in a marked increase in aureusidin production, supporting plastid-based biosynthesis.Endogenous PPO Candidates in P. patens

[0077] To explore whether P. patens possesses endogenous enzymes capable of catalyzing aurone biosynthesis, a comprehensive bioinformatic screen of the polyphenol oxidase (PPO) gene family was conducted. Basic Local Alignment Search Tool (BLAST) analysis of the P.patens genome using previously characterized aurone synthases from Antirrhinum majus, Marchantia polymorpha, and Coreopsis grandiflora as queries identified 13 annotated polyphenol oxidase (PPO) genes (SEQ ID NO: 19 - 31), consistent with prior reports. Referring to Figure 10, candidate sequences were aligned with previously characterized aurone synthases from A. majus (SEQ ID NO: 16), M. polymorpha (SEQ ID NO: 17), and C. grandiflora (SEQ ID NO: 18). Referring to Figure 6, candidates that retained key diphenolase-specific residues, such as the Gatekeeper (Phe or Leu), Waterkeeper (Glu), and First Activity Controller (HisB+1), as well as those predicted to localize to the plastid based on Plant-mSubP prediction algorithms were prioritized. Referring to Figure 6, candidates localized to the plastid (see Table 1 below) and with co-expression to chaicone synthase the upstream enzyme responsible for the first committed step in flavonoid biosynthesis were prioritized.Table 1 : Summary of polyphenol oxidase family (PPO) family in P. patens including predicted localization sequences, co-expression with chaicone synthases (CHS), conservation of key residues in plant catechol oxidases, and relative expression levels protonema tissue.

[0078] From this bioinformatic analysis, two genes, PpAS1 (Pp3c5_6310V3.1.p) and PpAS2 (Pp3c18_12100V3.1.p), emerged as the strongest candidates. Both genes, expressed in protonema, share structural similarity with MpAS1 and CgAS1 , and co-cluster with chaicone synthase (CHS) genes in co-expression analyses (see Table 1).

[0079] Referring to Figures 11 and 12, the genes were codon-optimized (SEQ ID NO: 12 and 14), cloned under the maize ubiquitin promoter, and stably integrated into the P. patens genome (also see SEQ ID NO. 13 and 15). Transgene insertion was validated by PCR. Referring to Figure 13, transgenic lines overexpressing PpAS1 and PpAS2 showed modest increases in aureusidin but accumulated notably high levels of luteolin compared to all other lines, including those expressing CgAS1 or the AmAUS-2A-4'GT cassette. This profile suggests that PpAS1 and PpAS2 may function through hydroxylation at the 3’ position of both flavones and aurones. The elevated accumulation of luteolin in CgAS1 , PpAS1 , and PpAS2 expressing lines, relative to WT, may indicate a shift in flavonoid pathway flux toward flavone biosynthesis. While these PPOs were originally annotated as aurone synthase-like, the observed metabolic profile raises the possibilitythat they do not catalyze aurone formation efficiently under current expression conditions. Instead, their expression may indirectly promote luteolin biosynthesis, potentially by influencing substrate availability or redox balance in the pathway. There is currently no biochemical evidence that these PPOs directly catalyze 3'- hydroxylation of apigenin to luteolin, a reaction canonically performed by cytochrome P450- dependent F3'H enzymes.

[0080] According to another embodiment, in order to increase production of aureusidin, P. patens was transformed with linearized construct comprising a bicistronic construct comprising SEQ ID NO. 5 (AUS isolated from snapdragon) and SEQ ID NO. 7 (its companion enzyme 4'GT) separated by SEQ ID NO. 6 (a 2A cleavage peptide) using the BSI digested fragment (see in Figure 14) from SEQ ID NO. 9 (moss expression vector ptH-AUS, as shown in Figure 15) which conferred hygromycin resistance to successfully transformed moss lines. According to another embodiment, insertion of SEQ ID NO. 8 into moss was confirmed and the expression of the bicistronic construct in protonema tissue was also confirmed. Surprisingly, LC / MS analysis of the resulting soluble phenolic fraction extracted from transgenic lines revealed substantial changes to the overall metabolic profile compared to wild-type moss (see, for example, Figure 16). While an increase in the accumulation of aureusidin was not observed, an increase in naringenin was observed, as confirmed by comparison to a purified standard and several ions with a m / z charge equal to that reported for both naringenin and naringenin chaicone (namely, m / z 271.06) (see, for example, Figure 16). MS / MS analysis of these distinctive ions at 271 confirmed with greater confidence that these ions were either naringenin or naringenin chaicone. On at least this basis, it is therefore believed that the expression of the AUS-2A-4GT (SEQ ID NO. 8) leads to an accumulation of naringenin and / or naringenin chaicone derivatives in P. patens.

[0081] Polyphenol oxidases (PPOs), particularly catechol oxidases, with aureusidin synthase (AUS) activity have been characterized in diverse plant species, including Antirrhinum majus (AmAUSI), Coreopsis grandiflora (CgAS1 , CgAS2), and Marchantia polymorpha (MpAS1), where they catalyze the oxidative cyclization of chaicones to aurones such as aureusidin (Nakayama et al., 2000; Sato et al., 2001 ; Molitor et al., 2015; Kampatsikas & Rompel, 2021 ; Furudate et al., 2023). Across these enzymes, at least six of eight key catalytic residues (H93, H116, H125, H252, H256, H286, plus F273 and C97 in CgAS1) are conserved and essential for aurone synthase activity (Kaintz et al., 2015; Molitor et al., 2016). SEQ ID NOS: 16, 17, and 18 have all beencharacterized as having aureusidin synthase activity. Given that aureusidin synthases from phylogenetically distant species share this conserved catalytic core, and that functional AUS enzymes operate across varied plant lineages and cellular localizations, it is reasonable to soundly predict that a protein with >80% sequence identity to, for example, SEQ ID NO: 32, retaining at least six of these essential residues, will catalyze aurone formation in a similar manner. This conclusion is supported by established biochemical characterizations of AUS orthologs and the demonstrated transferability of their activity into heterologous hosts.

[0082] SEQ ID NO: 32 is the translation of SEQ ID NO: 4 and SEQ ID NO: 8. SEQ ID NO: 33 is the translation of SEQ ID NO: 10. SEQ ID NO: 34 is the translation of SEQ ID NO: 12.Materials and Methods

[0083] Wild type strain of P. patens subculture 16 / 14 collected in Gransden Wood, Huntingdonshire, United Kingdom, is used, and such P. patens is propagated in accordance with the techniques taught in Engel (1968) Am J Bot 55, 438-446. For example, cultures may be propagated axenically in sterile BCD medium supplemented with ammonium tartrate (BCDAT) medium (1 mM MgS04, 10 mM KNO3, 45 pM FeS04, 1.8 mM KH2PO4at pH 6.5, supplemented with 0.22 pM CuS04, 0.19 pM ZnS04, 10 pM H3BO3, 0.1 pM Na2MoO4, 2 pM MnCI2, 0.23 pM CoCI2, and 0.17 pM KI). Cultures may be maintained in continuous light at 25 ± 3°C on an orbital shaker (120 rpm), and tissues may be harvested after seven days of growth for metabolite extraction. Protonema tissue used for transformations was grown on solidified BCDAT plates.

[0084] A codon-optimized bicistronic expression cassette, for example, one that is encoding a AUS-2A-4’GT sequence (aureusidin synthase-2A-chalcone-4'-glycosyltransferase) or SEQ ID NO: 4, is synthesized using the Geneous™ codon optimization software and DNA fragments provided by Twist Biosciences. Geneous™ codon optimization software draws from publicly available codon usage tables specific to P. patens to recommend codons that may uptake at high levels in P. patens. Simultaneous sequence optimization can be achieved while avoiding including or introducing forbidden motifs uses the algorithm described by Condon and Thachuk 2012. The construct was inserted into the pTH-UBI-GATE moss overexpression vector, which includes flanking sequences from the P. patens Pp108 locus to enable targeted integration via homologous recombination. Expression was driven by the maize ubiquitin promoter, and a hygromycin resistance cassette under a 35S promoter for selection.

[0085] Protoplasts were prepared from seven-day-old protonema pre-treated with 0.5 M mannitol and digested with 1 % Driselase™ (Sigma) for two hours in the dark. Protoplasts were filtered, centrifuged, and washed in W5 medium. Transformation was carried out using PEG-mediated DNA uptake: 250 pL protoplast suspension (1.2 x 106cells / mL) was incubated with 50 pL linearized DNA and 250 pL PEG solution (40% PEG 4000, 0.4 M mannitol, 0.1 M Ca(NO3)2). After 6 minutes at room temperature, the mixture was diluted gradually and centrifuged. Protoplasts were cultured in the regeneration medium and later transferred to selection plates containing 30 pg / mL hygromycin B. Colonies were screened by PCR and qPCR for transgene insertion and expression..

[0086] Total flavonoid content was quantified using a colorimetric aluminum chloride method adapted from Farooq et al. (2020). Samples (50 mg) were extracted in 8 mL of 80% methanol and filtered. For each assay, 300 pL of extract was mixed with 3 mL of 30% methanol, 125 pL of 0.5 M NaNO2, and incubated for 5 minutes. Then, 125 pL of 0.3 M AICI36H2O was added, followed by another 5-minute incubation. Subsequently, 1 mL of 1 M NaOH was added, and the absorbance was measured at 510 nm in triplicate using a 96-well plate reader. A standard curve prepared with naringenin was used to calculate flavonoid content, reported as mg naringenin equivalents per g dry extract.

[0087] Following separation of biomass from liquid media, harvested moss is snap-frozen using liquid nitrogen and ground into a powder. For each sample, 25 mg of tissue is extracted sequentially with 50%, 80%, and 100% methanol (0.01 % TFA). Each sample is heated at 70°C for 15 minutes. An internal standard (5 pL of 2-3 mg / mL o-anisic acid) is added. Samples are analysed on the Agilent 1290 Infinity II UPLC with the 1290 Infinity II Diode Array Detector (DAD) fit with an EclipsePlus C18 column (Agilent, Santa Clara, CA) eluting at a rate of 0.3 mL / min with a water / acetonitrile / methanol gradient. For example, the gradient transition may be from a mixture of 95% water (0.1 % TFA): 5% acetonitrile to a mixture of 60% water (0.1% TFA): 20% MeOH : 20% acetonitrile over 2 minutes, followed by a gradient transition to 50% MeOH: 50% acetonitrile over 6 minutes and 2 minute wash of 5% water (0.1% TFA): 95% acetonitrile. Target compounds are quantified using a standard curves generated from a dilution series of an external standard, and calculations are normalized to an internal standard, o-anisic acid.

[0088] For further analysis and profiling, extracts may be analyzed using LC / MS analysis on the Agilent 1200 SL LC System combined with a Bruker Maxis II equipped with a quadrupole-time-of-flight (Q-TOF) detector following protocols previously described in Dudley et al. 2022. Briefly, Chromatographic separation is conducted utilizing a C18 column (100 x 2.10mm, 2.6 pm particle size) at 40°C flow rate at 0.6mL / min, with a two solvent elution system: solvent A (H2O+0.1% formic acid) and solvent B (acetonitrile). Solvent B is held at 5% for the first minute, followed by a linear increase to 40% solvent B over 5 minutes followed by a wash step with 100% solvent B for 1.5 minutes and then re-equilibrated to 1 % solvent B. Injection volume is 2 pL. Mass spectrometry analysis is performed in both positive and negative ion modes with a scan range of m / z 100- 1000. Source settings as follows: capillary voltage set at 3.5 kV, nebulizer pressure at 2.5 Bar, dry gas flow rate at 11.0 L / min, and dry gas temperature at 250°C. Quantification is performed using calibration curves generated from purified standards (for those standards that are commercially available). Data analysis is conducted using the Bruker Data Analysis software.Moss growth and moss transformation protocols and cloning and vector construction

[0089] In some parts, moss protocols contemplated in this patent application are adapted from the Moss Methods Manual prepared by the Bezanilla Lab at Dartmouth College, Department of Biological Sciences (Bezanilla lab-Moss Methods 2018).

[0090] In order to overexpress aureusidin synthase genes (or other genes) in P. patens, overexpression promoters CaMV 35S and the ubiquitin promoter from Zea mays can be used to drive expression of each identified candidate AS gene. Candidate AS genes from P. patens identified through bioinformatic analysis can be codon optimized for P. patens and synthesized de novo in Gateway™ entry vectors. The sequences constructs can be inserted into the moss overexpression vector pTH-UBI-GATE using the Gateway™ LR enzyme reaction. The pTH-ll BIGATE vector (or other vector) can be modified to contain the DNA sequences originating from the Pp108 locus which flank a non-disruptive locus in the P. patens genome, as moss can be genetically engineered by homologous recombination. Positive transformants exhibiting hygromycin resistance can be selected for further analysis. All of the target flanking regions contemplated herein can be determined in advance to be non-disruptive when inserting ectopic genes (Bezanilla lab-Moss Methods 2018). All cloning reactions are carried out using Gateway™ cloning methods. To analyze subcellular localization, sequences constructs can be inserted into the moss overexpression vector using the Gateway™ LR enzyme reaction. Candidate AS genes are knocked-out using CRISPer knock out methods outlined by Wu et al. 2023, and in particular using homology mediated repair to insert a “stop cassette” into a target gene. Protospacersequences can be identified within candidate AS genes using CISPOR (crispor.tefor.net) (Haeussler et al., 2016) to target candidate AS genes identified through bioinformatic assays using P. patens (Phytozome V11) and S. pyogenes (5' NGG 3') as the genome and PAM parameters, respectively. Entry clones containing homology fragments can be generated upstream and downstream of the desired insertion site and can be subsequently recombined into pENTR221-P1 P4 and pENTR221 P3P2 respectively using the Gateway™ BP clonase reaction. The resulting vectors containing the 5' homology arm (pENTR-L1 L4-5arm), a stop cassette (pENTR-R4R3-stop cassette) which contains three stop codons in each reading from, and 3' homology fragments (pENTR-L3L2-3arm) can be combined into one stop codon doner vector using multisite Gateway™ cloning method (Invitrogen). Moss can then be co-transformed into moss protoplasts with the pMH-Cas9-gate which contains the Cas9 expression cassette driven by a Maize UBI promoter Cas9 / sgRNA and the DNA donor vector. A similar approach can be used to generate GFP tagging of a target AS sequences. Entry resulting vectors containing the 5' homology arm (pENTR-L1 L4-5arm), green fluorescent protein (pENTR-R4R3-mEGFP) and 3' homology fragments (pENTR-L3L2-3arm) can be transformed into one donor vector using a multisite Gateway™ cloning method (Invitrogen). Moss is then co-transformed into moss protoplasts with the pMH-Cas9-gate which contains the Cas9 expression cassette driven by a Maize UBI promoter and the DNA donor vector.Moss Transformation

[0091] Moss can be transformed using a polyethylene glycol (PEG) mediated transformation of P. patens protoplasts (Schaefer et al. 1991 ; Bezanilla Moss Manual). The cloned vector can be linearized using restriction enzyme digestion. For transient transformants, supercoiled plasmids containing the promoter, the target gene, an antibiotic resistance gene and flanking P. patens DNA sequences can be mixed with protoplasts. For stable transformants, only the linearized DNA containing the promoter, the target gene, an antibiotic resistance gene and flanking P. patens DNA sequences is required, and after gel electrophoresis the DNA of the appropriate length is purified and mixed with protoplasts. Protoplasts can be prepared from moss protonema tissue by exposure to a cell wall digestion mixture (Driselase™ from Basidiomycetes sp., Sigma-Aldrich™, D9515). The resulting cells can be separated from the cell wall remnants by filtration and centrifugation. The purified cells can be mixed with the PEG solution and the linearized DNA and can then be incubated at 45°C for 3 minutes. The protoplasts can be left to regenerate and grow on mannitol-containing agar media for several days. After that, the protoplasts can be transferredto complete growth media supplemented with the appropriate antibiotic. Transient transformations can be genotyped by PCR and screened after two weeks, while stable transformants can be left to grow for several weeks prior to genotyping. In the case that gene cassette size is too large for efficient insertion into the genome through homologous repair, agrobacterium mediated transformation technique would be employed as outlined in Cove 2009.Transcriptomic analysis of moss lines

[0092] In a non-limiting example, 100 mg of moss tissue is collected, flash-frozen in liquid nitrogen, and stored at -80°C. Frozen tissue is ground to a fine powder in liquid nitrogen, and total RNA is extracted using the TRIzol™ reagent (Invitrogen™, USA) according to the manufacturer's instructions. RNA quality and concentration is assessed using a NanoDrop™ spectrophotometer (Thermo Scientific™) and a Qubit™ Fluorometer (Invitrogen™). RNA integrity is confirmed using an Agilent™ 2100 Bioanalyzer (Agilent™ Technologies). Poly(A) mRNA is isolated from total RNA using oligo(dT) beads (Dynabeads™ mRNA Purification Kit, Invitrogen™), and cDNA libraries are prepared using the NEBNext™ Ultra II RNA Library Prep Kit (New England Biolabs) following the manufacturer's protocol. Prepared libraries are sequenced on an Illumina™ sequencing platform (for example, NextSeq™ or HiSeq™) to generate 150 bp paired-end reads. Raw sequencing reads are quality-checked using the FastQC™ software and trimmed for adapter sequences and low-quality bases using the Trimmomatic™ software. Clean reads are aligned to the reference moss genome using HISAT2™ or STAR™, and transcript abundance are quantified using featureCounts™ or HTSeq™. Differential expression analysis are performed using DESeq2™ or edgeR™, and functional annotation are conducted using GOseq™ and KEGG™. Selected differentially expressed genes are validated by qRT-PCR using specific primers. Relative expression levels are calculated using the 2A-AACt method with housekeeping genes as controls. Expression levels in members of the glycosyltransferase family identified are investigated to determine the identity of putative 4’GT responsible for producing naringenin chaicone 4’glucoside, the precursor to aureusidin synthase.Cloning and vector construction

[0093] In an example, in order to overexpress aureusidin synthase genes in P. patens, overexpression promoters CaMV 35S and the ubiquitin promoter from Zea mays are used to drive expression of each candidate AS gene. Previous sequences from characterized AS genes (AmAUS1 |ABR57233.1 , CgAS|AHN09736.1 and MpAS1 |Mapoly0021s004) are codon optimizedfor P. patens and synthesized de novo in Gateway™ entry vectors. In addition, transcription factors previously described to increase overall flavonoid production such as, but not limited to, MYBTT2 (Pp3c11_10350) (Luiet et al. 2015) and bhLHL transcription factors TT8 (Pp3c25_13850, Pp3c6_3190) and bHLH27 (Pp3c11_15370) (Otero-Blanca 2021 ; Reboledo 2021) are utilized. The sequences constructs are inserted into the moss overexpression vector pTH-UBI-GATE using the Gateway™ LR enzyme reaction. The pTH-UBI-GATE vector is modified to contain the DNA sequences originating from the Pp108 locus which flank a non-disruptive locus in the P. patens genome, as moss can be genetically engineered by homologous recombination.

[0094] Positive transformants exhibiting hygromycin resistance are selected for further analysis. All of the target flanking regions contemplated herein are determined in advance to be non- disruptive when inserting ectopic genes (Bezanilla lab-Moss Methods 2018). All cloning reactions are carried out using Gateway™ cloning methods. It is anticipated that expression levels of putative aurone synthases require optimization through promoter swapping. In the case aurone production, expression is increased through swapping of promoters. For example, the ubiquitin promoter derived from Zea mays is swapped out with a single and double repeat of the 35S universal promoter derived from the cauliflower mosaic virus. In the case that moss expressing a heterologous construct identified herein is unable to grow, the universal ubiquitin promoter is swapped with the estradiol inducible pGX promoter. Estradiol inducible pGX promoters are shown in advance to be effective at inducing target gene production in moss (Kubo et al. 2013). It is anticipated that such swapping would allow positive transformants time to regenerate from protoplast without interference from potentially toxic heterologous gene expression. Promoter swapping can also be achieved through ligation-digestion cloning methods.Moss Transformation

[0095] In an example, moss is transformed using a polyethylene glycol (PEG) mediated transformation of P. patens protoplasts as previously described by Schaefer et al. 1991 and in the Bezanilla Moss Manual. The cloned vector is linearized using restriction enzyme digestion. For transient transformants, supercoiled plasmids containing the promoter, the target gene, an antibiotic resistance gene and flanking P. patens DNA sequences are mixed with protoplasts. For stable transformants, only the linearized DNA containing the promoter, the target gene, an antibiotic resistance gene and flanking P. patens DNA sequences is required. After gel electrophoresis, the DNA of the appropriate length is purified and mixed with protoplasts.Protoplasts are prepared from moss protonema tissue by exposure to a cell wall digestion mixture (Driselase™ from Basidiomycetes sp., Sigma-Aldrich™, D9515). The resulting cells are separated from the cell wall remnants by filtration and centrifugation. The purified cells are mixed with the PEG solution and the linearized DNA and then incubated at 45°C for 3 minutes. The protoplasts are left to regenerate and grow on mannitol-containing agar media for several days. After that, the protoplasts are transferred to complete growth media supplemented with the appropriate antibiotic. Transient transformations are genotyped by PCR and screened after two weeks, while stable transformants are left to grow for several weeks prior to genotyping. In the case that gene cassette size is too large for efficient insertion into the genome through homologous repair, agrobacterium mediated transformation technique can be employed as outlined in Cove 2009.Moss growth in Bubble Reactors

[0096] In an example, bubble reactor set up is performed according to the following protocol: moss grown on sterile PpNH4(103 mM MgSO4, 1.86 mM KH2PO4, 3.3 mM Ca(NC>3)2, 2.72 mM (NH4)2-tartrate, 45 pM FeSO4, 9.93 pM H3BO3, 220 nM CuSO4, 1.966 pM MnCI2, 231 nM C0CI2, 191 nM ZnSO4, 169 nM KI, and 103 nM Na2MoO4)) agar media is homogenized using a Powergen™ 125 homogenizer (Fisher Scientific™) and inoculated in shaking flasks (for example, beveled shaking flasks) containing 100 mL PpNR4 liquid media. Following four days of growth, the starting moss culture is used to inoculate a 1 L GL45 bottle fit with 2 port Duran® GL 45 connection system caps. Compressed air is fed into the liquid media at a rate of 0.3 L / min through a 0.45 uM vent filter. Following 4 days of growth, moss is subjected to abiotic treatments to increase expression of flavonoids biosynthetic pathway.Metabolite Extraction & Chemical Analysis

[0097] In an example, to analyze resulting metabolites from tissue, the tissue is lyophilized overnight using the Virtis Genesis™ 25 ES freeze dryer. Dried tissue is ground using liquid nitrogen and extracted with 70% methanol + 0.01% formic acid (1 :100 w-v) for one hour, (10 minutes sonication followed by 50 minutes of shaking at room temperature (Dudley et al. 2022). Extracts are then analyzed using LC / MS analysis on the Agilent™ 1200 SL LC System combined with a Bruker Maxis™ II MS equipped with a quadrupole-time-of-flight (Q-TOF) detector following protocols previously described in Dudley et al. 2022. Chromatographic separation are conducted utilizing a C18 column (100 x 2.10mm, 2.6 pm particle size) at 40°C flow rate at 0.6mL / min, witha two solvent elution system: solvent A (H2O+0.1 % formic acid) and solvent B (acetonitrile). Solvent B is held at 5% for the first minute, followed by a linear increase to 40% solvent B over 5 minutes followed by a wash step with 100% solvent B for 1.5 minutes and then re-equilibrated to 1% solvent B. Injection volume are 2 pL. Mass spectrometry analysis is performed in both positive and negative ion modes with a scan range of m / z 100-1000. Source settings as follows: capillary voltage set at 3.5 kV, nebulizer pressure at 2.5 Bar, dry gas flow rate at 11.0 L / min, and dry gas temperature at 250°C. Quantification are performed using calibration curves generated from purified standards (for those standards that are commercially available). Data analysis is conducted using the Bruker™ Data Analysis software. Putative identification of metabolites is achieved using Bruker™ data analysis software.Transcriptomic analysis of moss lines producing higher amounts of aureusidin

[0098] In an example, approximately 100 mg of moss tissue found to be producing higher amounts of aureusidin either through abiotic stress or over expression of MYB transcription factors responsible for upregulating expression of flavonoid biosynthetic genes are collected, flash-frozen in liquid nitrogen, and stored at -80°C. Frozen tissue is ground to a fine powder in liquid nitrogen, and total RNA is extracted using the TRIzol™ reagent (Invitrogen™, USA) according to the manufacturer's instructions. RNA quality and concentration are assessed using a NanoDrop™ spectrophotometer (Thermo Scientific™) and a Qubit™ Fluorometer (Invitrogen™). RNA integrity is confirmed using an Agilent™ 2100 Bioanalyzer (Agilent™ Technologies). Poly(A) mRNA is isolated from total RNA using oligo(dT) beads (Dynabeads™ mRNA Purification Kit, Invitrogen™). cDNA libraries are prepared using the NEBNext Ultra™ II RNA Library Prep Kit (New England Biolabs) following the manufacturer's protocol. Prepared libraries are sequenced on an Illumina™ sequencing platform (for example, NextSeq™ or HiSeq™) to generate 150 bp paired-end reads. Raw sequencing reads are quality-checked using FastQC™ and trimmed for adapter sequences and low-quality bases using Trimmomatic™. Clean reads are aligned to the reference moss genome using HISAT2™ or STAR™ and transcript abundance is quantified using featureCounts™ or HTSeq™. Differential expression analysis is performed using DESeq2™ or edgeR™, and functional annotation is conducted using GOseq™ and KEGG™. Selected differentially expressed genes are validated by qRT-PCR using specific primers. Relative expression levels are calculated using the 2A-AACt method with housekeeping genes as controls.GENERAL:

[0099] It is contemplated that any part of any aspect or embodiment discussed in this specification may be implemented or combined with any part of any other aspect or embodiment discussed in this specification. While particular embodiments have been described in the foregoing, it is to be understood that other embodiments are possible and are intended to be included herein. It will be clear to any person skilled in the art that modification of and adjustment to the foregoing embodiments, not shown, is possible.

[0100] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any citation of references herein is not to be construed nor considered as an admission that such references are prior art to the present invention.

[0101] The scope of the claims should not be limited by the example embodiments set forth herein, but should be given the broadest interpretation consistent with the description as a whole.REFERENCES:

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Claims

1. WHAT IS CLAIMED IS:1 . A genetically modified bryophyte cell comprising a gene cassette encoding:(a) any one of: (i) a biological sequence set forth in SEQ ID NO: 1 and a biological sequence set forth in SEQ ID NO: 3; (ii) a biological sequence set forth in SEQ ID NO: 5 and a biological sequence set forth in SEQ ID NO: 7; (iii) a biological sequence set forth in SEQ ID NO: 10; (iv) a biological sequence set forth in SEQ ID NO: 12; and (v) a biological sequence set forth in SEQ ID NO: 14; or(b) any biological sequence having at least 80% sequence identity to any of the foregoing biological sequences identified in (a)(i) to (a)(v).

2. The cell as claimed in claim 1 , wherein the biological sequence set forth in SEQ ID NO: 1 and the biological sequence set forth in SEQ ID NO: 3 are linked by a biological sequence set forth in SEQ ID NO: 2.

3. The cell as claimed in claim 1 , wherein the biological sequence set forth in SEQ ID NO: 5 and the biological sequence set forth in SEQ ID NO: 7 are linked by a biological sequence set forth in SEQ ID NO: 6.

4. The cell as claimed in any one of claims 1 to 3, further comprising a promoter for promoting the combined sequence within the cell.

5. The cell as claimed in claim 4, wherein the promoter is a maize ubiquitin promoter.

6. A method of enhancing a production of naringenin in the cell as claimed in claim 1 , comprising introducing into a vacuole of the cell and integrating into a genome of the cell a biological sequence set forth in SEQ ID NO: 4, or any biological sequence having at least 80% sequence identity thereto, via homologous recombination using flanking sequences from P. patens genome locus 108.

7. A method of enhancing a production of naringenin in the cell as claimed in claim 1 , comprising introducing into the cell and integrating into a genome of the cell a biological sequence set forth in SEQ ID NO: 8, or any biological sequence having at least 80%sequence identity thereto, via homologous recombination using flanking sequences from P. patens genome locus 108.

8. A method of enhancing a production of aureusidin in the cell as claimed in claim 1 , comprising introducing into the cell and integrating into a genome of the cell the biological sequence set forth in SEQ ID NO: 10, or any biological sequence having at least 80% sequence identity thereto, via homologous recombination using flanking sequences from P. patens genome locus 108.

9. A method of enhancing a production of luteolin in the cell as claimed in claim 1 , comprising introducing into the cell and integrating into a genome of the cell either the biological sequence set forth in SEQ ID NO: 12, the biological sequence set forth in SEQ ID NO: 14, or any biological sequence having at least 80% sequence identity to SEQ ID NO: 12 or SEQ ID NO: 14, via homologous recombination using flanking sequences from P. patens genome locus 108.

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