Chlorophyll c synthase, variants thereof, and heterologous production in plants

Abstract

The invention pertains to the field of biochemical and molecular biology, specifically in the area of photosynthetic pigment biosynthesis, including heterologous expression of chlorophyll c synthase enzymes in plants.

Description

[0001] CHLOROPHYLL C SYNTHASE, VARIANTS THEREOF, AND HETEROLOGOUS PRODUCTION IN PLANTS

[0002] Cross-reference to related applications;

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 621,235, filed January 4, 2024, which is incorporated by reference herein in its entirety.

[0004] Government License Right:

[0005] This invention was made with government support under contract / grant number 2308644 awarded by the National Science Foundation (NSF). The government has certain rights in the invention.

[0006] Reference to a sequence listing:

[0007] This application contains a sequence listing, which is submitted electronically as a XML formatted sequence listing with a file name “UC 2024-731 Sequence Listing”, creation date January 5, 2025, and having a size of 47,334 bytes. The sequence listing submitted electronically is part of the specification and is herein incorporated by reference in its entirety.

[0008] Field of Invention:

[0009] The invention pertains to the field of biochemical and molecular biology, specifically in the area of photosynthetic pigment biosynthesis, including heterologous expression of chlorophyll c synthase enzymes in plants.

[0010] Background of the Invention

[0011] Photosynthetic organisms have evolved a diverse array of pigments to effectively capture light across a wide range of wavelengths. Chlorophyll c is a blue-green pigment found in diatoms, brown algae, dinoflagellates, and many other lineages of eukaryotic algae. These chlorophyll c containing algae perform a major fraction of primary production in marine ecosy stems. (1,2) Chlorophyll c has been known since 1864,(38) but not until 1968 was it determined that the observed ‘chlorophyll c’ peak was actually made up of both chlorophyll cl and c2 which have different spectral properties. (39) At that time it was also shown that different species of algae possess different ratios of chlorophyll cl and c2, for example both chlorophylls cl and c2 are often present in brown algae and diatoms while only chlorophyll c2 is typically found in dinoflagellates and cryptomonads. (5, 10, 40)

[0012] The presence of chlorophyll c has historically been used to classify and infer the evolutionary relationships of heterokont, haptophyte, cryptophyte, and dinoflagellate algae, making chlorophyll c a defining characteristic of the proposed kingdom Chromista.(3) However, the biosynthetic pathways involved in chlorophyll c production have been poorly characterized, which has made heterologous expression of chlorophyll c, and the use of chlorophyll c that stems therefrom, difficult.

[0013] Dinoflagellates are unique organisms that have had a non-conventional evolutionary trajectory and occupy key ecological niches. (29-32) The lack of genetic tools to probe their distinct biology has limited our ability to make discoveries about these organisms. Recently we have shown that UV exposure can generate mutants with a variety of phenotypes in the dinoflagellate B. minutum.(6) However, this advance is limited without the ability to link a mutant phenotype to a genotype, something commonly done with other organisms, but not yet demonstrated in dinoflagellates.

[0014] There remains a need for further elucidation of the biosynthetic pathways of chlorophyll c, production of variant nucleic acids and peptides thereof, heterologous expression of the nucleic acids and peptides thereof, and use of cells and organisms capable of such heterologous expression.

[0015] Summary of the Invention

[0016] In one aspect, the present application discloses a plant or plant part comprising a nucleic acid encoding a heterologous chlorophyll c synthase (CHLCS) enzyme. In certain embodiments the plant part comprises one or more plant cells. In some embodiments, the heterologous CHLCS enzyme is derived from an organism classified in the Chromista kingdom. In particular embodiments, the heterologous CHLCS enzyme is derived from a brown algae, diatom, or dinoflagellate. In other embodiments, the heterologous CHLCS enzyme comprises a mutant or variant CHLCS enzyme. In certain embodiments, the heterologous CHLCS enzyme comprises an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 1,

[0017] 5, 7, 9, 11, 13, 15, 17, 19, 21, and / or 23. In further embodiments, the nucleic acid encoding a heterologous CHLCS enzyme comprises a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and / or 24.

[0018] In another aspect, the present application discloses a mutant or variant chlorophyll c synthase (CHLCS) enzyme. In some embodiments, the mutant or variant CHLCS enzyme is derived from a CHLCS enzyme of an organism classified in the Chromista kingdom. In particular embodiments, the mutant or variant CHLCS enzyme is derived from a CHLCS enzyme of a brown algae, diatom, or dinoflagellate. In certain embodiments, the mutant or variant CHLCS enzyme comprising an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, and / or 23.

[0019] In a further aspect, the present application discloses a nucleic acid encoding a mutant or variant chlorophyll c synthase (CHLCS) enzyme. In some embodiments, the nucleic acid encoding the mutant or variant CHLCS enzyme is derived from a nucleic acid encoding a CHLCS enzyme of an organism classified in the Chromista kingdom. In particular embodiments, the nucleic acid encoding the mutant or variant CHLCS enzyme is derived from a nucleic acid encoding a CHLCS enzyme of a brown algae, diatom, or dinoflagellate. In certain embodiments, the nucleic acid encoding the mutant or variant CHLCS enzyme comprises a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 2,

[0020] 6, 8, 10, 12, 14, 16, 18, 20, 22, and / or 24.

[0021] In another aspect, the present application discloses a method of heterologously producing chlorophyll c in a plant or a part thereof. In certain embodiments, the method comprises introducing into the plant or part thereof a nucleic acid encoding a heterologous chlorophyll c synthase (CHLCS) enzyme and expressing the heterologous CHLCS enzyme in the plant or part thereof. In certain embodiments, the plant part comprises one or more plant cells. In some embodiments, the heterologous CHLCS enzyme is derived from an organism classified in the Chromista kingdom. In particular embodiments, the heterologous CHLCS enzyme is derived from a brown algae, diatom, or dinoflagellate. In further embodiments the heterologous CHLCS enzyme comprises a mutant or variant CHLCS enzyme. In additional embodiments the heterologous CHLCS enzyme comprises an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, and / or 23. In additional embodiments, the nucleic acid encoding a heterologous CHLCS enzyme comprises a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and / or 24.

[0022] Brief Description of Figures

[0023] FIG. 1A: Breviolum minutum wild type (WT) and Ibrl mutant grown heterotrophically on solid agar media (left). Representative microscopy images of WT and Ibrl mutant cells showing bright-field (middle) and chlorophyll autofluorescence (right). Scale bar, 5 pm.

[0024] FIG. IB: UHPLC-DAD analysis of pigment extracts from WT and Ibrl mutant and authentic standard of chlorophyll cl. Identity of chlorophyll cl was confirmed by high resolution mass spectrometry and authentic standards (Figure SI). N.D., not detected.

[0025] FIG. 1C: Analysis of the B. minutum genome with the deletion site in Ibrl mutant indicated as black solid triangle in the genomic region of s6_3623 with exons shown as black.

[0026] FIG. ID: Graphic representation of the primary structure of the s6_3623 protein divided into the predicted chloroplast transit peptide (eTP, black), chlorophyll-a / b binding domain (Chl_a-b, green), and 2 oxoglutarate-Fe(II) dioxygenase superfamily domain (2OGD, grey). Arrow indicates where the protein is altered in Ibrl due to the frame shift.

[0027] FIG. 2A: Normalized absorbance spectra of pigments extracted from Breviolum minutum WT and Ibrl mutant. FIG. 2B: UHPLC-DAD analysis of WT and Ibrl . Compounds identified and confirmed by authentic standards: (1) Chlorophyll cl and c2; (2) Peridinin; (3) Dinoxanthin; (4) Diadinoxanthin; (5) Diatoxanthin; (7) Chlorophyll a and (8) P-carotene. (6) 8-apocarotenal was used as an internal standard (IS). Chlorophyll cl and c2 was not detected (N.D.) in extracts from the Ibrl mutant.

[0028] FIG. 2B: UPLC-HRMS analysis of pigment extracts from WT and Ibrl. Extracted ion chromatogram of m / z 611.2139± 0.01 specific for chlorophyll cl and m / z 609.1982± 0.01 specific for chlorophyll c2.

[0029] FIG. 3: Summary of predicted effects and frequency of mutations detected in Ibrl.

[0030] FIG. 4: Annotated nucleotide sequence of the transcript s6_3623 with the predicted protein domains and motifs indicated. The AXA motif was selected as the truncation site for in planta expression studies.

[0031] FIG. 5A: Schematic representation of the experimental setup for in planta characterization of CHLCS.

[0032] FIG. 5B: A representative image of N. benthamiana leaf discs transiently expressing CHLCS placed in a buffer supplemented with 8-aminolevulinic acid (ALA) overnight in the dark or light. Scale bar, 1 cm.

[0033] FIG. 5C: FLAG-tagged BmCHLCS expressed in N. benthamiana leaves (BmCHLCS- FLAG) is detected in the chloroplast fraction by anti -FLAG immunoblot. Empty vector pl9 was used as a control. T, total protein extracts, C, chloroplast fraction.

[0034] FIGS. 6A and 6B: UHPLC-HRMS analysis of pigment extracts from Nicotiana benthamiana leaf disks expressing constructs indicated in the insets. For each sample absorbance at 445 nm and the corresponding extracted ion chromatogram for chlorophyll cl and c2 are shown. Chlorophyll c peak indicated with (7); trace amount detected was indicated as *; not detected as N.D.. Leaf disk incubation conditions are indicated in plots, such as with 6- aminolevulinic acid (ALA) or in the dark. The empty vector pl9 served as a control.

[0035] FIG. 6C: Chlorophyll biosynthesis pathway in N. benthamiana with BmCHLCS expression. Chemical changes driven by BmCHLCS, protochlorophyllide oxidoreductase (POR), divinyl reductase (DVR), chlorophyll synthase (CHLG), and chlorophyllide a oxygenase (CAO) are highlighted in red. POR is a light-dependent enzyme. Interventions made to the pathway are indicated in blue. FIG. 7A: Absorption spectra (upper) and mass spectrum (lower) of an authentic chlorophyll cl standard.

[0036] FIG. 7B: Absorption spectra (upper) and mass spectrum (lower) of an authentic chlorophyll c2 standard.

[0037] FIG. 7C: UHPLC-DAD chromatogram at 445 nm of authentic chlorophyll cl and c2 standards and pigment extract from B. mimitiim WT.

[0038] FIG. 8A: UHPLC-HRMS analysis of pigment extracts from N. benthamiana leaf disks expressing constructs indicated in the insets. For each sample absorbance at 445 nm is shown. Chlorophyll c peak indicated with (7); not detected as N.D.. Leaf disks were incubated with 8- aminolevulinic acid (ALA) and in the dark. The empty vector pl9 served as a control. Black box represents the native B. minutum chloroplast transit peptide (eTP) sequence. Grey boxes indicate chloroplast transit peptide (eTP) of plant origin.

[0039] FIG. 8B: Representative confocal microscopy images of N. benthamiana epidermal cells expressing full length or truncated constructs of BmCHLCS:mCitrine, as indicated at top. The empty vector pl9 was used as a control. Scale bars, 10 pm.

[0040] FIG. 9A: Collapsed phylogenetic tree of 2-oxoglutarate dioxygenase (20GD) domains identified in chlorophyll c containing eukaryotic algae. Scale bar indicates the estimated number of amino acid substitutions per site and shows the model used to infer the tree.

[0041] FIG. 9B: UHPLC-DAD chromatograms of N. benthamiana extracts expressing CHLCS or CHLC from select species, representing the groups depicted in the phylogeny. Species include Emiliania huxleyi (Eh), Diacronema lutheri (DI), Guillardia theta (Gt), Thalassiosira pseudonana (Tp), Phaeodactyhim tricornutum (Pt , Amphidinium carterae (Ac), and Biecheleriopsis adriatica (Ba . For each sample absorbance at 445 nm is shown.

[0042] FIG. 9C: Level of chlorophyll c2 as a fraction of total chlorophyll c (chlorophyll cl + chlorophyll c2) in extracts of N. benthamiana expressing the selected CHLCSs and CHLCs and in extracts from algae grown in culture. Fraction was based on peak areas from extracted ion chromatograms specific for chlorophyll c2 (m / z 609.1982± 0.01) and chlorophyll cl (m / z 611.2139±0.01), respectively. Dotted line indicates equal levels of chlorophyll c2 and chlorophyll cl. Error bars indicate standard deviation and gray dot individual replicates.

[0043] Brief Description of Sequences SEQ ID NO. 1 : Amino acid sequence of the peptide of wild-type Breviolum minutum transcript s6_3623, which has been identified as Breviolum minutum chlorophyll c synthase (BmCHLCS).

[0044] SEQ ID NO. 2 : Nucleotide sequence of wild-type B. minutum transcript s6_3623, which has been identified as B. minutum chlorophyll c synthase (BmCHLCS).

[0045] SEQ ID NO. 3: Amino acid sequence of the truncated peptide of the Ibrl mutant of B. minutum transcript s6_3623 / BmCHLCS.

[0046] SEQ ID NO. 4 : Nucleotide sequence of the Ibrl mutant of B. minutum transcript s6_3623 / BmCHLCS, which has a nucleotide deletion causing a frame-shift mutation.

[0047] SEQ ID NO. 5 : Amino acid sequence of the peptide of wild-type B. minutum chlorophyll c synthase (BmCHLCS) with additional amino acid residues.

[0048] SEQ ID NO. 6 : Nucleotide sequence of wild-type B. minutum chlorophyll c synthase (BmCHLCS) that has been codon optimized for production in Nicotiana benthamiana.

[0049] SEQ ID NO. 7 : Amino acid sequence of the peptide of wild-type Amphidinium carterae chlorophyll c synthase (AcCHLCS) from imicrobe.us data set MMETSP0258.

[0050] SEQ ID NO. 8 : Nucleotide sequence, codon optimized for production in Nicotiana benthamiana, of the peptide of wild-type Amphidinium carterae chlorophyll c synthase (AcCHLCS) from imicrobe.us data set MMETSP0258.

[0051] SEQ ID NO. 9 : Amino acid sequence of the peptide of wild-type Biecheleriopsis adriatica chlorophyll c synthase (BaCHLCS) from data set SRR11947552 8524.

[0052] SEQ ID NO. 10: Nucleotide sequence, codon optimized for production in Nicotiana benthamiana, of the peptide of wild-type Biecheleriopsis adriatica chlorophyll c synthase (BaCHLCS) from data set SRR11947552 8524.

[0053] SEQ ID NO. 11: Amino acid sequence of the peptide of wild-type Biecheleriopsis adriatica chlorophyll c synthase paralog (BaCHLCS paralog) from data set SRR11947552_37834.

[0054] SEQ ID NO. 12: Nucleotide sequence, codon optimized for production in Nicotiana benthamiana, of the peptide of wild-type Biecheleriopsis adriatica chlorophyll c synthase paralog (BaCHLCS paralog) from data set SRR11947552 37834. SEQ ID NO. 13: Amino acid sequence of the peptide of wild-type Karenia brevis putative chlorophyll c synthase with 20GD domain (KbCHLC) from imicrobe.us data set MMETSP0030.

[0055] SEQ ID NO. 14: Nucleotide sequence, codon optimized for production in Nicotiana benthamiana, of the peptide of wild-type Karenia brevis putative chlorophyll c synthase with 20GD domain (KbCHLC) from imicrobe.us data set MMETSP0030.

[0056] SEQ ID NO. 15: Amino acid sequence of the peptide of wild-type Phaeodactylum tricornutum putative chlorophyll c synthase with 20GD domain (PtCHLC) from ensemble.org data set ASM15095v2.

[0057] SEQ ID NO. 16: Nucleotide sequence, codon optimized for production in Nicotiana benthamiana, of the peptide of wild-type Phaeodactylum tricornutum putative chlorophyll c synthase with 20GD domain (PtCHLC) from ensemble.org data set ASM15095v2.

[0058] SEQ ID NO. 17: Amino acid sequence of the peptide of wild-type Thalassiosira pseudonana putative chlorophyll c synthase with 20GD domain (TpCHLC) from GenBank data set EED88104.

[0059] SEQ ID NO. 18: Nucleotide sequence, codon optimized for production in Nicotiana benthamiana, of the peptide of wild-type Thalassiosira pseudonana putative chlorophyll c synthase with 20GD domain (TpCHLC) from GenBank data set EED88104.

[0060] SEQ ID NO. 19: Amino acid sequence of the peptide of wild-type Emiliana Huxleyi putative chlorophyll c synthase with 20GD domain (EhCHLC) from imicrobe.us data set MMETSP0995.

[0061] SEQ ID NO. 20: Nucleotide sequence, codon optimized for production in Nicotiana benthamiana, of the peptide of wild-type Emiliana Huxleyi putative chlorophyll c synthase with 20GD domain (EhCHLC) from imicrobe.us data set MMETSP0995.

[0062] SEQ ID NO. 21: Amino acid sequence of the peptide of wild-type Diacronema lutheri putative chlorophyll c synthase with 20GD domain (D1CHLC) from GenBank data set KAG8460781.

[0063] SEQ ID NO. 22: Nucleotide sequence, codon optimized for production in Nicotiana benthamiana, of the peptide of wild-type Diacronema lutheri putative chlorophyll c synthase with 20GD domain (D1CHLC) from GenBank data set KAG8460781. SEQ ID NO. 23: Amino acid sequence of the peptide of wild-type Guillardia theta putative chlorophyll c synthase with 20GD domain (GtCHLC) from GenBank data set XP 005836611.

[0064] SEQ ID NO. 24: Nucleotide sequence, codon optimized for production in Nicotiana benthamiana, of the peptide of wild-type Guillardia theta putative chlorophyll c synthase with 2OGD domain (GtCHLC) from GenBank data set XP 005836611.

[0065] Detailed Description

[0066] Given the current need to further improve agricultural yields in the face of escalating climate perturbations and global population growth, the optimization of photosynthesis has emerged as a critical goal. Diversifying the photosynthetic pigment portfolio of plants by drawing from the broad light-harvesting strategies of various photosynthetic organisms offers a promising approach. Provided herein is a description of the CHLCS enzyme, its functional heterologous expression, and ultimate production of chlorophyll c in a plant, as well as mutants / variants of the CHLCS enzyme. Heterologous expression of the CHLCS enzyme can be employed to augment the photosynthetic palette of plants. To build an operational lightharvesting complex that utilizes chlorophyll c, heterologous production in planta of fucoxanthin- chlorophyll a / c binding protein (FCP) and fucoxanthin could be coupled with heterologous production in planta of CHLCS.

[0067] The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein and to the Figures and their previous and following description. Such description is intended to be illustrative and not limiting with respect to the scope of the present invention. It will be understood that other embodiments may be practiced with some variations without departing from the spirit or scope of the subject invention.

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0069] As used in this application and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, reference to a component is intended also to include composition of a plurality of components. References to a composition containing “a” constituent is intended to include other constituents in addition to the one named.

[0070] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “and / or” should be understood to mean either one, or both of the alternatives.

[0071] By “comprising” or “containing” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named. Additionally, the term “comprises” is intended to include embodiments where the method, apparatus, composition, etc., consists essentially of and / or consists of the listed steps, components, etc. Similarly, the term “consists essentially of’ is intended to include embodiments where the method, apparatus, composition, etc., consists of the listed steps, components, etc.

[0072] As used herein, the term “about” indicates a close range around a numerical value when used to modify that specific value. If “X” were the value, for example, “about X” would indicate a value from 0.75X to 1.25X, 0.8X to 1.2X, or0.9X to 1.1X, e.g., a value from 0.95X to 1.05X, or a value from 0.98X to 1.02X, or a value from 0.99X to 1.01X. Any reference to “about X” specifically indicates at least the values X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.1X, and values within this range.

[0073] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the foregoing phrases in various places throughout this application are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in a particular embodiment.

[0074] As used herein, a “heterologous” product (e.g., a nucleic acid sequence or peptide) refers to a product that is not naturally occurring in the relevant organism (e.g., the plant), but which instead has been artificially added to or produced in the relevant organism. For example, a nucleic acid sequence is heterologous to a plant if that plant, as it occurs in nature, would not possess that nucleic acid sequence.

[0075] As used herein, “expression” of a nucleic acid sequence refers to the conversion of the information contained in the nucleic acid sequence into a transcriptional product (e.g., mRNA, antisense RNA, or any other type of RNA). Expression of a peptide / protein in an organism refers to translation of a nucleic acid into a peptide product.

[0076] The term “plant” includes whole plants, shoot vegetative organs and / or structures (e. ., leaves, stems and tubers), roots, flowers and floral organs (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg and central cells), seed (including zygote, embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), seedlings, plant tissue (e.g., vascular tissue, ground tissue, and the like), cells (e.g., guard cells, egg cells, trichomes and the like), and progeny of same. A particular plant may be, for example, an angiosperm (a monocotyledonous or dicotyledonous plant), a gymnosperm, a fern, or a multicellular alga. Plants may be of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid, and hemizygous.

[0077] As used herein, the term “chlorophyll c synthase” and related terms, such as “CHLCS” and “CHLCS enzyme,” abbreviated throughout as CHLCS, shall include any synthase enzyme capable of generating chlorophyll c, including but not limited to the chlorophyll c synthases provided as SEQ ID NOS: 1, 5, 7, and 9, the chlorophyll c synthase paralog provided as SEQ ID NO: 11, and the chlorophyll synthases that possess a 20GD domain provided herein as SEQ ID NOS: 13, 15, 17, 19, 21, and 23, and homologs and variants thereof. Nucleotide sequences encoding chlorophyll c synthases shall include any nucleotide sequence that encodes any synthase enzyme capable of generating chlorophyll c, which may include wild-type, variant, and codon-optimized nucleic acid sequences.

[0078] To facilitate an understanding of the principles and features of the various embodiments of the disclosure, various illustrative embodiments are explained herein. Although exemplary embodiments of the disclosure are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the disclosure is limited in its scope to the details of construction and arrangement of components set forth in the description or examples. The disclosure is capable of other embodiments and of being practiced or carried out in various ways. The present application discloses enzymes capable of producing chlorophyll c, referred to herein as chlorophyll c synthase enzymes (CHLCS), as well as nucleotide sequences encoding such enzymes. The specific CHLCS enzymes disclosed herein include SEQ ID NOS: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, and / or 23. The specific nucleotide sequences encoding CHLCS enzymes disclosed herein include SEQ ID NOS: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and / or 24. However, the present application is not limited to these particular CHLCS enzymes and the nucleotide sequences encoding them, as a person of ordinary skill in the art, using the disclosure herein, would be able to readily identify additional CHLCS enzymes and the nucleotide sequences encoding them that could be used in the methods described herein.

[0079] The present application further describes mutants / variants of CHLCS enzymes and the nucleotide sequences encoding them, including the truncated mutant peptide provided herein as SEQ ID NO: 3 and the nucleotide sequence encoding such mutant, provided herein as SEQ ID NO: 4. However, the present application is not limited to these particular CHLCS mutants / variants and the nucleotide sequences encoding them, as a person of ordinary skill in the art, using the disclosure herein, would be able to readily identify additional CHLCS mutants / variants and the nucleotide sequences encoding them that could be used in the methods described herein.

[0080] The CHLCS enzymes useful in the methods described herein can include any suitable enzyme capable of producing chlorophyll c, including CHLCS enzymes derived from any suitable organism (and the nucleotide sequences encoding CHLCS enzymes from any suitable organism). In some embodiments, the CHLCS enzyme and / or nucleotide sequence encoding the CHLCS enzyme is derived from an organism classified in the Chromista kingdom. In particular embodiments, the CHLCS enzyme and / or nucleotide sequence encoding the CHLCS enzyme is derived from a brown algae, diatom, or dinoflagellate. For example, the CHLCS enzyme and / or nucleotide sequence encoding the CHLCS enzyme may be derived from Breviolum minutum, Amphidinhim carterae, Biecheleriopsis adriciticci, Karenia brevis, Phaeodactylum tricormitum, Thalassiosira pseudonana, Emiliana Huxley i, Diacronema hitheri, or Guillardia theta.

[0081] In certain embodiments, the nucleotide sequence encoding the CHLCS enzyme may be codon optimized for expression in a particular organism, such as a plant, for example Nicotiana benthamiana. In certain embodiments, the nucleotide sequence is codon optimized for expression in the particular organism , for example the particular plant species, in which the enzyme will be heterologously produced.

[0082] In certain embodiments, the CHLCS enzyme, which may be a mutant / variant CHLCS enzyme, comprises an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21 , and / or 23.

[0083] In further embodiments, the nucleic acid encoding a heterologous CHLCS enzyme, which may be a mutant / variant CHLCS enzyme, comprises a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and / or 24.

[0084] In certain embodiments, the present application discloses a plant or plant part comprising a nucleic acid encoding a CHLCS enzyme. In another aspect, the present application discloses a method of heterologously producing chlorophyll c in a plant or a part thereof. In certain embodiments, the method comprises introducing into the plant or part thereof a nucleic acid encoding a heterologous chlorophyll c synthase (CHLCS) enzyme and expressing the heterologous CHLCS enzyme in the plant or part thereof. Any suitable plant species may be used, and a person of ordinary skill in the art would be able to readily select an appropriate plant species for heterologous production depending on their particular needs. Moreover, heterologous expression of genes in plants is well known in the art, as are the methods and reagents required to do so, and a person of ordinary skill in the art would be able to readily select an appropriate heterologous expression system based on their particular needs.

[0085] Examples

[0086] Example 1 : Characterization of aBreviolum mutant deficient in chlorophyll c production

[0087] Breviolum minutum is a chlorophyll c2 containing dinoflagellate alga that is brown in color. In a mutagenesis screen we isolated a series of B. minutum mutants with altered color phenotypes. One pale yellow mutant, LESS BROWN 1 (lbrl could not grow photoautotrophically, had disrupted photosynthetic function, and exhibited less absorbance in the blue-green region (FIGS. 1A and 2A). Evaluation of the pigment composition of Ibrl using ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC- HRMS) revealed that it does not produce chlorophyll c2 (FIGS. IB and 2B). These pigments were also validated by MS (data not shown).

[0088] To identify the causative mutation responsible for the loss of chlorophyll c phenotype we sequenced the transcriptome of Ibrl. Analysis of sequencing data revealed at least 79 mutations identifiable in the transcriptome (FIG. 3). One of the mutations, a single nucleotide deletion, caused a frame shift in a gene (s6_3623) encoding a peptide with a predicted chloroplast targeting sequence and putative chlorophyll a / b binding and 2 oxoglutarate-Fe(II) dioxygenase (20GD) superfamily domains (FIGS. 1C, ID, and 4). The full-length nucleotide sequence of the wild-type transcript is provided as SEQ ID NO: 2 while the wild-type peptide is provided as SEQ ID NOS: 1 and 5. The full-length nucleotide sequence of the Ibrl mutant transcript is provided as SEQ ID NO: 4 while the truncated mutant peptide sequence is provided as SEQ ID NO: 3.

[0089] Example 2: Heterologous in planta biosynthesis of chlorophyll c

[0090] To elucidate the potential role of s6_3623 in chlorophyll c2 biogenesis, we employed a heterologous plant expression system. We transiently expressed a codon-optimized version of the gene (SEQ ID NO: 6, which encodes the peptide of SEQ ID NO: 5) in Nicotiana benthamiana (FIG. 5A). (8,9) Subsequent UHPLC-HRMS analysis of pigment extracts from N. benthamiana leaves expressing the s6_3623 coding sequence revealed a small peak displaying an absorption spectrum characteristic of porphyrin-type chlorophyll which was absent in the negative control (FIG. 6). The new peak had the same retention time and absorption spectra similar to chlorophyll cl and chlorophyll c2 authentic standards, which coelute during our chromatographic separation (FIG. 7). This correspondence in retention time and spectral properties indicates that the peak represents chlorophyll c. The detection of chlorophyll c supported that s6_3623 encodes an enzyme capable of catalyzing the formation of chlorophyll c. We therefore designated the gene linked to the s6_3623 transcript as chlorophyll c synthase (BmCHLCS). This represents the first documented case of heterologous biosynthesis of chlorophyll c in a phototrophic organism and the inaugural instance of a non-plant accessory pigment being synthesized in planta. Example 3: 8-vinyl-protochlorophyllide a and protochlorophyllide a are substrates for BmCHLCS

[0091] Because only trace amounts of chlorophyll c were found in the N. benthamiana extracts, we postulated that this limited accumulation might be attributable to the lack of substrate availability for the BmCHLCS enzyme. It has been hypothesized that 8-vinyl- protochlorophyllide a (8vPC) and protochlorophyllide a (PC) are precursors for chlorophyll c2 and chlorophyll cl, respectively. (10,11) To increase the concentration of 8vPC and PC, we immersed N. benthamiana leaf discs in a buffer supplemented with the protochlorophyllide precursor 8- Aminolevulinic acid (ALA) 12 (FIG. 5B). This, however, did not enhance chlorophyll c production (FIG. 6A). It is notable that both 8vPC and PC are substrates for the protochlorophyllide oxidoreductase (POR) in planta, catalyzing their conversion to 8-vinyl- Chlorophyllide and Chlorophyllide a, respectively 13 (FIG. 6C). POR is a light dependent enzyme so in addition to supplementing with ALA, N. benthamiana leaf discs expressing BmCHLCS were also placed in the dark to facilitate accumulation of 8vPC and PC. This dual intervention led to a pronounced increase in the chlorophyll c peak (FIG. 6A), suggesting that substrate availability in planta directly impacts the ability of BmCHLCS to catalyze the formation of chlorophyll c.

[0092] This increase also enabled mass spectrometry detection of adducts with mass-to-charge ratios of m / z 611.2139 and m / z 609.1982. This aligns with the pseudo molecular ions [M+H+] of chlorophyll cl and chlorophyll c2, respectively, with their identities verified against authentic standards (FIG. 7). Notably, whereas only chlorophyll c2 is observed in B. minutum wild type (WT) (FIG. 2C), in planta expression of BmCHLCS results in both chlorophyll cl and c2 production. Overall, these results indicate that BmCHLCS operates as a light-independent enzyme and, within the context of N. benthamiana, utilizes 8vPC and possibly PC as substrates for synthesizing chlorophyll cl and chlorophyll c2, respectively.

[0093] Example 4: 2OG dioxygenase domain is the catalytic domain that produces chlorophyll c

[0094] BmCHLCS encodes two distinct domains: a putative chlorophyll a / b binding domainl4 and a domain belonging to the 2-oxoglutarate-Fe(II) dioxygenase (20GD) superfamily domain. (15) However, it was unclear whether the chlorophyll a / b binding or the 2OGD domain, if not both, is essential for BmCHLCS catalytic activity. To resolve this ambiguity, we selectively deleted each domain to assess their individual functional contributions to chlorophyll c production in N. benthamiana. When the 20GD domain was deleted, no chlorophyll cl or c2 was detected in extracts from leaves (FIG. 6B). In contrast, deletion of the chlorophyll a / b binding domain had no effect on chlorophyll cl and c2 production, with levels produced similar to the full length BmCHLCS (FIG. 6B). These results indicate that, in planta, the 20GD domain functions as the catalytic site for chlorophyll cl and c2 biosynthesis, while the chlorophyll a / b binding domain is non-essential.

[0095] The 20GD domain of chlorophyll c synthase shares weak homology (E-value: 2.02e-05 for BmCHLCS, NCBI Conserved Domain Database) to a 20GD domain from phytanoyl-CoA dioxygenase enzymes (EC 1.14.11.18). (15) 2OGDs have been identified in all kingdoms of life and are commonly involved in catalyzing diverse oxidative reactions across nature, such as hydroxylation of aliphatic or aromatic C-H Bonds, forming or cleaving C-C bonds, demethylation, ring formation or cleavage, and desaturation. (15, 33-35) Formation of chlorophyll c2 and chlorophyll cl from 8vPC and PC, respectively, requires a desaturation of Cl 71 -Cl 72 (FIG. 6C). Desaturation of aliphatic carbons with 20GD typically requires an adjacent heteroatom, (36) although none are present in 8vPC or PC. While examples are limited, 20GD catalyzed desaturation independent of the presence of adjacent heteroatoms have been reported for a gibberellin oxidase / desaturase (CsGAlox / ds) involved in plant hormone biosynthesis. (37) The present that the 20GD domain from CHLCS catalyzes the requisite desaturation to form chlorophyll c broadens our understanding of 20GD enzymatic versatility.

[0096] Example 5: Chloroplast localization of BmCHLCS is required for chlorophyll c biosynthesis Subcellular localization of BmCHLCS is likely foundational to its functional role, especially as the late stages of chlorophyll biosynthesis occur within the chloroplast in plants. In contrast to plants, marine eukaryotic algae derived from a secondary or more endosymbiotic event(s), including dinoflagellates, utilize a bipartite N-terminal pre-sequence consisting of a signal peptide followed by a chloroplast transit peptide to facilitate protein import into the chloroplast. (16) Our predictive analyses, utilizing HECTAR17 and DeepLoc 2.0,18 identified a signal peptide followed by chloroplast transit peptide at BmCHLCS's N-terminus. At the C- terminal of the predicted chloroplast transit peptide a cleavage motif (AXA)(19) was observed (FIG. 4). Remarkably, BmCHLCS, even with its native dinoflagellate bipartite pre-sequence, can synthesize chlorophyll c in N. benthamiana (FIGS. 6A and 8A), suggesting the dinoflagellate bipartite pre-sequence can facilitate chloroplast localization in planta. To determine if BmCHLCS is actually localized to the chloroplast in TV. benthamiana, we expressed BmCHLCS fused with the yellow fluorescent protein mCitrine.(20) Epidermal cells imaged with confocal microscopy revealed localization of mCitrine fluorescence within chloroplasts (Figure 3B). This was further corroborated by identifying BmCHLCS in isolated chloroplasts (Figure S3C). This information prompted us to experiment with transit peptide modifications, revealing that even when swapping the native dinoflagellate bipartite pre-sequence with plantspecific stromal and luminal targeting sequences,(21) chlorophyll c was still produced (FIG.

[0097] 8A). Intriguingly, the omission of the dinoflagellate bipartite pre-sequence did not impede either chlorophyll c production or BmCHLCS's chloroplast localization. However, stripping both the bipartite pre-sequence and the chlorophyll a / b binding domain abolished chloroplast localization and chlorophyll c production (FIG. 8), solidifying that appropriate subcellular positioning of BmCHLCS is imperative for chlorophyll c synthesis. Fusion of the bipartite pre-sequence and the 20GD domain also lead to chlorophyll c accumulation, showing that the bipartite presequence facilitates chloroplast translocation independently from the chlorophyll a / b binding domain (FIG. 8A). In summary, BmCHLCS is directed to the chloroplast in planta via the dinoflagellate bipartite pre-sequence and BmCHLCS localization to the chloroplast is essential for chlorophyll c biosynthesis.

[0098] Example 6: Chlorophyll c biosynthesis across algal lineages

[0099] To gain insights into chlorophyll c biosynthesis across eukaryotic algae, we sought to examine CHLCS distribution and function in species harboring this pigment. Using BLAST to search data from the Marine Microbial Eukaryote Transcriptome Sequencing Project, (22) we identified transcripts that share sequence similarity to the full-length CHLCS in all peri dinincontaining dinoflagellate species available (FIG. 9A). To determine their chlorophyll c production capability, we chose transcripts from the extensively researched dinoflagellate, Amphidinium carterae, and Biecheleriopsis adriatica, which is deemed a basal lineage in this dinoflagellate clade. (23, 24) These were then codon-optimized and expressed in N. benthamiana. The codon-optimized nucleotide sequence for A. carterae CHLCS (AcCHLCS) is provided as SEQ ID NO: 8, which encodes the peptide sequence of AcCHLCS, provided as SEQ ID NO: 7. The codon-optimized nucleotide sequence for B. adriatica CHLCS (BaCHLCS) is provided as SEQ ID NO: 10, which encodes the peptide sequence of BaCHLCS, provided as SEQ ID NO: 9. Both genes proved successful in catalyzing the production of chlorophyll cl and c2 (FIG. 9B). A paralog of CHLCS was also identified in B. adriatica, the peptide sequence for which is provided as SEQ ID NO: 11 and the codo-optimized nucleotide sequence for which is provided as SEQ ID NO: 12.

[0100] A broader phylogenetic scan(25) revealed a conspicuous absence of genes outside of the peridinin- containing dinoflagellates that simultaneously harbor both the chlorophyll a / b binding and 2OGD domains (FIG. 9A). Significantly, however, all chlorophyll c dependent algal lineages were found to encode 2OGD-containing proteins, and in phylogenetic analyses these formed a monophyletic group with strong support (FIG. 9A). We evaluated the function of genes harboring the 2OGD domain across major algal lineages, given that the 2OGD domain of BmCHLCS alone can drive the biosynthesis of chlorophyll c. Genes were selected from representatives of major chlorophyll c-containing algal groups (Karenia brevis (Kareniaceae) [peptide sequence provided as SEQ ID NO: 13; codon-optimized nucleotide sequence provided as SEQ ID NO: 14] , Emiliana huxleyi (Haptophycae') [peptide sequence provided as SEQ ID NO: 19; codon-optimized nucleotide sequence provided as SEQ ID NO: 20], Diacronema lutheri (Haptophycae) [peptide sequence provided as SEQ ID NO: 21; codon-optimized nucleotide sequence provided as SEQ ID NO: 22], Phaeodactylum tricornutum (I)ialomisla) [peptide sequence provided as SEQ ID NO: 15; codon-optimized nucleotide sequence provided as SEQ ID NO: 16], Thalassiosira pseudonana (Diatomista) [peptide sequence provided as SEQ ID NO: 17; codon-optimized nucleotide sequence provided as SEQ ID NO: 18], and Guillardia theta (Cryptophycae) [peptide sequence provided as SEQ ID NO: 23; codon-optimized nucleotide sequence provided as SEQ ID NO: 24]), codon-optimized, and expressed in N. benthamiana. Both chlorophyll cl and chlorophyll c2 were detected in the N. benthamiana assays for all genes evaluated, thus we refer to these genes with only a 2OGD domain as CHLC (FIG. 9B). In total, we show that genes similar to BmCHLCS that possess both the chlorophyll a / b binding and 2OGD domain and those that only possess the 2OG dioxygenase domain, CHLC, catalyze the biosynthesis of chlorophyll c across diverse algal lineages. These algae all acquired their secondary plastids from red algae, so we searched all available genomic data from red algae, but recovered no CHLCS homologs. This suggests that the 20GD domain in CHLCS most likely arose after the acquisition of the red algal plastid and not from the red algal ancestor of the plastid.

[0101] Example 7: Chlorophyll c synthases influence the ratio of chlorophyll cl to c2

[0102] During the in planta expression of these three CHLCS and six CHLC genes, we observed variations in the ratio of chlorophyll cl to chlorophyll c2 produced across species. Given that all the heterologous expression was conducted within a constant host, N. benthamiana, the observed variability suggested there may be inherent differences among chlorophyll c synthases that give rise to different chlorophyll cl / c2 ratios. To evaluate if these distinct ratios observed in planta are naturally present in the algae that harbor these genes, pigments were extracted and analyzed from seven algal species grown in culture (FIG. 9C). Comparison of both sets of data, revealed a significant correlation between chlorophyll cl / c2 ratios in alga and in planta (Pearson correlation coefficient > 0.60, p-value = 0.0004). For D. lutheri and P tricornutum, the chlorophyll cl / c2 ratios observed in alga closely matched those in planta, and were statistically indistinguishable (p-value = 0.57, two-tailed t-test). Pigments extracted from B. minutum, A. carterae, K. brevis, and E. huxleyi cultures only contained chlorophyll c2 (FIG. 9C). When chlorophyll c synthase genes from these four species were expressed in planta, chlorophyll c2 was primarily produced. However, in a departure from what is observed in alga, chlorophyll cl was also detected (FIG. 9C). Interestingly, all four pigment extracts contained similar levels of chlorophyll cl, -29% of the total chlorophyll c (p-value = 0.74, one-way ANOVA). G. theta, however, diverged from this trend (FIG. 9C). This data collectively implies that observed variations in chlorophyll cl / c2 ratios among species appear to be influenced, in part, by enzymatic differences in their specific chlorophyll c synthases.

[0103] Example 8: CHLC has been lost in a subgroup of ochrophytes

[0104] The one exception to the strict correlation between the presence of chlorophyll c and CHLCS or CHLC homologs is found in a subset of ochrophytes. Ochrophytes encompass many subgroups and include both photosynthetic and non-photosynthetic species. One major subgroup is the Diatomista, which are chlorophyll c dependent(26) and encode CHLC26. The other major subgroup includes a diversity of both photosynthetic and non-photosynthetic lineages, some photosynthetic lineages are chlorophyll c dependent while others depend on chlorophyll a alone. No CHLC homolog was identified in any genome or transcriptome from any of these species, regardless of their pigment composition. This most likely suggests that alternative chlorophyll c biosynthetic enzyme(s) evolved in this lineage and, based on the scattered presence of chlorophyll cl and c2 in this group, that this pathway was subsequently lost in the Eugstigmatophyte and Xanthophyte lineages. Interestingly, in ochrophytes the presence of chlorophyll cl and c2 also correlates with the presence of the secondary pigment fucoxanthin. Recently, the ZEP1, VDL2, and CRTISO5 genes were all shown to be essential for fucoxanthin biosynthesis in Diatomista and Haplophyceae ll ,28) but we now find that these genes are also lacking in the same subgroup of ochrophytes lacking CHLC, suggesting an even more widespread remodeling of their pigment biosynthesis pathways took place after their divergence from Diatomista.

[0105] Methods used in examples

[0106] Ibrl mutant isolation and characterization

[0107] To identify and isolate mutant Ibrl, clonal and axenic liquid cultures of Breviolum minutum (Clade B) strain SSB01 were subjected to UV mutagenesis. (59) Cells were plated on agar plates of marine broth (MB) media (37.4 g / L, Millipore-Sigma 76448) supplemented with 10 g / L glucose (Millipore-Sigma G8270) and kept in the dark. Colonies with an altered color were isolated and transferred to autotrophic conditions (MB in the light) to identify mutants deficient in autotrophic growth. One mutant identified, Ibrl, had a light pale yellow color, was incapable of autotrophic growth, and then subjected to pigment analysis. Emiliania huxleyi CCMP375, Guillardia theta CCMP2712, Karenia brevis CCMP2281, Diacronema lutheri CCMP1251, Amphidinium carterae CCMP3177, Thalassiosira pseudonana CCMP1335 and Phaeodactylum tricornutum CCMP632 microalgae were purchased from the Bigelow collection (National Center for Marine Algae and Microbiota, https: / / ncma.bigelow.org / ) and grown according to Bigelow protocols.

[0108] Pigment extraction

[0109] For pigment extraction from algae, cells were grown in MB supplemented with 10 g / L glucose in the dark. Log phase cells were collected by centrifugation, and lyophilized in the dark. Extraction solvent (90% Me0H:H20 + 5 ppm 8-apo-carotenal) was added to lyophilized algal cells. Samples were incubated in brown glass vials at room temperature for 1 hour. For pigment extraction from Nicotiana benlhamiana. leaves expressing chlorophyll c synthase genes were extracted with 100% acetone. After extraction, the acetone was evaporated and the pigments were resuspended in 90% MeOH with 5 ppm 8-apocarotenal as an internal standard. All samples were filtered through a 96-well filter plate with 0.2 pm pore size polyvinylidene fluoride membrane (PVDF) membrane (Agilent, 203980-100) prior to analysis.

[0110] UHPLC-APCI-qTOF-MS analysis

[0111] Pigment extracts were analyzed using an Ultimate 3000 UHPLC+ Focused system (Dionex Corporation, Sunnyvale, CA, USA) coupled to a Bruker Compact APCI-QTOF-MS (Bruker, Billerica, MA, USA) system. Method for analysis was adopted as previously described. (60) Briefly, samples were separated on a ACQUITYUPLC HSS C18 SB Column (100 A, 1.8 pm, 2.1 mm X 100 mm; Phenomenex Inc., Torrance, CA, USA) maintained at 35 °C with a flow rate of 0.5 mL / min. Injection volume was 10 pL. Mobile phase consisted of A: 50:22.5:22.5:5 5 mM ammonium acetate dissolved in watermethanol: acetonitrile:ethyl acetate and B: 50:50 acetonitrile:ethyl acetate. The liquid chromatography (LC) procedure was outlined as following: 0-0.1 min, 10% B; 0.1-0.8 min, linear increase from 10 to 30% B; 0.8-20 min, increase 30% to 91% B; 20-20.1 min, increase from 91% to 100% B; 20.1-20.4 min isocratic; 20.4-20.5 min linear decrease from 100% to 10% B; 20.5-23 min isocratic. Mass spectra were acquired in positive ion mode over a scan range of 50-1200 mass-to-charge ratios (m / z) and 2 Hz sample rate, with the following settings for MS and atmospheric pressure chemical ionization (APCI): Capillary voltage, 4000 V; end plate offset, 500 V; corona, 4 pA; Vaporizer temperature, 400 °C; dry gas temperature, 250 °C; dry gas flow, 5 L min-1; and nebulizer pressure, 3 bar. Acquired chromatogram data were calibrated using an internal standard, APCI-L (low concentration tuning mix, Agilent technologies). Data analysis was performed with DataAnalysis 4.3 (Bruker, Billerica, MA USA).

[0112] Bioinformatic identi fication of chlorophyll c synthase

[0113] Total RNAs from B. minutum WT strain SSB01 and Ibrl were isolated as previously described. (61) RNA Sequencing was subsequently carried out by Novogene using an Illumina NovaSeq 6000 Sequencing System. Reads were mapped to the B. minutum WT strain SSB01 Symb6 transcriptome assembly (GenBank: GICE00000000)62,63 using BWA.(64) Variants were called using bcftools mpileup (-max-depth 250 — adjust-MQ 50) and bcftools call (-m — ploidy 1 ).(65) The functional effects of the variants identified were called with SnpEff 66 and filtered with SnpSift 67 and the Pandas python package. (68) Two wild type samples (denoted as WT and WT2) were included in the analysis to help with variant identification in Ibrl. Variants were filtered to only include those that met the following criteria: (1) Ibrl called the alternative genotype; (2) WT and WT2 called the reference genotype; (3) WT and WT2 had greater than 10 read depth of coverage for the reference genotype; (4) WT and WT2 had less than 10 read depth of coverage for the alternative genotype; (5) Ibrl had greater than 10 read depth of coverage for the alternative genotype; and (6) Ibrl had less than 10 read depth of coverage for the reference genotype. 79 variants from 64 transcripts met all criteria and were manually reviewed.

[0114] Transcript s6_3623 (GenBank: GICE01003545.1), which had a 1-bp deletion resulting in a frame shift, was identified as a candidate for further investigation.

[0115] Heterologous expression of chlorophyll c synthase

[0116] The function of different candidate chlorophyll c synthase genes was characterized by transient expression in Nicotiana benthamiana using established protocols. (8) CHLCS coding sequences from Breviolum minutum SSB01, Emiliania huxleyi (CCMP1516, XP 005758961), Karenia brevis (CCMP2229, CAMPEP_0173950170 22), Diacronema lutheri (KAG8460781), Guillardia theta (CCMP2712 XP_005836611), Thalassiosira pseudonana (CCMP1335 XP 002294744), Phaeodactylum tricornutum (CCAP1055 / 1 XP 002177807), Amphidinium carterae (CCMP1314, CAMPEP_0176533696 22) and Biecheler iopsi s adriatica (BATY0608, transcript assembled from SRR11947552) were used for transient expression. Peptide sequences and sequences codon optimized for A. benthamiana expression can be found in SEQ ID NOS. 7- 24. The transformation was performed with Agrobacterium tumefaciens AGL1 harboring new_pLIFE and pEAQ-HT vectors. (69, 70) Native genes, or those with plant chloroplast targeting signals, were cloned into the vectors. The PGK (phosphoglycerate kinase) promoter drove expression in the New pLIFE vector, while the CaMV (Cauliflower Mosaic Virus) 35S promoter was utilized for the pEAQ-HT vector. The N-terminal chloroplast targeting sequence from N. tabacum VDE was used to target the stroma side. The N-terminal signal from Arabidopsis thaliana ZEP was used for chloroplast lumen localization. (21, 71) Tobacco transient expression was performed on 4 to 6-week-old N. benthamiana plants. After infdtration of Agrobacterium into tobacco leaves and expressing the desired genes for 4 days, the leaves were harvested for LC-MS analysis. For experiments involving 8-aminolevulinic acid (ALA) feeding, (72) leaf discs (diameter 0 = 2 cm) were extracted on the third day post-infiltration. Leaf discs were then incubated overnight in the dark, submerged in a buffer solution containing 10 mM 8-aminolevulinic acid, 5 mM MgC12 and 10 mM potassium phosphate pH 7.0.

[0117] Localization of BmCHLCS in N, benthamiana

[0118] To determine if chloroplast transit peptide exists in BmCHLCS, HECTAR17 was employed which identified a 22 amino acid signal peptide sequence. Using A22BmCHLCS as a query, the subcellular localization prediction method DeepLoc 2.018 predicted the presence of a plastid targeting sequence and a ASAFAP motif was observed at the alanine in position 67. To visualize CHLCS proteins, mCitrine was tagged to the C-terminal of the full-length or truncated forms of the protein using the new_pLIFE vector for expression. (8) Constructs were generated via USER cloning. N. benthamiana leaf discs after 4 days of agroinfiltration were mounted on a microscope slide using perfluorodecalin (Sigma, P9900) and visualized with a Leica SP5 X confocal microscope. Citrine was excited at 510 nm using a white laser and its emission was captured at 520-560 nm. Chlorophyll autofluorescence was excited using a 458 nm argon laser and detected at 650-700 nm.

[0119] Chloroplast isolation from tobacco leaves

[0120] Tobacco leaves were washed with distilled water and sectioned into fragments measuring approximately 1-3 cm in size. For every 5 mg of tissue, 5 mL of cold, sterile-filtered HS buffer (50 mM HEPES / KOH pH 8.0, 0.33 M sorbitol) was added. The tissue was homogenized in a blender using two 2-second pulses, separated by a 5-second break. The resulting homogenate was filtered through a two-layer nylon mesh into a chilled beaker. This homogenate was centrifuged at 1,000 g at 4 °C for 7 minutes. After discarding the supernatant, the pellet was slowly resuspended in 4 mL of HS buffer. This was achieved by rolling the tube gently on ice, and a portion was aliquoted and set aside as the total protein. To isolate chloroplasts from the homogenate, a Percoll gradient was prepared in 15 mL round-bottom tubes by first adding 2.5 mL of an 80% Percoll solution in the HS buffer. This was carefully overlaid with 5 mL of a 40% Percoll solution in the HS buffer. The required percentages of Percoll in lx HS buffer were achieved using 5x HS buffer stock and water. The homogenate was then introduced to this gradient using wide orifice tips and centrifuged at 3,200 g for 15 minutes at 4 °C. The interface between 40% and 80% Percoll was collected as the intact chloroplast fraction. Subsequently, 3 volumes of lx HS buffer were added, and the intact chloroplasts were centrifuged for 1,700 g for 2 minutes at 4 °C. Protein concentration for each fraction was determined via Bradford assay. The isolated chloroplast fraction was aliquoted, snap frozen, and stored at -80 °C. (73)

[0121] Signal peptide and chloroplast transit peptide analysis

[0122] Protein sequence of BmCHLCS was analyzed using the HECTAR ver 1.3 for identification of heterokont type signal peptide. (17) DeepLoc ver. 2.018 was used for chloroplast transit peptide prediction using A22BmCHLCS, peptide sequence without the predicted signal peptide, as a query. TMHMM ver. 2.074 was used for prediction of transmembrane domains.

[0123] Western blot of isolated chloroplasts

[0124] The total or chloroplast fractions were individually centrifuged at 20000 g for 10 minutes at 4 °C. The pellet was resuspended in IX SDS loading buffer (2% [w / v] SDS, 10% [v / v] glycerol, 0.02% [w / v] bromophenol blue, 62.5 mM Tris-HCl, pH 6.8, 1% [v / v] P- mercaptoethanol) and incubated at 95 °C for 5 minutes. Samples were analyzed by SDS-PAGE 12% and immunodetection using a monoclonal a-FLAG antibody (F3165 Sigma-Aldrich) in a dilution of 1 : 1000 in Tris-Buffered Saline (TBS) 3% milk and a secondary anti-mouse HRP conjugated antibody (DAKO P0161).

[0125] Phylogenetic analysis

[0126] For chlorophyll c synthase, the query fde consisted of the sequences identified in the present study. Using these query files, BLASTp (v2.9)(75) searches were performed (E-value threshold <1 e- 25) against the Marine Microbial Eukaryotic Transcriptome Sequencing Project (MMETSP) transcriptome database. (22) The retrieved sequences were aligned with the original query sequences using MAFFT (v7.481)(76) and trimmed using trimAl (vl.4)(77) with a gap threshold of 0.8. Phylogenetic trees were then constructed from the trimmed alignments using FastTree (v2.1.11),(78) followed by a thorough inspection to eliminate any contaminant sequences. Subsequently, the purified gene collections were used to initiate another round of BLASTp searches (E-value threshold <1 e— 25) against a curated database of stram enopiles, which include select ochrophytes and major stram enopile groups that are non-photosynthetic, compiled from Eukprot (v3),(79) PhyloFisher (vl.l.2),(80) and additional published resources. (81-84) For each gene, ochrophyte sequences were combined with their query file, and then aligned and trimmed as described above. The final phylogenetic trees were deduced using IQ-TREE2 (v2.1.0)(85) employing the ultrafast bootstrap approximation (UFBoot)(86) and ModelFinder(87) to determine the most suitable model for each alignment. For ZEP1 and VDL2, query files of known amino acid sequence homologs from diverse eukaryotes were curated.

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Claims

We claim;1. A plant or plant part comprising a nucleic acid encoding a heterologous chlorophyll c synthase (CHLCS) enzyme.

2. The plant or plant part of claim 1, wherein the plant part comprises one or more plant cells.

3. The plant or plant part of claim 1 or 2, wherein the heterologous CHLCS enzyme is derived from an organism classified in the Chromista kingdom, optionally from a brown algae, diatom, or dinoflagellate.

4. The plant or plant part of any one of claims 1-3, wherein the heterologous CHLCS enzyme comprises a mutant or variant CHLCS enzyme.

5. The plant or plant part of any one of claims 1-4, wherein the heterologous CHLCS enzyme comprises an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, and / or 23.

6. The plant or plant part of any one of claims 1-4, wherein the nucleic acid encoding a heterologous CHLCS enzyme comprises a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and / or 24.

7. A mutant or variant chlorophyll c synthase (CHLCS) enzyme, wherein said mutant or variant CHLCS enzyme is derived from an organism classified in the Chromista kingdom, optionally from a brown algae, diatom, or dinoflagellate.

8. The mutant or variant CHLCS enzyme of claim 7, wherein said mutant or variant CHLCS enzyme comprises an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, and / or 23.

9. A nucleic acid encoding a mutant or variant chlorophyll c synthase (CHLCS) enzyme, wherein said nucleic acid encoding a mutant or variant CHLCS enzyme is derived from an organism classified in the Chromista kingdom, optionally from a brown algae, diatom, or dinoflagellate.

10. The nucleic acid encoding a mutant or variant CHLCS enzyme of claim 9, wherein nucleic acid comprises a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and / or 24.

11. A method of heterologously producing chlorophyll c in a plant or a part thereof, the method comprising introducing into the plant or part thereof a nucleic acid encoding a heterologous chlorophyll c synthase (CHLCS) enzyme and expressing the heterologous CHLCS enzyme in the plant or part thereof.

12. The method of claim 11, wherein the plant part comprises one or more plant cells.

13. The method of claim 11 or 12, wherein the heterologous CHLCS enzyme is derived from an organism classified in the Chromista kingdom, optionally from a brown algae, diatom, or dinoflagellate.

14. The method of any one of claims 11-13, wherein the heterologous CHLCS enzyme comprises a mutant or variant CHLCS enzyme.

15. The method of any one of claims 11-14, wherein the heterologous CHLCS enzyme comprises an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, and / or 23.

16. The method of any one of claims 11-14, wherein the nucleic acid encoding a heterologous CHLCS enzyme comprises a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and / or 24.

Images