A method to avoid epigenetic silencing of exogenous nucleic acids in algae

Abstract

This application relates to the identification of novel DNA methyltransferases, including CHG methylation, in algal species. This application relates to algal mutants that allow expression of exogenous genes by alleviating the epigenetic mechanisms of CHG and CHH methylation of exogenous DNA and mono- and trimethylation of histone 3 lysine 9 (H3K9). This is achieved by mutating or attenuating algal methyltransferase (MTase) genes. This application also relates to methods for efficiently expressing exogenous genes in algal species.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. patent application Ser. No. 62 / 779,364, filed December 13, 2018, the entire contents of which are incorporated herein by reference in their entirety.

[0002] Incorporating sequence tables The data in the attached sequence listing is incorporated by reference into this application. The attached sequence listing text file, filename SGI2210_1WO_Sequence_Listing.txt, was created on December 11, 2019 and is 79kb. This file can be accessed using Microsoft Word on a computer using the Windows OS.

[0003] FIELD OF THEINVENTION This application relates to algal mutants that allow expression of exogenous genes by relaxing the epigenetic mechanisms of methylation of exogenous DNA. This is accomplished by mutating or attenuating algal methyltransferase (MTase) genes. This application also relates to methods for efficiently expressing exogenous genes in algal species. [Background technology]

[0004] background DNA methylation is a common epigenetic signaling tool used by cells to fix genes in an "off" position. Generally, DNA methylation occurs at cytosine bases in eukaryotic DNA and is converted to 5-methylcytosine by DNA methyltransferase (DNMT) enzymes. DNA methylation can be classified into three types depending on the sequence context of the cytosine: CG, CHG, and CHH (H=A, C, or T). Typically, in eukaryotes, methylation is sparse but global, distributed in well-defined CpG sequences throughout the genome, except for CpG islands.

[0005] Cytosine methylation is more frequent in CpG sequences than in CHG or CHH sequences in the algal species Chlamydomonas reinhardtii (Feng. et al., Proc. Natl. Acad. Sci. USA 2010 May 11;107(19):8689-94).

[0006] Besides DNA methylation, histone methylation can also silence genes. For example, monomethylation of histone 3 lysine (H3K9) has been found to mark transgene tandem repeats in Chlamydomonas reinhardtii. SET3, an H3K9 monomethyltransferase involved in H3K9 methylation, has been identified. The role of H3K9 mono-, di-, and trimethylation is species-specific and serves different functions (Caras-Mollano et al., Nucleic Acids Res. 2007; 35(3):939-50). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Feng. et al., Proc. Natl. Acad. Sci. USA 2010 May 11;107(19):8689-94 [Non-Patent Document 2] Caras-Mollano et al., Nucleic Acids Res. 2007; 35(3):939-50 Summary of the Invention

[0008] overview Provided herein are algae mutants that allow expression of exogenous genes by alleviating epigenetic mechanisms that methylate exogenous or foreign DNA. In some embodiments, the methyltransferase is a CHG DNA methyltransferase. In some embodiments, the methyltransferase is a CHH DNA methyltransferase. Also provided are methods for efficiently expressing exogenous DNA in algae species.

[0009] CHG methylation (and silencing) has been observed when certain exogenous genes are expressed in organisms. Moreover, targeting the MTase gene offers the advantage of being specific for targeting the silencing mechanism without the need to deploy other tools for gene refactoring.

[0010] In one embodiment, a mutant photosynthetic organism is provided that comprises a mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity, the mutant photosynthetic organism having reduced CHG DNA methylation compared to a control photosynthetic organism that does not have the mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity.

[0011] In one embodiment, a method for enhancing expression of exogenous DNA in a photosynthetic organism is provided, the method comprising: a) introducing exogenous DNA into the photosynthetic organism; b) mutating or attenuating a gene encoding a polypeptide having CHG DNA methyltransferase activity, such that expression of the exogenous DNA is enhanced in the photosynthetic organism compared to a control photosynthetic organism, wherein the mutant microorganism has reduced CHG DNA methylation of the exogenous DNA compared to the control organism, wherein the control organism contains the exogenous DNA but does not have the mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity.

[0012] In one aspect, a method is provided for reducing methylation (e.g., monomethylation, trimethylation) of lysine 9 on histone H3 (H3K9) in a photosynthetic organism. The method includes mutating or attenuating a gene encoding a polypeptide having CHG DNA methyltransferase activity, wherein the mutant microorganism has reduced CHG DNA methylation, compared to a control photosynthetic organism that does not have the mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity, wherein the photosynthetic organism has reduced monomethylation of lysine 9 on histone H3 (H3K9me1) or reduced trimethylation of lysine 9 on histone H3 (H3K9me3).

[0013] In some embodiments, the mutant photosynthetic organism is a genetically engineered mutant. In some embodiments, the mutant is genetically engineered by insertion mutagenesis, gene replacement, RNAi, antisense RNA, meganuclease genome engineering, one or more ribozymes, and / or CRISPR / Cas system. In some embodiments, the mutant is genetically engineered by CRISPR / Cas system. In some embodiments, the mutant photosynthetic organism is generated by UV irradiation, gamma irradiation, or chemical mutagenesis.

[0014] In some embodiments, the mutant photosynthetic organism comprises a mutation or attenuation in a gene encoding a polypeptide having CHG DNA methyltransferase activity, the polypeptide comprising an amino acid sequence having at least 65% identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:28 prior to the mutation or attenuation of the gene. In some embodiments, the polypeptide having CHG DNA methyltransferase activity comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:28 prior to the mutation or attenuation of the gene.

[0015] In some embodiments, the mutant photosynthetic organism comprises exogenous DNA, and reduced CHG DNA methylation is in the exogenous DNA. In some embodiments, the exogenous DNA is integrated into the genome of the photosynthetic organism.

[0016] In some embodiments, reduced CHG DNA methylation can be in a DNA sequence native to the photosynthetic organism. The reduced DNA methylation can be in a centromere or in a highly repetitive DNA region of the mutant photosynthetic organism.

[0017] In some embodiments, expression of the exogenous nucleic acid is improved (e.g., increased) compared to a control photosynthetic organism, where the control organism contains the exogenous nucleic acid but does not have a mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity.

[0018] In some embodiments, the mutant photosynthetic organism has reduced methylation (e.g., mono-methylation, tri-methylation) of lysine 9 (H3K9) of histone H3. In some embodiments, the mutant photosynthetic organism has reduced CHH DNA methylation compared to a control photosynthetic organism, where the control organism does not have a mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity.

[0019] In some embodiments, the photosynthetic organism is an algae. In some embodiments, the algae is selected from the genera Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteo, and the like. Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium ium), Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Gloeothamnio Gloeothamnion, Haematococcus, Halocafeteria, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monodus, Monoraphidium, Nannochloris,Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella, Pascheria, Pelagomonas, Phaeodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleuroch ... Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Skeleton The mutant photosynthetic organism belongs to the genera Skeletonema, Spyrogyra, Stichococcus, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, and Volvox. In some embodiments, the mutant photosynthetic organism is an alga of the Chlorophyte or Charophyte classes. In some embodiments, the organism is an alga of the Chlorophyceae, Trebouxiophyceae, Chlorodendrophyceae, Ulvophyceae,The organism may be from any of the classes of algae, Pedinophyceae or Prasinophyceae. The organism may belong to the family Chlorellales, Oocystaceae, or Chlorodendraceae. In some embodiments, the mutant algal cell is a chlorophyte algal cell from the Trebouxiophyceae class, such as an algal cell from a species of a genera such as Botryococcus, Chlorella, Auxenochlorella, Heveochlorella, Marinichlorella, Parachlorella, Pseudochlorella, Tetrachlorella, Eremosfera, Frankeia, Michractinium, Nanochloris, Oocystis, Picochlorum, or Prototheca. In some embodiments, the mutant algae can be mutant algae of species of the genera Auxenochlorella, Chlorella, Heveochlorella, Marinichlorella, Parachlorella, Pseudochlorella, or Tetrachlorella. In other embodiments, the mutant algae can be algae of the Chlorodendronychophyceae class (e.g., algae of the genus Tetraselmis).

[0020] In some embodiments, the mutant photosynthetic microorganism is a cyanobacterium. In some embodiments, the cyanobacterium is selected from the genera Acaryochloris, Agmenellum, Anabaena, Anabaenopsis, Anacystis, Aphanizomenon, Arthrospira, Asterocapsa, Borzia, Calothrix, Chamaesiphon, and the like. amaesiphon, Chlorogloeopsis, Chroococcidiopsis, Chroococcus, Crinalium, Cyanobacterium, Cyanobium, Cyanocystis, Cyanospira, Cyanothece, Cylindrospermopsis, lindrospermopsis, Cylindrospermum, Dactylococcopsis, Dermocarpella, Fischerella, Fremyella, Geitleria, Geitlerinema, Gloeobacter, Gloeocapsa, Gloeotheca, thece), Halospirulina, Iyengariella, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Microcystis, Myxosarcina, Nodularia, Nostoc, Nostochopsis,The genera Oscillatoria, Phormidium, Planktothrix, Pleurocapsa, Prochlorococcus, Prochloron, Prochlorothrix, Pseudanabaena, Rivularia, Schizothrix, Scytonema, Spirulina, Spirulina, Stanieria, Starria, Stigonema, Symploca, Synechococcus, Synechocystis, Thermosynechocystis, Tolypothrix, Trichodesmium, Tychonema, or Xenococcus species. [The present invention 1001] 1. A mutant photosynthetic organism comprising a mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity, Control photosynthetic organisms that do not contain a mutated or attenuated gene encoding a polypeptide with CHG DNA methyltransferase activity The mutant photosynthetic organism, having reduced CHG DNA methylation as compared to said mutant photosynthetic organism. [The present invention 1002] 1001. The mutant photosynthetic organism of the present invention, wherein said mutant is a genetically engineered mutant. [The present invention 1003] 1002. The mutant photosynthetic organism of the present invention, wherein said mutant is genetically engineered by insertional mutagenesis, gene replacement, RNAi, antisense RNA, meganuclease genome engineering, one or more ribozymes, and / or a CRISPR / Cas system. [The present invention 1004] 1003. A mutant photosynthetic organism of the present invention, wherein said mutant is genetically engineered by a CRISPR / Cas system. [The present invention 1005] 1001. The mutant photosynthetic organism of the present invention, wherein said mutant is generated by UV irradiation, gamma irradiation, or chemical mutagenesis. [The present invention 1006] The mutant photosynthetic organism of any of claims 1001 to 1004, wherein the mutation or attenuation is in a gene encoding a polypeptide having CHG DNA methyltransferase activity, and the polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 28 prior to the mutation or attenuation of the gene. [The present invention 1007] The mutant photosynthetic organism of the present invention 1006, wherein the polypeptide having CHG DNA methyltransferase activity comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 28 prior to said mutation or attenuation of said gene. [The present invention 1008] The mutant photosynthetic organism of any of claims 1001 to 1007, wherein the mutant photosynthetic organism comprises exogenous DNA, and wherein reduced CHG DNA methylation is in the exogenous DNA. [The present invention 1009] The mutant photosynthetic organism of the present invention, wherein said exogenous DNA is integrated into the genome of said photosynthetic organism. [The present invention 1010] The mutant photosynthetic organism of any of claims 1001 to 1007, wherein said reduced CHG DNA methylation is in DNA native to said photosynthetic organism. [The present invention 1011] The mutant photosynthetic organism of the present invention, wherein said reduced CHG DNA methylation is in a centromere or highly repetitive DNA region of said mutant photosynthetic organism. [The present invention 1012] A control photosynthetic organism that contains the exogenous nucleic acid but does not have a mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity. The mutant photosynthetic organism of any of claims 1008 to 1009, wherein expression of an exogenous nucleic acid is improved as compared to the mutant photosynthetic organism of claim 1008. [The present invention 1013] 13. The mutant photosynthetic organism of any of claims 1001 to 1012, having reduced mono- or tri-methylation of lysine 9 of histone H3 (H3K9). [The present invention 1014] Control photosynthetic organisms that do not have a mutated or attenuated gene encoding a polypeptide with CHG DNA methyltransferase activity The mutant photosynthetic organism of any of claims 1001 to 1013, having reduced CHH DNA methylation as compared to said mutant photosynthetic organism. [The present invention 1015] The mutant photosynthetic organism of any one of claims 1001 to 1014, wherein the photosynthetic organism is an algae. [The present invention 1016] 1. A method for enhancing expression of exogenous DNA in a photosynthetic organism, comprising: a) introducing exogenous DNA into said photosynthetic organism; b) mutating or attenuating a gene encoding a polypeptide having CHG DNA methyltransferase activity, A control photosynthetic organism containing exogenous DNA but not having a mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity. the mutant microorganism has reduced CHG DNA methylation of exogenous DNA compared to the wherein expression of the exogenous DNA is enhanced in the photosynthetic organism compared to the control photosynthetic organism. [The present invention 1017] The method of claim 1016, wherein the gene encoding a polypeptide having CHG DNA methyltransferase activity is mutated or attenuated by genetic manipulation. [The present invention 1018] The method of claim 1017, wherein said genetic manipulation is by insertional mutagenesis, gene replacement, RNAi, antisense RNA, meganuclease genome manipulation, one or more ribozymes, and / or a CRISPR / Cas system. [The present invention 1019] The method of claim 1018, wherein the genetic manipulation is by a CRISPR / Cas system. [The present invention 1020] The method of claim 1016, wherein mutating or attenuating the gene encoding a polypeptide having CHG DNA methyltransferase activity is by UV irradiation, gamma irradiation, or chemical mutagenesis. [The present invention 1021] Any of the methods of claims 1016 to 1020, wherein the mutation or attenuation is in a gene encoding a polypeptide having CHG DNA methyltransferase activity, and the polypeptide comprises an amino acid sequence having at least 65% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 28 prior to the mutation or attenuation of the gene. [The present invention 1022] The method of claim 1021, wherein the polypeptide having CHG DNA methyltransferase activity comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 28 prior to mutation or attenuation of said gene. [The present invention 1023] Control photosynthetic organisms that do not have a mutated or attenuated gene encoding a polypeptide with CHG DNA methyltransferase activity 3. The method of any of claims 1016 to 1022, wherein the mutant photosynthetic organism has reduced CHH DNA methylation compared to. [The present invention 1024] The method according to any one of claims 1016 to 1023, wherein the exogenous DNA is integrated into the genome of the photosynthetic organism. [The present invention 1025] The method according to any one of claims 1016 to 1024, wherein the photosynthetic organism is an algae. [The present invention 1026] 1. A method for reducing mono- or tri-methylation of lysine 9 on histone H3 (H3K9) in a photosynthetic organism, comprising: Mutating or attenuating a gene encoding a polypeptide having CHG DNA methyltransferase activity Including, Control photosynthetic organisms that do not have a mutated or attenuated gene encoding a polypeptide with CHG DNA methyltransferase activity The method, wherein the mutant microorganism has reduced CHG DNA methylation, and the mono- or tri-methylation of lysine 9 on histone H3 (H3K9) of the photosynthetic organism is reduced compared to the mutant microorganism. [The present invention 1027] The method of claim 1026, wherein the gene encoding a polypeptide having CHG DNA methyltransferase activity is mutated or attenuated by genetic manipulation. [The present invention 1028] 1027. The method of claim 1027, wherein said genetic manipulation is by insertional mutagenesis, gene replacement, RNAi, antisense RNA, meganuclease genome manipulation, one or more ribozymes, and / or a CRISPR / Cas system. [The present invention 1029] The method of claim 1028, wherein the genetic manipulation is by the CRISPR / Cas system. [The present invention 1030] The method of claim 1026, wherein mutating or attenuating the gene encoding a polypeptide having CHG DNA methyltransferase activity is by UV irradiation, gamma irradiation, or chemical mutagenesis. [The present invention 1031] Any of the methods of claims 1026 to 1030, wherein the mutation or attenuation is in a gene encoding a polypeptide having CHG DNA methyltransferase activity, and the polypeptide comprises an amino acid sequence having at least 65% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 28 prior to the mutation or attenuation of the gene. [The present invention 1032] The method of claim 1031, wherein the polypeptide having CHG DNA methyltransferase activity comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 28 prior to mutation or attenuation of said gene. [The present invention 1033] Control photosynthetic organisms that do not have a mutated or attenuated gene encoding a polypeptide with CHG DNA methyltransferase activity 33. The method of any of claims 1026 to 1032, wherein the mutant photosynthetic organism has reduced CHH DNA methylation compared to. [The present invention 1034] The method according to any one of claims 1026 to 1033, wherein the photosynthetic organism is an algae. [The present invention 1035] The method according to any one of claims 1026 to 1034, wherein the photosynthetic organism comprises exogenous DNA. [The present invention 1036] The method of claim 1035, wherein said exogenous DNA is integrated into the genome of said photosynthetic organism. [The present invention 1037] The method of claim 1036, wherein the reduced mono- or trimethylation of lysine 9 on histone H3 (H3K9) of the photosynthetic organism is in a portion of a chromosome that contains the exogenous DNA. [Brief description of the drawings]

[0021] [Figure 1]Figure 1 is a genome track showing the presence of CpG, CHG, and CHH DNA methylation and H3K9 mono- and tri-methylation of exogenous DNA integrated into the genome of Parachlorella species, as well as the reduction of these methylations by knocking out CHG methyltransferase. The exogenous DNA methylation and H3K9 mono- and tri-methylation status of Parachlorella knockout strains STR03778 and STR03749 were compared to the Parachlorella control strain STR00014. Table 1 further identifies these strains. All three strains contain integrated blasticidin, Cas-9, and GFP genes. Representative exogenous DNA genome tracks containing the blasticidin, Cas-9, and GFP genes showing percent DNA methylation (first set of columns) and genome coverage (second set of columns) are shown, as well as sequence information from chromatin immunoprecipitation (ChIP) with three Parachlorella strains isolated with antibodies specific for H3K9me1 and H3K9me3. Tracks are scaled to allow comparison between different samples. Coverage tracks for histone 3 (H3) are also shown. Chromosomal locations are shown at the top and gene models at the bottom. [Diagram 2]Figure 2 shows a general lack of CHG and CHH DNA methylation and H3K9 mono- and trimethylation outside of repetitive and centromeric regions of native Parachlorella DNA. The native DNA methylation and H3K9 mono- and trimethylation status of Parachlorella knockout strains STR03778 and STR03749 were compared to the Parachlorella control strain STR00014 (see Table 1 for additional information). Representative native DNA genomic tracks showing the percent DNA methylation (first set of columns) and genome coverage (second set of columns) are shown, as well as sequence information from ChIP with three Parachlorella strains isolated with antibodies specific for H3K9me1 and H3K9me3. Tracks are scaled to allow comparison between different samples. Histone 3 (H3) coverage tracks are also shown. Chromosomal locations are shown at the top and gene models at the bottom. [Diagram 3] Figure 3 shows a general lack of CHG and CHH DNA methylation and H3K9 mono- and trimethylation outside of repeat, transposable element, and centromeric regions of chromosome 2 in native Parachlorella DNA. DNA methylation and H3K9 mono- and trimethylation status of Parachlorella knockout strains STR03778 and STR03749 were compared to the Parachlorella control strain STR00014. Representative native DNA genomic tracks showing percent DNA methylation (first set of columns) and genome coverage (second set of columns) are shown, as well as sequencing reads from ChIP with the three Parachlorella strains pulled down with antibodies specific for H3K9me1 and H3K9me3. Tracks are scaled to allow comparison between different samples. Histone 3 (H3) coverage tracks are also shown. Chromosomal locations are shown at the top and gene models at the bottom. [Figure 4]FIG. 4 shows a comparison of exogenous gene expression in selected Parachlorella knockout strains STR03749, STR03778, STR03779 compared to the Parachlorella control strain STR00014 as measured by Western blot. [Diagram 5] Figure 5 shows genome tracks showing the presence of DNA methylation (CpG, CHG, and CHH) of the exogenous DNA (blasticidin gene) integrated into the Ocystis genome for the indicated strains. The first set of rows shows the percent DNA methylation, and the second set of rows shows the genome coverage. STR24194 is the background strain, and STR28031 is a knockout strain of SEQ ID NO:29 (encoding SEQ ID NO:28) with blasticidin as the selection marker. [Figure 6] Figure 6 is a zoomed-in view of the genome track distribution of DNA methylation (CpG, CHG, and CHH) in the background strain (STR24194) of the Ocystis genome. DNA showing the percent DNA methylation (first set of columns) and genome coverage (second set of columns) are shown. [Figure 7] Figure 7 shows a zoomed-in view of genome track distribution of DNA methylation (CpG, CHG, CHH) of native DNA of the Hypericum genome. DNA showing percent DNA methylation (first set of columns) and genome coverage (second set of columns) are shown. STR28031 is a knockout strain of SEQ ID NO:29 (encoding SEQ ID NO:28) with blasticidin as a selection marker; STR24194 is the background strain. [Figure 8]Figures 8A and 8B show agarose gels showing expression of the transgenes (Cre recombinase and Ble) in Hylocytis spp. Figure 8A shows the STR24194 (background strain) strain of the five Cre constructs. Figure 8B shows the same data for Hylocytis spp. with a deletion of the methyltransferase coding sequence of SEQ ID NO: 28 (STR29997). The data show that the cre recombinase transgene is expressed in Hylocytis spp. background strain (STR24194). [Figure 9] 9 shows the results of a productivity assay test in Parachlorella sp. (STR03778 with deletion of methyltransferase of SEQ ID NO:2) measured as total organic carbon (TOC) as an indicator of productivity under semi-continuous area culture. The data shows that there is no defect in the productivity of the cells compared to the wild-type Parachlorella sp. strain (STR0010). [Figure 10] 10 shows the results of a productivity assay in Ocystis sp. (STR29997, carrying a deletion of the methyltransferase of SEQ ID NO: 29) measured as total organic carbon (TOC) as an indicator of productivity under semi-continuous area culture. The data shows that there is no defect in the productivity of the cells compared to a background strain (STR24194) that was improved and selected from the wild type for growth characteristics. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description of the Invention This application discloses epigenetic mechanisms for silencing the expression of exogenous genes in algae. This application identifies the role of CHG methylation of exogenous DNA in epigenetic silencing in algae. Therefore, the role of CHG methylation in exogenous gene silencing is generally unknown in algae because of the total absence of CHG methylation even from endogenous elements (Chlamydomonas, Volvox) or because only native but not exogenous DNA elements were examined (Chlorella). Furthermore, this application also identifies the role of histone 3, lysine 9 (H3K9) methylation (e.g., mono- or tri-methylation) in epigenetic silencing of exogenous genes in algal species.

[0023] The present application identifies a novel putative methyltransferase gene (Pfam PF00145, C-5 cytosine-specific DNA methyltransferase) in Trebouxiophyceae algae. The activity of the newly identified gene was analyzed by knocking out the gene individually and assessing the effect of the knockout on DNA methylation in native and exogenous DNA sequences, and the protein expression levels of the exogenous gene. The effect of these knockouts on the methylation of cytosines in CpG, CHG, and CHH DNA sequences, where H = A, T, or C, is disclosed. Also disclosed are the levels of histone 3, lysine 9 (H3K9) methylation (e.g., monomethylation, trimethylation) in chromatin regions of native DNA and in chromatin regions containing exogenous DNA. Also disclosed is an orthologous CHG DNA methyltransferase in Ocystis species.

[0024] The present inventors unexpectedly discovered that mutating or attenuating DNA methyltransferase reduces CHG and / or CHH methylation of exogenous DNA, which may occur by epigenetic mechanisms. The present inventors also discovered that mutating or attenuating DNA methyltransferase enhances protein expression of exogenous genes. In addition, the levels of mono- and trimethylation of histone 3, lysine 9 (H3K9) in chromatin regions containing exogenous DNA are also reduced. In addition, it was discovered that mutating or attenuating DNA methyltransferase reduces CHH methylation of exogenous DNA. Thus, the present invention provides mutant chlorophyte algae having attenuation of at least one CHG and / or CHH methyltransferase, as disclosed herein.

[0025] DNA methylation of exogenous DNA in chlorophyte algae can include DNA methylation of CHG sequences, where "C" is a methylated cytosine and "H" is A, T, or C). In various embodiments, the mutant photosynthetic organisms of the invention can have an attenuation or deletion of at least one gene encoding a methyltransferase. In some embodiments, the encoded methyltransferase is a sequence encoding any of SEQ ID NOs: 1, 3, 5, 7, 28, or any combination or subcombination thereof, or a variant thereof, having at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or 100% amino acid sequence identity to any of them, or to a fragment thereof comprising a contiguous sequence of at least 50, or at least 100, at least 125, at least 150 or more amino acid residues of the entire protein, or to any combination or subcombination thereof. In other embodiments, the methyltransferase is encoded by any of the sequences disclosed herein, or a variant thereof.

[0026] The present invention also includes a method for enhancing the expression of exogenous DNA in mutant photosynthetic organisms of the present invention. The method includes a) introducing exogenous DNA into the photosynthetic organism; and b) mutating, attenuating, or deleting a gene encoding a polypeptide having CHG and / or CHH DNA methyltransferase activity. The exogenous DNA introduced into the organism can be a DNA construct that includes a sequence for editing, attenuating, or deleting a gene encoding a methyltransferase activity. The mutation, attenuation, or deletion of the sequence can be achieved by any suitable method known to those skilled in the art. For example, CRISPR Cas9 gene editing, Cre-Lox recombination, or other gene editing techniques can be used. The mutant organism produced by this method can have reduced CHG and / or CHH DNA methylation of the exogenous DNA compared to a control photosynthetic organism that has exogenous DNA but does not have a mutated or attenuated gene encoding a polypeptide having CHG and / or CHH DNA methyltransferase activity. Thus, the expression of exogenous DNA is enhanced in the mutant organism compared to the control organism. The encoded methyltransferase that is mutated, attenuated or deleted in the organism may have at least 80% or at least 90% or at least 95% or at least 97% or at least 98% or 100% sequence identity to any of SEQ ID NOs: 1, 3, 5, 7, 28, or any combination or subcombination thereof, or a (variant) sequence thereof, or to a fragment thereof comprising a consecutive sequence of at least 50 or at least 100, at least 125, at least 150 or more amino acid residues of the entire protein or nucleotide of the nucleic acid sequence, or any combination or subcombination thereof. In other embodiments, the methyltransferase is encoded by any sequence disclosed herein or a variant thereof.For example, the methyltransferase may be encoded by any of SEQ ID NOs: 2, 4, 6, 8, or 29, or a (variant) sequence thereof having at least 80% or at least 90% or at least 95% or at least 97% or at least 98% or 100% sequence identity to any of them, or to a fragment thereof comprising a contiguous sequence of at least 200, at least 300, at least 500 or more nucleotide residues of the entire nucleic acid sequence, or any combination or subcombination thereof. In some embodiments, the reduction of CHG and / or CHH methylation occurs in highly repetitive regions of the exogenous DNA and / or in centromeric regions of the exogenous DNA. Highly repetitive regions or sequences of DNA do not usually code for polypeptides. In some embodiments, the highly repetitive regions or sequences are short sequences of 5-100 or 150-300 nucleotides. In some embodiments, the sequence is repeated at least 10,000 times, or at least 50,000 times, or at least 100,000 times, or at least 500,000 times, or at least 1 million times in a region of DNA, which can be a chromosome or a section of DNA less than 1 Mb, or less than 25 Mb, or less than 50 Mb, or less than 100 Mb, or less than 250 Mb.

[0027] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. In case of conflict, the present application, including definitions, will control. Unless otherwise required by context, singular terms include plurals and plural terms include the singular. All ranges provided within this application include the upper and lower end values ​​of the range unless otherwise specified.

[0028] All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0029] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A," and "B."

[0030] "About" can mean either within 10% of the stated value, or within 5% of the stated value, or in some cases within 2.5% of the stated value, or "about" can mean rounded to the nearest significant figure.

[0031] The term "gene" is used broadly to refer to any segment of a nucleic acid molecule (typically DNA, but optionally RNA) that codes for a polypeptide or expressed RNA. Thus, a gene includes a sequence that codes for an expressed RNA (which may include a polypeptide coding sequence or a functional RNA, such as, for example, ribosomal RNA, tRNA, antisense RNA, microRNA, short hairpin RNA, ribozyme, etc.). Genes may further include regulatory sequences that are necessary for or affect their expression, as well as sequences associated with the protein or RNA coding sequence in its natural state, such as, for example, intron sequences, 5' or 3' untranslated sequences. In some examples, a "gene" may refer to only the protein coding portion of a DNA or RNA molecule, which may or may not include introns. A gene is preferably more than 50 nucleotides in length, more preferably more than 100 nucleotides in length, and may be between 50 and 500,000 nucleotides in length, such as, for example, between 100 and 100,000 nucleotides in length, or between about 200 and about 50,000 nucleotides in length, or between about 200 and about 20,000 nucleotides in length. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information.

[0032] The term "nucleic acid" or "nucleic acid molecule" refers to a segment of DNA or RNA (e.g., mRNA), including nucleic acids with modified backbones (e.g., peptide nucleic acids, locked nucleic acids) or modified or non-natural nucleobases. Nucleic acid molecules can be double-stranded or single-stranded, and single-stranded nucleic acid molecules, including genes or portions thereof, can be the coding (sense) strand or non-coding (antisense) strand.

[0033] A nucleic acid molecule or polypeptide may be "derived" from an indicated source, including the isolation (in whole or in part) of a nucleic acid segment or polypeptide from an indicated source. A nucleic acid molecule may also be derived from an indicated source, for example, by direct cloning, PCR amplification, or artificial synthesis from an indicated polynucleotide source, or based on a sequence associated with an indicated polynucleotide source, which may be, for example, a biological species.

[0034] Genes or nucleic acid molecules derived from a particular source or species also include genes or nucleic acid molecules with sequence modifications relative to the source nucleic acid molecule, i.e., the sequence of the gene or nucleic acid molecule is derived from the sequence of the gene or nucleic acid molecule derived from the referenced source or species, but may have modifications. For example, a gene or nucleic acid molecule derived from a source (e.g., a particular reference gene) may contain one or more mutations that are unintentionally or intentionally introduced relative to the source gene or nucleic acid molecule, and when one or more mutations, including substitutions, deletions, or insertions, are intentionally introduced, the sequence modifications may be introduced by random or targeted mutation of cells or nucleic acids, by amplification or other gene synthesis or molecular biology techniques, or by chemical synthesis, or by any combination thereof. A gene or nucleic acid molecule derived from a reference gene or nucleic acid molecule that encodes a functional RNA or polypeptide may encode a functional RNA or polypeptide that has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the reference or source functional RNA or polypeptide, or a functional fragment thereof. For example, a gene or nucleic acid molecule derived from a reference gene or nucleic acid molecule that encodes a functional RNA or polypeptide can encode a functional RNA or polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the reference or source functional RNA or polypeptide, or a functional fragment thereof.

[0035] Similarly, a polypeptide or protein derived from a particular source or species includes a polypeptide or protein with sequence modification with respect to the source polypeptide, i.e., the polypeptide is derived from the sequence of the polypeptide derived from the reference source or species, but may have modification.For example, a polypeptide or protein derived from a source (e.g., a particular reference protein) can contain one or more mutations (amino acid differences) with respect to the source polypeptide, which are unintentional or are intentionally introduced (e.g., by mutation of the encoding nucleic acid molecule).A polypeptide derived from a reference polypeptide can have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the reference or source polypeptide, or its functional fragment.For example, a polypeptide derived from a reference polypeptide can have at least 80%, or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the reference or source polypeptide, or its functional fragment.

[0036] The terms "natural," "native," and "wild-type" refer to a form found in nature. For example, a naturally-occurring or wild-type nucleic acid molecule, nucleotide sequence, or protein can be found in a natural source, has been isolated from a natural source, and has not been intentionally modified by human manipulation.

[0037] "Attenuated" as used herein means a reduction in amount, degree, intensity, or strength. Attenuated gene expression can mean a significantly reduced amount and / or rate of transcription of the gene in question, or of translation, folding, or assembly of the encoded protein. As a non-limiting example, an attenuated gene can be a mutated or disrupted gene (e.g., a gene disrupted by partial or full deletion, truncation, frameshift, or insertion mutation) that does not encode a complete functional open reading frame or whose expression is reduced by alteration or disruption of gene regulatory sequences. An attenuated gene can also be a gene that is targeted by a construct that reduces the expression of the gene, such as, for example, an antisense RNA, a microRNA, an RNAi molecule, or a ribozyme. Attenuated gene expression can be, for example, gene expression that is reduced to an amount that is insignificant or undetectable. Attenuated gene expression can be gene expression that results in a non-fully functional or non-functional RNA or protein, for example, attenuated gene expression can be gene expression that results in a truncated RNA and / or polypeptide.

[0038] An "exogenous nucleic acid molecule," "transgene," or "exogenous gene" refers to a nucleic acid molecule or gene that has been introduced (e.g., transformed) into a cell. A transformed cell may be referred to as a recombinant cell into which additional exogenous genes may be introduced. Progeny of a cell transformed with a nucleic acid molecule are also referred to as "transformed" if they inherit the exogenous nucleic acid molecule. An "endogenous" nucleic acid molecule, gene, or protein is a native nucleic acid molecule, gene, or protein that occurs in or is naturally produced by a host.

[0039] A "recombinant" or "engineered" nucleic acid molecule is a nucleic acid molecule that has been modified by human manipulation. As a non-limiting example, a recombinant nucleic acid molecule includes any of the following nucleic acid molecules: (1) partially or completely synthesized or modified in vitro using, for example, chemical or enzymatic techniques (e.g., by using chemical nucleic acid synthesis or by using enzymes for the replication, polymerization, digestion (exonuclease or endonuclease degradation), ligation, reverse transcription, transcription, base modification (including, for example, methylation), integration or recombination (including homologous and site-specific recombination) of the nucleic acid molecule; (2) containing a linked nucleotide sequence that is not linked in nature; (3) engineered using molecular cloning techniques to lack one or more nucleotides relative to the naturally occurring nucleic acid molecule sequence; and / or (4) engineered using molecular cloning techniques to have one or more sequence changes or rearrangements relative to the naturally occurring nucleic acid sequence. As a non-limiting example, a cDNA is a recombinant DNA molecule such as one produced by an in vitro polymerase reaction or any nucleic acid molecule to which a linker has been attached or which has been incorporated into a vector, such as a cloning vector or an expression vector.

[0040] As used herein, the term "recombinant protein" refers to a protein produced by genetic engineering, regardless of whether or not the amino acids differ from those of the wild-type protein.

[0041] The terms "recombinant," "engineered," or "genetically engineered," as applied to an organism, refer to an organism that has been engineered by introducing a heterologous or exogenous recombinant nucleic acid sequence into the organism (e.g., a non-native nucleic acid sequence), including gene knockouts, targeted mutations, gene replacements, and promoter replacements, deletions, disruptions, or insertions, as well as the introduction of a transgene or synthetic gene or nucleic acid sequence into the organism. That is, recombinant, engineered, or engineered refers to an organism that has been modified by human intervention. A recombinant or engineered organism may be an organism into which a construct has been introduced to reduce gene expression or a gene "knockdown." Such constructs include, but are not limited to, RNAi, microRNA, shRNA, siRNA, antisense, and ribozyme constructs. Also included are organisms whose genomes have been altered by the activity of meganucleases, zinc finger nucleases, TALENs, or Cas / CRISPR systems. An exogenous or recombinant nucleic acid molecule may be integrated into the genome of the recombinant / engineered organism or, in other cases, may not be integrated into the host genome. As used herein, a "recombinant microorganism" or "recombinant host cell" includes the progeny or derivatives of a recombinant microorganism of the invention. Because certain modifications may occur in successive generations, either due to mutation or environmental influences, such progeny or derivatives may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0042] The term "promoter" refers to a nucleic acid sequence capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. A promoter includes a minimum number of bases or elements necessary to initiate transcription at a level detectable above background. A promoter can include a transcription initiation site as well as a protein binding domain (consensus sequence) responsible for binding RNA polymerase. Eukaryotic promoters often, but not always, include "TATA" boxes and "CAT" boxes. Prokaryotic promoters can include -10 and -35 prokaryotic promoter consensus sequences. Numerous promoters, including constitutive, inducible and repressible promoters from a variety of different sources, are well known in the art. Representative sources include, for example, algal, viral, mammalian, insect, plant, yeast and bacterial cell types, and suitable promoters from these sources are readily available or can be synthetically produced, for example, based on publicly available sequences online or from depositories such as the ATCC, as well as other commercial or individual sources. Promoters can be unidirectional (transcription initiation in one direction) or bidirectional (transcription initiation in both directions). Promoters can be constitutive, repressible, or inducible. In addition to the gene-proximal promoter to which RNA polymerase binds to initiate transcription, a promoter region can include additional sequences upstream of the gene that can be within 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, or more of the gene's transcription start site, which can affect the transcription rate of downstream genes and, optionally, the responsiveness of the promoter to developmental, environmental, or biochemical (e.g., metabolic) conditions.

[0043] The term "heterologous" when used with respect to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme, refers to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is derived from a source other than the host organism. In contrast, a "homologous" polynucleotide, gene, nucleic acid, polypeptide, or enzyme is used herein to refer to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is derived from the host organism. When referring to gene regulatory sequences or auxiliary nucleic acid sequences used to maintain or manipulate a gene sequence (e.g., promoters, 5' untranslated regions, 3' untranslated regions, poly A addition sequences, intron sequences, splice sites, ribosome binding sites, internal ribosome entry sequences, regions of genomic homology, recombination sites, etc.), "heterologous" means that the regulatory or auxiliary sequences are not naturally associated with the gene to which they are juxtaposed in a construct, genome, chromosome, episome, etc. Thus, a promoter operably linked to a gene to which it is not operably linked in its native state (i.e., in the genome of a non-genetically engineered organism) is referred to herein as a "heterologous promoter," even if the promoter is derived from the same species (or, in some cases, the same organism) as the gene to which it is linked.

[0044] The term "protein" or "polypeptide" as used herein is intended to encompass the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to one or more chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms.

[0045] Gene and protein accession numbers, typically provided in parentheses after the gene or species name, are unique identifiers of sequence records publicly available at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov), maintained by the National Institutes of Health. "GenInfo Identifier" (GI) sequence identification numbers are specific to a nucleotide or amino acid sequence. If the sequence changes in any way, a new GI number is assigned. The sequence revision history tool can be used to track the various GI numbers, version numbers, and update dates of a sequence that appears in a particular GenBank record. Searching and retrieving nucleic acid or gene sequences or protein sequences based on accession numbers and GI numbers is well known, for example, in the fields of cell biology, biochemistry, molecular biology, and molecular genetics.

[0046] As used herein, the term "percent identity" or "homology" with respect to a nucleic acid or polypeptide sequence is defined as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical to a known polypeptide after aligning the sequences for the maximum percent identity and introducing gaps, if necessary, to achieve the maximum percent homology. N- or C-terminal insertions or deletions shall not be construed as affecting homology, and internal deletions and / or insertions into a polypeptide sequence of less than about 30, less than about 20, or less than about 10 amino acid residues shall not be construed as affecting homology. Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithms adopted by the programs blastp, blastn, blastx, tblastn, and tblastx (Altschul (1997), Nucleic Acids Res. 25, 3389-3402, and Karlin (1990), Proc Natl. Acad. Sci. USA 87, 2264-2268), which are tailored for sequence similarity searches. The approach used by the BLAST program is to first consider similar segments with or without gaps between the query sequence and the database sequence, then evaluate the statistical significance of all identified matches, and finally summarize only those matches that meet a preselected significance threshold. For a discussion of basic issues in similarity searching of sequence databases, see Altschul (1994), Nature Genetics 6, 119-129. The search parameters of histogram, description, alignment, expectation (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix, and filter (low complexity) can be set to default.The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff (1992), Proc. Natl. Acad. Sci. USA 89, 10915-10919), which is recommended for query sequences of 85 or more nucleotide bases or amino acids in length.

[0047] For blastn, designed to compare nucleotide sequences, the scoring matrix is ​​set by the ratio of M (i.e., reward score for a pair of matching residues) to N (i.e., penalty score for mismatched residues), where the default values ​​for M and N can be +5 and -4, respectively. Four blastn parameters can be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (create word hits at every winkth position along the query); and gapw=16 (sets the window width within which gapped alignments are created). Equivalent Blastp parameter settings for comparison of amino acid sequences can be Q=9; R=2; wink=1; and gapw=32. For best-fit comparison between sequences available in the GCG package version 10.0, DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) can be used, and equivalent settings in protein comparisons are GAP=8 and LEN=2.

[0048] Disclosed herein are polypeptide or nucleic acid sequences of the invention that are variants of any of the sequences disclosed herein (e.g., any of SEQ ID NOs: 1-29) that have at least 40%, at least 45%, at least 50%, at least 55%, at least 70%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to the full-length polypeptide or nucleic acid sequence, or to a fragment thereof that comprises at least 50, or at least 100, at least 125, at least 150 or more consecutive amino acid residues or nucleotides of the entire protein or nucleic acid sequence. Variants of the disclosed sequences can have at least one amino acid residue or nucleotide inserted at and / or within the N-terminus and / or C-terminus of the disclosed sequences, including insertions and substitutions. Variants also include sequences having at least one substitution of an amino acid or nucleotide, e.g., a substitution in any of SEQ ID NOs: 1-29, which may be a conservative amino acid substitution. Variants can also be nucleotide sequences encoding any of the amino acid sequences disclosed herein, or variants thereof. Contemplated variants can additionally or alternatively include those that contain predetermined mutations, e.g., by homologous recombination or site-directed or PCR mutagenesis, as well as other naturally occurring variants of a family of polypeptides or nucleic acids, including corresponding polypeptides or nucleic acids of other species, alleles, or insertions and substitutions, including, but not limited to, those described herein, and / or derivatives in which the polypeptide has been covalently modified by chemical, enzymatic, or other suitable means, substitutions with moieties (e.g., detectable moieties such as enzymes) that contain insertions and substitutions other than naturally occurring amino acids.In either embodiment, the disclosed polypeptide sequence may be the encoded methyltransferase in a mutant organism of the invention, or may be a nucleic acid sequence that encodes such a methyltransferase in an organism.

[0049] The phrase "conservative amino acid substitution" or "conservative mutation" as used herein refers to the replacement of one amino acid with another amino acid that has common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz (1979) Principles of Protein Structure, Springer-Verlag). According to such analysis, it is possible to define groups of amino acids in which the amino acids in the group are preferentially exchanged with each other and thus most similar to each other in their effect on the overall protein structure (Schulz (1979) Principles of Protein Structure, Springer-Verlag). Examples of amino acid groups defined in this way include the "charged / polar group" which includes Glu, Asp, Asn, Gln, Lys, Arg, and His, the "aromatic or cyclic group" which includes Pro, Phe, Tyr, and Trp, and the "aliphatic group" which includes Gly, Ala, Val, Leu, Ile, Met, Ser, Thr, and Cys. Within the groups, subgroups can be distinguished. For example, the group of charged / polar amino acids can be subdivided into subgroups including a "positively charged subgroup" containing Lys, Arg, and His, a "negatively charged subgroup" containing Glu and Asp, and a "polar subgroup" containing Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into subgroups including a "nitrogen ring subgroup" containing Pro, His, and Trp, and a "phenyl subgroup" containing Phe and Tyr. In another further example, the aliphatic group can be subdivided into subgroups including a "large aliphatic non-polar subgroup" containing Val, Leu, and Ile, an "aliphatic mildly polar subgroup" containing Met, Ser, Thr, and Cys, and a "small residue subgroup" containing Gly and Ala.Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, including, but not limited to, for example, Arg to Lys or vice versa to maintain a positive charge, Asp to Glu or vice versa to maintain a negative charge, Thr to Ser or vice versa to maintain a free -OH, and Asn to Gln or vice versa to maintain a free -NH2. A "conservative variant" is a polypeptide that contains one or more amino acids substituted to replace one or more amino acids of a reference polypeptide (e.g., a polypeptide whose sequence has been disclosed in a publication or sequence database or whose sequence has been determined by nucleic acid sequencing) with an amino acid that shares common properties, e.g., belongs to the same amino acid group or subgroup as delineated above.

[0050] As used herein, "expression" includes expression of a gene at least at the level of RNA production, "expression product" includes the resulting product, e.g., a polypeptide or functional RNA (e.g., ribosomal RNA, tRNA, antisense RNA, microRNA, shRNA, ribozymes, etc.) of the expressed gene, and the term "increased expression" includes alterations in gene expression to promote increased mRNA production and / or increased polypeptide expression. "Increased production" includes an increase in the amount of polypeptide expression, an increase in the level of enzymatic activity of a polypeptide, or a combination of both, compared to the native production or enzymatic activity of the polypeptide.

[0051] Some aspects of the present invention include partial, substantial or complete attenuation, deletion, silencing, inactivation, or downregulation of the expression of a particular polynucleotide sequence. Genes can be partially, substantially or completely deleted, silenced, inactivated, or their expression can be downregulated to affect the activity carried out by the polypeptides they encode, such as the activity of an enzyme. Genes can be partially, substantially or completely deleted, silenced, inactivated, or downregulated by the insertion of a nucleic acid sequence that disrupts the function and / or expression of the gene (e.g., viral insertion, transposon mutagenesis, meganuclease engineering, homologous recombination, or other methods known in the art). The terms "remove", "remove", and "knockout" can be used interchangeably with the terms "deletion", "partial deletion", "substantial deletion", or "complete deletion". In certain embodiments, the microorganism of interest can be engineered by site-specific homologous recombination or targeted recombination or mutation using the Cas / CRISPR system to knock out a particular gene of interest. In yet other embodiments, targeted insertion or mutation into gene regulatory regions using Cas / CRISPR systems, RNAi, or antisense DNA (asDNA) constructs can be used to partially, substantially, or completely silence, inactivate, or downregulate specific genes of interest.

[0052] These insertions, deletions, or other modifications of a particular nucleic acid molecule or particular polynucleotide sequence may be understood to encompass "genetic modification" or "transformation," such that the resulting strain of a microorganism or host cell may be understood to be "genetically modified," "genetically engineered," or "transformed."

[0053] As used herein, "enhancing expression" includes increasing the expression or enzymatic activity of a gene or nucleic acid molecule of interest in a photosynthetic organism containing one or more genetic modifications compared to the expression or activity in a control photosynthetic organism without such genetic modifications.

[0054] "Mutant" as used herein refers to an organism that has a mutation in a gene that is the result of classical mutagenesis, for example, using gamma irradiation, UV, or chemical mutagenesis. "Mutant" as used herein also refers to a recombinant cell that has a change in the structure or expression of a gene as a result of genetic engineering, which may include, but are not limited to, overexpression, including expression of a gene under different temporal, biological, or environmental regulation and / or to a degree different from that which occurs in nature, and / or expression of a gene that is not naturally expressed in the recombinant cell; homologous recombination, including knock-out and knock-in (e.g., gene replacement with a gene that encodes a polypeptide that is more or less active than the wild-type polypeptide, and / or a dominant negative polypeptide); attenuation of a gene via RNAi, antisense RNA, or ribozyme, etc.; and genome engineering, such as using meganuclease, TALEN, and / or CRISPR technology. Thus, a mutant is not an organism that exists in nature. A mutant organism of interest typically has a phenotype that differs from that of the corresponding wild-type or ancestral strain lacking the mutation, where the phenotype can be evaluated by growth assays, product analysis, photosynthetic characteristics, biochemical assays, and the like. When referring to a gene "mutant," it means that the gene has at least one base (nucleotide) change, deletion, or insertion compared to the native or wild-type gene. The mutation (one or more nucleotide changes, deletions, and / or insertions) may be in the coding region of the gene, in an intron, 3'UTR, 5'UTR, or promoter region, for example, within 2 kb, or within 3 kb, of the transcription start site, or at the translation start site.For example, a mutant with attenuated expression of a gene as disclosed herein can have a mutation that can be one or more nucleobase changes and / or one or more nucleobase deletions and / or one or more nucleobase insertions in a region of the gene 5' of the transcription start site, such as within 2 kb, 1.5 kb, 1 kb, or 0.5 kb of a known or putative transcription start site, or within 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, or 0.5 kb of the translation start site. As non-limiting examples, a mutant gene can be a gene with a mutation, insertion, or deletion in the promoter region that can increase or decrease the expression of the gene; a gene with a deletion that results in the production of a non-functional protein, a truncated protein, a dominant negative protein, or a non-protein; a gene with one or more point mutations that lead to changes in the amino acids of the encoded protein or that result in aberrant splicing of the gene transcript, etc.

[0055] Conserved domains of polypeptides include those identified in the "cd" (conserved domain) database, the COG database, the SMART database, the PRK database, the TIGRFAM database, or others known in the art. The National Center for Biotechnology Information WEB site provides the Conserved Domain Database (CDD), which it describes as "a protein annotation resource consisting of a collection of well-annotated multiple sequence alignment models for classical domains and full-length proteins." These are available as position-specific score matrices (PSSMs) for rapid identification of conserved domains in protein sequences via RPS-BLAST. CDD content includes NCBI cured domains, which explicitly define domain boundaries and use 3D structural information to provide insight into sequence / structure / function relationships, and domain models imported from many external source databases (Pfam, SMART, COG, PRK, TIGRFAM). Any of these resources can be used to identify conserved domains.

[0056] The term "Pfam" refers to a large collection of protein domains and protein families maintained by the Pfam Consortium and available at several sponsored worldwide websites, including Pfam.sanger.ac.uk / (Welcome Trust, Sanger Institute), pfam.sbc.su.se (Stockholm Bioinformatics Center), pfam.janelia.org / (Janelia Farm, Howard Hughes Medical Institute), pfam.jouy.inra.fr / (Institut national de la Recherche Agronomique), and pfam.ccbb.re.kr. The latest release of Pfam is Pfam 32.0 (September 2018). Pfam domains and families are identified using multiple sequence alignments and hidden Markov models (HMMs). Pfam-A family or domain assignments are high quality assignments generated by curated seed alignments with representative members of the protein family and profile hidden Markov models based on the seed alignments (unless otherwise stated, a match between a queried protein and a Pfam domain or family is a Pfam-A match). All identified sequences belonging to a family are then used to automatically generate a complete alignment of the family (Sonnhammer (1998) Nucleic Acids Research 26, 320-322; Bateman (2000) Nucleic Acids Research 26, 263-266; Bateman (2004) Nucleic Acids Research 32, Database Issue, D138-D141; Finn (2006) Nucleic Acids Research Database Issue 34, D247-251; Finn (2010) Nucleic Acids Research Database Issue 38, D211-222).By accessing the Pfam database, for example, using any of the websites mentioned above, protein sequences can be queried against HMMs using HMMER homology search software (e.g., HMMER2, HMMER3, or higher versions at hmmer.janelia.org / ). Significant matches that identify a queried protein as belonging to a Pfam family (or as having a particular Pfam domain) are matches whose bit score is equal to or greater than the collection threshold for the Pfam domain. Expectation values ​​(e-values) can also be used as a criterion for including a queried protein in a Pfam or for determining whether a queried protein has a particular Pfam domain, with low e-values ​​(less than 1.0, e.g., less than 0.1, or 0.01 or less) representing a low probability of matching by chance.

[0057] Reference to a property being "substantially the same" or "substantially identical" indicates minor and unrelated deviations that are not important to the property considered important in the context of the present invention. In various embodiments, this can mean that the property is within 10%, preferably within 5%, or within 2.5% of a reference value.

[0058] A "control cell" or "control microorganism" is either a wild-type cell or microorganism from which a mutant microorganism (genetically engineered or mutagenized microorganism) is derived directly or indirectly, or a cell or microorganism that is substantially identical to the referenced mutant cell or microorganism, except that the control cell or microorganism does not have a mutation that results in increased lipid production, e.g., the control cell or microorganism has not been genetically engineered or mutagenized to increase lipid production. For example, if a recombinant algae contains an exogenous gene encoding a Cas9 gene and a knockout of the native CHG methyltransferase gene, the control algae can be substantially identical to the recombinant algae, except that the control algae does not contain a knockout of the CHG methyltransferase gene.

[0059] Disclosed herein are methods for manipulating, assaying, culturing, and analyzing microorganisms. The invention described herein also uses standard methods, techniques, and reagents for cell culture, microbial transformation, genetic manipulation, and biochemical analysis known in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the specification and claims. EXAMPLES

[0060] Example 1. Development of a fully systemic CAS9-expressing Parachlorella strain A Parachlorella strain was engineered to express the Streptococcus pyogenes Cas9 gene using methods essentially as described in PCT Publication WO2016109840, which is incorporated by reference in its entirety. Briefly, Parachlorella strain WT-1185 was transformed with a vector containing the Streptococcus pyogenes Cas9 gene operably linked to the Parachlorella RPS17 promoter, a Parachlorella-optimized Aspergillus terreus codon-derived blasticidin resistance gene, and a GFP reporter expression cassette operably linked to the Parachlorella ACP1 promoter to generate Parachlorella strain STR00014. Integration of the Cas9 and GFP genes into the Parachlorella genome was confirmed by sequencing, a shift in fluorescence by flow cytometry, and demonstration of Cas9 protein expression by Western blotting.

[0061] Example 2. Identification of DNA methyltransferase in Parachlorella Four novel putative DNA methyltransferase genes, including Pfam PF00145 (C-5 cytosine-specific DNA methyltransferase) in Parachlorella species, were identified from sequence databases using Pfam analysis, BLAST searches, and HMMER. Each of these four putative methyltransferase genes contains Pfam PF00145, which corresponds to a C-5 cytosine-specific DNA methyltransferase. The amino acid sequences of the Parachlorella putative DNA methyltransferases and their corresponding cDNA sequences are shown as SEQ ID NOs: 1 to 8.

[0062] Example 3. Knockout of each of four putative DNA methyltransferases using the fully permeable Parachlorella CAS9 editor strain STR00014 Each of the individual putative DNA methyltransferases was knocked out using CRISPR Cas9 integrated into the Parachlorella Cas9 editor background strain STR00014 (which is a wild-type strain carrying the Cas9 cassette) and chimeric gRNAs for each gene. Chimeric gRNAs were designed and synthesized in vitro to target the four DNA methyltransferase genes of Parachlorella: EMRE3EUKT598198, EMRE3EUKT590754, EMRE3EUKT596408, and EMRE3EUKT596208 (these respective chimeric gRNA sequences are shown as SEQ ID NOs: 9-12).

[0063] Parachlorella STR00014 was transformed by electroporation with 1-2 μg of purified chimeric guide RNA and 1 μg of selectable marker DNA (SEQ ID NO: 13) containing the bleomycin resistance "BleR" gene codon-optimized for Parachlorella and containing a Parachlorella-derived intron. The BleR gene was operably linked to the Parachlorella RPS4 promoter (SEQ ID NO: 14) and terminated by the Parachlorella RPS4 terminator (SEQ ID NO: 15).

[0064] 1 × 106 A 100 mL seed culture was inoculated to 1 × 10 cells / mL 2 days before transformation. 6 Electroporation was performed by seeding, which was used to inoculate a 1 L culture to 5 × 10 cells / mL. On the day of transformation, cells were pelleted by centrifugation at 5000 × g for 20 min, washed three times with 0.1 um filtered 385 mM sorbitol, and diluted with 5 × 10 9 The cells were resuspended in 385 mM sorbitol to 100 μL cells / mL. Electroporation of 100 μL concentrated cells was performed in a 0.2 cm cuvette in a Bio-Rad® Gene Pulser Xcell™ under various conditions. The DNA used for electroporation optimization was linearized pSG6640 containing ble and TurboGFP expression cassettes. The TurboGFP cassette contained the Parachlorella ACP promoter (SEQ ID NO: 16) operably linked to the TurboGFP gene (SEQ ID NO: 17) and the Parachlorella ACP terminator (SEQ ID NO: 18). Immediately after electroporation of the pre-chilled cells and cuvette, 1 mL of cold sorbitol was added and used to transfer the cells into 10 mL of commercial algal growth medium. After overnight recovery, the cells were concentrated and spread to a diameter of 13 cm in growth medium containing 250 mg / L Zeocin and grown under the conditions described in the Biolistics section. The sequences of the Parachlorella ACP promoter, ACP terminator, and TurboGFP gene are shown below.

[0065] After testing a range of voltages, resistances, and capacitances, optimal electroporation conditions were determined to be 1.0-1.2 kV (5000-6000 V / cm), 200-300 ohms, and 25-50 μF. Use of larger amounts of DNA increased the resulting number of Zeocin-resistant colonies, although the effect plateaued at amounts above 4 μg.

[0066] Following electroporation, cells were plated on agar medium (commercial algal growth medium supplemented with 10 mM ammonium and 15 mM HEPES pH 8) containing 250 μg / ml Zeocin to select for transformants that had integrated the bleR cassette. EMRE3EUKT598198 knockout transformants were screened by colony PCR using primers (DNA_oligoST106; SEQ ID NO: 19, and DNA_oligoST107; SEQ ID NO: 20) designed to amplify the entire native target locus. Primers were designed to produce a band of approximately 400 bp in the absence of integration into the locus (e.g., a "knock-in" of the BleR cassette), or a band of approximately 5.1 kb in the case of integration of a single BleR cassette into the target locus, or likely no band in the case of multiple integrations into the target locus. In addition, two additional colony PCR reactions were performed using one each of the primers mentioned above flanking the target and an internal BleR primer (DNA_oligoST078; SEQ ID NO: 21) designed to amplify the selectable marker from the chromosome. Regardless of the orientation of the integratable cassette, if integration of the target was successful, a band of approximately 800 bp would result from amplification with either the flanking primers and the internal primer DNA_oligoST078. The sequences of the primers are shown below. TIFF0007608339000001.tif38128The resulting EMRE3EUKT598198 knockout strain is called STR03778.

[0067] EMRE3EUKT590754 knockout transformants were screened by colony PCR using primers (DNA_oligoST258; SEQ ID NO: 22; and DNA_oligoST259; SEQ ID NO: 23) designed to amplify the entire native target locus. Primers were designed to produce a band of approximately 400 bp in the absence of integration into the locus (e.g., a "knock-in" of the BleR cassette), or a band of approximately 5.1 kb in the case of integration of a single BleR cassette into the target locus, or possibly no band in the case of multiple integrations into the target locus. In addition, two additional colony PCR reactions were performed using one of each of the primers mentioned above flanking the target and an internal BleR primer (SEQ ID NO: 21) designed to amplify from the chromosome to the selection marker. Regardless of the orientation of the integratable cassette, if integration of the target was successful, a band of approximately 800 bp would result from amplification with either the flanking primers and the internal primer DNA_oligoST078. The sequences of the primers are shown below. TIFF0007608339000002.tif25128The resulting EMRE3EUKT590754 knockout strain is called STR03826.

[0068] EMRE3EUKT596408 knockout transformants were screened by colony PCR using primers (DNA_oligoST108; SEQ ID NO: 24, and DNA_oligoST109; SEQ ID NO: 25) designed to amplify the entire native target locus. Primers were designed to generate a band of about 400 bp in the absence of integration into the locus (e.g., a "knock-in" of the BleR cassette), or a band of about 5.1 kb in the case of integration of a single BleR cassette into the target locus, or likely no band in the case of multiple integrations into the target locus. In addition, two additional colony PCR reactions were performed using one each of the primers mentioned above flanking the target and an internal BleR primer (DNA_oligoST078; SEQ ID NO: DNA_oligoST078) designed to amplify from the chromosome to the selectable marker. Regardless of the orientation of the integratable cassette, if targeted integration is successful, an approximately 800 bp band will result from amplification with either the flanking primers and the internal primer DNA_oligoST078. TIFF0007608339000003.tif25128The resulting EMRE3EUKT596408 knockout strain is called STR03749.

[0069] EMRE3EUKT596208 knockout transformants were screened by colony PCR using primers (DNA_oligoST110; SEQ ID NO: 26, and DNA_oligoST111; SEQ ID NO: 27) designed to amplify the entire native target locus. Primers were designed to generate a band of about 400 bp in the absence of integration into the locus (e.g., a "knock-in" of the BleR cassette), or a band of about 5.1 kb in the case of integration of a single BleR cassette into the target locus, or likely no band in the case of multiple integrations into the target locus. In addition, two additional colony PCR reactions were performed using one each of the primers mentioned above flanking the target and an internal BleR primer (DNA_oligoST078; SEQ ID NO: DNA_oligoST078) designed to amplify from the chromosome to the selectable marker. Regardless of the orientation of the integratable cassette, if the targeted integration is successful, a band of approximately 800 bp will result from amplification with either the flanking primers or the internal primer DNA_oligoST078. The sequences of the primers are shown below. TIFF0007608339000004.tif25128The resulting EMRE3EUKT596208 knockout strain is called STR03779.

[0070] SEQ ID NOs for Parachlorella DNA methyltransferase genes, amino acid and DNA sequences, corresponding gRNA sequences used to knock out these genes, and corresponding knockout strains are summarized below.

[0071] (Table 1) Parachlorella DNA methyltransferase and corresponding sequences TIFF0007608339000005.tif67150

[0072] Example 4. Analysis of knockout strains for DNA methyltransferase activity DNA methylation status The cystine methylation status of CpG, CHG, and CHH of Parachlorella native DNA as well as exogenous DNA integrated into the Parachlorella spp. genome (e.g., DNA sequences for blasticidin, Cas-9, and GFP) was assessed for Parachlorella DNA methyltransferase knockout strains STR03749, STR03826, STR03779, and STR03778 and compared to the control Parachlorella strain STR00014, which contains genes for blasticidin, Cas9, and GFP integrated into the genome and an intact DNA methyltransferase.

[0073] Briefly, Parachlorella chromosomal DNA was isolated using standard techniques. The methylation status of the isolated DNA was analyzed using MethylSeq™ (Illumina Inc., San Diego, Calif.).

[0074] Parachlorella knockout strain STR03778 The Parachlorella DNA methyltransferase knockout strain STR03778 showed significantly reduced CHG DNA methylation levels of the exogenous genes blasticidin, Cas-9, and GFP integrated into the Parachlorella sp. genome compared to the control Parachlorella strain STR00014 without such a knockout (Figure 1 ).

[0075] Additionally, the CHH DNA methylation levels of the exogenous genes blasticidin, Cas-9, and GFP integrated into the Parachlorella sp. genome were reduced in the Parachlorella DNA methyltransferase knockout strain STR03778 compared to the control Parachlorella sp. strain STR00014 without such a knockout (Figure 1). However, the CpG DNA methylation levels of the exogenous genes blasticidin, Cas-9, and GFP integrated into the Parachlorella sp. genome were relatively unchanged in the Parachlorella DNA methyltransferase knockout strain STR03778 compared to the control Parachlorella sp. strain STR00014 without such a knockout (Figure 1).

[0076] The levels of CHG, CHH, and CpG DNA methylation of native Parachlorella DNA sequences were also assessed. The Parachlorella DNA methyltransferase knockout strain STR03778 exhibited lower CHG methylation in highly repetitive and centromeric regions of its genome compared to the control Parachlorella strain STR00014 without such a knockout, whereas the levels of CHH and CpG DNA methylation of native Parachlorella DNA sequences remained relatively unchanged (Figures 2 and 3).

[0077] Parachlorella knockout strain STR03749 In the Parachlorella DNA methyltransferase knockout strain STR03749, the CHG, CpG, and CHH DNA methylation levels of the exogenous genes blasticidin, Cas-9, and GFP integrated into the Parachlorella sp. genome were relatively unchanged compared to the control Parachlorella strain STR00014 without such a knockout ( Figure 1 ).

[0078] The levels of CHG, CHH, and CpG DNA methylation of the native Parachlorella DNA sequence were also evaluated. The levels of CHG, CHH, and CpG DNA methylation of the native Parachlorella DNA sequence were relatively unchanged (Figures 2 and 3).

[0079] Parachlorella knockout strains STR03826 and STR03779 The Parachlorella knockout strains STR03826 and STR03779 showed similar methylation patterns to the Parachlorella knockout strain STR03749 (data not shown).

[0080] Thus, the Parachlorella gene EMRE3EUKT598198 is responsible for methylation of exogenous DNA.

[0081] Example 5. Analysis of H3K9 mono- and trimethylation knockout lines Mono- and trimethylation of histone 3 lysine 9 (H3K9) was assessed for control Parachlorella strain STR00014 and DNA methyltransferase knockout strains STR03749 and STR03778 using chromatin immunoprecipitation (ChIP) using active Motif® (Carlsbad, CA, Catalog No. 53040) technology reagents. An antibody specific for trimethylated lysine 9 of histone 3 was purchased from Abcam® (Cambridge, MA) and used for the ChIP assay.

[0082] The Parachlorella DNA methyltransferase knockout strain STR03778 (knockout of the EMRE3EUKT598198 gene encoding SEQ ID NO:1) showed a significant reduction in mono- and trimethylation of H3K9 in the chromosomal portion containing the exogenous genes blasticidin, Cas-9, and GFP integrated into the Parachlorella genome compared to the control Parachlorella strain STR00014 without such a knockout (Figure 1).

[0083] In the Parachlorella DNA methyltransferase knockout strain STR03778 (knockout of the EMRE3EUKT598198 gene), a slight reduction in mono- and trimethylation of H3K9 in native chromosomes was observed (Figures 2 and 3).

[0084] Parachlorella strain STR03749 did not show significant changes in mono- and trimethylation of H3K9 (Figures 1-3). Thus, the Parachlorella gene EMRE3EUKT598198 is indirectly involved in the mono- and trimethylation of histone H3K9 of integrated exogenous DNA.

[0085] Figure 9 also shows that the knockout strains were tested for productivity (as total organic carbon), and no productivity defect was observed compared to the wild-type strain.

[0086] Example 6. Evaluation of protein expression of foreign genes Expression of the foreign gene Cas-9 integrated into the Parachlorella genome was evaluated by Western blot analysis in three knockout strains, STR03778, STR03749, and STR03779, and compared with the control strain, STR00014. Anti-Cas-9 antibody was used for Western blot analysis.

[0087] Both knockout strains STR03778 and STR03749 had higher expression levels of Cas-9 protein compared to the control strain STR00014, regardless of the presence or absence of blasticidin selection pressure (Fig. 4 ).

[0088] Thus, mutating or attenuating the Parachlorella gene EMRE3EUKT598198 increases the expression of exogenous DNA in Parachlorella species.

[0089] Example 7. Identification of orthologous DNA methyltransferases in other algal species The amino acid sequence of the Parachlorella gene EMRE3EUKT598198 (SEQ ID NO: 1) was used to identify orthologous DNA methyltransferases in the algae Ocystis sp. using BLAST analysis. The amino acid and cDNA sequences of the identified DNA methyltransferases are shown as SEQ ID NOs: 28-29.

[0090] CHG DNA methylation of the exogenous DNA (blasticidin gene) integrated into the Ocystis spp. genome was identified in addition to some CpG and CHH DNA methylation (Figures 5-7). Furthermore, the present application identifies CHG methylation in repetitive regions and centromeres of Ocystis spp. DNA (Figures 6-7).

[0091] Example 8. Attenuation of CHG methyltransferase in Ocystis sp. The methyltransferase gene was identified in Oxytis sp. as described above. Deletions of the sequence encoding the methyltransferase SEQ ID NO:28 were generated using RNP / DNA coated bullets using the Helios® Gene Gun System (Bio-Rad, Hercules, Calif., USA). Selection marker DNA and Cas9 ribonucleoprotein (RNP) targeting the gene to be knocked out are deposited on gold particles, which are attached to the inside of a tube through which a burst of helium gas fired by a gene gun launches the coated gold particles into the cells of Oxytis sp. that are attached to solid non-selective medium. The next day, the cells were transferred to selective medium for the growth of transformed colonies.

[0092] Cas9 RNPs were prepared using the Alt-R CRISPR-Cas9 system (Integrated DNA Technologies, Inc., Coralville, Iowa, USA). The crRNA XT targeting the gene of interest was annealed to the tracrRNA, and the resulting guide RNA duplex was complexed with Cas9 V3 to form the Cas9 RNP. Selectable marker DNA was prepared from E. coli and restriction digested to separate the backbone. Either NAS16305 (vector encoding blasticidin resistance) or NAS15142 (vector encoding nourseothricin resistance) was used. Both markers were codon-optimized for Ocystis spp., contained an endogenous intron from Ocystis spp., operably linked to an endogenous Ocystis spp. promoter, and terminated by an endogenous Ocystis spp. terminator.

[0093] 0.6um gold particles were resuspended in protamine sulfate salt solution and sonicated. DNA markers were mixed with Cas9 RNP (62pmol Cas9 V3 and 500pmol guide RNA duplex) in PBS, and the DNA-RNP mixture was added to the protamine-gold solution and precipitated on ice for 2 hours.

[0094] For each sample, a 7'' length of Tefzel™ (ethylene tetrafluoroethylene) (EI du Pont de Nemours, Wilmington, Del.) tubing was inserted into flexible tubing attached to a manifold dryer (e.g., as described in published U.S. patent application 2017 / 0130238). The flexible tubing was removed from the manifold dryer at the Leur lock and attached to a 10 mL syringe. The DNA-RNP / gold suspension was mixed thoroughly and drawn into the Tefzel™ tube by applying suction with the syringe. While still connected to the syringe, the Tefzel™ tube was placed on a flat surface for 2 minutes, during which time the gold was allowed to settle out of solution and adhere to the inside of the tube. The syringe was then used to apply pressure to gently push the PBS solution out of the tube. The tube was quickly inverted to allow the remaining gold slurry to be applied to the side of the Tefzel™ tube opposite where the gold was originally precipitated. The Tefzel™ tubing was then removed from the syringe and placed back onto the manifold dryer under a nitrogen flow of 0.5-0.6 LPM. Once the gold was completely dry, as evidenced by a visible color change from dark to pale yellow, the Tefzel™ tubing was removed from the flexible tubing and cut into half-inch pieces for use in the Helios Gene Gun™.

[0095] Transformation To prepare cells for transformation, a 100 ml seed culture inoculated to 0.05 OD730 6 days prior to transformation was used to inoculate a 500 ml culture to 0.2 OD730 1 day prior to transformation. Cultures were grown in commercial algal growth medium with half-strength salts in a plant growth chamber with shaking at 130 rpm, 25°C, 1% CO2, 16:8 light:dark cycle.

[0096] On the day of transformation, cell cultures were pelleted by centrifugation at 5000 x g for 20 min. Cells were resuspended in 50 mL osmolyte (250 mM mannitol / 250 mM sorbitol, 0.1 um filter sterilized) and incubated at room temperature for 1-2 h. After osmotic pretreatment, cells were concentrated in osmolyte to 20.0 OD730 / mL and 200 uL of cell suspension was spread onto each of five 4 cm diameter circles on a 13 cm diameter shooting plate containing 2% agar PM147 solid medium. When the cells were completely dry, a gene gun was used to fire two bullets per cell circle at 400 psi from a distance of 3-6 cm from the plate. A total of 10 replicate bullets were fired for each sample, aimed at the 20.0 OD730 cells split among the five cell circles. Cells were left on the shooting plate overnight in a dark 30°C incubator.

[0097] The day after transformation, cells from the replicate cell circles were pooled together by washing the shooting plates with liquid commercial algal growth medium. The harvested cells were plated at the intended density of 10.0 OD730 per 13 cm diameter plate onto growth medium containing either 425 mg / L blasticidin or 80 mg / L nourseothricin sulfate.

[0098] Transformation of MTase knockout and parental strains with DNA-coated bullets The Hypericus sp. methyltransferase (MT) knockout strain and parental strain were transformed with DNA using the Helios® Gene Gun System (Bio-Rad, Hercules, Calif., USA). DNA was deposited on gold particles, which were attached to the inside of a tube through which a burst of helium gas fired by a gene gun launched the DNA-coated gold particles into the Hypericus sp. cells that were attached on solid non-selective medium. The next day, the cells were transferred onto selective medium for growth of transformed colonies.

[0099] Five DNA vectors (CRE1-5) were tested that encoded the same BSD selection marker, but with different versions of the CRE recombinase (Figure 8). The different versions of CRE shared the same CDS sequence codons optimized for Ocystis spp., but each contained a different endogenous intron than Ocystis spp. All CRE versions were operably linked to the same endogenous Ocystis spp. promoter and terminated by the same endogenous Ocystis spp. terminator. Vector DNA was prepared from E. coli and restriction digested to separate the backbone prior to transformation. The data show partial or complete floxing in the knockout strains. CHG methylation was extensive for the parental strain and absent in the knockouts, as shown in Figures 5-7.

[0100] DNA (2-10 μg) was deposited onto the gold particles and resuspended in 100% ethanol solution. The volume was calculated to make 10 bullets; no PVP was used, but protamine sulfate solution was used. While the DNA / gold suspension was being prepared, for each sample, one 7'' length of Tefzel™ (ethylene tetrafluoroethylene) tubing was pre-dried by inserting it into flexible tubing attached to a manifold dryer (as described in U.S. Patent Application No. 2017 / 0130238, published May 11, 2017) and left for at least 15 minutes with a flow of 0.5-0.6 LPM nitrogen passed through to remove any environmental humidity buildup from inside the Tefzel™ tubing.

[0101] After the DNA / gold suspension was prepared and the Tefzel™ tube was pre-dried, the flexible tubing was removed from the manifold dryer at the Leur lock and attached to a 10 mL syringe. The DNA / gold suspension was mixed thoroughly and drawn into the Tefzel™ tube by application of suction with the syringe. While still connected to the syringe, the Tefzel™ tube was placed on a flat surface for 5 minutes, during which time the gold was allowed to settle out of solution and adhere to the inside of the tube. After the 5 minute settling period, pressure was applied with the syringe to gently push the ethanol out of the tube. The tube was quickly inverted to allow the remaining gold slurry to be applied to the side of the Tefzel™ tube opposite where the gold was initially precipitated. After a 2-5 minute air drying period, the Tefzel™ tube was removed from the syringe and placed back onto the manifold dryer under a nitrogen flow of 0.5-0.6 LPM. Once the gold was completely dry, as evidenced by a visible color change from dark to pale yellow, the Tefzel™ tube was removed from the flexible tubing and cut to half inch pieces for use in the Helios® Gene Gun.

[0102] To prepare cells for transformation, a 100 ml seed culture inoculated to 0.05 OD730 6 days prior to transformation was used to inoculate a 500 ml culture to 0.2 OD730 1 day prior to transformation. Cultures were grown in commercial algal growth medium in a Conviron™ incubator with shaking at 130 rpm, 16:8 light:dark cycle, 25° C., 1% CO2.

[0103] On the day of transformation, cell cultures were pelleted by centrifugation at 5000 x g for 20 minutes. Cells were resuspended in 50 mL osmolyte (250 mM mannitol / 250 mM sorbitol 0.1 um filter sterilized) and incubated at room temperature for 1-2 hours.

[0104] After osmotic pretreatment, cells were concentrated in osmolytes to 20.0 OD730 / mL and 200uL of cell suspension was spread onto each of five 4cm diameter circles on a 13cm diameter shooting plate containing 2% agar PM147 solid medium. When the cells were completely dried, a Helios® gene gun was used to fire two bullets per cell circle at 400psi from a distance of 3-6cm from the plate. A total of 10 replicate bullets were fired for each sample, aimed at the 20.0 OD730 cells split among the five cell circles. Cells were left on the shooting plate overnight in a dark 30C incubator.

[0105] The day after transformation, cells from the replicate cell circles were pooled together by washing the shot plates with liquid standard algal growth medium. The harvested cells were plated onto selection medium (standard algal growth medium containing 425 mg / L blasticidin) at the intended density of 10.0 OD730 per 13 cm diameter plate.

[0106] Strains of Ocystis sp. carrying a deletion of the methyltransferase-encoding sequence of SEQ ID NO:28 were identified as STR28031 and STR29997. Note that these strains differ only in that '031 contains BSD as a selection marker and '997 contains nourseothricin as a selection marker. The deletion strains showed a significant reduction in CHG and CHH methylation as shown in Figure 5, the background control strain was STR24194 (but had the corresponding selection marker for comparison of methylation in the transgene).

[0107] Productivity analysis (Figure 10) of Ocystis sp. (STR29997) was performed and showed no defect in the productivity of the cells as measured as TOC compared to the background strain (STR24194).

[0108] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the scope of the following claims.

Claims

1. A mutant photosynthetic organism comprising exogenous DNA and a mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity, The polypeptide having CHG DNA methyltransferase activity has at least 90% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 28, and The mutant photosynthetic organism, wherein the mutant photosynthetic organism has reduced CHG DNA methylation compared to a control photosynthetic organism that does not contain a mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity.

2. The mutant photosynthetic organism of claim 1 , wherein the mutant is a genetically engineered mutant.

3. 3. The mutant photosynthetic organism of claim 2, wherein the mutant is genetically engineered by insertional mutagenesis, gene replacement, RNAi, antisense RNA, meganuclease genome engineering, one or more ribozymes, and / or a CRISPR / Cas system.

4. 2. The mutant photosynthetic organism of claim 1, wherein the mutant is generated by UV irradiation, gamma irradiation, or chemical mutagenesis.

5. The mutant photosynthetic organism of claim 1, wherein the reduced CHG DNA methylation is in the exogenous DNA.

6. The mutant photosynthetic organism of claim 5 , wherein the exogenous DNA is integrated into the genome of the photosynthetic organism.

7. The mutant photosynthetic organism of any one of claims 1 to 4, wherein said reduced CHG DNA methylation is present in a DNA sequence native to said photosynthetic organism.

8. 8. The mutant photosynthetic organism of claim 7, wherein the reduced CHG DNA methylation is in a centromere or highly repetitive DNA region of the mutant photosynthetic organism.

9. The mutant photosynthetic organism of any one of claims 5 to 6, wherein expression of the exogenous DNA is increased compared to a control photosynthetic organism comprising the exogenous DNA but not having a mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity.

10. 7. The mutant photosynthetic organism of any one of claims 1 to 6, having reduced mono- or trimethylation of lysine 9 of histone H3 (H3K9).

11. The method of claim 10, wherein the reduction in mono- or trimethylation of lysine 9 on histone H3 (H3K9) in the photosynthetic organism occurs in a portion of a chromosome that contains the exogenous DNA.

12. The mutant photosynthetic organism of any one of claims 1 to 6, wherein the photosynthetic organism is an algae.

13. The mutant photosynthetic organism of claim 12, wherein the algae is derived from the genus Parachlorella or Oocystis.

14. 1. A method for enhancing expression of exogenous DNA in a photosynthetic organism, comprising: a) introducing exogenous DNA into said photosynthetic organism; b) mutating or attenuating a gene encoding a polypeptide having CHG DNA methyltransferase activity having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 28, The mutant organism has reduced CHG DNA methylation of exogenous DNA compared to a control photosynthetic organism. wherein expression of the exogenous DNA is enhanced in the photosynthetic organism compared to the control photosynthetic organism.

15. The method according to claim 14, wherein the gene encoding a polypeptide having CHG DNA methyltransferase activity is mutated or attenuated by genetic manipulation.

16. 16. The method of claim 15, wherein the genetic manipulation is by insertional mutagenesis, gene replacement, RNAi, antisense RNA, meganuclease genome manipulation, one or more ribozymes, and / or a CRISPR / Cas system.

17. The method of claim 16, wherein the genetic manipulation is by the CRISPR / Cas system.

18. The method of claim 14, wherein the gene encoding a polypeptide having CHG DNA methyltransferase activity is mutated or attenuated by UV irradiation, gamma irradiation, or chemical mutagenesis.

19. 19. The method of any one of claims 14 to 18, wherein the mutant photosynthetic organism has reduced CHG DNA methylation compared to a control photosynthetic organism that does not have a mutated or attenuated gene encoding a polypeptide having CHG DNA methyltransferase activity.

20. The method of any one of claims 14 to 18, wherein the exogenous DNA is integrated into the genome of the photosynthetic organism.

21. The method according to any one of claims 14 to 18, wherein the photosynthetic organism is an algae.

22. The method of claim 21, wherein the algae is derived from the genus Parachlorella or Oocystis.

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