Inducible expression of algal genes

Abstract

The present application provides novel algae regulatory elements, including nitrate / nitrite inducible promoter sequences and terminator sequences. The present application also discloses DNA constructs comprising these novel regulatory elements, and recombinant microorganisms comprising these regulatory elements. Methods for modifying, preparing and using regulatory elements are also disclosed. The methods disclosed in the present application are applicable to the inducible expression of genes such as transgenic or natural genes in algae species.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority under 35 USC §119(e) to U.S. application serial number 62 / 782,152, filed on December 19, 2018, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Incorporation of Sequence Listing

[0004] The materials in the attached sequence listing are hereby incorporated by reference into this application. The attached sequence listing text file named SGI2220_1WO_sequencelisting.txt was created on December 18, 2019 and is 107kb in size. The file can be accessed using Microsoft Word on a computer using Windows OS. Technical Field

[0005] The present invention relates generally to the field of genetic engineering of algal cells for selective expression of genes of interest. Background Art

[0006] Algae cells are a promising source of biofuels (Wijffels & Barbosa (2010) Science 329:796-799). Their ability to use solar energy to convert carbon dioxide into carbon-rich lipids exceeds that of oil-producing crops, with the added advantage that algae used for biofuels do not compete with oil-producing crops for agricultural land (Wijffels & Barbosa, 2010). To maximize algae fuel production, new algae strains need to be engineered to achieve industrial-scale growth and carbon fixation (Wijffels & Barbosa, 2010).

[0007] Furthermore, the development of modern recombinant strains requires powerful and efficient tools to express transgenes as well as endogenous genes to alter the metabolism and physiology of the cell in a desired manner. An essential component of any genetic engineering "toolkit" is a set of functional promoters and terminators that drive the expression of transgenes or endogenous genes. Endogenous promoters cloned and validated from strains being developed for recombinant DNA technology are needed, as well as other strategies to increase transformation of microorganisms (such as algae) and improve expression of heterologous genes. Summary of the invention

[0008] Provided herein are novel algae-inducible promoters and terminator sequences for inducing expression of native and heterologous DNA sequences in algae cells. Also provided are DNA constructs and expression cassettes comprising the novel algae-inducible promoters and / or terminator sequences. Also provided are algae mutants comprising DNA constructs comprising the novel algae-inducible promoters and / or terminator sequences, and methods for selectively expressing target DNA in algae cells.

[0009] In one aspect, the disclosure provides algae-inducible nitrate reductase and nitrite reductase promoter sequences comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity (but optionally in any embodiment less than 100% sequence identity) to at least 100, at least 200, at least 300, at least 400, or at least 500 contiguous nucleotides, or the entire sequence, of a sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, and 51. For example, the promoter may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% (but optionally less than 100%) identity to at least 100, at least 200, at least 300, at least 400 or at least 500 consecutive nucleotides extending from the 3' end in the 5' direction (or alternatively from the 5' end in the 3' direction) of a sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51. In another example, the promoter may comprise a sequence having at least 90% but less than 100% identity to any of the named sequences or at least 500 consecutive nucleotides extending from the 3' end in the 5' direction. In some embodiments, the nitrate reductase and nitrite reductase promoters are located in the intergenic region between the nitrate reductase and nitrite reductase genes. In some embodiments, the nitrate and nitrite reductase promoters are located in the 5'-UTR region of the nitrate and nitrite reductase genes, respectively.

[0010] In one aspect, the disclosure provides algal nitrate reductase and nitrite reductase terminator sequences comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity (but optionally less than 100% in any embodiment) to at least 25, at least 50, at least 75, at least 100, or at least 150 consecutive nucleotides or the full-length sequence of a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52. In some embodiments, the nitrate and nitrite reductase terminators are located in the 3'-UTR region of the nitrate and nitrite reductase genes, respectively.

[0011] In one aspect, the present disclosure provides an isolated DNA molecule comprising an algal nitrate reductase or nitrite reductase inducible promoter operably linked to a target DNA encoding a polypeptide or a functional RNA, wherein the target DNA encoding the polypeptide or the functional RNA is not regulated by a promoter in nature or is not operably linked to a promoter. The algal nitrate reductase or nitrite reductase inducible promoter has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity (but optionally in any embodiment less than 100% sequence identity) to at least 100, at least 200, at least 300, at least 400 or at least 500 consecutive nucleotides (or the full length sequence) of a sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51. For example, the isolated DNA molecule may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% (but optionally less than 100%) sequence identity to at least 100, at least 200, at least 300, at least 400 or at least 500 consecutive nucleotides extending from the 3' end in the 5' direction (or alternatively from the 5' end in the 3' direction) of a sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51. The algae nitrate reductase or nitrite reductase promoter of the present application can be operably linked to any DNA of interest that is heterologous or homologous to an algae species. For DNA targets that are homologous to algae, these promoters are not juxtaposed to these DNA targets in nature and do not regulate the expression of these DNA targets in nature.

[0012] In some embodiments, the isolated DNA molecule comprises an algal nitrate reductase or nitrite reductase terminator sequence operably linked to a DNA of interest encoding a polypeptide or functional RNA, the terminator sequence comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (and optionally in any embodiment less than 100%) to at least 25, at least 50, at least 75, at least 100, or at least 150 consecutive nucleotides (or the full-length sequence) of a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52.

[0013] On the one hand, genetically engineered algae are provided herein, comprising a DNA molecule or sequence operably connected to a target DNA, the DNA molecule or sequence comprising an algae nitrate reductase or nitrite reductase inducible promoter, wherein the target DNA is not regulated by a promoter in nature. In some embodiments, the DNA molecule is integrated into the algae genome. The target DNA can be heterologous or homologous to an algae species. For a target DNA homologous to algae, the promoter is not juxtaposed with the target DNA in nature, and does not regulate the expression of the target DNA in nature.

[0014] In one aspect, an expression cassette comprising a DNA molecule is provided herein, the DNA molecule comprising an algae nitrate reductase or nitrite reductase inducible promoter operably linked to a target DNA encoding a polypeptide or functional RNA, wherein the target DNA encoding the polypeptide or functional RNA is not regulated by a promoter in nature, wherein the target DNA encodes (a) a protein associated with lipid biosynthesis, (b) a lipase, (c) a protein involved in photosynthesis, (d) a protein associated with carbon fixation, (e) a transporter, (f) a dehydrogenase, (g) a transcription factor, (h) a transcription activator, (i) a cell signaling protein, (j) a metabolic enzyme, (k) a reporter protein, (l) a selection marker, (m) a recombinase, n) an antisense sequence, (o) a short hairpin RNA (shRNA), (p) a small interfering RNA (siRNA), (q) a gRNA (gRNA) or (r) a ribozyme. In some embodiments, the expression cassette further comprises an algae nitrate reductase or nitrite reductase terminator sequence operably linked to the target DNA encoding the polypeptide or functional RNA. The target DNA can be heterologous or homologous to the algae species. For target DNAs that are homologous to algae, these promoters are not juxtaposed with these target DNAs in nature and do not regulate the expression of these target DNAs in nature.

[0015] In one aspect, a method for selectively expressing a target DNA in an algae cell is provided herein, the method comprising: a) transforming an algae cell with an isolated DNA molecule comprising an algae nitrate reductase or nitrite reductase inducible promoter operably linked to a target DNA encoding the target DNA, wherein the target DNA encoding the target DNA is not regulated by the promoter in nature, thereby producing a transformed algae cell, or any DNA molecule or sequence described herein; and b) growing the transformed algae cell in a culture medium that selectively allows the expression of the target DNA in the algae cell. In some embodiments, the isolated DNA molecule is introduced by particle bombardment. In some embodiments, the isolated DNA molecule is introduced by electroporation. In some embodiments, the promoter sequence is nitrite reductase, and wherein the algae cell is grown in a culture medium comprising nitrates, wherein the expression of the target DNA is induced. In some embodiments, the promoter sequence is nitrite reductase, and wherein the algae cell is grown in a culture medium comprising ammonium salts, wherein the expression of the target DNA is suppressed.

[0016] In some embodiments of the above aspects, the algae nitrate reductase or nitrite reductase terminator is from the same species as the promoter. In some embodiments of the above aspects, the target DNA encoding the polypeptide or functional RNA is heterologous to the promoter sequence. In some embodiments of the above aspects, the target DNA encoding the polypeptide or functional RNA and the promoter are from the same algae species, wherein the encoding target DNA and the promoter are not juxtaposed in nature.

[0017] In some embodiments of the above aspects, the target DNA encoding the polypeptide or functional RNA is genetically engineered to include at least one, at least two, at least three, at least four, at least five introns, wherein the introns are heterologous to the target DNA encoding the polypeptide or functional RNA. In some embodiments of the above aspects, the introns and the promoter are from the same algae species. In some embodiments of the above aspects, two or more heterologous introns, for example, at least two, at least three, at least four, at least five introns can be from the same gene. In some embodiments of the above aspects, one or more introns and the promoter can be from the same gene.

[0018] In some embodiments of the above aspects, the target DNA encodes a functional RNA selected from the group consisting of antisense sequences, microRNA (miRNA), shRNA, siRNA, gRNA and ribozyme.

[0019] In some embodiments of the above aspects, the promoter and terminator are from the same gene. In some embodiments of the above aspects, the promoter and terminator are from different genes.

[0020] In some embodiments of the above aspects, the target DNA encodes (a) a protein associated with lipid biosynthesis, (b) a lipase, (c) a protein involved in photosynthesis, (d) a protein associated with carbon fixation, (e) a transporter, (f) a dehydrogenase, (g) a transcription factor, (h) a transcription activator, (i) a cell signaling protein, (j) an enzyme, (k) a reporter protein, (l) a selection marker, or (m) a recombinase. In some embodiments of the above aspects, the target DNA encodes a Cre recombinase.

[0021] In some embodiments, the mutant algae belongs to a genus selected from any one or more of the group consisting of: Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria ), Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ern odesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Gloeothamnion, Haematococcus, Halocafeteria, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monodus, Monoraphid um), Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella, Pascheria,Pelagomonas, Phaeodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Skeletonema, Spyrogyra, Stichococcus, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, and Volvox. These genera are disclosed herein in every possible combination and subcombination, as if fully set forth herein.

[0022] In some embodiments of the above aspects, the algae inducible nitrate reductase or nitrite reductase promoter sequence is operably linked to the target DNA. In some embodiments, in the presence of nitrate ions, the expression of the target DNA operably linked to the nitrate reductase or nitrite reductase promoter sequence is increased. In some embodiments, in the presence of ammonium ions, the expression of the target DNA operably linked to the nitrate reductase or nitrite reductase promoter sequence is inhibited.

[0023] In some embodiments of the above aspects, the algal nitrate reductase and nitrite reductase terminator sequences comprise a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to at least 25, at least 50, at least 75, at least 100, or at least 150 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52. In some embodiments of the above aspects, the algal nitrate reductase or nitrite reductase terminator sequence is operably linked to the DNA of interest.

[0024] In one aspect, the disclosure provides a vector comprising an expression cassette as disclosed herein and one or both of an autonomously replicating sequence and a selectable marker gene. In some embodiments, the vector comprises at least one origin of replication. In some embodiments, the vector further comprises an additional promoter operably connected to a selectable marker or reporter gene, such as, but not limited to, a promoter disclosed herein.

[0025] In some embodiments, the vector is used for transformation of eukaryotic cells (such as, but not limited to, eukaryotic microalgae cells or phytoplankton cells), wherein the vector includes a selectable marker gene operably linked to a promoter as provided herein, for example, a promoter comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity (and optionally in any embodiment less than 100%) to at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 consecutive nucleotides (or the full-length sequence) of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51. The transformation vector may further include one or more additional genes or constructs for transfer into the host cell, such as a gene encoding a polypeptide, such as but not limited to any gene disclosed above, or a construct encoding a functional RNA, wherein the gene encoding the polypeptide or the functional RNA may optionally be operably linked to a promoter described herein, or may optionally be operably linked to another promoter.

[0026] In addition, or alternatively, carrier provided herein may include terminator provided herein. For example, carrier of the present invention may include at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700 or at least 800 continuous nucleotides (or full-length sequence) with SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50 and 52, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity (and optionally in any embodiment less than 100%) nucleotide sequence. DNA of interest or selection marker gene on carrier of the present invention may be operably connected to terminator sequence provided herein.

[0027] In some embodiments, the selectable marker gene is selected from the group consisting of: resistance to antibiotics (e.g., tetracycline, doxycycline or its analogs, puromycin, hygromycin, blasticidin, bleomycin, or phleomycin (Zeocin TM ), norstromycin); genes conferring resistance to herbicides; genes encoding acetyl-CoA carboxylase (ACCase); genes encoding acetohydroxyacid synthase (ahas); genes encoding acetolactate synthase; genes encoding aminoglycoside phosphotransferase; genes encoding anthranilate synthase; genes encoding bromoxynil nitrile hydrolase; genes encoding cytochrome P450-NADH-cytochrome P450 oxidoreductase; genes encoding dalapon dehalogenase; genes encoding dihydropteroate synthase; genes encoding class I 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS); genes encoding class II EPSPS (aroA); genes encoding non-class I / II EPSPS; genes encoding glutathione reductase; genes encoding grass Gene encoding glyphosate acetyltransferase; gene encoding glyphosate oxidoreductase; gene encoding hydroxyphenylpyruvate dehydrogenase; gene encoding hydroxyphenylpyruvate dioxygenase; gene encoding isopentenyl pyrophosphate isomerase; gene encoding lycopene cyclase; gene encoding phosphinothricin acetyltransferase; gene encoding phytoene desaturase; gene encoding isopentenyl transferase; gene encoding protoporphyrin oxidase; gene encoding superoxide dismutase arg7, his3, hisD, hisG, manA, nit1, trpB, uidA, xylA; dihydrofolate reductase gene; mannose 6-phosphate isomerase gene; nitrate reductase gene; ornithine decarboxylase gene; thymidine kinase gene; 2-deoxyglucose resistance gene or R locus gene. The detectable marker gene can be, for example, a tyrosinase gene, lacZ, an alkaline phosphatase gene, an α-amylase gene, a horseradish peroxidase gene, an α-galactosidase gene, a luciferin / luciferase gene, a β-glucuronidase gene (GUS), or a gene encoding a fluorescent protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

[0029] Figure 1 . Figure 1qRT-PCR results showing induction and repression of Cre recombinase gene expression operably linked to the Parachlorella nitrite reductase promoter in Parachlorella cells expressing Cre recombinase in selective medium are shown. Recombinant Parachlorella cells grown in repressive medium (RM-NH4+ / NO3-) were repressed (left column), while recombinant Parachlorella cells grown in induction medium (IM-NO3-) were induced (right column).

[0030] Figure 2 . Figure 2 Western Blot results showing induction and inhibition of Cre recombinase gene expression operably linked to the Chlorella nitrite reductase promoter in Chlorella cells expressing Cre recombinase in selective medium. Recombinant Chlorella cells grown in repressive medium (RM-NH4+ / NO3-) were inhibited (middle column), while recombinant Chlorella cells grown in induction medium (IM-NO3-) were induced (right column). Results for wild-type Chlorella cells are shown in the left column.

[0031] Figure 3 . Figure 3 The Blast alignment results of nitrite / sulfite reductase genes in Pseudochlorella are shown. The results show that the most Pfam hit fragments are nitrite / sulfite reductase genes.

[0032] Figure 4 . Figure 4 Shown are the coding sequences of the Pseudochlorella nitrate and nitrite reductase genes and a schematic diagram of the intergenic untranslated region between the two genes, which contain the nitrate and nitrite reductase promoter sequences, respectively, in opposite orientations. Figure 4 The nitrite reductase terminator in the 3'-UTR region of the nitrite reductase gene is also shown.

[0033] Figure 5 . Figure 5 The plasmid map of plasmid pSGE06785, which expresses Cre recombinase (containing the native C. vulgaris intron) in the absence of ammonium by using the nitrite reductase promoter / terminator, is shown. The expression of the BleR and GFP genes is driven by a constitutive promoter / terminator. DETAILED DESCRIPTION

[0034] Based on RNA sequencing data, hidden Markov model analysis, BLAST analysis, and Pfam analysis of PfamPF01077 and PF03460, the present application identified novel algal nitrate and nitrite / sulfite reductase promoter and terminator sequences in the 5'- and 3'-untranslated regions (UTRs) of algal nitrate reductase and nitrite reductase genes. In some embodiments, the nitrite reductase and nitrite reductase genes are in opposite directions on the same chromosome of the algae. In some embodiments, the nitrite reductase and nitrite reductase promoters are located in the intergenic region of the two genes ( Figure 4 In some embodiments, the nitrite reductase and nitrite reductase terminators are located in the 3'-UTR region of the nitrite reductase and nitrite reductase genes, respectively ( Figure 4 ).

[0035] The present application discloses several novel algae inducible nitrate reductase or nitrite / sulfite reductase promoter sequences from various algae such as Pseudochlorella, Oocystis, Microchlorophyta and Tetraselmis. Non-limiting examples of such algae nitrate reductase or nitrite / sulfite reductase promoter sequences are listed in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51, as shown below.

[0036] The present application discloses several novel algal nitrate reductase or nitrite reductase terminator sequences from various algae such as Pseudochlorella, Oocystis, Microchlorophyta and Tetraselmis. Non-limiting examples of such algal nitrate reductase or nitrite / sulfite reductase terminator sequences are listed in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52, as shown below.

[0037] The application also discloses a DNA construct, the DNA construct comprises a novel algae inducible nitrate reductase or nitrite reductase promoter sequence as described herein, the promoter sequence is operably connected to a target DNA encoding a polypeptide or a functional RNA, wherein the polypeptide or functional RNA encoded by the target DNA is not regulated by a promoter in nature (e.g., in a wild-type organism) or cannot be operably connected. In some embodiments, the promoter can be a heterologous promoter. In some embodiments, the DNA construct also comprises an algae nitrate reductase or nitrite reductase terminator sequence operably connected to the target DNA. The application also discloses an expression vector comprising the DNA construct. Whether a regulatory sequence regulates a nucleic acid sequence in nature can be determined by whether the regulatory sequence regulates a nucleic acid sequence in a wild-type organism.

[0038] The present application also discloses a method for selectively expressing a target DNA in algae using a novel algae inducible nitrate reductase or nitrite reductase promoter sequence operably linked to the target DNA. Genetically engineered algae comprising a novel algae inducible nitrate reductase or nitrite reductase promoter operably linked to the target DNA are grown in a selective medium (e.g., a medium containing nitrates) to induce or express the genetically engineered algae of the target DNA, or the genetically engineered algae can be grown in a medium containing ammonium ions to inhibit the expression of the target DNA.

[0039] Listed below are exemplary novel algal nitrate reductase or nitrite reductase promoter and terminator sequences from various algal species.

[0040] Unless otherwise defined, all technical terms, symbols and other scientific terms or proper nouns used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, for the sake of clarity and / or for timely reference, terms with commonly understood meanings are defined herein, and the inclusion of these definitions herein should not necessarily be interpreted as representing substantial differences relative to the meanings generally understood in the art. Many of the techniques and procedures described or mentioned herein are well understood by those skilled in the art and are typically utilized using conventional methods.

[0041] Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. For example, the term "a cell" includes one or more cells, including mixtures thereof. "A and / or B" is used herein to include all of the following alternatives: "A," "B," and "A and B."

[0042] As used herein, when referring to any numerical value, the term "about" or "approximately" means within the provided value plus or minus 10%. For example, "about 50 degrees Celsius" (or "approximately 50 degrees Celsius") covers a temperature range from 45 degrees Celsius to 55 degrees Celsius (including end values). Similarly, "about 100mM" (or "about 100mM") covers a concentration range from 90mM to 110mM (including end values). Alternatively, "about" or "approximately" can be expressed as within 5% of the value, or within 2.5% of the value in some cases, or, "about" can represent rounding to the nearest significant figure. All ranges provided in the present application include values ​​at the upper and lower ends of the range.

[0043] As used herein, the terms "cell," "cell culture," "cell line," "recombinant host cell," "recipient cell," and "host cell" include the primary subject cell and any progeny thereof, without regard to the number of transfers. It should be understood that not all progeny will be identical to the parent cell (due to intentional or unintentional mutations or differences in the environment). However, as long as the progeny retain the same functions as the originally transformed cell, these terms include such altered progeny.

[0044] As used herein, the term "construct" is intended to mean any recombinant nucleic acid molecule, such as an expression cassette, a plasmid, a cosmid, a virus, an autonomously replicating polynucleotide molecule, a bacteriophage, or a linear or circular, single-stranded or double-stranded, DNA or RNA polynucleotide molecule capable of genomic integration or autonomous replication derived from any source, which comprises a nucleic acid molecule in which one or more nucleic acid sequences have been linked in a functionally operative manner (i.e., operably linked).

[0045] As used herein, "control organism," "control microorganism," or "control cell" refers to an organism, microorganism, or cell that is substantially identical to a test organism, microorganism, or cell (except for the engineered genetic manipulation or introduced mutation disclosed for the test organism, microorganism, or cell) and that provides a reference point for measuring changes in the test organism or cell phenotype. Thus, "substantially identical" includes, for example, small random variations in genomic sequence ("SNPs") that are unrelated to the target genotype, phenotype, parameter, or gene expression level in the test microorganism. Depending on the specific purpose of its use, a control organism or cell may include, for example, (a) a progenitor cell strain or species, cell or microorganism population or organism relative to the test organism, microorganism or cell, wherein the progenitor cell lacks the genetic engineering construct or change introduced into the progenitor cell strain, species, organism or cell or microorganism population to produce the test organism, microorganism or cell; (b) a wild-type organism or cell, i.e., the same genotype as the starting material that caused the genetic change in the test organism or cell; (c) an organism or cell with the same genotype as the starting material but transformed with an ineffective construct (i.e., a construct that has no known effect on the trait of interest, such as a construct containing a reporter gene); (d) an organism or cell that is an untransformed segregant in the progeny of the test organism, microorganism or cell; or (e) the target organism or cell itself, in the case of not expressing the gene of interest. In some cases, a "control organism" may refer to an organism that does not contain the exogenous nucleic acid present in the transgenic organism of interest, but has the same or very similar genetic background as such transgenic organism.

[0046] As used herein, "genetically engineered" algae refers to non-naturally occurring recombinant algae cells with altered nucleotide composition of the genome or altered gene expression, including overexpression or inhibition of gene expression under different temporal, biological or environmental regulation and / or to a degree different from that occurring naturally, and / or expression of genes that are not naturally expressed in recombinant cells. The altered nucleotide composition (alteration, deletion and / or insertion of one or more nucleotides) can be in the coding region of the gene, or can be in an intron, 3'UTR, 5'UTR or promoter region, for example within 2 kb of the transcription start site or within 3 kb of the translation start site. For example, genetically engineered algae with altered gene expression as disclosed herein can have altered nucleotide composition, which can be one or more nucleobase changes and / or one or more nucleobase deletions and / or one or more nucleobase insertions in the region 5' of the transcription start site, such as, in non-limiting examples, within about 2 kb, within 1.5 kb, within 1 kb, or within 0.5 kb of a known or putative transcription start site, or within about 3 kb, within 2.5 kb, within 2 kb, within 1.5 kb, within 1 kb, or within about 0.5 kb of a translation start site. A genetically modified algae cell refers to an algae cell that has been manipulated by introducing a heterologous or exogenous (e.g., non-natural) recombinant nucleic acid sequence into an organism, and includes, but is not limited to, gene knockout, targeted mutation, and gene replacement, promoter replacement, deletion or insertion, or the transfer of a nucleic acid molecule such as a transgene, synthetic gene, promoter or other sequence into an organism. A genetically modified algae cell also includes the offspring of a genetically modified parent cell.

[0047] The term "expression cassette" as used herein refers to a nucleic acid construct encoding a protein or functional RNA operably linked to an expression regulatory element (such as a promoter), and optionally, a combination of any or other nucleic acid sequences that affect gene transcription or translation, such as but not limited to transcription terminators, ribosome binding sites, splice sites or splice recognition sequences, introns, enhancers, polyadenylation signals, internal ribosome entry sites, etc.

[0048] A "functional RNA molecule" is an RNA molecule that can interact with one or more proteins or nucleic acid molecules to perform or participate in a structural, catalytic or regulatory function that affects the expression or activity of a gene or gene product other than the gene that produces the functional RNA. The functional RNA can be, for example, a transfer RNA (tRNA), a ribosomal RNA (rRNA), an antisense RNA (asRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a guide RNA (gRNA), a CRISPR RNA (crRNA) or a trans-activating RNA (tracrRNA) of a CRISPR system, a small nucleolar RNA (snoRNA), a piwi-interacting RNA (piRNA) or a ribozyme.

[0049] The term "target DNA" is widely used to refer to any fragment of a DNA molecule encoding a polypeptide or an expressed RNA. Therefore, the target DNA includes a sequence encoding an expressed RNA, which may include a polypeptide coding sequence or, for example, a functional RNA. The target DNA may further include a regulatory sequence that requires or affects its expression, and a sequence related to a protein or RNA coding sequence in its native state, such as, for example, an intron sequence, a 5' or 3' non-translated sequence, etc. In some examples, "target DNA" may refer only to a protein coding portion of a DNA or RNA molecule, which may or may not include introns. The target DNA may optionally include heterologous introns, i.e., introns that are not natural to the gene from which the protein or functional RNA coding sequence is derived. The length of the target gene is preferably greater than 50 nucleotides, more preferably greater than 100 nucleotides, and for example, the length can be 50 nucleotides to 500,000 nucleotides, such as 100 nucleotides to 100,000 nucleotides or about 200 nucleotides to about 50,000 nucleotides, or about 200 nucleotides to about 20,000 nucleotides. The target DNA can be obtained from a variety of sources, including cloning from a target source or synthesizing from known or predicted sequence information.

[0050] Non-limiting examples of proteins encoded by the DNA of interest include protein lipases associated with lipid biosynthesis, proteins involved in photosynthesis, proteins associated with carbon fixation, transporters, dehydrogenases, transcription factors, transcription activators, cell signaling proteins, enzymes, reporter proteins, selection markers, and recombinases.

[0051] Non-limiting examples of proteins associated with lipid biosynthesis, proteins associated with carbon fixation and / or photosynthesis include those described in U.S. Application Publication No. 20140220638, U.S. Application Publication No. 20160304896, U.S. Application Publication No. 2017005830303, and U.S. Application Publication No. 20180186842. Each of these patent application publications is incorporated herein by reference in its entirety.

[0052] Non-limiting examples of enzymes include recombinases such as Cre (NCBI Protein Database Accession Nos.: YP_006472.1, WP_063075144, WP_052200029.1), CRISPR Cas9 (NCBI Protein Database Accession No. WP_117329810).

[0053] An exemplary nucleic acid sequence of a Cre recombinase comprising an N-terminal nuclear localization signal and six introns of Pseudomonas nitrite reductase is shown below.

[0054] Non-limiting examples of reporter proteins include (NCBI Protein Database Accession Number: YP_002302326.1). An exemplary sequence of Cre recombinase is shown below.

[0055] As used herein, the terms "protein" or "polypeptide" are intended to encompass the singular "polypeptide" as well as the plural "polypeptides", and refer to molecules composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, and does not refer to a specific length of the product. Thus, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to one or more chains of two or more amino acids, and the term "polypeptide" may be used instead of or interchangeably with any of these terms.

[0056] A DNA molecule can be "derived from" a specified source, which includes isolating a nucleic acid fragment (in whole or in part) from a specified source. A DNA molecule can also be derived from a specified source, for example, by direct cloning, PCR amplification, or artificial synthesis from a specified polynucleotide source, or based on a sequence associated with a specified polynucleotide source. A DNA molecule can be a part of an algae genome, or it can be an exogenous DNA sequence. A DNA molecule can be exogenous DNA integrated into an algae genome. A DNA molecule can include one or more genes, 5'- and 3'-untranslated regions (UTRs). In some embodiments, a 5'- or 3'-UTR can include one or more regulatory elements.

[0057] A DNA molecule or a target DNA derived from a particular source or species also includes a gene or nucleic acid molecule with a sequence modification relative to a source nucleic acid molecule. For example, a DNA molecule or a target DNA derived from a source (e.g., a specific reference gene) may include one or more mutations relative to a source gene or nucleic acid molecule, the mutations being unintentional or intentionally introduced, and if one or more mutations are intentionally introduced, including substitutions, deletions, or insertions, the sequence changes may be introduced by random or targeted mutations of cells or nucleic acids, by amplification or other molecular biology techniques, or by chemical synthesis, or any combination thereof.

[0058] As used herein, "isolated" nucleic acid or protein is removed from its natural environment or the naturally occurring background of nucleic acid or protein. For example, an isolated protein or nucleic acid molecule is removed from a cell or organism associated with it in its natural or natural environment. In some cases, an isolated nucleic acid or protein can be partially or substantially purified, but separation does not require a specific level of purification. Therefore, for example, an isolated nucleic acid molecule can be a nucleotide sequence excised from a chromosome, genome or episome of its natural integration.

[0059] A "purified" nucleic acid molecule or nucleotide sequence or protein or polypeptide sequence is substantially free of cellular material and cellular components. For example, a purified nucleic acid molecule or protein may be free of chemicals other than a buffer or solvent. "Substantially free" is not intended to mean that other components other than the new nucleic acid molecule cannot be detected.

[0060] The terms "naturally occurring" and "wild-type" refer to forms found in nature. For example, a naturally occurring or wild-type nucleic acid molecule, nucleotide sequence, or protein can be present in or isolated from a natural source and has not been intentionally modified by human manipulation.

[0061] As used herein, "expression" includes gene expression at least at the level of RNA production, and "expression product" includes the resulting product, such as a polypeptide or functional RNA (e.g., ribosomal RNA, tRNA, antisense RNA, microRNA, shRNA, ribozyme, etc.) of the expressed gene. The term "increased expression" includes changes in gene expression to promote an increase in mRNA production and / or an increase in polypeptide expression. When referring to protein abundance or active protein abundance produced by gene expression, protein turnover rate, protein activation state, etc., "increased production" includes an increase in polypeptide expression, a level of enzymatic activity of a polypeptide, or a combination of the two compared to the natural production or enzymatic activity of the polypeptide.

[0062] As used herein, the term "induce expression of a target DNA" refers to a selective increase in expression of a target DNA under given conditions compared to expression of the target DNA in the absence of such conditions. For example, when algae comprising a target DNA regulated by an algal nitrite reductase promoter are grown in a medium comprising nitrate ions, expression of the target DNA is increased compared to the expression level of the target DNA when grown in a medium without nitrate ions.

[0063] As used herein, the term "inhibiting expression of a target DNA" refers to a selective reduction in expression of a target DNA under given conditions compared to expression of the target DNA in the absence of such conditions. For example, when algae comprising a target DNA regulated by an algal nitrite reductase promoter are grown in a medium comprising ammonium ions, expression of the target DNA is reduced compared to the expression level of the target DNA when grown in a medium without ammonium ions.

[0064] Further, the term "exogenous" as used herein in the context of a gene or protein refers to a gene or protein that is not derived from the host organism species.

[0065] As used herein, the term "transgene" refers to an exogenous gene, ie, a gene introduced into a microorganism or progenitor cell by human intervention.

[0066] As used herein, the term "ortholog" of a gene or protein refers to its functional equivalent in another species.

[0067] Gene and protein accession numbers (usually provided herein in parentheses followed by gene or species names) are unique identifiers for publicly available sequence records at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov) maintained by the United States National Institutes of Health. "Gene Information Identifier" (GenInfoIdentifier, GI) sequence identification numbers are specific to nucleotide or amino acid sequences. If the sequence is changed 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 sequences that appear in a particular GenBank record. Searching and obtaining nucleic acid or gene sequences or protein sequences based on accession numbers and GI numbers is well known in the fields of, for example, cell biology, biochemistry, molecular biology, and molecular genetics.

[0068] As used herein, the term "percent identity" or "homology" with respect to nucleic acid or polypeptide sequences 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 maximum percent identity and introducing gaps, if necessary, to achieve maximum percent homology. N-terminal or C-terminal insertions or deletions should not be construed as affecting homology, and internal deletions and / or insertions of less than about 30, less than about 20, or less than about 10 amino acid residues into a polypeptide sequence should 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 (Altschul (1997), Nucleic Acids Res. 25, 3389-3402, and Karlin (1990), Proc. Natl. Acad. Sci. USA 87, 2264-2268) using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx, which are designed for sequence similarity searches. The method used by the BLAST program is to first consider similar fragments with and 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 discussion of the basic issues in sequence database similarity searches, see Altschul (1994), Nature Genetics 6, 119-129. Search parameters for histograms, descriptions, comparisons, expected values ​​(i.e., statistically significant thresholds for reporting compatibility for database sequences), truncation, matrices, and filters (low complexity) can be default settings. 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), and the recommended query sequence length exceeds 85 (nucleotide bases or amino acids).

[0069] For blastn, in a design for comparison of nucleotide sequences, the scoring matrix is ​​set by the ratio of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), where the default values ​​for M and N can be +5 and -4, respectively. The four blastn parameters can be adjusted as follows: Q = 10 (gap creation penalty); R = 10 (gap extension penalty); flicker = 1 (generate word hits at each flicker position along each query); and gapw = 16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings for comparison of amino acid sequences can be: Q = 9; R = 2; flicker = 1; and gapw = 32. The best fit comparison between sequences available in the GCG software package version 10.0 can use the DNA parameters GAP = 50 (gap creation penalty) and LEN = 3 (gap extension penalty), and the equivalent settings for protein comparisons can be GAP = 8 and LEN = 2.

[0070] Thus, when referring to a polypeptide or nucleic acid sequence of the invention, sequences having 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%, such as 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 a fragment thereof comprising a contiguous sequence of at least 100, at least 125, at least 150 or more amino acid residues of the entire protein; variants of such sequences, for example, wherein at least one amino acid residue has been inserted into the N- and / or C-terminus, and / or within the disclosed sequences containing insertions and substitutions. Contemplated variants may additionally or alternatively include those containing predetermined mutations achieved by, for example, homologous recombination or site-directed or PCR mutagenesis; as well as corresponding polypeptides or nucleic acids from other species, including, but not limited to, those described herein; alleles or other naturally occurring variants of polypeptide or nucleic acid families containing insertions and substitutions; and / or derivatives in which the polypeptide has been covalently modified by substitution, chemical, enzymatic or other appropriate means at a portion other than the naturally occurring amino acids containing insertions and substitutions (e.g., a detectable moiety, such as an enzyme).

[0071] The term "natural" as used herein refers to a nucleic acid sequence or amino acid sequence that is naturally present in a host. The term "non-natural" as used herein refers to a nucleic acid sequence or amino acid sequence that is not naturally present in a host. A nucleic acid sequence or amino acid sequence that has been removed from a cell and introduced or reintroduced into a host cell through laboratory manipulation is considered to be "non-natural". Synthetic genes or partially synthetic genes introduced into a host cell are "non-natural". Non-natural genes further include genes that are endogenous to a host microorganism that is operably linked to one or more heterologous regulatory sequences that have been recombined into the host genome.

[0072] A "recombinant" or "engineered" nucleic acid molecule is a nucleic acid molecule that has been altered by artificial 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, for example, using chemical or enzymatic techniques (e.g., by utilizing chemical nucleic acid synthesis or by utilizing enzymes for replication, polymerization, digestion (exonucleoside or endonucleoside), ligation, reverse transcription, transcription, base modification (including, for example, methylation), integration, or recombination (including homologous and specific site recombination) of nucleic acid molecules); 2) including those binding nucleotide sequences that do not bind in nature; 3) engineered using molecular cloning techniques to lack one or more nucleotides relative to the sequence of naturally occurring nucleic acid molecules; and / or 4) manipulated using molecular cloning techniques to have one or more sequence changes or rearrangements relative to the sequence of naturally occurring nucleic acid molecules. As a non-limiting example, a cDNA is a recombinant DNA molecule, such as any nucleic acid molecule that has been produced by an in vitro polymerase reaction, or a molecule to which a linker has been attached, or a molecule that has been integrated into a vector (such as a cloning vector or an expression vector).

[0073] As used herein, the term "recombinant protein" refers to a protein produced by genetic engineering.

[0074] When used with respect to polynucleotides, genes, nucleic acids, polypeptides or enzymes, the term "heterologous" refers to polynucleotides, genes, nucleic acids, polypeptides or enzymes that are not derived from a host species. For example, "heterologous genes" or "heterologous nucleic acid sequences" as used herein refer to genes or nucleic acid sequences from species different from the species of the host organism into which they are introduced. When it comes to gene regulatory sequences or auxiliary nucleic acid sequences (e.g., 5' untranslated regions, 3' untranslated regions, polyadenylation increase sequences, intron sequences, splice sites, ribosome binding sites, internal ribosome entry sequences, genome homology regions, recombination sites, etc.) or nucleic acid sequences encoding protein domains or protein localization sequences used to manipulate gene sequences, "heterologous" means that the regulatory sequence or auxiliary sequence or the sequence encoding the protein domain or the localization sequence is different from the gene source of the regulatory or auxiliary nucleic acid sequence or the nucleic acid sequence encoding the protein domain or the localization sequence in the genome, chromosome or episome. Thus, a promoter operably linked to a gene that is not operably linked in nature (e.g., in the genome of a non-genetically engineered organism) is referred to herein as a "heterologous promoter," even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked. Introns inserted into genes that are not related in nature (e.g., introns derived from different genes) are also referred to herein as "heterologous introns," even though the promoter may be derived from the same species (or, in some cases, the same organism). Similarly, "heterologous," when referring to a protein localization sequence or protein domain of an engineered protein, means that the localization sequence or protein domain is derived from a different protein than the protein into which it is incorporated by genetic engineering.

[0075] "Regulatory sequence", "regulatory element" or "regulatory element sequence" refers to a nucleotide sequence located upstream (5'), within or downstream (3') of a coding sequence. The transcription of a coding sequence and / or the translation of an RNA molecule produced by the transcription of a coding sequence is generally affected by the presence or absence of a regulatory sequence. These regulatory element sequences may include promoters, cis-elements, enhancers, terminators or introns. Regulatory elements can be isolated or identified from the untranslated region (UTR) of a particular polynucleotide sequence. Any regulatory element described herein may be present in a chimeric or hybrid regulatory expression element. Any regulatory element described herein may be present in a recombinant construct of the present invention.

[0076] The term "promoter", "promoter region" or "promoter sequence" refers to a nucleic acid sequence that can bind RNA polymerase to initiate transcription of a gene in the 5' to 3' ("downstream") direction. When the binding of RNA polymerase to the promoter is the most direct cause of gene transcription, the gene is in a "promoter-controlled" or "regulated" state. The promoter or promoter region generally provides recognition sites for RNA polymerase and other factors necessary for proper transcription initiation. The promoter can be isolated from the 5' untranslated region (5'UTR) of the genomic copy of the gene. Alternatively, the promoter can be synthesized or designed by changing known DNA elements. Chimeric promoters that combine the sequence of one promoter with the sequence of another promoter are also considered. Promoters can be defined by their expression pattern based on, for example, metabolic, environmental or developmental conditions. Promoters can be used as regulatory elements for regulating the expression of operably linked transcribable polynucleotide molecules (e.g., coding sequences). In addition to sequences recognized by RNA polymerase and preferably other transcription factors, promoters can also contain regulatory sequence elements, such as cis elements or enhancer domains that affect transcription of operably linked genes. An "algal promoter" is a native or non-native promoter that functions in algal cells.

[0077] As used herein, the term "operably connected" refers to such a configuration, wherein a control sequence is placed in a suitable position relative to the coding sequence of a polynucleotide sequence, so that the control sequence instructs or regulates the expression of the coding sequence of a polypeptide and / or a functional RNA. Therefore, if a promoter can mediate the transcription of a nucleic acid sequence, it is operably connected to the nucleic acid sequence. If a terminator can mediate the termination of a nucleic acid sequence, it is operably connected to the nucleic acid sequence. When introduced into a host cell, an expression cassette can cause transcription and / or translation of the encoded RNA or polypeptide under appropriate conditions. This definition does not exclude untranslated or untranslatable sense or antisense structures. In the case of transgenic expression and suppression of endogenous genes (e.g., by antisense or RNAi), those of ordinary skill in the art will recognize that the inserted polynucleotide sequences do not have to be identical, but may only be substantially identical to the gene sequence derived therefrom. As described herein, these substantially identical variants are specifically covered by reference to a specific nucleic acid sequence. When a control sequence (e.g., a promoter or terminator) regulates the transcription or transcription termination of a nucleic acid sequence, it is operably connected to the sequence it regulates.

[0078] As used herein, the term "selectable marker" or "selectable marker gene" includes any gene that confers a phenotype on a cell expressing it, so as to facilitate selection of cells transfected or transformed with a nucleic acid construct of the invention. The term may also be used to refer to a gene product that achieves the phenotype. Non-limiting examples of selectable markers include: 1) antibiotic resistance genes such as amikacin (aphA6), ampicillin (ampR), blastomycin (bis, bsr, bsd), bleomycin or phleomycin (ZEOCIN TM )(ble), chloramphenicol (cat), emetine (RBS14p or cry1-1), erythromycin (ermE), G418 (GENETICIN TM)(neo), gentamicin (aac3 or aacC4), hygromycin B (aphIV, hph, hpt), kanamycin (nptll), methotrexate (DHFR-mtxR), penicillin and other β-lactams (β-lactamase), streptomycin or spectinomycin (aadA, spec / strep), and tetracycline (tetA, tetM, tetQ); 2) genes for herbicide resistance, such as aminotriazole, cypermethrin, andrimid, aryloxyphenoxypropionate, atrazine, bipyridine, bromoxynil, cyclohexanedione oxime, cypermethrin, dicamba, diclfop, dichlorophenyl dimethyl urea (DCMU), furanones, diketonitriles, Diuron, fluridone, glyphosate, halogenated hydrogenated benzonitrile, flupyrazole, 4-hydroxypyridine, imidazolinone, isoxazoline, isoxazolidinone, miloamide B, p-nitrodiphenyl ether, norchloride, oxadiazole, m-phenoxybenzamide, N-phenylimide, pinoxadin, protoporphyrinogen oxidase inhibitors, pyridazinones, pyrazolinate, sulfonylurea, 1,2,4-triazolopyrimidine, triketone or urea; acetyl-CoA carboxylase (ACCase); acetohydroxyacid synthase (AHAS); acetolactate synthase (ALS, CSR1-1, CSR1-2, IMR1, IMR2), aminoglycoside phosphotransferase (APPT), anthranilate synthase, bromoxynil nitrile hydrolase (bxn), cytochrome P450-NADH-cytochrome P450 oxidoreductase, dalapon dehalogenase (dehal), dihydropterin synthase (sul), class I 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), class II EPSPS (aroA), non-class I / II EPSPS, glutathione reductase, glyphosate acetyltransferase (gat), glyphosate oxidoreductase (gox), hydroxyphenylpyruvate dehydrogenase, hydroxyphenylpyruvate dioxygenase (hppd), isoprenyl pyrophosphate isomerase, lycopene cyclase, phosphinothricin acetyltransferase (pat, bar), octadecene phytoene desaturase (crtl), prenyltransferase, protoporphyrin oxidase, psbA photosystem II polypeptide (psbAase) and SMM esterase (SulE) superoxide dismutase (sod); 3) genes that can be used for auxotrophic strains or to confer other metabolic functions, such as arg7, his3, hisD, hisG, lysA, manA, metE, nitl, trpB, ura3, xylA, dihydrofolate reductase gene, mannose 6-phosphate isomerase gene, nitrate reductase gene or ornithine decarboxylase gene; negative selection factors, such as thymidine kinase; or toxin resistance factors, such as 2-deoxyglucose resistance gene.

[0079] "Reporter gene" is a gene encoding a detectable protein or a protein having an activity that produces a detectable product. The reporter gene can encode a visual marker or enzyme that produces a detectable signal, such as cat, lacZ, uidA, xylE, alkaline phosphatase gene, α-amylase gene, α-galactosidase gene, β-glucuronidase gene, β-lactamase gene, horseradish peroxidase gene, luciferin / luciferase gene, R-locus gene, tyrosinase gene or a gene encoding a fluorescent protein, including but not limited to blue, cyan, green, red or yellow fluorescent protein, light conversion, light-operated switch or high-brightness fluorescent protein or any variant thereof, including but not limited to codon optimization, fast folding, monomer, increased stability and enhanced fluorescence variants.

[0080] As used herein, the term "terminator" or "terminator sequence" or "transcription terminator" refers to a regulatory portion of a gene sequence that signals RNA polymerase to terminate transcription, usually in a conventional manner. A terminator can generally mark the end of a gene, coding sequence, or operon in DNA.

[0081] As used herein, the term "transformation" refers to the introduction of one or more exogenous nucleic acid sequences or polynucleotides into a host cell or organism by using one or more physical, chemical or biological methods. As non-limiting examples, physical and chemical methods of conversion (i.e., "transfection") include electroporation, particle bombardment, and liposome delivery. Biological methods of conversion (i.e., "transduction") include the use of engineered viruses or microorganisms (e.g., Agrobacterium) to transfer DNA.

[0082] As used herein, the term "intron" refers to a nucleotide sequence within a gene that is removed from the RNA transcribed from the gene by RNA splicing (the term intron is used to refer to the RNA sequence that appears in the RNA molecule before splicing, as well as the DNA sequence that appears in the gene.). Introns disclosed herein are "splicing introns" naturally present in the nuclear genes of eukaryotic organisms, and spliced ​​by the splicing mechanism (spliceosome) of eukaryotic cells. Introns derived from naturally occurring introns are also contemplated, such as introns that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of a naturally occurring intron or a variant thereof deleted internally, for example, variants that are deleted from 1 to 1000bp within the intron boundary. Chimeric introns comprising intron sequences of two or more naturally occurring introns are also contemplated. Introns include a GT at the 5' end (GU in the primary RNA transcript), a branch site sequence near the 3' end of the intron, and an AG acceptor site at the 3' end of the intron. The surrounding exon sequence includes a GG at the 5' boundary of the intron and a G after the AG at the 3' end of the intron. Such sequences can be optionally engineered into the coding sequence of a gene as provided herein at the intron insertion site.

[0083] Intronized genes as provided herein are engineered to include at least one heterologous intron, i.e., at least one intron that is not naturally present in the gene encoding the polypeptide encoded by the engineered gene, and in some embodiments, the intronized gene is preferably engineered to include at least three, at least four or at least five heterologous introns, i.e., at least three, at least four or at least five introns that are not naturally present in the gene. For example, the amino acid coding sequence of the engineered gene can encode a polypeptide that is not encoded by the gene from which the heterologous intron is derived. For example, using genetic engineering or gene synthesis technology, heterologous introns are inserted into genes that do not naturally exist. The amino acid coding sequence of the engineered gene can be optionally changed, for example, to produce a sequence that is immediately adjacent to the heterologous intron, thereby allowing the correct splicing of the introduced intron and / or changing codon usage (e.g., to reflect the codon preference of the host) and / or introducing mutations. In some embodiments, at least three heterologous introns are derived from one or more genes (except for the gene from which the amino acid coding sequence of the engineered gene is derived), for example, at least three exogenous introns can be derived from naturally occurring introns. In various embodiments, at least three, at least four or at least five exogenous introns can be naturally occurring introns from another gene (amino acid coding sequence of the engineered gene derived therefrom) of the same or different organism, or can be, for example, derived from naturally occurring introns from another gene (amino acid coding sequence of the engineered gene derived therefrom) of the same or different organism by one or more sequence modifications or internal deletions of sequences in naturally occurring introns. In some embodiments, at least three, at least four or at least five exogenous introns inserted into the engineered gene are all naturally occurring introns of the same gene, and in some embodiments, multiple introns of the same naturally occurring gene can be introduced into the engineered gene in the same order in which they appear in the naturally occurring gene from which they are derived. In some embodiments, the engineered gene is operably connected to a promoter, and the promoter and the exogenous intron can be optionally derived from the same organism. In some embodiments, the engineered gene is operably connected to a promoter and a terminator, and the promoter, the terminator and the exogenous intron can all be derived from the same organism, and can all be derived from the same gene. Further, in various embodiments, the amino acid coding sequence of the engineered gene can be codon optimized, and in some instances can be codon optimized for expression in the organism from which the exogenous introns were derived.

[0084] Expression cassette

[0085] The expression cassette disclosed herein comprises one or more regulatory elements as described herein to drive the expression of the target DNA. These boxes comprise a DNA molecule, and the DNA molecule comprises any one of the algae nitrate reductase or nitrite reductase promoter sequences as described herein that are operably connected to the target DNA, wherein the target DNA is located downstream of the promoter sequence, and optionally any one of the algae nitrate reductase or nitrite reductase terminator sequences as described herein or any combination thereof is operably connected downstream of the target DNA. The algae nitrate reductase and nitrite reductase promoters of the present invention can be used together with any heterologous or homologous DNA of the target. In the case of homologous genes, these promoters are not juxtaposed with these homologous genes of the target in nature. Therefore, the algae nitrate reductase and nitrite reductase promoters cannot regulate the expression of these homologous genes of the target in nature. The target DNA can optionally comprise a heterologous intron, i.e., a natural intron of a gene that is not derived from a protein or functional RNA coding sequence. In some embodiments, the expression cassette can be integrated into the genome of an algae cell or organism. In some embodiments, integration occurs by the transformation of a cell or organism.

[0086] The basic techniques for operably linking two or more DNA sequences together are familiar to those skilled in the art, and these methods are described in many standard textbooks on molecular biology procedures (e.g., see Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Gibson et al. (2009) Nature Methods 6:343-345).

[0087] Carrier

[0088] The present invention also provides vectors that can include regulatory elements and / or expression cassettes as described herein. The vector can further optionally include at least one replication origin ("ORI") sequence for replication in the cell. The vector can further optionally include one or more selection markers controlled by one or more eukaryotic promoters, one or more selection markers controlled by one or more prokaryotic promoters and / or one or more sequences that mediate the recombination of exogenous nucleic acid sequences into the target cell genome. In some embodiments, the vector can be integrated into the genome of an algae cell or organism. In some embodiments, integration occurs by transformation of a cell or organism.

[0089] Additionally, the vectors described herein may further comprise a selectable marker as described above.

[0090] The selection marker gene can be operably connected to a promoter provided herein and / or under its control. The promoter regulating the expression of the selection marker can be conditional or inducible, but preferably constitutive, and can be any promoter or another promoter disclosed herein, for example. Alternatively, the selection marker can be placed under the control of an expression cassette promoter. If the selection marker is placed under the control of an expression cassette promoter, the selection marker and the expression cassette can be operably connected to an internal ribosome entry site ("IRES") element (Komar & Hatzoglou (2011) "Cell Cycle (Cell Cycle)" 10:229-240 and Hellen & Samow (2001) "Gene and Development (Genes & Dev.)" 15:1593-1612, incorporated by reference as a whole) or "2A" sequence (Kim et al., (2011) American Public Library of Science (PLoS One) 6 (4): e18556, incorporated by reference as a whole) between the expression cassette and the selection marker.

[0091] Conversion method

[0092] The present invention also provides a transformation method, wherein a eukaryotic cell is transformed with an expression vector as described herein. The method comprises introducing an expression vector as provided herein, the expression vector comprising at least one promoter or DNA sequence as provided herein; and then selecting a transformant. The expression vector can be introduced by many methods familiar to those skilled in the art, including those described in U.S. Patent No. 10,041,079 and U.S. Patent Application Publication No. 2017 / 0073695, the entire contents of which are incorporated herein by reference.

[0093] The algal cell can be a green algae, such as Agaricus, Cocoon, Amphiphytum, Fibrophytum, Astrocystis, Golden Chromophytum, Bolidomonas, Borodinella, Balloon Algae, Botrytis, Bracteococcus, Chaetococcus, Tetradinium, Chlamydomonas, Chlorocapra, Chlorofusobacterium, Chlorella, Cryptophyte, Cryptophyte, Cricosphaera, Cryptodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Colobus, Unicoccella, Ernodesmius, Euglena, True Eyed Algae, Phalarophyte, Fragilis, Lissium, Haematococcus, Halocafeteria , Heterosigma, Hymenium, Isochrysis, Leptopora, Microsporum, Mononema, Microchlorophyll, Microchlorophyll, Navicula, Neochlorophyll, Nephrodinium, Nephrodinium, Nitzschia, Brown Dinophyll, Sheath, Oocystis, Oystercospermum, Pavlova, Pseudochlorella, Parrella, Pelagomonas, Brown Finger, Bacteriophage, Microchlorophyll, Platymonas, Coccolithophore, Costalcohol, Protocystis, Pseudochlorococcus, Pseudoneocereus, Pyramidalis, Pyramidalis, Morus, Scenedesmus, Skeleton, Spyrogyra, Schizothrix, Tetraselmis, Thalassiosira, Yellow Tricholoma, Asterum, Viridiella, Weissella, and Volvox. For example, the eukaryotic cells transformed using the methods provided herein can optionally be of a species of Pseudochlorella, such as the non-limiting examples of Pseudochlorella kessleri, Pseudochlorella hussii, Pseudochlorella beijerinckii, Pseudochlorella sp. CCAP 206 / 1, or Pseudochlorella sp. pgu003.

[0094] In other embodiments, the algae cell can be any eukaryotic microalgae, such as, but not limited to, green algae, Ochrophyte or Chara. In some embodiments, the algae can be a green algae of the taxonomy Chlorophyceaceae, Chlorodendrophyceae, Prasinophyceae, Trebouxiophyceae or Eustigmatophyceae. In some embodiments, the algae can be a member of the Chlorophyceae, such as Asterosporea, Cellulose, Tetradinium, Chlamydomonas, Chlorococcus, Chlorofusium, Chlorodendrales, Chloroellales, Chrysosphaera, Dunaliella, Haematococcus, Monospinyta, Neochlorophyta, Sheath, Pelagomonas, Coleococcus, Synechococcus, Morus, Scenedesmus, Volvox, any one or more species. In other embodiments, the algae may be a member of the class Tetraselmis, such as species of any one or more of the genera Prasinocladus, Scherffelia, or Tetraselmis. In further alternative embodiments, the algae may be a member of the class Chlorophyceae, optionally species of any one or more of the genera Ostreococcus or Micromonas. Further alternatively, the algae may be a member of the class Symphycophyceae, and optionally a member of the order Chlorellales, and optionally a genus selected from any one or more of the genera Staphylococcus, Chlorella, Auxenochlorella, Heveochlorella, Marinicholella, Oocystis, Pseudochlorella, Pseudochlorococcus, Tetrachlorella, Unicocystis, Pseudochlorococcus, Microchlorophyceae, Microchlorophyceae, Protocystis, Schizothrix, or Viridiella, or a combination or subcombination of all possible genera. In another embodiment, the algae is a green algae of the class Symcochaetes, the orders Chlorellae, the family Oocystaceae, the family Chlorellaceae, or the class Euopsoma, and is optionally selected from one or more of Oocystaceae, Pseudochlorella, Nannochloropsis, Nannochloropsis, and Tetraselmis. The algae may also be from the genus Oocystaceae, Pseudochlorella, or Nannochloropsis, or Tetraselmis, or from any of all possible combinations and subcombinations of the disclosed genera. Any algae described herein may comprise a DNA molecule or sequence of the invention, such as comprising an algal nitrate reductase or nitrite reductase promoter operably linked to a DNA of interest, as described herein.

[0095] nourish

[0096] The transformed algal cell culture can be diluted, plated on agar, and allowed to grow until isolated colonies can be selected for further propagation as clonal strains.

[0097] The transformed algae cells can be cultured in an induction medium (IM), for example, in the presence of nitrate ions or nitrite ions, to induce the expression of the target DNA. The transformed algae cells can also be cultured in an inhibitory medium (RM), such as in the presence of ammonium salts, to inhibit the expression of the target DNA.

[0098] In addition, the photosynthetic organisms can be cultured in a mixotrophic culture, wherein the organisms are grown in the presence of light for at least a portion of the day and are also provided with one or more reducing carbon sources. The photosynthetic organisms can be grown mixotrophically for a period of time and then phototrophically for a period of time, or vice versa.

[0099] Media for phototrophic or mixotrophic growth of algae are known in the art, and the media can be optimized to enhance growth or production of fatty acid products of a particular species.Artificial light sources can be used as the sole light source or to enhance or extend natural light.

[0100] The growth of algae can be in an open area, such as, for example, a pond, canal, river, raceway, or tank, or can be in a bioreactor. Bioreactors are preferred for mixotrophic growth, and can also be used for phototrophic growth. The bioreactor can be of any size and form, and can include a system for providing nutrients, additives, or gases (such as, for example, but not limited to, air or CO). 2 ). The bioreactor preferably also has an outlet for sampling the culture. The bioreactor can be configured to mix the algae culture during growth, for example, by stirring, shaking, vibrating, inverting, bubbling, etc., with gases in the culture. Outdoor ponds, raceways, tanks, canals, etc. can also be designed for mixing the culture, for example, by paddles, pumps, hoses, or nozzles for circulating the culture medium, or for supplying air or CO to the culture. 2 pipe, hose or inlet.

[0101] Example

[0102] Example 1 Identification of regulatory sequences of Pseudochlorella vulgaris

[0103] To evaluate the ability of multiple sequences to act as promoters or terminators, promoter sequences were detected in intergenic untranslated nucleic acid sequences flanking the nitrate reductase and nitrite reductase genes based on the genome assembly of the wild-type Pseudomonas sp. strain WT-1185, RNA sequencing data, hidden Markov model analysis, BLAST analysis, and Pfam analysis of Pfam PF01077 and PF03460.

[0104] Blast sequence alignment of Pseudomonas showed that the most Pfam hits (PF01077 and PF03460) were nitrite / sulfite reductase genes ( Figure 3 ). In the Pseudomonas aeruginosa strain WT-1185, the nitrite reductase and nitrate reductase genes are located in opposite directions on the same chromosome ( Figure 4 The nitrite reductase and nitrite reductase promoters were identified in the intergenic region of the nitrite reductase and nitrate reductase genes ( Figure 4 ). The nitrite reductase and nitrite reductase terminators were identified in the 3'-UTR region of the nitrite reductase and nitrite reductase genes, respectively ( Figure 4 ).

[0105] Example 2 Generation of expression cassette

[0106] The ParaCreXP vector construct was used to successfully generate recombinant strains of Chlorella vulgaris ( Figure 5 ). The vector construct contains expression cassettes for the selection markers bleomycin (Ble), Cre recombinase, and TurboGFP. The Ble and Cre genes were optimized for Chlorella codon usage, while TurboGFP was amplified directly from pTurboGFP-C purchased from Evrogen (Moscow, Russia). The Ble gene contains 5 introns from the Chlorella 40S ribosomal protein S4 (RPS4) gene, which is controlled by the constitutive RPS4 promoter and terminator. The expression cassette also includes a green fluorescent protein (TurboGFP) reporter gene. The expression of the GFP gene is regulated by the constitutive acyl carrier protein (ACP) promoter and terminator. The Cre coding sequence (SEQ ID NO: 53) contains an N-terminal NLS (sv40) and 6 introns from the Chlorella nitrite reductase (NIR) gene, and is controlled by the inducible / repressible NIR promoter and terminator. The expression of Cre gene was regulated by the promoter of Pseudochlorophyll nitrite reductase (SEQ ID NO: 1) and the terminator of nitrite reductase (SEQ ID NO: 2). The vector construct was assembled from these parts into the puc19 vector backbone using the HiFi 1-step kit (Synthetic Genomics, La Jolla, CA).

[0107] Example 3 Transformation by electroporation

[0108] The ParaCreXP vector construct was linearized with Ascl / Notl restriction endonucleases. The linearized vector was used to transform the Pseudochloropsis WT-1185 strain using the transformation method described in US20170073695A1, which is incorporated herein by reference in its entirety. TMSeveral C. pseudochloasma transformants containing randomly integrated constructs in their genome were analyzed on a C6 cytometer (BD Biosciences, Franklin Lakes, NJ, USA) for GFP fluorescence analysis and Western Blot analysis.

[0109] Example 4 Culture medium formulation and culture conditions

[0110] Induction medium (IM-NO3-) contained 35 g / L aquarium salts 10x F / 2 trace metals and vitamins, and 0.361 mM NaH2PO4. The N source was 15 mM NaNO3. Repression medium (RM-NH4+ / NO3-) consisted of the same ingredients as IM medium, but was further supplemented with 10 mM NH4Cl and buffered with 15 mM HEPES, pH 8.0. Cells were cultured in vented cap flasks in a 1% CO atmosphere. 2 The cells were cultured for 3 days on an orbital shaker in a growth chamber (25°C) under continuous light from cool white fluorescent lamps (50 μmol photons m -2 s -1 ) under lighting.

[0111] Example 5 RNA extraction and QRT-PCR

[0112] Grow the strains in IM or RM medium to OD 730 was 2.0, and 5 ml of the culture was pelleted by centrifugation. The cell pellet was resuspended in 1.8 ml of extraction solution (5 ml of grinding buffer, 5 ml of phenol, 1 ml of 1-bromo-3-chloropropane and 20 μL of mercaptoethanol, wherein the grinding buffer contained 9 ml of 1 M Tris (pH 8), 5 ml of 10% SDS, 0.6 ml of 7.5 M LiCl and 450 μl of 0.5 M EDTA (final volume of 50 ml) and vortexed vigorously for 5 minutes at 4°C in the presence of 200 pm zirconium beads. After centrifugation, 1 ml of 25:24:1 phenol extraction solution (25 ml of phenol (pH 8.1); 24 ml of 1-bromo-3-chloropropane and 1 ml of isoamyl alcohol) was added to the aqueous phase in another test tube. The test tube was shaken vigorously and centrifuged at 21,000 g for 2 minutes. The extraction was repeated with 1 ml of 1-bromo-3-chloropropane and the resulting aqueous layer was washed with 0.356 volumes of 7.5 M LiCl treatment was performed to precipitate RNA overnight at -20°C. After LiCl precipitation, the RNA pellet was resuspended in 50 μl HO. 2 O, and RNA quality was assessed by chip gel electrophoresis using a 2100 Bioanalyzer according to the manufacturer's instructions (Agilent Technologies, La Jolla, CA).

[0113] Using iScript TMcDNA was prepared using the Reverse Transcription Supermix Kit (Bio-Rad, Hercules, CA) and used as a Ssofast TM Supermix (Bio-Rad) qRT-PCR template. The primer sequence of Cre is F: 5'-GATCTTTGAGGCAACACATCG-3' (SEQ ID NO: 54); R: 5'-AATGCTCACTCCAGCTCTTG-3' (SEQ ID NO: 55). The efficiency of qRT-PCR primers was evaluated, and the 2-ΔΔCT method was used to evaluate the gene expression standardized for the control gene with a lower coefficient of variation under different conditions determined empirically (EMRE3EUKT595283; primer sequence is F: 5'-GCCTTTGGTTATCGTGCTTTAG-3 (SEQ ID NO: 56); R: 5'-TCCCTCCGATCCTTTACTCTC-3) (SEQID NO: 57).

[0114] qRT-PCR results showed that the expression of Cre in the recombinant Chlorella vulgaris cell line was induced in the presence of nitrate ions and inhibited in the presence of ammonium ions ( Figure 1 ).

[0115] Example 6 Western Blot

[0116] Grow the Cre-expressing Chlorella strain in IM or RM medium to an OD of 730 The pH was 2.0 and 5 ml of the culture was pelleted by centrifugation. The pellet was washed once with TBS buffer (50 mM Tris-Cl (pH 7.6), 150 mM NaCl) and then resuspended in 300 ml of SDS-PAGE extraction buffer, which consisted of 125 mM Tris (pH 8.8), 10% glycerol and 2% SDS. 100 μl of zirconium beads were added to the cell slurry, the cells were vortexed for 30 seconds and then incubated at 85°C for 10 minutes. During the incubation at 85°C, the lysate was vortexed for another 30 seconds, three times, and then centrifuged and the supernatant was collected. The supernatant was mixed with Sample buffer (Thermo Fisher Scientific, Waltham, MA) was mixed in a 3:1 ratio and incubated at 85°C for 10 min, and 25 μl of the mixture was loaded into each well of the gel. For CRE detection, 4-12% Bis-Tris gels were used and electrophoresis was performed using MOPS running buffer. iBind TMThe blots were incubated with primary and secondary antibodies using the Western Blotting Instrument (Thermo Fisher Scientific, Waltham, MA). The CRE blots were incubated with primary antibodies (rabbit anti-CRE, Millipore, at a dilution of 1:1000) and secondary antibodies (goat anti-rabbit AP, Novex TM , diluted 1.5:1000). TM The AP chromogenic substrate BCIP / NBT kit (Thermo Fisher Scientific, Waltham, MA) was used to detect immune signals.

[0117] The expression of Cre in the presence of nitrate and ammonium ions was compared in a wild-type strain lacking the Cre gene. The results showed that Cre expression was induced in the presence of nitrate ions and inhibited in the presence of ammonium ions ( Figure 2 ).

[0118] Although the present invention has been described with reference to the above embodiments, it will be understood that modifications and variations are encompassed within the spirit and scope of the present invention. Accordingly, the present invention is limited only by the appended claims.

Claims

1. A genetically engineered Chlorella vulgaris, comprising a DNA molecule, wherein the DNA molecule comprises an algae nitrite reductase inducible promoter of SEQ ID NO: 1 and a terminator sequence of SEQ ID NO: 2, wherein the promoter and the terminator are operably linked to a target DNA encoding a polypeptide or a functional RNA, and wherein the target DNA encoding the polypeptide or the functional RNA is not regulated by the promoter in nature, and wherein the DNA molecule is integrated into the algae genome.

2. The genetically engineered Chlorella vulgaris of claim 1, wherein the DNA of interest encodes a polypeptide, and wherein the polypeptide is heterologous to the algal species.

3. The genetically engineered Chlorella vulgaris according to any one of claims 1 to 2, wherein the target DNA comprises at least one intron, wherein the intron is heterologous to the target DNA encoding a polypeptide or a functional RNA.

4. The genetically engineered Chlorella vulgaris according to any one of claims 1 to 2, wherein the target DNA encodes a transcription factor, a transcription activator, a cell signaling protein, an enzyme or a selection marker.

5. The genetically engineered Chlorella vulgaris according to claim 3, wherein the target DNA encodes a transcription factor, a transcription activator, a cell signaling protein, an enzyme or a selection marker.

6. The genetically engineered Chlorella vulgaris according to claim 4, wherein the target DNA encodes Cre recombinase.

7. An expression cassette comprising a DNA molecule, wherein the DNA molecule comprises an algae nitrite reductase inducible promoter of SEQ ID NO: 1 and a terminator sequence of SEQ ID NO: 2, wherein the promoter and terminator are operably linked to a target DNA encoding a polypeptide or a functional RNA, and wherein the target DNA encoding the polypeptide or the functional RNA is not regulated by a promoter in nature.

8. The expression cassette according to claim 7, wherein the DNA molecule encoding the polypeptide or functional RNA comprises at least one intron, wherein the intron is heterologous to the target DNA encoding the polypeptide or functional RNA.

9. A method for selectively expressing a target DNA in an algae cell, comprising: a) transforming a Chlorella cell with an isolated DNA molecule, the DNA molecule comprising an algal nitrite reductase inducible promoter of SEQ ID NO: 1 and a terminator of SEQ ID NO: 2, wherein the promoter and the terminator are operably linked to a target DNA encoding a polypeptide or a functional RNA, and wherein the target DNA encoding the polypeptide or the functional RNA is not regulated by a promoter in nature to produce a transformed Chlorella cell; and b) growing the transformed Chlorella cells in a culture medium that selectively allows expression of the DNA of interest in the algal cells.

10. The method of claim 9, wherein the isolated DNA molecule is introduced by particle bombardment.

11. The method of claim 9, wherein the isolated DNA molecule is introduced by electroporation.

12. The method according to any one of claims 9 to 11, wherein the Pseudocerulina cells are grown in a medium comprising nitrates, thereby inducing expression of the DNA of interest.

13. The method of claim 9, wherein the Chlorella cells are grown in a medium comprising ammonium salts, thereby inhibiting expression of the DNA of interest.

14. The method according to any one of claims 9 to 11 and 13, wherein the target DNA encodes a transcription factor, a transcription activator, a cell signaling protein, an enzyme or a selection marker.

15. The method of claim 12, wherein the target DNA encodes a transcription factor, a transcription activator, a cell signaling protein, an enzyme, or a selection marker.

16. according to the method described in any one of claims 9 to 11, 13 and 15, wherein said target DNA molecule encoding polypeptide or functional RNA comprises at least one intron, wherein said intron is not native to said DNA molecule encoding said polypeptide or functional RNA.

17. The method of claim 12, wherein the target DNA molecule encoding a polypeptide or a functional RNA comprises at least one intron, wherein the intron is not native to the DNA molecule encoding the polypeptide or the functional RNA.

18. The method of claim 14, wherein the target DNA molecule encoding a polypeptide or a functional RNA comprises at least one intron, wherein the intron is not native to the DNA molecule encoding the polypeptide or the functional RNA.

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