# Patent

## Title
Autonomous replication sequences and episomal DNA molecules

## Abstract

The present invention provides autonomous replication sequences (ARSs) isolated from Nannochloropsis that support the replication of episomal DNA molecules (EDMs) in eukaryotic cells. The ARSs and EDMs provided herein can be used for expressing genes in organisms including algae and heterokonts.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/734,285 filed Dec. 6, 2012, the entire contents of which is herein incorporated by reference.


REFERENCE TO A SEQUENCE LISTING
This application contains references to nucleic acid sequences which have been submitted concurrently herewith as the sequence listing text file “Sequence Listing_SGI-060.20US.txt”, file size 4 kilobytes (kb), created on 6 Dec. 2012. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. §1.52(e)(iii)(5).
FIELD OF THE INVENTION
The present invention relates, in some embodiments, to genetic elements for the manipulation of eukaryotic cells in biotech applications. The present invention also relates, in some embodiments, to methods of identifying ARS elements from organisms of interest.
BACKGROUND
An Autonomously Replicating Sequence (ARS) is a sequence that serves as an origin of DNA replication on eukaryotic chromosomes. An ARS is required for DNA replication during cell division, and is a necessary part of a functional synthetic chromosome (Gilbert (2001) Science 294:96-100). Additionally, some ARS sequences have been shown to function as matrix attachment regions (MARs), which can improve heterologous genes' amount of expression, consistency of expression over time, and degree of repression by heterochromatin formation (Kim et al. 2007).
While DNA sequences that constitute the origins of DNA replication are readily identifiable in bacteria, origins of replication in eukaryotes have been more difficult to identify (Gilbert 2001). ARS primary sequences are not significantly conserved, either within or across eukaryotic species, although the location of some ARS elements is conserved across species (Gilbert 2001). For example, Saccharomyces cerevisiae (budding yeast) (Smith & Boeke (1997) Genes Dev. 11:241-54) and Pisum sativum (garden pea) (Van't Hof (1996) “DNA Replication in Eukaryotic Cells,” CSHL Press) both have an ARS between the 18S and 5S ribosomal RNA genes in their respective ribosomal repeat regions.
The current methodology for acquiring non-Saccharomyces cerevisiae ARS elements for use in artificial chromosomes is through random acquisition with large ( ̃100 kbp) pieces of genomic DNA (Basu et al. (2005) BMC Biotechnol. 5:21). This process is both inefficient and liable to import sequences that are deleterious to optimal industrial performance, but which happen to be located proximal on a chromosome to an ARS. Once an ARS is located, it can be placed into artificial chromosomes or plasmids to permit those constructs to replicate in a host of interest (Henning et al. (1999) Proc. Nat'l. Acad. Sci. USA 96:592-97). At present, there are no known Nannochloropsis sp.—or heterokont—ARS elements available.
SUMMARY
The present invention provides a number of nucleic acid sequences that were isolated from the Nannochloropsis genome by analyzing the region of ribosomal RNA gene repeats (see Example 1) and identifying AT-rich relative to surrounding sequences. Insertion of these sequences into plasmids that carried selectable marker genes demonstrated that the sequences were necessary and sufficient for replication of plasmids (Episomal DNA Molecules or EDMs) in cells maintained under selection. Genes maintained on EDMs were demonstrated to have better expression of the encoded polypeptides than genes integrated into the genome.
The sequences were tested characterized as Autonomously Replicating Sequences (ARSs) by their ability to transform Nannochloropsis and to allow for 1) recovery of DNA molecules from uncut genomic DNA that a) migrated as circular molecules in gel electrophoresis, b) persisted in algal cultures under selection, and c) were intact. EDMs that included these ARSs were recovered from uncut isolated DNA from EDM-transformed cells were used to transform E. coli, where the EDMs were also found to reside intact.
Thus, provided herein are ARSs isolated from Nannochloropsis sp. that can support independent replication of episomal DNA molecules (EDMs) in eukaryotic cells, including microorganisms such as algae and heterokonts. Also provided are EDMs that include an ARS as provided herein, and methods of their use in expressing genes in algae and heterokonts.
In a first aspect, provided herein is an isolated or recombinant nucleic acid molecule that includes an ARS having a nucleic acid sequence having at least 85% identity to SEQ ID NO:13 or SEQ ID NO:14. The nucleic acid molecule that includes the ARS further includes at least one nucleic acid sequence that is heterologous with respect to the ARS, for example, where the ARS is identical to a naturally-occurring sequence of an organism, the ARS is not adjacent to sequences it is found adjacent to in the organism from which the sequence is isolated. For example, if identical to a naturally-occurring genomic sequence, the ARS can be provided in a nucleic acid molecule where the ARS is juxtaposed with at least one nucleic acid sequence not isolated from the rDNA region of the genome of the organism from which ARS has been isolated. A nucleic acid molecule as provided herein that includes a sequence having at least 85% identity to SEQ ID NO:13 or SEQ ID NO:14 can further comprise, for example, a reporter gene, selectable marker gene, a gene encoding a polypeptide of interest, or a gene encoding a functional RNA that is not a ribosomal RNA, where any of the foregoing may not be from genome of the organism from which the ARS has been isolated. The ARS that includes a nucleic acid sequence having at least 85% identity to SEQ ID NO:13 or SEQ ID NO:14 can have, for example, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:13 or SEQ ID NO:14. In some examples, an isolated or recombinant nucleic acid molecule includes an ARS having a nucleic acid sequence having at least 85% identity to SEQ ID NO:13 and a nucleic acid sequence having at least 85% identity to SEQ ID NO:14 juxtaposed with at least one nucleic acid sequence that is heterologous with respect to SEQ ID NO:13 and/or SEQ ID NO:14.
An ARS as provided herein is functional in a eukaryotic cell, for example, a microbial cell such as a fungal, algal, or heterokont cell. In various examples, an ARS supports replication of an episomal DNA molecule (EDM) in diatom and/or Eustigmatophyte cells.
In some examples, a nucleic acid molecule provided herein includes an ARS having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to at least 150 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; or SEQ ID NO:10, in which the nucleic acid sequence also has at least 85% identity to SEQ ID NO:13. In some examples, a nucleic acid molecule provided herein includes an ARS having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to at least 150 contiguous nucleotides of SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:11; or SEQ ID NO:12 and also has at least 85% identity to SEQ ID NO:14.
A nucleic acid molecule as provided herein in various examples can include a nucleic acid sequence having at least 85% identity to SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:11; or SEQ ID NO:12, juxtaposed with a heterologous nucleic acid sequence, e.g., juxtaposed with a nucleic acid sequence not derived from the rDNA region of the genome of an organism, such as the rDNA region of the genome of Nannochloropsis. 
In another aspect, provided herein are episomal DNA molecules (EDMs) that include an ARS having at least 85% identity to at least 150 contiguous nucleotides of SEQ ID NO:1; at least 150 contiguous nucleotides of SEQ ID NO:5; at least 150 contiguous nucleotides of SEQ ID NO:6; at least 150 contiguous nucleotides of SEQ ID NO:7; at least 150 contiguous nucleotides of SEQ ID NO: 8; at least 150 contiguous nucleotides of SEQ ID NO:9; at least 150 contiguous nucleotides of SEQ ID NO:10; at least 150 contiguous nucleotides of SEQ ID NO:11; or at least 150 contiguous nucleotides of SEQ ID NO:12. For example, an EDM as provided herein can include an ARS having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to at least 150 contiguous nucleotides of SEQ ID NO:1; at least 150 contiguous nucleotides of SEQ ID NO:5; at least 150 contiguous nucleotides of SEQ ID NO:6; at least 150 contiguous nucleotides of SEQ ID NO:7; at least 150 contiguous nucleotides of SEQ ID NO:8; at least 150 contiguous nucleotides of SEQ ID NO:9; at least 150 contiguous nucleotides of SEQ ID NO:10; at least 150 contiguous nucleotides of SEQ ID NO:11; or at least 150 contiguous nucleotides of SEQ ID NO:12. An EDM as provided herein can be circular. The EDM can replicate autonomously in a eukaryotic host cell, such as a cell of a microorganism, such as, for example, a fungal, heterokont, or algal cell.
In various examples, an EDM as provided herein can include an ARS having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, or at least 750 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. For example, an EDM as provided herein can include an ARS having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, or at least 750 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:12. In various examples, an EDM as provided herein can include an ARS having the sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:12.
An EDM as provided herein can further comprise a nucleic acid sequence encoding a polypeptide. For example, an EDM can include a nucleic acid sequence encoding a reporter protein, such as a fluorescent protein or signal-producing enzyme, or selectable marker protein, such as a protein that confers resistance to a drug, antibiotic, toxin, or herbicide or a protein that allows autotrophic growth on stringent media.
Alternatively or in addition, an EDM as provided herein can include a gene encoding a metabolic enzyme, structural protein, kinase, phosphatase, nucleotide cyclase, phosphodiesterase, transcriptional regulator, transcriptional activator, transporter, secretory protein, ion channel, receptor, photosynthetic protein, chaperonin, ribosomal protein, or nuclear scaffold protein. In various examples, an EDM as provided herein can include a gene encoding a meganuclease, a zinc finger nuclease, a TALEN protein, a cas protein, a recombinase, a topoisomerase, or a transposase.
Further alternatively or additionally, an EDM as provided herein can comprise a nucleic acid sequence encoding a functional RNA, such as, for example, an interfering RNA or a precursor thereof, an antisense RNA, a ribozyme, a micro RNA, or a guide RNA of a CRISPR system. Preferably, the functional RNA is not a ribosomal RNA. In various examples, the functional RNA can be a functional RNA designed to attenuate expression of a targeted gene.
In further examples, alternatively or in addition to any of the above, an EDM can include sequences for mediating homologous or site specific recombination, wherein the sequences for homologous recombination include genomic sequences that flank or comprise at least a portion of a gene targeted for gene replacement or disruption, or can include sequences recognized by site-specific recombination systems.
In another aspect of the invention, methods are provided for propagating a gene of interest through at least 8 generations of cell division, in which the method includes incorporating the gene of interest into an EDM as provided herein, transforming the EDM into a host cell, and propagating the host cell for at least 8 generations. In exemplary embodiments, the EDM includes a selectable marker gene, and the host cell is propagated under selection. The host cell can be, in various examples, an algal, heterokont, plant, animal, fungal, or yeast cell, and may be a diatom or Eustigmatophyte cell, for example, a Nannochloropsis cell.
In yet another aspect of the invention, methods are provided for expressing a gene or sequence of interest in a host cell, comprising transforming an EDM as provided herein that includes a gene or sequence of interest into a host cell and culturing the host cell under conditions in which the gene or sequence is expressed. In exemplary embodiments the EDM includes a selectable marker gene in addition to the gene or sequence of interest, and the host cell is cultured in the presence of a selective agent for one or more generations. In some examples, the method is for transiently expressing the gene or sequence of interest in a host strain. For example, the host cell transformed with the EDM may be cultured in the presence of the selective agent for one or more generations during which the gene or sequence of interest is expressed, and subsequently cultured in the absence of the selective agent for one or more additional generations, during or after which the gene or sequence of interest is not expressed.
In yet a further aspect a method is provided for isolating an autonomous replication sequence (ARS) in a species of interest, in which the method includes sequencing an sequence amplified from a region of the species' genome between 35S ribosomal DNA (rDNA) repeats and a 28S rDNA repeat; and selecting a portion of the amplified sequence with the lowest percentage guanidine/cytosine content, in which the selected portion is at least 100 nucleotides, at least 200 nucleotides, at least 500 nucleotides, or at least 700 nucleotides in length. The method can further include testing the ARS for the ability to support replication of an EDM in a host cell. Also provided herein are methods for effecting homologous recombination in a host cell of interest, in which the method includes introducing an EDM that includes a selectable marker and a construct for homologous recombination into a host cell.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph depicting a BLAST alignment of regions from the Nannochloropsis sp. putative ARS elements. The minor lines in parallel of the major diagonal line are indicative of tandem repeats. An inverted repeat would appear at a 90 degree angle to the major diagonal line.
FIG. 2 shows an alignment of portions of putative ARSs isolated from Nannochloropsis gaditana. The aligned ARSs are, from top to bottom: NgARS1 (SEQ ID NO:5), NgARS3 (SEQ ID NO:7), NgARS5 (SEQ ID NO:9), NgARS4 (SEQ ID NO:8), NgARS2 (SEQ ID NO:6), NgARS7 (SEQ ID NO:10), NgARS7 (SEQ ID NO:10), and NgARS8 (SEQ ID NO:11).
FIG. 3 is a vector map of pSGE-05959 that included an AfeI site for insertion of putative ARSs. The vector included a blasticidin resistance gene (“Blast 150”; SEQ ID NO:19) driven by the Nannochloropsis TCTP promoter (SEQ ID NO:18) and also included a PMB1 ori and ampicillin resistance gene (Apr) for replication and selection in E. coli. 
FIG. 4 are photographs of transformation plates showing E. coli colonies resulting from transformation with uncut DNA isolated with Nannochloropsis lines that had been transformed with EDMs that included NgARS2.
FIG. 5 is a vector map of pSGE-06067 that included NgARS2. The vector included a blasticidin resistance gene (“Blast 150”; SEQ ID NO:19) driven by the Nannochloropsis TCTP promoter (SEQ ID NO:18). Also included in the vector was a gene encoding TurboGFP (SEQ ID NO:21) driven by Nannochloropsis promoter 5303630 (SEQ ID NO:22) and a gene encoding a Cyclotella enoyl ACP reductase (293 FabI; SEQ ID NO:24). The vector also included a PMB1 ori and ampicillin resistance gene (Apr) for replication and selection in E. coli. 
FIG. 6 is a photograph of a Southern blot of DNA extracted from Nannochloropsis lines transformed with a vector (EDM) that included NgARS2 and digested with an enzyme that does not cut the EDM. Above the lanes designated for each transformed Nannochloropsis line, data is provided on the number of colonies that resulted from transforming E. coli with the DNA extracted from that line, along with the results of PCR to detect the GFP gene and ARS, and a determination of whether the line was considered to be carrying an EDM (“EDM call”).
FIG. 7 is a photograph of a Western blot of extracts from Nannochloropsis lines transformed with a vector that included NgARS2. Separated proteins were detected with anti-GFP or anti-ENR antibodies. Above the lanes designated for each transformed Nannochloropsis line, data is provided on the number of colonies that resulted from transforming E. coli with the DNA extracted from that line, along with the results of PCR to detect the GFP gene and ARS, and a determination of whether the line was considered to be carrying an EDM (“EDM call”).
FIG. 8 is a graph showing that Nannochloropsis lines transformed with an EDM that included NgARS2 retained the EDM when maintained under blasticidin selection, as evaluated by the expression of the GFP protein, and lost expression of the GFP protein when blasticidin selection was removed.



DETAILED DESCRIPTION
Definitions
Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.
The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. 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”.
“About” means within plus or minus 10% of the provided value, inclusive of the provided value. Where ranges are provided, they are inclusive of the boundary values.
As used herein, “amino acid” refers to naturally-occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally-occurring amino acids. Naturally-occurring amino acids are those encoded by the genetic code, including D/L optical isomers, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally-occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally-occurring amino acid. Amino acid mimetics, as used herein, refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally-occurring amino acid.
An “Autonomously Replicating Sequence” or “ARS”, sometimes referred to as an “Autonomous Replication Sequence” is a sequence that serves as an origin of DNA replication on eukaryotic chromosomes. An ARS, when incorporated into a DNA molecule, supports replication of the DNA molecule by binding a protein complex that unwinds and replicates the DNA. An ARS can be confirmed (functionally validated) by incorporating the sequence into a DNA molecule that is not self-replicating in a given host and demonstrating that the DNA molecule replicates autonomously in the host only when the ARS is present.
A “nucleic acid construct”, “DNA construct” or simply “construct” is a nucleic acid molecule produced by recombinant means that includes at least two operably linked nucleic acid sequences that are not operably linked to one another in nature.
An “episomal DNA molecule” or “EDM” is an independently replicating nucleic acid molecule that is not integrated into the genome of the host organism in which the EDM resides and replicates. An EDM may be stable, in which it persists for many generations or unstable, where the EDM is gradually diluted out of the population by successive cell divisions. A stable EDM may be maintained in a cell population by selective pressure (e.g., the presence of an antibiotic).
A “detectable marker” is a gene or the polypeptide encoded by the gene that confers some detectable phenotype on a cell that expresses the gene. Detection can be colorometric (for example, the blue color by expression of beta galactosidease or beta-glucorinidase in the present of a colorometric substrate) or by detection of luminescence or fluorescence. A detectable marker generally encodes a detectable polypeptide, for example, a green fluorescent protein or a signal producing enzyme such as luciferase, which, when contacted with an appropriate agent (a particular, wavelength of light or luciferin, respectively) generates a signal that can be detected by eye or using appropriate instrumentation (Giacomin, Plant Sci. 116:59-72, 1996; Scikantha, J. Bacteriol. 178:121, 1996; Gerdes, FEBS Lett. 389:44-47, 1996; see, also, Jefferson, EMBO J. 6:3901-3907, 1997, fl-glucuronidase).
The term or “selectable marker” or “selection marker” refers to a gene (or the encoded polypeptide) that confers a phenotype that allow the organism expressing the gene to survive under selective conditions. For example, a selectable marker generally is a molecule that, when present or expressed in a cell, provides a selective advantage (or disadvantage) to the cell containing the marker, for example, the ability to grow in the presence of an agent that otherwise would kill the cell, or the ability to grow in the absence of a particular nutrient.
A “cDNA” is a DNA molecule that comprises at least a portion the nucleotide sequence of an mRNA molecule, with the exception that the DNA molecule substitutes the nucleobase thymine, or T, in place of uridine, or U, occurring in the mRNA sequence. A cDNA can be single-stranded or double-stranded, and can be the complement of the mRNA sequence. In preferred embodiments, a cDNA does not include one or more intron sequences that occur in the naturally-occurring gene (in the genome of an organism) that the cDNA corresponds to. For example, a cDNA can have sequences from upstream (5′) of an intron of a naturally-occurring gene juxtaposed to sequences downstream (3′) of the intron of the naturally-occurring gene, where the upstream and downstream sequences are not juxtaposed in a DNA molecule (i.e., the naturally occurring gene) in nature. A cDNA can be produced by reverse transcription of mRNA molecules by a polymerase (e.g., a reverse transcriptase), or can be synthesized, for example, by chemical synthesis and/or by using one or more restriction enzymes, one or more ligases, one or more polymerases (including, but not limited to, high temperature tolerant polymerases that can be used in polymerase chain reactions (PCRs)), one or more recombinases, etc., based on knowledge of the cDNA sequence, where the knowledge of the cDNA sequence can optionally be based on the identification of coding regions from genome sequences or compiled from the sequences of multiple partial cDNAs.
A “coding sequence” or “coding region”, as used herein in reference to an mRNA or DNA molecule, refers to the portion of the mRNA or DNA molecule that codes for a polypeptide. It typically consists of the nucleotide residues of the molecule which are matched with an anticodon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding sequence may thus include nucleotide residues corresponding to amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).
“Down-regulation” refers to regulation that decreases production of expression products (mRNA, polypeptide, biological activity, or combinations of any thereof) relative to basal or native states.
“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting there from. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
The term “endogenous,” within the context of the present disclosure refers to any polynucleotide, polypeptide or protein sequence which is a natural part of a cell or organism.
“Exogenous” with respect to a nucleic acid or gene indicates that the nucleic acid or gene is part of a recombinant nucleic acid construct and is not in its natural environment. For example, an exogenous nucleic acid or gene is from one species and has been introduced (“transformed”) into another organism, microorganism, or cell by human intervention. Typically, such an exogenous nucleic acid or gene into a cell or organism via a recombinant nucleic acid construct. An exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. An exogenous nucleic acid can also be a sequence that is native or endogenous to an organism (i.e., the nucleic acid sequence occurs naturally in that species or encodes a polypeptide that occurs naturally in the host species) and that has been reintroduced into cells of that organism. An exogenous nucleic acid that includes an endogenous sequence can often be distinguished from the naturally-occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking an endogenous gene sequence in a recombinant nucleic acid construct. Alternatively or in addition, a stably transformed exogenous nucleic acid can be detected or distinguished from a native gene by its juxtaposition to sequences in the genome where it has integrated. A nucleic acid is considered exogenous if it has been introduced into a progenitor of the cell, organism, or strain under consideration.
As used herein, “expression” refers to the process of converting genetic information of a polynucleotide into RNA through transcription, which is typically catalyzed by an enzyme, RNA polymerase, and into protein, through translation of mRNA on ribosomes.
“Expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to a nucleotide sequence of interest, which is—optionally—operably linked to termination signals and/or other regulatory elements. An expression cassette may also comprise sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a non-translated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one, which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. An expression cassette may be assembled entirely extra-cellularly (e.g., by recombinant cloning techniques). However, an expression cassette may also be assembled using in part endogenous components. For example, an expression cassette may be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence, which thereby becomes functionally linked and controlled by said promoter sequences. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus.
“Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Examples of expression vectors known in the art include cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
“Fragment”, as applied to a nucleic acid, refers to a clone or any part of a polynucleotide molecule that retains a usable, functional characteristic. Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation. In the context of the present disclosure, a fragment may ordinarily be any subsequence of a nucleic acid, typically of at least about 9 consecutive nucleotides, at least about 12 consecutive nucleotides, at least about 14 consecutive nucleotides, at least about 16 consecutive nucleotides, more typically from about 10 to 18 consecutive nucleotides, typically from at least about 12 to 16 consecutives nucleotides, even more typically from at least about 10 to 16 consecutive nucleotides, of any one of the nucleotide sequences provided herein in the Sequence Listing.
An “oligonucleotide”, as used herein, is a nucleic acid molecule 200 or fewer nucleotides in length. An oligonucleotide can be RNA, DNA, or a combination of DNA and RNA, a nucleic acid derivative, or a synthetic nucleic acid, for example, an oligonucleotide can be a peptide nucleic acid or a locked nucleic acid, and can be single-stranded, double-stranded, or partially single-stranded and partially double-stranded. An oligonucleotide can be, for example, between about 4 and about 200 nucleotides in length, between about 6 and about 200 nucleotides in length, between about 10 and about 200 nucleotides in length, between about 15 and about 200 nucleotides in length, between about 17 and about 200 nucleotides in length, between about 20 and about 200 nucleotides in length, or between about 40 and about 200 nucleotides in length. In additional examples, an oligonucleotide can be between about 15 and about 180 nucleotides in length, between about 15 and about 160 nucleotides in length, between about 15 and about 140 nucleotides in length, between about 15 and about 120 nucleotides in length, between about 17 and about 100 nucleotides in length, between about 17 and about 80 nucleotides in length, or between about 17 and about 70 nucleotides in length, for example between about 20 and about 65 nucleotides in length.
When used in reference to a polynucleotide, a gene, a nucleic acid, a polypeptide, or an enzyme, the term “heterologous” refers to a polynucleotide, gene, a nucleic acid, polypeptide, or an enzyme not derived from the host species, e.g., is from a different species with respect to the host cell. For example, a transgenic Nannochloropsis microorganism transformed with the coding sequence for a fatty acid desaturase from a Tetraselmis microorganism or from a plant is transformed with a heterologous desaturase gene. When referring to nucleic acid sequences operably linked or otherwise joined to one another (“juxtaposed”) in a nucleic acid construct or molecule, “heterologous sequences”, as used herein, are those that are not operably linked or are not in proximity or contiguous to each other in nature. As such, elements operably linked or contiguous to each other in nature are not heterologous to each other. For example, a promoter from Tetraselmis sp. is considered heterologous to a Nannochloropsis coding region sequence. Also, a promoter from a gene encoding a tubulin gene from Nannochloropsis is considered heterologous to a sequence encoding a Nannochloropsis fatty acid desaturase. Similarly, when referring to a gene regulatory sequence or to an auxiliary nucleic acid sequence used for maintaining or manipulating a gene sequence (e.g. a 5′ un-translated region, 3′ un-translated region, Kozak sequence, poly A addition sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genome homology region, recombination site, etc.), “heterologous” means that the regulatory sequence or auxiliary sequence is from a different source (e.g., different gene, whether from the same or different species as the host organisms) than the gene with which the regulatory or auxiliary nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome.
The term “hybridization”, as used herein, refers generally to the ability of nucleic acid molecules to join via complementary base strand pairing. Such hybridization may occur when nucleic acid molecules are contacted under appropriate conditions and/or circumstances. As used herein, two nucleic acid molecules are said to be capable of specifically hybridizing to one another if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure. A nucleic acid molecule is said to be the “complement” of another nucleic acid molecule if they exhibit complete complementarity. As used herein, nucleic acid molecules are said to exhibit “complete complementarity” when every nucleotide of one of the molecules is complementary to its base pairing partner nucleotide of the other. Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional “low-stringency” conditions. Similarly, the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional “high-stringency” conditions. Nucleic acid molecules that hybridize to other nucleic acid molecules, e.g., at least under low stringency conditions are said to be “hybridizable cognates” of the other nucleic acid molecules. Conventional stringency conditions are described by Sambrook et al., 1989, supra), and by Haymes et al. In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985). Departures from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure. Thus, in order for a nucleic acid molecule or fragment thereof of the present invention to serve as a primer or probe it needs only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed.
Appropriate stringency conditions which promote DNA hybridization include, for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at about 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed. These conditions are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, low stringency conditions may be used to select nucleic acid sequences with lower sequence identities to a target nucleic acid sequence. One may wish to employ conditions such as about 0.15 M to about 0.9 M sodium chloride, at temperatures ranging from about 20° C. to about 55° C. High stringency conditions may be used to select for nucleic acid sequences with higher degrees of identity to the disclosed nucleic acid sequences (Sambrook et al., 1989, supra). In one embodiment of the present invention, high stringency conditions involve nucleic acid hybridization in about 2×SSC to about 10×SSC (diluted from a 20×SSC stock solution containing 3 M sodium chloride and 0.3 M sodium citrate, pH 7.0 in distilled water), about 2.5× to about 5×Denhardt's solution (diluted from a 50× stock solution containing 1% (w/v) bovine serum albumin, 1% (w/v) ficoll, and 1% (w/v) polyvinylpyrrolidone in distilled water), about 10 mg/mL to about 100 mg/mL fish sperm DNA, and about 0.02% (w/v) to about 0.1% (w/v) SDS, with an incubation at about 50° C. to about 70° C. for several hours to overnight. High stringency conditions are typically provided by 6×SSC, 5×Denhardt's solution, 100 mg/mL fish sperm DNA, and 0.1% (w/v) SDS, with incubation at 55×C for several hours. Hybridization is generally followed by several wash steps. The wash compositions generally comprise 0.5×SSC to about 10×SSC, and 0.01% (w/v) to about 0.5% (w/v) SDS with an incubation for 15-min at about 20° C. to about 70° C. Typically, the nucleic acid segments remain hybridized after washing at least one time in 0.1×SSC at 65° C.
“Percentage of sequence identity,” as used herein, is determined by comparing two optimally locally aligned sequences over a comparison window defined by the length of the local alignment between the two sequences. The amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Local alignment between two sequences only includes segments of each sequence that are deemed to be sufficiently similar according to a criterion that depends on the algorithm used to perform the alignment (e.g. BLAST). The percentage identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (Add. APL. Math. 2:482, 1981), by the global homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85: 2444, 1988), by heuristic implementations of these algorithms (NCBI BLAST, WU-BLAST, BLAT, SIM, BLASTZ), or by inspection. GAP and BESTFIT, for example, can be employed to determine their optimal alignment of two sequences that have been identified for comparison. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. The term “substantial sequence identity” between polynucleotide or polypeptide sequences refers to polynucleotide or polypeptide comprising a sequence that has at least 50% sequence identity, for example, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using the programs. In addition, pairwise sequence homology or sequence similarity, as used refers to the percentage of residues that are similar between two sequences aligned. Families of amino acid residues having similar side chains have been well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
For example, query nucleic acid and amino acid sequences can be searched against subject nucleic acid or amino acid sequences residing in public or proprietary databases. Such searches can be done using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST v 2.18) program. The NCBI BLAST program is available on the interne from the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov/Blast.cgi). Exemplary parameters for NCBI BLAST include: Filter options set at “default”, the Comparison Matrix set to “BLOSUM62”, the Gap Costs set to “Existence: 11, Extension: 1”, the Word Size set to 3, the Expect (E threshold) set to 1e-3, and the minimum length of the local alignment set to 50% of the query sequence length. Sequence identity and similarity may also be determined using GENOMEQUESTTM software (Gene-IT, Worcester, Mass. USA).
As used herein, an “isolated” nucleic acid or protein is removed from its natural milieu or the context in which the nucleic acid or protein exists in nature. For example, an isolated protein or nucleic acid molecule is removed from the cell or organism with which it is associated in its native or natural environment. As such, an “isolated” nucleic acid typically is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the cell of the organism from which the nucleic acid is derived. An isolated nucleic acid or protein can be, in some instances, partially or substantially purified, but no particular level of purification is required for isolation. For example, an isolated nucleic acid molecule can be a nucleic acid sequence that has been excised from the chromosome, genome, or episome that it is integrated into in nature. Thus, an isolated nucleic acid includes, without limitation, a nucleic acid that exists as a purified molecule, or a nucleic acid molecule that is incorporated into a vector or a recombinant cell.
A “purified” nucleic acid molecule or nucleotide sequence is substantially free of cellular material and cellular components. The purified nucleic acid molecule may be free of chemicals beyond buffer or solvent, for example. “Substantially free” is not intended to mean that other components beyond the novel nucleic acid molecules are undetectable. In some circumstances “substantially free” may mean that the nucleic acid molecule or nucleotide sequence is free of at least 95% (w/w) of cellular material and components.
The term “mis-expression” refers to an increase or decrease in the transcription of a coding region into a complementary RNA sequence as compared to the parental wild-type, for example, alga or plant. This term also encompasses expression of a gene or coding region for a different time period as compared to the wild-type and/or from a non-natural location within the parental genome. The term “overexpression” or “increased expression” as used herein refers to a greater expression level of a gene, a polynucleotide sequence, or a polypeptide, in a host cell compared to a wild-type cell or a wild-type organism, at any developmental or temporal stage. Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters (e.g. constitutive promoters), the use of transcription enhancers or translation enhancers. Overexpression may also under control of an inducible or a growth-phase specific promoter. These or other inducible or phase-specific promoters may be incorporated into an expression cassette comprising a transcription factor polynucleotide of the invention, where the promoter is operably linked to the transcription factor polynucleotide, can be envisioned and produced. Thus, overexpression may occur throughout an algal cell for example, in specific growth phases of the algal cell or in the presence or absence of particular environmental signals, depending on the promoter used.
The term “native” is used herein to refer to nucleic acid sequences or amino acid sequences as they naturally occur in the host. The term “non-native” is used herein to refer to nucleic acid sequences or amino acid sequences that do not occur naturally in the host, or are not configured as they are naturally configured in the host. A nucleic acid sequence or amino acid sequence that has been removed from a host cell, subjected to laboratory manipulation, and introduced or reintroduced into a host cell is considered “non-native.” Synthetic or partially synthetic genes introduced into a host cell are “non-native.” Non-native genes further include genes endogenous to the host microorganism operably linked to one or more heterologous regulatory sequences that have been recombined into the host genome, or genes endogenous to the host organism that are in a locus of the genome other than that where they naturally occur.
In reference to a nucleic acid molecule or a polypeptide, the term's “naturally-occurring” 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 may be present in and isolated from a natural source, and is not intentionally modified by human manipulation.
The term's “nucleic acid molecule” and “polynucleotide molecule” are used interchangeably herein, and refer to both DNA and RNA molecule, including cDNA, genomic DNA, synthetic DNA, and DNA or RNA containing nucleic acid analogs. Polynucleotides can have any three-dimensional structure. Polynucleotides can be natural-occurring or synthetic origin. A nucleic acid molecule can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand). Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, nucleic acid probes and nucleic acid primers. A polynucleotide may contain unconventional or modified nucleotides.
As used herein, “operably linked” is intended to mean a functional linkage between two or more sequences such that activity at or on one sequence affects activity at or on the other sequence(s). For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the polynucleotide of interest. In this sense, the tend “operably linked” refers to the positioning of a regulatory region and a coding sequence to be transcribed so that the regulatory region is effective for regulating transcription or translation of the coding sequence of interest. For example, to operably link a coding sequence and a regulatory region, the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the regulatory region. A regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by “operably linked” is intended that the coding regions are in the same reading frame. When used to refer to the effect of an enhancer, “operably linked” indicated that the enhancer increases the expression of a particular polypeptide or polynucleotides of interest. “Juxtaposed with” in the context of nucleic acid sequences, means the referenced sequences are part of the same continuous nucleic acid molecule.
The tends “polynucleotide sequence” and “nucleic acid sequence” as used herein interchangeably refer to a sequence of a polynucleotide molecule, and can refer, for example, to DNA or RNA sequences. The nomenclature for nucleotide bases as set forth in 37 CFR §1.822 is used herein.
A “promoter” refers to a transcription control sequence that is capable of initiating transcription in a host cell and can drive or facilitate transcription of a nucleotide sequence or fragment thereof of the instant invention. Such promoters need not be of naturally-occurring sequences. In addition, it will be understood that such promoters need not be derived from the target host cell or host organism.
“Polypeptide” and “protein” are used interchangeably herein and refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics, regardless of post-translational modification, e.g., phosphorylation or glycosylation. The subunits may be linked by peptide bonds or other bonds such as, for example, ester or ether bonds. Full-length polypeptides, truncated polypeptides, point mutants, insertion mutants, splice variants, chimeric proteins, and fragments thereof are encompassed by this definition. In various embodiments the polypeptides can have at least 10 amino acids or at least 25, or at least 50 or at least 75 or at least 100 or at least 125 or at least 150 or at least 175 or at least 200 amino acids.
As used herein “progeny” means a descendant, offspring, or derivative of an organism. For example, daughter cells from a transgenic alga are progeny of the transgenic alga. Because certain modifications may occur in succeeding generations due to mutations or environmental influences, such progeny, descendant, 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.
The terms “recombinant” or “engineered” as used herein in reference to a nucleic acid molecule, refer to a nucleic acid molecule that has been altered through human intervention. As non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector. As non-limiting examples, a recombinant nucleic acid molecule: 1) has been synthesized or modified in vitro, for example, using chemical or enzymatic techniques (for example, by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, e.g., methylation), or recombination (including homologous and site-specific recombination)) of nucleic acid molecules; 2) includes conjoined nucleotide sequences that are not conjoined in nature; 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleic acid molecule sequence; and/or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleic acid sequence.
The term “recombinant protein” as used herein refers to a protein produced by genetic engineering, for example, by expression of a genetically engineered nucleic acid molecule in a cell.
The term “regulatory region” “regulatory sequence”, “regulatory element”, or “regulatory element sequence”, as used in the present invention, refer to a nucleotide sequence that influences transcription or translation initiation or rate, and stability and/or mobility of a transcription or translation product. Such regulatory regions need not be of naturally-occurring sequences. Regulatory sequences include but are not limited to promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ un-translated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. A regulatory region typically comprises at least a core (basal) promoter. A regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
As used herein, “transgenic organism” refers to an organism which comprises a heterologous polynucleotide. When applied to organisms, the terms “transgenic” or “recombinant” or “engineered” or “genetically engineered,” used interchangeably herein, refer to organisms that have been manipulated by introduction into the organism of an exogenous or recombinant nucleic acid sequence. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations, although it can also be present on an episome, and may be present on a synthetic chromosome of the transgenic organism. The non-native polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. In additional examples, a transgenic microorganism can include an introduced exogenous regulatory sequence operably linked to an endogenous gene of the transgenic microorganism. Non-limiting examples of such manipulations include gene knockouts, targeted mutations and gene replacement, promoter replacement, deletion, or insertion, as well as introduction of transgenes into the organism. Recombinant or genetically engineered organisms can also be organisms into which constructs for gene “knock down” have been introduced. Such constructs include, but are not limited to, RNAi, microRNA, shRNA, antisense, and ribozyme constructs. Also included are organisms whose genomes have been altered by the activity of meganucleases, zinc finger nucleases, or Crisper nucleases. As used herein, “recombinant microorganism” or “recombinant host cell” includes progeny or derivatives of the recombinant microorganisms of the invention. Because certain modifications may occur in succeeding generations from either 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.
For nucleic acids and polypeptides, the term “variant” is used herein to denote a polypeptide, protein, or polynucleotide molecule with some differences, generated synthetically or naturally, in their base or amino acid sequences as compared to a reference polypeptide or polynucleotide, respectively, such that the variant has at least 70% sequence identity with the reference polypeptide or polynucleotide. In other embodiments the variant can have at least 80% or at least 90% or at least 95% or at least 97% or at least 98% or at least 99% sequence identity with the reference polypeptide or polynucleotide. For example, these differences include substitutions, insertions, deletions or any desired combinations of such changes in a reference polypeptide or polypeptide. Polypeptide and protein variants can further consist of changes in charge and/or post-translational modifications (such as glycosylation, methylation. phosphorylation, etc.). When the term “variant” is used in reference to a microorganism, it typically refers to a strain microbial strain having identifying characteristics of the species to which it belongs, while having at least one nucleotide sequence variation or identifiably different trait with respect to the parental strain, where the trait is genetically based (heritable). For example, for a Nannochloropsis strain, identifiable traits include the lack of chlorophyll b and c, which is different from other related microalgae.
A “vector” is any genetic element capable of serving as a vehicle of genetic transfer, expression, or replication for a foreign polynucleotide in a host cell. For example, a vector may be an artificial chromosome or a plasmid, and may be capable of stable integration into a host cell genome, or it may exist as an independent genetic element (e.g., episome, plasmid). A vector may exist as a single polynucleotide or as two or more separate polynucleotides. Vectors may be single copy vectors or multicopy vectors when present in a host cell. Preferred vectors for use in the present invention are expression vector molecules in which one or more functional genes can be inserted into the vector molecule, in proper orientation and proximity to expression control elements resident in the expression vector molecule so as to direct expression of one or more proteins when the vector molecule resides in an appropriate (e.g. homologous) host cell.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and embodiments will be apparent to those of skill in the art upon review of this disclosure.
Autonomously Replicating Sequences (ARSs)
The invention provides ARSs isolated from the heterokont (stramenopile) alga Nannochloropsis. 
In a first aspect, provided herein is an isolated or recombinant nucleic acid molecule that includes an ARS, in which the ARS includes a nucleic acid sequence having at least 85% identity to SEQ ID NO:13 or SEQ ID NO:14. The ARS can be juxtaposed with or adjacent to at least one heterologous nucleic acid sequence, i.e., a sequence not juxtaposed with or adjacent to the ARS in the genomic of the organism from which the ARS is derived. In some examples, provided herein is a nucleic acid molecule that includes an ARS having a nucleic acid sequence with at least 85% identity to SEQ ID NO:13 or SEQ ID NO:14 that also includes one or more nucleic acid sequence s that are not derived from the organism from with the ARS is derived. For example, an ARS as provided herein can be juxtaposed with or adjacent to a nucleic acid sequence not isolated or derived from an rDNA repeat region of the genome of an organism from which the ARS is derived. In this context, juxtaposed means the ARS is on the same nucleic acid molecule, i.e., is part of the same nucleotide polymer that includes the heterologous sequence. For example an ARS having a nucleic acid sequence having homology to SEQ ID NO:13 or SEQ ID NO:14 can be directly or indirectly linked to a heterologous sequence such as a reporter gene, a selectable marker gene, a gene encoding a polypeptide of interest, or a gene encoding a functional RNA that is preferably not a ribosomal RNA. The nucleic acid sequence having at least 85% identity to SEQ ID NO:13 or SEQ ID NO:14 can have, for example, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:13 or SEQ ID NO:14. In some examples, an isolated or recombinant nucleic acid molecule includes a nucleic acid sequence having at least 85% identity to SEQ ID NO:13 and additionally includes a nucleic acid sequence having at least 85% identity to SEQ ID NO:14. The nucleic acid molecule can be a DNA molecule.
An ARS having at least 85% identity to SEQ ID NO:13 or SEQ ID NO:14 can be a naturally-occurring sequence, for example, a nucleic acid sequence isolated from a plant, animal, fungal, algal, or heterokont cell, or can be a variant of a naturally-occurring sequence. For example, an ARS as provided herein can be a naturally-occurring sequence isolated from an algal or heterokont species. Isolated sequences can be tested for ARS function using methods provided herein, and can be truncated to test for ARS function of fragments of reduced size.
An ARS as provided herein is functional in a eukaryotic cell, for example, a microbial cell such as a plant, animal, fungal, algal, or heterokont cell. For example, an ARS as provided herein and may be functional in a diatom or Eustigmatophyte cell, for example, a Nannochloropsis cell.
Nonlimiting examples of algal cells in which an ARS as provided herein can be functional include Amphora, Ankistrodesmus, Aplanochytrium, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Carteria, Chaetoceros, Chlamydomonas, Chlorella, Chlorococcum, Chlorogonium, Chrococcidiopsis, Chroomonas, Chrysophyceae, Chrysosphaera, Colwellia, Cricosphaera, Crypthecodinium, Cryptococcus, Cryptomonas, Cunninghamella, Cyclotella, Desmodesmus, Dunaliella, Elina, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Fragilaria, Fragilariopsis, Franceia, Gloeothamnion, Haematococcus, Hantzschia, Heterosigma, Hymenomonas, Isochrysis, Japanochytrium, Labrinthula, Labyrinthomyxa, Labyrinthula, Lepocinclis, Micractinium, Monodus, Monoraphidium, Moritella, Mortierella, Mucor, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Parachlorella, Parietochloris, Pascheria, Pavlova, Pelagomonas, Phaeodactylum, Phagus, Pichia, Picochlorum, Pithium, Platymonas, Pleurochrysis, Pleurococcus, Porphyridium, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Rhodosporidium, Scenedesmus, Schizochlamydella, Schizochytrium, Skeletonema, Spirulina, Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira, Thraustochytrium, Tribonema, Ulkenia, Vaucheria, Vibrio, Viridiella, Vischeria, and Volvox. 
Heterokont species in which an ARS as disclosed herein may be functional include, but are not limited to, species of the taxonomic groups Bacillariophytes (diatoms), Eustigmatophytes, Labrinthulids, and Thraustochytrids. In some examples, a strain used in the invention may be a species of Labrinthulid or Thraustochytrid such as Labryinthula, Labiyinthuloides, Thraustochytrium, Schizochytrium, Aplanochytrium, Aurantiochytrium, Japonochytrium, Diplophrys, or Ulkenia. 
Exemplary diatoms may include members of the genera Achnanthes, Amphora, Chaetoceros, Coscinodiscus, Cylindrotheca, Cyclotella, Cymbella, Hantzschia, Navicula, Nitzschia, Pavlova, Pseudo-Nitzschia, Phaeodactylum, Psammodictyon, Skeletonema, Thalassionema, and Thallasiosira. Eustigmatophytes that can be used for genetic modification or nucleic acid isolation include, for example, species of Eustigmatos, Monodus, Nannochloropsis, and Vischeria. For example, microorganisms for genetic modification or nucleic acid isolation as disclosed herein include members of the genus Nannochloropsis. Particularly suitable species include N. gaditana, N. granulata, N. limnetica, N. maritime, N. oceanica, N. oculata, and N. salina. Preferred species within the genus Nannochloropsis include, but are not limited to, N. gaditana, N. oceanica, N. oculata, and N. salina. 
Functionality of an ARS can be tested using the methods provided in the Examples herein, for example, an ARS can be tested by inserting the ARS into a circular vector that includes a selectable marker, and verifying that the circular vector replicates independently (i.e., is an EDM) with, but not without, the ARS. For example, an independently replicating vector having a selectable marker can be tested by isolating uncut DNA from the host grown under selection and demonstrating transformation of E. coli with the vector (where the vector also includes an origin of replication and selectable marker functional in E. coli), demonstration by gel electrophoresis that the vector is maintained in the host in circular form, and/or demonstration (by PCR or DNA sequencing) that vector sequences are maintained intact in the host.
In some examples, the nucleic acid molecule that includes an ARS that includes a nucleic acid sequence having at least 85% identity to SEQ ID NO:13 is an ARS having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1, SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; or SEQ ID NO:10. In some examples, the nucleic acid molecule includes an ARS that includes a nucleic acid sequence that has at least 85% identity to SEQ ID NO:14 and has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:11; or SEQ ID NO:12.
For example, a nucleic acid molecule as provided herein in various examples can include an ARS having at least 85% or at least 90% identity to SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:11; or SEQ ID NO:12, juxtaposed with a heterologous nucleic acid sequence, e.g., a nucleic acid sequence not derived from the rDNA region of the genome of an organism, such as the rDNA region of the genome of Nannochloropsis. In exemplary embodiments, a nucleic acid molecule as provided herein can include an ARS having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:11; or SEQ ID NO:12, juxtaposed with a heterologous nucleic acid sequence, e.g., a nucleic acid sequence not derived from the rDNA region of the genome of an organism, such as the rDNA region of the genome of Nannochloropsis. In various examples, the ARS has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:6.
In another aspect, provided herein are episomal DNA molecules (EDMs) that include an ARS having at least 85% identity to at least 150 contiguous nucleotides of SEQ ID NO:1; at least 150 contiguous nucleotides of SEQ ID NO:5; at least 150 contiguous nucleotides of SEQ ID NO:6; at least 150 contiguous nucleotides of SEQ ID NO:7; at least 150 contiguous nucleotides of SEQ ID NO:8; at least 150 contiguous nucleotides of SEQ ID NO:9; at least 150 contiguous nucleotides of SEQ ID NO:10; at least 150 contiguous nucleotides of SEQ ID NO:11; or at least 150 contiguous nucleotides of SEQ ID NO:12. For example, an EDM as provided herein can include an ARS having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to at least 150 contiguous nucleotides of SEQ ID NO:1; at least 150 contiguous nucleotides of SEQ ID NO:5; at least 150 contiguous nucleotides of SEQ ID NO:6; at least 150 contiguous nucleotides of SEQ ID NO:7; at least 150 contiguous nucleotides of SEQ ID NO:8; at least 150 contiguous nucleotides of SEQ ID NO:9; at least 150 contiguous nucleotides of SEQ ID NO:10; at least 150 contiguous nucleotides of SEQ ID NO:11; or at least 150 contiguous nucleotides of SEQ ID NO:12. An EDM as provided herein can be circular. The EDM can further include a selectable marker gene. The EDM can replicate autonomously in a eukaryotic host cell, such as a plant or animal cell or a cell of a microorganism, such as, for example, a fungal, heterokont, or algal cell, including but not limited to those disclosed hereinabove.
In various examples, an EDM as provided herein can include an ARS having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, or at least 750 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. For example, an EDM as provided herein can include an ARS having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, or at least 750 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:12. In particular examples, an EDM as provided herein can include an ARS having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to at least at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, or at least 750 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:12. In further examples, an EDM as provided herein can include an ARS having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:12. In yet further examples, an EDM can include the ARS of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:12. For example, provided herein is an EDM comprising the ARS of SEQ ID NO:6.
An EDM as provided herein can further comprise a nucleic acid sequence encoding a polypeptide. For example, an EDM can include a nucleic acid sequence encoding a reporter protein, such as a fluorescent protein or signal-producing enzyme, or a selectable marker protein, such as a protein that confers resistance to a drug, antibiotic, toxin, or herbicide or a protein that allows autotrophic growth on stringent media.
Detectable markers or reporter genes can include genes encoding a variety of fluorescent proteins, including without limitation green, cyan, blue, yellow, and red fluorescent proteins and their variants. Other fluorescent or chromogenic markers that have been used include luciferase (Falciatore et al., J. Mar. Biotechnol., 1: 239-251, 1999; Fuhrmann et al., Plant Mol. Biol., 2004; Jarvis and Brown, Curr. Genet., 19: 317-322, 1991), β-glucuronidase (Chen et al., 2001, supra; Cheney et al., 2001, supra; Chow and Tung, 1999, supra; El-Sheekh, 1999, supra; Falciatore et al., 1999, supra; Kubler et al., J. Mar. Biotechnol., 1:165-169, 1994), β-galactosidase (Gan et al., J. Appl. Phycol., 15:345-349, 2003; Jiang et al., Plant Cell Rep., 21:1211-1216, 2003; Qin et al., High Technol. Lett., 13:87-89, 2003). Further nonlimiting examples of enzymes that can be used for detecting a colored or labeled product include aryl sulfatase and chloramphenicol acetyl transferase.
A selectable marker can provide a means to obtain heterokont cells, algal cells, yeast cell, plant cells or any combination that express the marker and, therefore, include the EDM and can therefor be useful as a component of an EDM of the present disclosure (see, for example, Bock, supra, 2001). Examples of selectable markers include deaminase, such as the deaminase from Aspergillus terreus, which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59:2336-2338, 1995), as well as genes conferring resistance to antibiotics such as bleomycin, gentamycin, glyphosate, hygromycin, kanamycin, neomycin, phleomycin, puromycin, spectinomycin, and streptomycin. For example, neomycin phosphotransferase confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2:987-995, 1983) and the “hygro” gene confers resistance to hygromycin (Marsh, Gene 32:481-485, 1984). Several different antibiotic resistance genes have been used successfully for selection of microalgal transformants, including blastocydin, bleomycin (see, for example, Apt et al., 1996, supra; Fischer et al., 1999, supra; Fuhrmann et al., Plant J., 19, 353-61, 1999, Lumbreras et al., Plant J., 14(4):441-447, 1998; Zaslayskaia et al., J. Phycol., 36:379-386, 2000), spectinomycin (Cerutti et al., Genetics, 145: 97-110, 1997; Doetsch et al., Curr. Genet., 39, 49-60, 2001; Fargo, Mol. Cell. Biol., 19:6980-90, 1999), streptomycin (Berthold et al., Protist, 153:401-412, 2002), paromomycin (Jakobiak et al., Protist, supra.; Sizova et al., Gene, 277:221-229, 2001), nourseothricin (Zaslayskaia et al., 2000, supra), G418 (Dunahay et al., 1995, supra; Poulsen and Kroger, FEBS Lett., 272:3413-3423, 2005, Zaslayskaia et al., 2000, supra), hygromycin (Berthold et al., 2002, supra), chloramphenicol (Poulsen and Kroger, 2005, supra), and many others. Additional selectable markers for use in microalgae such as Chlamydomonas can be markers that provide resistance to kanamycin and amikacin resistance (Bateman, Mol. Gen. Genet. 263:404-10, 2000), zeomycin and phleomycin (e.g., ZEOCINTM pheomycin D1) resistance (Stevens, Mol. Gen. Genet. 251:23-30, 1996), and paramomycin and neomycin resistance (Sizova et al., 2001, supra).
Also considered are genes conferring resistance to antimetabolites such as methotrexate, e.g., genes encoding dihydrofolate reductase, (Reiss, Plant Physiol. (Life Sci. Adv.) 13:143-149, 1994); trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci., USA 85:8047, 1988); mannose-6-phosphate isomerase which allows cells to utilize mannose (WO 94/20627); ornithine decarboxylase, which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-omithine (DFMO; McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.). Additional selectable markers include those that confer herbicide resistance, for example, phosphinothricin acetyltransferase gene, which confers resistance to phosphinothricin (White et al., Nucl. Acids Res. 18:1062, 1990; Spencer et al., Theor. Appl. Genet. 79:625-631, 1990), a mutant EPSPV-synthase, which confers glyphosate resistance (Hinchee et al., BioTechnology 91:915-922, 1998), a mutant acetolactate synthase, which confers imidazolione or sulfonylurea resistance (Lee et al., EMBO J. 7:1241-1248, 1988), a mutant psbA, which confers resistance to atrazine (Smeda et al., Plant Physiol. 103:911-917, 1993), or a mutant protoporphyrinogen oxidase (see U.S. Pat. No. 5,767,373), or other markers conferring resistance to an herbicide such as glufosinate, sulfonamide, or phosphinotricin and sulfonylurea resistance (see, for example, Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Laboratory Press, 1995, page 39). Genes conferring resistance to antibiotics such as tetracycline; ampicillin, kanamycin, and chloramphenicol can be used for selection of the plasmid in prokaryotes such as E. coli. 
Auxotrophic markers are selectable markers that can be used in a host having a mutation in a gene encoding a metabolic enzyme, such as, for example, arginosuccinate lyase, for arginine synthesis, nitrate reductase for nitrogen assimilation (ability to utilize nitrate), thi10 for thiamine biosynthesis, and nic for nicotinamide biosynthesis.
Negative selection markers that may be included on an EDM include, without limitation, thymidine kinase (Lupton et al. (1991) Molecular and Cellular Biology 11: 3374-3378), DAOO (Erikson et al. (2004) Nature Biotechnology 22: 455-458) URA, and sacB (Quenee et al. (2005) Biotechniques 38: 63-67).
Alternatively or in addition to including a reporter gene or selectable marker gene, an EDM as provided herein can include a gene encoding a metabolic enzyme, structural protein, kinase, phosphatase, nucleotide cyclase, phosphodiesterase, transcriptional regulator, transcriptional activator, transporter, secretory protein, ion channel, receptor, photosynthetic protein, chaperonin, ribosomal protein, or nuclear scaffold protein. In various examples, an EDM as provided herein can include a gene encoding a meganuclease, a zinc finger nuclease, a TALEN protein, a cas protein, a recombinase, a topoisomerase, or a transposase.
Further alternatively or additionally, an EDM as provided herein can comprise a nucleic acid sequence encoding a functional RNA, such as, for example, an interfering RNA or a precursor thereof, an antisense RNA, a ribozyme, a micro RNA, or a guide RNA of a CRISPR system. Preferably, the functional RNA is not a ribosomal RNA. In various examples, the functional RNA is designed to attenuate expression of a targeted gene.
In further examples, alternatively or in addition to any of the above, an EDM can include sequences for mediating homologous or site specific recombination, wherein the sequences for homologous recombination include genomic sequences that flank or comprise at least a portion of a gene targeted for gene replacement or disruption, or can include sequences recognized by site-specific recombination systems.
The ARSs of the present invention may be used in DNA constructs to enhance replication. Therefore, methods of enhancing replication are also provided herein utilizing the ARSs described herein. A method of enhancing replication of a DNA construct can comprise, for example: (1) providing an ARS of the present invention in a DNA construct; and (2) transforming the DNA construct into a host cell.
The ARSs of the present invention may be used in artificial chromosomes and episomal nucleic acid molecules such as plasmids to facilitate their replication in host cells. ARS elements can often function in non-native species (Kim et al. (2007) Plasmid 58:228-39). Therefore, ARSs or consensus ARS elements disclosed herein may facilitate replication of a chromosome (synthetic or naturally derived) or plasmid (synthetic or naturally derived) in species beyond those of the Nannochloropsis genus.
For example, a host cell for an artificial chromosome or EDM comprising an ARS of the present invention may optionally be an algal cell, such as a cell of a species of Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Ghroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Fragilaropsis, Gloeothamnion, Haematococcus, Halocafeteria, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monoraphidium, 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, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, or Volvox. 
Exemplary diatoms include members of the genera Achnanthes, Amphora, Chaetoceros, Coscinodiscus, Cylindrotheca, Cyclotella, Cymbella, Fragilaria, Fragilaropsis, Hantzschia, Navicula, Nitzschia, Pavlova, Pseudo-Nitzschia, Phaeodactylum, Psammodictyon, Skeletonema, Thalassionema, Thallasiosira, and Vischeria. Examples of eustigmatophytes that may be hosts for a nucleic acid construct that includes an ARS as provided herein include not only Nannochloropsis species but also species of Monodus and Eustigmatos. In some examples, an alga of a species of the genus Nannochloropsis such as, but are not limited to, N. gaditana, N. granulata, N. limnetica, N. oceanica, N. oculata, and N. salina is transformed with a nucleic acid construct that includes an ARS as provided herein.
Alternatively or in addition, a host cell for an artificial chromosome or EDM comprising an ARS of the present invention may optionally be a heterokont cell, an animal cell, a plant cell, a yeast cell, a fungal cell, or a protist. For example, heterokonts include not only eustigmatophytes and diatoms such as those listed above but also chytrid species, including labrinthulids. In some examples, heterokont species considered for use in the invention include, but are not limited to, Bacillariophytes, Eustigmatophytes, Labrinthulids, and Thraustochytrids. In some examples, the strain may be a species of Labryinthula, Labryinthuloides, Thraustochytrium, Schizochytrium, Aplanochytrium, Aurantiochytrium, Japonochytrium, Diplophrys, or Ulkenia. For example, the strain may be a species of Thraustochytrium, Schizochytrium, or Aurantiochytrium. 
For example, an ARS sequence or variant, including a truncated variant of a validated ARS, or a region of DNA that includes a known or putative ARS can be assessed for function in a host species of interest by incorporating the known ARS, ARS sequence variant, or putative ARS into a circular DNA construct that includes a selectable marker functional in the host species of interest, and also includes, preferably an origin of replication for a bacterial strain that does not function in the host species of interest as well as a selectable marker that is functional in the bacterial strain. The circular DNA construct that includes the putative ARS or ARS variant can then be transformed into the host species of interest and transformants can be selected. Transformants can be analyzed to verify the presence of the circular molecule, for example, using PCR, restriction mapping, and/or Southern hybridization. Preferably the transformants are analyzed after multiple generations to verify the persistence of the construct, and further, the replication of the circular molecule can be further verified by isolating DNA from host species transformants under conditions in which circular DNA remains largely intact, transforming the isolated DNA into cells of the bacterial strain (for which the construct includes an origin of replication) and selecting bacterial transformants.
DNA can then be isolated from the bacterial transformants and analyzed by restriction digests and/or PCR for circular conformation, and used to retransform either or both of bacteria and the host species of interest. The persistence of the construct in cells of the host of interest together with the ability to transform bacteria (e.g., E. coli) with DNA isolated from the host species after multiple generations, indicates that the construct has been maintained in a non-integrated state, and therefore was able to replicate autonomously in the host species of interest.
Methods of Expressing a Gene
Methods are provided herein for propagating a gene of interest through at least eight generations of cell division, in which the method includes incorporating the gene of interest into and EDM as provided herein, transforming the EDM into a host organism, and propagating the host organism for at least eight generations under conditions in which the gene is expressed. In exemplary embodiments, the EDM includes a selectable marker gene, and the host organism is propagated under selection. The host organism can be, in various examples, an algal, heterokont, plant, animal, fungal organism, as described herein. For example, the host organism can be a microorganism, and may be a heterokont microorganism or microalga, including a diatom or Eustigmatophyte, for example, the microorganism may be a species of Nannochloropsis. In these methods, the EDM preferably includes a selectable marker gene, and the host organism is preferably propagated under selection, for example, in the presence of an antibiotic or toxin to which the selectable marker gene confers resistance.
In a related aspect of the invention, methods are provided for transiently expressing a gene or sequence of interest in a host cell, comprising transforming an EDM as provided herein into a host cell and culturing the host cell under conditions in which the gene or sequence is expressed. In exemplary embodiments the EDM includes a selectable marker gene, and the host cell is cultured in the presence of a selective agent for one or more generations. For example, the host cell may be cultured in the presence of the selective agent for one or more generations during which the gene or sequence of interest is expressed, and subsequently cultured in the absence of the selective agent for one or more additional generations, during or after which the gene or sequence of interest is not expressed.
EDM constructs that include a gene of interest for expression in a host cell can include regulatory sequences such as but not limited to promoter sequences and terminator sequences that are operably linked to the gene of interest. Such regulatory sequences, for example, promoter sequences, may be regulatable, e.g., inducible by environmental conditions or media components. Where a promoter is inducible, the method can further include inducing expression of the gene of interest. The promoters may be from the same genus or species as the host organism, or may be of a different species than the host organism.
A variety of known promoter sequences can be usefully deployed for transformation systems of microalgal and heterokont species. For example, the promoters commonly used to drive transgene expression in microalgae include various versions of the of cauliflower mosaic virus promoter 35S (CaMV35S), which has been used in both dinoflagellates and chlorophyta (Chow et al, Plant Cell Rep., 18:778-780, 1999; Jarvis and Brown, Curr. Genet., 317-321, 1991; Lohuis and Miller, Plant J., 13:427-435, 1998). The SV40 promoter from simian virus has also reported to be active in several algae (Gan et al., J. Appl. Phycol., 151 345-349, 2003; Qin et al., Hydrobiologia 398-399, 469-472, 1999). The promoters of RBCS2 (ribulose bisphosphate carboxylase, small subunit) (Fuhrmann et al., Plant J., 19:353-361, 1999) and PsaD (abundant protein of photosystem I complex; Fischer and Rochaix, FEBS Lett. 581:5555-5560, 2001) from Chlamydomonas can also be useful. The fusion promoters of HSP70A/RBCS2 and HSP70A/β2TUB (tubulin) (Schroda et al., Plant J., 21:121-131, 2000) can also be useful for an improved expression of transgenes, in which HSP70A promoter may serve as a transcriptional activator when placed upstream of other promoters. High-level expression of a gene of interest can also be achieved in heterokonts, for example diatoms species, under the control of a promoter of an fcp gene encoding a diatom fucoxanthin-chlorophyll a/b binding protein (Falciatore et al., Mar. Biotechnol., 1:239-251, 1999; Zaslayskaia et al., J. Phycol. 36:379-386, 2000) or the vcp gene encoding a eustigmatophyte violaxanthin-chlorophyll a/b binding protein (see U.S. Pat. No. 8,318,482). If so desired, inducible promoters can provide rapid and tightly controlled expression of genes in transgenic microalgae. For example, promoter regions of the NR genes encoding nitrate reductase can be used as such inducible promoters. The NR promoter activity is typically suppressed by ammonium and induced when ammonium is replaced by nitrate (Poulsen and Kroger, FEBS Lett 272:3413-3423, 2005), thus gene expression can be switched off or on when macroalgal cells are grown in the presence of ammonium/nitrate. Other regulatable promoters from Nannochloropsis include those disclosed in U.S. Patent Appl. Pub. No. US 2013/0023035, incorporated by reference herein. Additional Nannochloropsis algal promoters that can find use in the constructs and transformation systems provided herein include those disclosed in U.S. patent application Ser. No. 13/486,930, filed Jun. 1, 2012; U.S. patent application Ser. No. 13/693,585, filed Dec. 4, 2012; and U.S. patent application Ser. No. 13/915,522, filed Jun. 11, 2013, all incorporated by reference herein.
The gene of interest can be any gene of interest, including, without limitation, a growth regulator, a photosynthetic protein, a metabolic enzyme, a structural protein, a kinase, a phosphatase, a nucleotide cyclase, a phosphodiesterase, a transcriptional regulator, a transcriptional activator, a transporter, a secretory protein, an ion channel protein, a receptor, a chaperonin, a ribosomal protein, or a nuclear scaffold protein. Additional genes that can be included on an EDM as provided herein include genes encoding proteins that can be used to modify the genome. For example, a meganuclease, a zinc finger nuclease, a TALEN protein, a cas protein, a recombinase, a topoisomerase, or a transposase.
Also provided herein are methods for effecting homologous recombination or site-specific recombination in a host cell of interest, in which the method includes introducing an EDM that includes a selectable marker and a construct for homologous recombination or site specific recombination into a host cell. A construct for homologous recombination can include, for example, a nucleic acid sequence from a genetic locus of interest having at least one alteration with respect to the locus of interest. For example, a construct for homologous recombination can include at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1200, at least 1400, at least 1600, at least 1800, at least 2000, at least 2200, at least 2400, at least 2600 at least 2800, or at least 3000 base pairs of sequence from the genomic locus to be targeted, where the genomic sequence differs from the native sequence by at least one mutation, deletion, or insertion. For example, the genomic sequence in the homologous recombination construct can be interrupted by a selectable marker gene. A construct for site specific recombination can include, for example, sites recognized by recombinases, such as but not limited to lox or frt sequences, that may flank a gene sequence that may be mutated with respect to the native gene of the host organism. An EDM that includes a homologous recombination construct can be propagated in a host cell for multiple generations, for example, 10, 20, 30, 40, 50, or more generations, for example, by using selective media on which the EDM confers the ability to grow. Selection can be removed to allow the recombination construct to be lost from the cultured cells, and the cultured cells can be tested for the presence of the integrated gene knock out or gene replacement construct, for example, by PCR, Southern blot, and/or DNA sequencing. Optionally, the recombination construct can include a second selectable marker and selection for integration of the construct can be performed using the second marker, subsequent to removing selection for the presence of the EDM.
Methods of Identifying ARS Elements
The present invention also provides methods of identifying ARS elements from organisms of interest. A method for identifying an ARS element can comprise, for example: (1) sequencing the region between the 18S and 28S repeats in a chromosome from an organism of interest; and (2) selecting a portion, for example 5 kbp or less, 4 kbp or less, 2 kbp or less, or 1 kbp or less, of a nucleic acid sequence having the lowest guanidine/cytosine content within the sequenced region. For example, the methods of the present invention may optionally be used to identify an ARS from an algal cell, such as a cell of a species 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, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Gloeothamnion, Haematococcus, 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, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Skeletonema, Spyrogyra, Stichococcus, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, or Volvox. Alternatively or in addition, the methods of the present invention may optionally be used to identify an ARS from a heterokont, an animal, a plant, a fungus, or a protist.
For example, the method may be used to isolate an ARS from a heterokont such as a Eustigmatophyte (including, but not limited to a species of Eustigmatos, Monodus, Nannochloropsis, or Vischeria); a diatom (including, but not limited to a species of Achnanthes, Amphora, Chaetoceros, Cyclotella, Fragilaria, Navicula, Nitzschia, Phaeodactylum, or Thalassiosira. Monodus, Nannochloropsis, or Vischeria); or a labrinthulid or thraustochytrid, such as, but not limited to a species of Labryinthula, Labryinthuloides, Thraustochytrium, Schizochytrium, Aplanochytrium, Aurantiochytrium, Japonochytrium, Diplophrys, or Ulkenia. 
The following examples illustrate but do not limit the invention.
EXAMPLES
These examples describe a novel process used to identify ARS elements in a species of interest. It was hypothesized that an ARS exists between the 18S, 5.8S, and 28S rDNA repeat regions, known as 35S rDNA repeat units, in Nannochloropsis genomes. These types of repeat regions cannot be assembled in genome sequence databases because of their repetitive nature and the general inability to integrate repetitive elements into genome assemblies. The examples further demonstrate the functionality of these sequences in supporting autonomous replication of an episomal DNA molecule (EDM) in Nannochloropsis, and various uses of the EDM for delivery and expression of transgenes.
Example 1
Identification of Algal Autonomous Replication Sequences
Whole genomic DNA of Nannochloropsis gaditana was prepared for shotgun 454-pyrosequencing. Genomic DNA was used for library construction according to the recommended protocol (454 Life Sciences) for single long reads. The sequences were generated by GS FLX Titanium series sequencing runs. Mate-pair and paired-end genomic DNA library construction was also performed for Illumina short-read (100 bp) sequencing of the Nannochloropsis genome. Genome sequence assemblies were carried out using Newbler assembler version 2.0.00.20 for the 454-sequence data and using ALLPATHS-LG for the Illumina mate-pair and paired-end data.
A repetitive ribosomal DNA region of the genome that was assembled as a unit was found to lack 5S ribosomal DNA sequences that typically are found between the ribosomal RNA gene repeat units that include the 18S, 5.8S, and 28S ribosomal RNA genes, referred to as 35S ribosomal RNA gene repeats. To isolate the 5S ribosomal DNA region that is typically found between 35S ribosomal DNA repeat units, the region between Nannochloropsis gaditana 35S ribosomal DNA gene repeat units was amplified from genomic DNA by PCR, isolated, and sequenced. As anticipated, several slightly different DNA sequences were acquired, at least one of which included a sequence having homology to the Cyanidioschyzon merolae (red alga) 5S rDNA sequence (Genbank: NC_010138 REGION: 84222 . . . 84389). The GC content of the isolated inter-35S rDNA repeat DNA sequences showed regions with high AT. content, a characteristic of some ARSs, adjacent to GC-rich regions that included 5S rDNA-homologous sequences. A  ̃1.6 kpb A/T rich region (SEQ ID NO:1) of one of the inter-35S rDNA repeat amplified regions was isolated for further characterization.
The 1.6 kbp sequence, referred to as “NgARS1.6” (SEQ ID NO:1), was analyzed for internal repeats by comparing the sequence with itself using the National Center for Biotechnology Information (NCBI) BLAST program (graphically depicted in FIG. 1). This demonstrated that the sequence included imperfect tandem repeats of approximately 85 bp. Imperfect tandem sequence repeats are also found in the S. cerevisiae ARS cluster, indicating, along with the localization to the inter-35S rDNA repeat region of the genome, that SEQ ID NO:1 included an ARS. Two approximately 85 bp regions of the AT-rich putative Nannochloropsis ARS with significant primary sequence identity (SEQ ID NO:2 and SEQ ID NO:3; consensus sequence provided as SEQ ID NO:4) were found to make up part of a repetitive and low GC area found in the 5S rDNA sequence region.
Example 2
Characteristics of Algal Autonomous Replication Sequences
Additional sequences (SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12) isolated from Nannochloropsis gaditana genomic DNA by amplification of genome regions between the 35S rDNA repeat units ranged in size from 770 bp (NgARS2; SEQ ID NO:6) to 4421 bp (NgARS1; SEQ ID NO:5). See Table 1.
These fragments were found to have a high degree of sequence homology with one another over a stretch of about 350 bp of AT rich sequence as shown in the sequence alignment of FIG. 2. With respect to nucleotides 1-323 of NgARS2 (SEQ ID NO:6), provided as SEQ ID NO:13, nucleotides 1-337 of NgARS1 (SEQ ID NO:5) were found to be 89% identical, NgARS1.6 (SEQ ID NO:1) nucleotides 301-622 were found to be 99% identical, nucleotides 780-1116 of NgARS3 (SEQ ID NO:7) were found to be 89% identical, nucleotides 784-1104 of NgARS4 (SEQ ID NO:8) were found to be 99% identical, nucleotides 614-935 of NgARS5 (SEQ ID NO:9) were found to be 99% identical, and nucleotides 784-1105 of NgARS7 (SEQ ID NO:10) were found to be 99% identical. With respect to nucleotides 322 to 770 of NgARS2 (SEQ ID NO:6), provided as SEQ ID NO:14, nucleotides 3973-4421 of NgARS1 (SEQ ID NO:5) were found to be 99% identical, nucleotides 2956-3404 of NgARS3 (SEQ ID NO:7) were found to be 98% identical, and nucleotides 2026-2474 of NgARS8 (SEQ ID NO:11) were found to be 99% identical. In addition, nucleotides 983-1183 of NgARS-Sm (SEQ ID NO:12) were found to be 100% identical to nucleotides 322-522 of NgARS2 (SEQ ID NO:6). Thus, all of the isolated fragments had a high percentage of sequence identity to at least 300 nucleotides of NgARS2. NgARS1, NgARS1.6, NgARS3, NgARS4, NgARS5, and NgARS7 were found to include sequences having at least 85% identity to SEQ ID NO:13 (nucleotides 1-323 of SEQ ID NO:6 (NgARS2)). NgARS8 was found to include sequences having at least 85% identity to SEQ ID NO:14 (nucleotides 322-770 of SEQ ID NO:6 (NgARS2)). NgARS1 and NgARS3 were found to have at least 85% sequence identity to both “halves” of NgARS2: at least 85% identity (at least 89% identity) to SEQ ID NO:13, and at least 95% identity (at least 98% identity) to SEQ ID NO:14.







TABLE 1







Nannochloropsis ARS Sequences












Homology to
Homology to




SEQ ID NO: 13
SEQ ID NO: 14




(nucleotides
(nucleotides


Putative ARS
Length of
1-323 of
322-770 of


Sequence
Fragment
SEQ ID NO: 6)
SEQ ID NO: 6)





NgARS1.6
1624 bp
nucleotides



(SEQ ID NO: 1)

301-622,




99% identical


NgARS1
4421 bp
nucleotides
nucleotides


(SEQ ID NO: 5)

1-337,
3973-4421,




89% identical
99% identical


NgARS2
 770 bp
Self
Self


(SEQ ID NO: 6)


NgARS3
3777 bp
nucleotides
nucleotides


(SEQ ID NO: 7)

780-1116,
2956-3404,




89% identical
98% identical


NgARS4
3093 bp
nucleotides


(SEQ ID NO: 8)

784-1104




99% identical


NgARS5
3722 bp
nucleotides


(SEQ ID NO: 9)

614-935,




99% identical


NgARS7
2543 bp
nucleotides


(SEQ ID NO: 10)

784-1105,




99% identical


NgARS8
2474 bp

nucleotides


(SEQ ID NO: 11)


2026-2474,





99% identical


NgARS-Sm
1183 bp

nucleotides


(SEQ ID NO: 12)


983-1183,





98% identical









Example 3
Validation of ARS Function in Replication in Nannochloropsis Gaditana 
A plasmid vector, pSGE-05959 (FIG. 3), was designed to test the putative NgARS sequences SEQ ID NO:5 (NgARS1), SEQ ID NO:6 (NgARS2), SEQ ID NO:7 (NgARS3), SEQ ID NO:8 (NgARS4), SEQ ID NO:9 (NgARS5), SEQ ID NO:10 (NgARS7), SEQ ID NO:11 (NgARS8), and SEQ ID NO:12 (NgARS-Sm) for their ability to support autonomous replication of an episomal DNA molecule by cloning the sequences into the unique AfeI site within the vector. The constructs in circular (uncut) form were used to transform Nannochloropsis as described below:
The pSGE-05959 based constructs were transformed into Nannochloropsis cells by electroporation. Briefly, Nannochloropsis gaditana cells were grown in PM032 media and harvested at a concentration between 1-3×107 cells/mL. Cells were centrifuged at 2500×g for 10 minutes at 25° C. to pellet the cells. Cells were then resuspended in a sterile solution of 385 mM sorbitol and centrifuged again, then washed two more times in sorbitol to remove all traces of media. The cell pellet was resuspended in sorbitol to a final concentration of 1×1010 cells/mL. 0.5-5 μg of Uncut (i.e., circular) vectors based on pSGE-05959 plasmid DNA that included one of the following putative NgARS elements (SEQ ID NO:5 (NgARS1), SEQ ID NO:6 (NgARS2), SEQ ID NO:7 (NgARS3), SEQ ID NO:8 (NgARS4), SEQ ID NO:9 (NgARS5), SEQ ID NO:10 (NgARS7), SEQ ID NO:11 (NgARS8), and SEQ ID NO:12 (NgARS-Sm)) were aliquoted into a microfuge tubes, and 100 μL of cell mixture was mixed with the DNA. The mixture was transferred to chilled 2 mm gap electroporation cuvette. The electroporator was set to 50 μF capacitance, 500 ohms resistance and a voltage of 2.2 kV. Following electroporation, samples were resuspended in 1 mL of sorbitol and incubated on ice for a few minutes. Cells were transferred to 15 mL conical tubes containing 10 mL of fresh media, and allowed to recover overnight in dim light ( ̃5 μmol photons m−2 sec−1). The next day, cells were plated at a concentration between 5-7×108 cells/mL on PM032 plates containing 100 μg/mL blasticidin. Plates were incubated under constant light ( ̃80 μmol photons m−2 sec−1) until colonies appeared (about 2-3 weeks).
PM032 media includes: 35 ppt Instant Ocean Salts (Aquatic Eco Systems; Apopka, Fla.), 10× Guillard's F/2 marine water enrichment solution (50× stock from Sigma-Aldrich, St. Louis, Mo., cat. No. G0154; final concentrations of components in media: 8.825 μM Sodium nitrate; 0.32 mM Sodium phosphate monobasic; 0.205 μM Biotin; 0.420 μM Cobalt chloride•6H2O; 0.400 μM Cupric sulfate•5H2O; 0.11713 mM Disodium EDTA•2H2O; 9.095 μM Manganese chloride•4H2O; 0.248 μM Sodium molybdate•2H2O; 2.965 μM Thiamine•HCl; 0.037 μM Vitamin B12; 0.765 μM Zinc sulfate•7H2O).
Blasticidin-resistant colonies were obtained from the transformation events. Non-homologous End Joining (NHEJ) based recombination into the algal genome (i.e. random integration) is exceedingly rare in the absence of linear DNA molecules, and thus the mere presence of the blasticidin resistant N. gaditana colonies is strong evidence for the function of the tested ARS element. Additionally, it was predicted that functional ARS elements would result in the production of an Episomal DNA Molecule (EDM) within the algae that could be recovered from the algae and, due to the presence of E. coli and/or Saccharomyces cerevisiae replication elements, directly transformed into E. coli or S. cerevisiae. DNA was therefore isolated from blasticidin-resistant Nannochloropsis lines as described below.
100-1000 uL of mid-log phase algal culture was placed into a 1.5 mL microcentrifuge tube and the algae were pelleted at 4,000×g for 2 minutes. The supernatant was removed and the pellet resuspended in 1 mL of sterile filtered milliQ (SFMQ) water. The mixture was pelleted as before and resuspended in 200 uL of SFMQ water. To this mixture, 200 uL of 2× Proteinase K buffer (100 mM Tris pH 8.0, 100 mM EDTA, 1% SDS) and 5 uL of Proteinase K solution (20 mg/mL, NEB P8102) were added and briefly vortexed. The mixture was placed on a vortexer heated to 50° C. at 1000 RPM for 1 hr. The samples were cooled to 37° C. and 5 uL of DNAse free RNAseA (10 mg/mL) was added. The samples were placed on the heated vortexer at 37° C. and 1000 RPM for 15 minutes. 400 uL of PCIA (25:24:1 Phenol:Chloroform:Isoamyl alcohol pH 8.0) was added and the samples were thoroughly vortexed at room temperature (RT). The samples were pelleted at 21,817×g for 5 min at RT. The upper aqueous layer was transferred to a new tube. 30 uL of 5M NaCl was added and mixed by vortexing. 1.1 mL of 100% ethanol was added and mixed by vortexing and the samples were placed at −20° C. for  ̃10 min. The samples were pelleted at 20,817×g at 4° C. for 20 min. All of the supernatant was removed and the pellet was vortexed thoroughly in the presence of 1 mL of 70% ethanol. The samples were pelleted at 20,817×g at RT for 5 min and the supernatant was completely removed. The pellet was briefly air dried and resuspended in 25 uL of SFMQ water. 2 uL was used for DNA quantification on the qBit DNA fluorometer.
An aliquot of between 1 ng to approximately 250 ng of DNA isolated from each of the blasticidin resistant Nannochloropsis transformants was used to separately transform E. coli ElectroSHOX cells (Bioline BIO-85038) using standard electroporation techniques (50 uL cells, 5 uL max DNA solution, 1 mm gap cuvette, 2.5 kV, 25 ohm, 50 uF, SOC resuspension). A ratio of E. coli colony count to electroporated DNA amount was used to determine which of the putative NgARSs conferred eukaryotic replicative function to the Episomal DNA Molecule (EDM). Five of the eight putative NgARSs (NgARS1, NgARS2, NgARS3, NgARS7, and NgARS-Sm) showed significantly higher values for the colony number per transformed DNA ratio than the control, with NgARS2 having the highest transformation efficiency (Table 2), strongly suggesting that the pSGE-05945 construct with NgARS2 was most efficiently replicating as an episome in these Nannochloropsis lines. Photographs of exemplary transformation plates are provided in FIG. 4. To confirm that the recovered colonies contained the EDM, a standard plasmid miniprep procedure (Qiagen) was performed on cultures that came from a sample of these transformants. The isolated plasmid DNA was used in PCR reactions to confirm the presence of the GFP gene (Primers were: GFP.ColCheck.f1, gttgctgtgatcctcctccac (SEQ ID NO:15) and GFP.ColCheck.r1 catgaccaacaag atgaagagcac (SEQ ID NO:16); generating a 512 bp PCR product that included most of the GFP coding region) and indicated that the initial EDM was providing the host E. coli with the necessary antibiotic resistance. Samples of the recovered vectors were fully resequenced and shown to have no changes from the parental strain, indicating that propagation of these EDMs within Nannochloropsis did not result in any changes to the EDM over the time course of the experiment.







TABLE 2







Results of transforming recovered


Nannochloropsis EDMs into E. coli













Recovered
Electroporated





Colony #
DNA Quantity


ARS ID
Plasmid ID
Average
(ng) Average
Col/DNA














NgARS1
pSGE05944
18.00
27.35
0.66


NgARS2
pSGE05945
64.00
39.78
1.61


NgARS3
pSGE05946
36.00
138.00
0.26


NgARS4
pSGE05947
12.14
131.47
0.09


NgARS5
pSGE05948
10.11
54.46
0.19


NgARS7
pSGE05949
41.33
128.33
0.32


NgARS8
pSGE05950
19.67
116.43
0.17


No ARS
pSGE05959
5.25
33.00
0.16


(control)


NgARS-Sm
pSGE05943
12.00
14.30
0.84









Example 4
Validation of ARS Function in Replication in Other Nannochloropsis Sp
Nannochloropsis oceanica, a species of Nannochloropsis distantly related to N. gaditana, was used to determine the species specificity of the isolated NgARS2 element. pSGE05945, and/or related NgARS2 containing vectors, was transformed into N. oceanica using similar protocols as for N. gaditana and using the same amount of vector DNA for each transformation event ( ̃2 ug). Table 3 provides the results and demonstrates that similar numbers of blasticidin resistant colonies arise from transforming N. oceanica as N. gaditana, thus demonstrating broad host range functionality for the isolated NgARS2 element. The slightly higher number of colonies seen on the N. oceanica plates is likely due to slightly higher transformation efficiencies of that strain of Nannochloropsis. Transformation of an NgARS2-containing EDM into both N. gaditana and N. oceanica has been performed multiple times, using EDM constructs up to 100 kb in size.







TABLE 3







Transformation of Nannochloropsis


species with EDM containing NgARS2











No. of blasticidin resistant




colonies, pSGE-05945



Species
vector







N. gaditana
~200



N. oceanica
~300










Example 5
Necessity of ARS Elements for Replicative Function of EDMs within N. gaditana 
NgARS2 was also cloned into a pUC-based plasmid vector that included a hygromycin resistance gene (SEQ ID NO:17) driven by the TCTP promoter (SEQ ID NO:18) for selection of Nannochloropsis transformants. The resulting construct, pSGE-6023, was used to transform Nannochloropsis gaditana essentially according to the methods provided in Example 3, resulting in approximately 100 colonies. A control transformation in which the same vector was used, except that the vector did not include NgARS2 (SEQ ID NO:6), resulted in only a single algal colony, presumably resulting from the relatively rare occurrence of integration of the EDM construct into the genome. This experiment was repeated with a similar construct that replaced the Hygromycin antibiotic resistance cassette with the Blasticidin resistance cassette and showed essentially the same results. These results showed that the NgARS2 sequence is necessary for EDM replication within N. gaditana. 
Example 6
Determination of Gene Expression Profiles within EDM Constructs
A plasmid vector, pSGE-6067 (FIG. 5), was designed to include NgARS2 (SEQ ID NO:6) previously isolated and described in Example 2 to determine if there are differences between randomly integrated and EDMs in the expression pattern of desired metabolic or non-metabolic genes. In addition to the NgARS2 sequence (SEQ ID NO:6), a gene conferring resistance to blasticidin (“Blast150”; SEQ ID NO:19) driven by the Nannochloropsis TCTP promoter (SEQ ID NO:18) and followed by a Nannochloropsis alpha tubulin terminator (SEQ ID NO:20) was included in the construct as a selectable marker. The plasmid construct further included as a reporter gene the green fluorescent protein TurboGFP (SEQ ID NO:21; Evrogen) driven by Nannochloropsis promoter 5303630 (SEQ ID NO:22) and linked to the Nannochloropsis eIf3a terminator (SEQ ID NO:23). For replication and selection in E. coli, the plasmid construct included the pMB1 ORI, an origin of replication for E. coli, and the beta lactamase or “bla” gene conferring ampicillin resistance (APr), all derived from the commonly used pUC based vectors. The construct additionally included a gene encoding the metabolic enzyme enoyl ACP reductase (ENR) from Cyclotella (SEQ ID NO:24) linked to the eIF3a promoter (SEQ ID NO:25) and T9 terminator (SEQ ID NO:26).
The pSGE-6067 construct was transformed into Nannochloropsis cells by electroporation as previously described. Blasticidin-resistant Nannochloropsis colonies from the transformations of Example 3 were streaked into patches on fresh blasticidin (100 μg/mL) plates, and those patches served as the seed source for initiating 10 ml volume PM074 liquid cultures that included 75 mg/mL blasticidin.
As previously described, cells were harvested for DNA isolation and the resultant DNA was used to transform E. coli. E. coli colonies were counted and screened for the presence of the GFP gene (SEQ ID NO:21) and the NgARS2 sequence (SEQ ID NO:6) by PCR. Table 4 shows that EDMs isolated from the transformed N. gaditana lines were positive for both the GFP gene and the requisite ARS element.
Based on this assessment, Nannochloropsis lines 101, 107, 108, 109, and 110 were considered to be carrying an EDM. To confirm this, DNA was also isolated from E. coli transformants using a commercial DNA “miniprep” kit (Qiagen) for isolating plasmid DNA. The isolated plasmid DNA was run on a gel in either digested or undigested form and was seen to be identical to the parental lines, with the exception of line 101, which is further discussed below.







TABLE 4







Transformation of E. coli with undigested DNA isolated from


Nannochloropsis lines transformed with NgARS2 Construct pSGE-6067











Number of




Nannochloropsis
E. coli transformants


Blasticidin-resistant
using extracted uncut


transformant line
DNA
GFP PCR
ARS PCR













101
1279
+
+


102
0
NA
NA


103
0
NA
NA


104
3
+
+


105
0
NA
NA


106
0
NA
NA


107
1376
+
+


108
1867
+
+


109
1985
+
+


110
2118
+
+









Aliquots of Nannochloropsis gDNA were digested with PsiI, an enzyme that does not cut pSGE06067, and run on 0.65% agarose gels, which were electrotransferred to a nylon membrane for a Southern Blot using the Blast150 (blasticidin resistance) gene sequence SEQ ID NO:19) as a probe. The results of the Southern blot are provided in FIG. 6. The Southern blot demonstrated that at least four of the five Nannochloropsis lines that carried DNA able to transform E. coli at a frequency indicative of plasmid DNA did in fact include pSGE-6067 DNA in open circular form. By contrast, blasticidin-resistant Nannochloropsis lines whose DNA was not able to transform E. coli (or transformed E. coli at a very low frequency inconsistent with undigested plasmid DNA, e.g., line 104), did not include pSGE-6067 DNA in circular form, but rather appeared to have pSGE-6067-hybridizing DNA in higher molecular weight DNA, indicating that pSGE-6067, or at least a portion thereof including the blasticidin resistance gene, had integrated into the Nannochloropsis genome. To locate possible integration sites, the genomic DNA for lines 101, 102, 107, 108, 109 and 110 were resequenced using a MiSeq deep sequencer (Illumina). Mapping the resultant data back to the genome assembly showed no detectable integration events for lines 101, 107-110, consistent with them being purely episomal, and a single integration point for line 102, indicating that the linear construct had integrated into the host genome. Line 101, which started out as a linear molecule, showed at least three whole molecule concatamerization events, with the construct sequences multimerized either in imperfect tandem and/or as inversions, leading to a circular EDM. When the Line 101 EDM was isolated from E. coli, and its uncut form was run on a gel, it showed a banding pattern consistent with unresolved strand breakage, which is consistent with the presence of multiple bacterial origins of replication that would be predicted to exist in that molecule.
In addition, Western blots were performed using extracts of the Nannochloropsis lines transformed with pSGE-6067 DNA. Briefly, Nannochloropsis cells were boiled in a lithium dodecyl sulfate/Urea/DTT protein loading solution and run on a NuPage gel, transferred to PVDF and probed with either anti-GFP or anti-ENR antibodies using standard techniques. FIG. 7 shows that the Nannochloropsis lines that included the pSGE-6067 construct in circular form (i.e., as an EDM) expressed GFP at a higher level than was found the blasticidin-resistant Nannochloropsis lines in which the pSGE-6067 was integrated into the genome. Further, Nannochloropsis lines that included the pSGE-6067 construct in circular form expressed the metabolically relevant ENR transgene, such that the protein was detectable, whereas no ENR protein was detected in the Nannochloropsis lines that included genome-integrated pSGE-6067 DNA. Thus, there appears to be a significant advantage for transgene expression in using EDMs in Nannochloropsis. 
To complement the analysis of relative protein expression levels as determined by the Western blot, direct GFP fluorometric measurements of the cells within the transgenic populations was measured using a BD Accuri C6 flow cytometer. (BD Biosciences). Table 5 gives the amount of fluorescence per cell of ten cell lines. The highest GFP fluorescence, indicative of high levels of GFP expression, are found in lines 2C, 3L, 5L, 12L, and 15L, in which the pSGE-6081 construct is maintained as an EDM.







TABLE 5







Fluorescence











Mean fluorescence
% cell in
Peak 2 Mean fluorescence


Strain
(FL1-A)
Peak 2
(FL1-A)













WE3730
740.53
0.83%
9,411.75


2C
51,871.35
50.97%
100,868.07


6C
4,882.05
81.20%
5,649.52


7C
4,339.33
16.55%
21,683.65


8C
2,090.80
9.60%
12,279.30


10C 
5,067.24
16.12%
12,549.03


11C 
2,533.12
11.43%
14,724.75


3L
12,357.68
33.16%
35,322.09


5L
13,205.75
31.4%
39,965.28


12L 
13,155.89
30.62%
40,833.77


15L 
11,732.72
23.59%
46,287.03









Example 7
Use of ARS Elements in Constructing Transient EDM Vectors
Nannochloropsis lines transformed with the EDM pSGE-06081, which contains the previously described blasticidin antibiotic resistance gene (“Blast 150”; SEQ ID NO:19) and TurboGFP reporter gene (SEQ ID NO:21) were generated. These lines were propagated in T75 tissue culture flasks containing 10 mL PM032 media with either 0 ug/mL or 75 ug/mL of blasticidin. The lines were passaged once per approximately 8 generations (approximately every 7 days) and the GFP fluorescence of the cells within the population at each of the passaging time periods was measured using the BD Accuri C6 Flow Cytometer. FIG. 8 shows the percent of the population that has GFP expression levels above background over time, with each date being a point where the cells were split into new media with the same antibiotic or no antibiotic composition. After approximately 2 weeks without selective pressure (no blasticidin in the media), the GFP expression levels were near background, and after approximately 4 weeks, no EDMs could be recovered. This demonstrates the potential of this EDM in providing a scaffold onto which genes can be deployed and maintained until antibiotic selective pressure is removed, at which point the EDM is diluted out of the population through cell division.
The results (Table 6) demonstrate that although cells of the culture that was under constant selection maintained the EDM, it was lost from cells of the culture that was released from antibiotic selection within 35 days. This result, together with the superior expression levels enabled by providing a gene on an EDM as compared to an integrated nucleic acid molecule (Example 6), indicates that transient expression of genes can be readily achieved by use of an EDM under selective conditions that maintain the EDM in the transformed line and allow for high expression levels, and subsequent removal of the selective agent to result in loss of the construct containing the transgene. Thus, EDMs as provided herein can be useful not only for testing functionality of genes and gene products (including gene attenuation constructs such as RNAi and antisense constructs), but also for transiently expressing genes for modifying the genome including, as nonlimiting examples, transposases, topoisomerases, recombinases, meganucleases, zinc finger nucleases, talens, CRISPR-associated endonucleases, etc.
Example embodiments are provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those who are skilled in the art. Numerous specific details are set forth such as examples of specific components and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known structures, and well-known technologies are not described in detail.

## Claims

1. An isolated or recombinant nucleic acid molecule comprising an autonomous replication sequence (ARS) juxtaposed with at least one heterologous nucleic acid sequence, wherein the ARS has at least 95% identity to a nucleic acid selected from the group consisting of SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:11; and SEQ ID NO:12, further wherein the ARS comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14.

2. An isolated or recombinant nucleic acid molecule according to claim 1, wherein the nucleic acid molecule does not comprise a 35S ribosomal RNA gene unit.

3. An episomal DNA molecule (EDM) comprising an ARS that comprises a sequence having at least 95% identity to a nucleotide sequence selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; and SEQ ID NO:12.

4. An EDM according to claim 3, wherein the ARS comprises a sequence having at least 98% identity to a nucleotide sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

5. An EDM according to claim 4, wherein the ARS comprises the polynucleotide sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; and SEQ ID NO: 12.

6. An EDM according to claim 3, further comprising a nucleic acid sequence encoding a polypeptide.

7. An EDM according to claim 6, wherein the polypeptide is a reporter protein or selectable marker protein.

8. An EDM according to claim 7, wherein the reporter protein is a fluorescent protein or a signal producing enzyme.

9. An EDM according to claim 7, wherein the selectable marker protein is a protein that confers resistance to a drug, antibiotic, toxin, or herbicide; a protein that allows autotrophic growth on particular media; or a protein that prohibits growth on particular media.

10. An EDM according to claim 6, wherein polypeptide is a metabolic enzyme, structural protein, kinase, phosphatase, nucleotide cyclase, phosphodiesterase, nucleotide diesterase, transcriptional regulator, transcriptional activator, transporter, secretory protein, ion channel, receptor, photosynthetic protein, chaperonin, ribosomal protein, or nuclear scaffold protein.

11. An EDM according to claim 6, wherein the polypeptide is a meganuclease, a zinc finger nuclease, a TALEN protein, a cas protein, a recombinase, a topoisomerase, or a transposase.

12. An EDM according to claim 3, further comprising a nucleic acid sequence encoding a functional RNA.

13. An EDM according to claim 12, wherein the functional RNA is selected from the group consisting of an interfering RNA or a precursor thereof, an antisense RNA, a ribozyme, a micro RNA, and a guide RNA of a CRISPR system.

14. An EDM according to claim 3, wherein the ARS is functional in an algal or heterokont cell.

15. An EDM according to claim 3, wherein the EDM is circular.

16. An EDM according to claim 3, comprising sequences for mediating homologous or site specific recombination, wherein the sequences for homologous recombination flank a gene, a gene disruption sequence, or a functional RNA.

17. A method for propagating a gene of interest through at least 8 generations of cell division, the method comprising incorporating the gene of interest into the episomal DNA molecule of claim 3, transforming the episomal DNA molecule into a cell, and transforming the EDM into a host cell, and propagating the host cell for at least 8 generations.

18. A method according to claim 17, wherein the transformed cell is an algal, heterokont, plant, animal, fungal, or yeast cell.

19. A method according to claim 17, wherein the transformed cell is a Nannochloropsis cell.

20. A method for transiently expressing a gene or sequence of interest in a microbial cell, comprising transforming the EDM of claim 3 into a microbial cell and culturing the microbial cell under conditions in which the gene or sequence is expressed.

21. The method of claim 20, wherein the episomal DNA molecule comprises a selectable marker, and further wherein the microbial cell is cultured in the presence of a selective agent for one or more generations.

22. The method of claim 21, wherein the microbial cell is cultured in the presence of the selective agent for one or more generations and subsequently cultured in the absence of the selective agent for and additional one or more generations.

23. A method of transforming a microorganism, comprising: contacting at least one cell of the microorganism with an EDM according to claim 3, and selecting transformed cells on a selective medium.

24. An isolated or recombinant nucleic acid molecule according to claim 1, wherein the ARS has at least 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:12.

25. An isolated or recombinant nucleic acid molecule according to claim 24, wherein the ARS is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:12.

26. An isolated or recombinant nucleic acid molecule according to claim 1, wherein the ARS has at least 95% identity to SEQ ID NO:6.

27. An isolated or recombinant nucleic acid molecule according to claim 26, wherein the ARS has at least 97% identity to SEQ ID NO:6.

28. An isolated or recombinant nucleic acid molecule according to claim 27, wherein the ARS comprises SEQ ID NO:6.