# Patent

## Title
Compositions and methods for conferring herbicide resistance

## Abstract

The present invention provides a protein, specifically Parietochloris incisa acetohydroxyacid synthase, and methods for producing branched-chain amino acid in a cell. The present invention further provides polypeptides and polynucleotides useful for conferring herbicide resistance in a cell or an organism. In particular, the present invention discloses a mutated acetohydroxyacid synthase which is resistant to herbicides.

## Background
REFERENCE TO CO-PENDING APPLICATIONS
Priority is claimed as a 371 of international application number PCT/IB2012/055347, filed on Oct. 4, 2012; which claims priority to U.S. provisional patent application Ser. No. 61/543,339, filed on Oct. 5, 2011.


FIELD OF THE INVENTION
The present invention provides a protein, specifically Parietochloris incisa acetohydroxyacid synthase, and methods for producing branched-chain amino acid in a cell. The present invention further provides polypeptides and polynucleotides useful for conferring herbicide resistance in a cell or an organism. In particular, the present invention discloses a mutated acetohydroxyacid synthase which is resistant to herbicides.
BACKGROUND OF THE INVENTION
Branched-chain amino acids (BCAAs), namely leucine, isoleucine and valine, are synthesized by plants, algae, fungi, bacteria and archaea, but not by animals. BCCAs are essential amino acids for humans and are used clinically for the treatment of burns and hepatic encephalopathy as well as for increasing muscle mass. The enzymes of the BCAA biosynthetic pathway are potential targets for the development of herbicides, fungicides, and antimicrobial compounds. Some of the most popular herbicides (e.g. sulfometuron methyl, SMM), act by inhibiting the first common enzyme in the BCAA biosynthetic pathway, acetohydroxyacid synthase (AHAS). Since AHAS is required for the synthesis of all three BCAAs, its inhibition is detrimental to the organism and effectively inhibits its growth. Plant and green algal AHAS are localized in the chloroplast and fungal AHAS in the mitochondria, although the genes may be present in the nuclear or organelle genome. In cases of nuclear-encoded genes, the enzyme is transported to the target subcellullar compartment by an additional, poorly conserved, N-terminal targeting peptide.
Microalgae are one of the richest sources of long-chain polyunsaturated fatty acids (LC-PUFAs). The green freshwater microalga Parietochloris incisa (Trebouxiophyceae) is the only microalga able to accumulate extraordinary high amounts of LC- PUFA arachidonic acid (ARA)-rich triacylglycerols (TAG). When P. incisa is cultivated under nitrogen starvation, the fatty acid (FA) content of the alga is over 35% of dry weight; ARA constitutes about 60% of total FAs, and over 90% of cell ARA is deposited in TAG, making it the richest green dietary source of ARA. LC-PUFAs include the ω3-fatty acids, eicosapentaenoic acid (EPA, 20:5ω3), docosahexaenoic acid (DHA, 22:6ω3), ω6-fatty acid, arachidonic acid (ARA, 20:4ω6) and dihomo-γ-linolenic acid (DGLA, 20:3ω6). LC-PUFA are major components of membrane phospholipids of the retina, brain and testis and are predominant in the human brain and breast milk (specifically ARA and DHA). ARA is necessary for normal fetal growth and for cognitive development in infants and is also the primary substrate in eicosanoids biosynthesis, which regulates many physiological processes such as homeostasis, reproduction, immune and inflammatory responses.
Use of antibiotic resistance genes as selection markers in transformation processes presents numerous environmental and health risks, as well as regulatory difficulties that define the organism as genetically modified (GM). Thus, a selection system based on mutation(s) of an endogenous gene(s) is highly advantageous. It has been reported that mutant forms of AHAS exhibit herbicide resistance in yeast, higher plants and green algae, where most of the characterized AHAS herbicide resistances is due to a single or double amino acid change from the wild-type enzyme sequence. At least 17 different amino acid substitutions in AHAS are known to confer resistance to growth inhibiting herbicides. For example, in tobacco, a resistant mutant with a single amino acid change of Tryptophan557, within a conserved region of AHAS, was found to be insensitive to inhibition by two sulfonylurea herbicides, chlorsulfuron and SMM. In addition, corresponding Trp residue mutations were shown to be important for AHAS SMM resistance of Escherichia coli, Mycobacterium tuberculosis, Brassica napus and the red microalga Porphyridium sp.
There remains an unmet need for the development of cells, in particular microalgal cells which are capable of producing LC-PUFA in large scale systems and which are resistant to herbicides, using methods which do not classify the organism as genetically modified.
SUMMARY OF THE INVENTION
According to one aspect, the present invention provides an isolated protein, comprising an amino acid sequence set forth in SEQ ID NO: 1.
According to one embodiment the present invention further provides a composition comprising the protein comprising an amino acid sequence set forth in SEQ ID NO: 1 and an acceptable carrier.
According to another embodiment the present invention further provides an isolated polynucleotide comprising a coding portion encoding the protein comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein said coding portion comprises a nucleic acid sequence set forth in SEQ ID NO: 3.
According to yet another embodiment the present invention further provides an expression vector comprising said polynucleotide.
According to yet another embodiment the present invention further provides a cell transformed by said polynucleotide.
According to yet another embodiment the present invention provides a method for enhancing the production of branched-chain amino acid (BCAA) in a cell comprising the step of transforming a cell with said polynucleotide operably linked to a constitutive promoter, thereby increasing the production of BCAA in a cell.
According to yet another embodiment the cell is an alga cell.
According to yet another embodiment said method further comprises the step of transforming said cell with a polynucleotide encoding an enzyme selected from the group consisting of: Threonine deaminase (TD), ketol-acid Reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD); transaminase (TA), 2-isopropylmalate synthase (2-IPMS), 3-isopropylmalate dehydratase (3-IPMD) and 3-isopropylmalate dehydrogenase (3-IPMDH), or a combination thereof.
According to another aspect, the present invention provides an isolated protein comprising an amino acid sequence set forth in SEQ ID NO: 2.
According to one embodiment, the present invention provides a composition comprising the protein comprising an amino acid sequence set forth in SEQ ID NO: 2 and an acceptable carrier.
According to another embodiment, the present invention further provides an isolated polynucleotide comprising a coding portion encoding the protein comprising an amino acid sequence set forth in SEQ ID NO: 1, wherein said coding portion comprises a nucleic acid sequence set forth in SEQ ID NO: 3.
According to yet another embodiment the present invention further provides an expression vector comprising said polynucleotide.
According to yet another embodiment, the present invention further provides a cell transformed by said polynucleotide.
According to yet another embodiment the present invention further provides a method for producing a cell having herbicide resistance comprising the step of transforming said cell with said polynucleotide, thereby producing a cell having herbicide resistance.
According to yet another embodiment said cell is an alga cell.
According to yet another embodiment, said alga is Parietochloris incisa. 
According to yet another embodiment, said herbicide is selected from the group consisting of: sulfonylureas, imidazolinones, triazolopyrimidines and combinations thereof.
According to yet another embodiment, said alga produces long-chain polyunsaturated fatty acid (LC-PUFA), arachidonic acid (ARA) and rich triacylglycerols (TAG).
According to yet another embodiment, said method further comprises the step of transforming said alga with a polynucleotide encoding an enzyme involved in the biosynthesis of ω-3 LC-PUFA in said alga.
According to yet another embodiment said enzyme involved in the biosynthesis of ω-3 LC-PUFA is ω-3 desaturase, C20 PUFA elongase, Δ4 desaturase or combinations thereof.
According to yet another embodiment, said ω-3 LC-PUFA is eicosapentaenoic acid (EPA, 20:5 ω-3) or docosahexaenoic acid (DHA, 22:6 ω-3).
According to yet another embodiment, the present invention provides a method for producing an alga having an endogenous selection marker for herbicide resistance, comprising the step of transforming said alga with said polynucleotide comprising a coding portion encoding the protein comprising an amino acid sequence set forth in SEQ ID NO: 1, wherein said coding portion comprises a nucleic acid sequence set forth in SEQ ID NO: 3, thereby producing an alga having an endogenous selection marker for herbicide resistance.
According to one embodiment, said alga is Parietochloris incisa. 
According to another embodiment, said herbicide inhibits acetohydroxyacid synthase.
Further embodiments, features, advantages and the full scope of applicability of the present invention will become apparent from the detailed description and drawings given hereinafter. However, it should be understood that the detailed description, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Is a multiple sequence alignment of known AHAS proteins. Alignment of five AHAS sequences from green algae (C. reinhardtii, C. variabilis and of V. carteri.), moss (P. patens subsp.) and higher plant (B. napus), showing the conserved “blocks” A-D, SEQ ID Nos: 29-32, accordingly, used for primer design.
FIG. 2. Provides multiple sequence alignment of PiAHAS with other green algae AHAS (C. reinhardtii, (SEQ ID NO: 33); C. variabilis (SEQ ID NO: 35); and of V. carteri. (SEQ ID NO: 34)). The poorly conserves N-terminus, is shown (grey highlight). The black triangle points to start of the “truncated” PiAHAS gene form. Mutated Tryptophan residue, involved in the herbicide binding and resistance, is shown as conserved (black highlight).
FIG. 3. Shows functional complementation of BUM1 by cloned PiAHAS genes. A single colony from each transformed cell lines was streaked onto M9 supplemented with valine, leucine and isoleucine (A), M9 (B) and M9 supplemented with 50 μM SMM (C) agar plates and incubated at 37° C. for 48 hrs. The sections are: 1. pEp-empty, 2. pAH29 (ilvGM), 3. pEp-ORF, 4. pEp-truncated and 5. pEp-mut.
FIG. 4. Is a micrograph of a SDS-PAGE analysis of IPTG induction of wild-type and mutant PiAHAS. Protein marker (M), Lane 1: wild-type PiAHAS before induction, lane 2: wild-type after induction, lane 3: mutant PiAHAS before induction, and lane 4: mutant PiAHAS after induction. 75 kD recombinant protein over-expression is visible.
FIG. 5. Is a graph showing pyruvate dependence for PiAHAS wild-type and its mutant. The reactions for AHAS wild-type (●) and its mutant (◯).
FIG. 6. Is a graph showing inhibition by SMM of AHAS wild-type (●) and its mutant (603 ).
FIGS. 7A and 7B collectively disclose, and referred to as FIG. 7, the DNA sequence encoding the genomic fragment of P. incisa, containing AHAS gene. (1) Loci of mutation are bold letters with gray background and (2) region of primers annealing used for cloning are underlined. (3) CDS region labeled with capital letters. (4) Region of primers annealing used for mutation introduction is double underlined.
FIG. 8. Is a scheme showing P. incisa gAHAS cloning in to pJET 2.1 for achieving stable nuclear transformation. The structure of P. incisa AHAS genomic loci is provided in 8A; the structure of the two fragments of AHAS gene, cloned into pJet 2.1 vector with overlapping regions, the insertion of 3′ AHAS fragment by Nhe I, Not I restriction sites are provided in 8B; the structure of the assembled genomic fragment of the P. incisa AHAS gene, cloned into pJet 2.1 vector is provided in 8C.



DETAILED DESCRIPTION OF THE INVENTION
In one aspect, the present invention provides an isolated protein. In one embodiment the present invention provides that the isolated protein is a polypeptide. In another embodiment, the present invention provides that the isolated protein is an enzyme. In another embodiment, the present invention provides that the isolated protein is acetohydroxyacid synthase (AHAS). In another embodiment, the present invention provides that the isolated protein is a microalgae AHAS. In another embodiment, the present invention provides that the isolated protein is Parietochloris incisa (Trebouxiophyceae) AHAS.
In another embodiment, the present invention provides an isolated acetohydroxyacid synthase (AHAS) comprising the amino acid sequence:







(SEQ ID NO: 1)


MQGTMRPTAGALQQTVGCWHVPAGIPHAQQALRGRILPEELKQRCSATKP


 


RAARQSAVTAAKLAEGKAGTPSRSLRQQPAAPQQQQQQQDSNELVALREA


 


AKASLSSPAPAEWVDRFGSEPRKGADILVQCLEREGAFRVFAYPGGASME


 


IHQALTRSGIIRNILCRHEQGEIFAAEGYAKCTGDVGVCIATSGPGATNL


 


VTGLADAMLDSVPLVAITGQVPRKMIGTDGFQETPIVEVTRQITKHNFLV


 


MDLDDLPRIMKEAFYLARTGRPGPVLVDVPKDIQQQLAVPDWDTPMAISG


 


YMSRLPAPPNPSQLAAVVRALKEAKRPTLYVGGGALDSSAELREFVRLTG


 


IPVAQTLMGLGTFPEEDPLALQMLGMHGTVYANYAVNDSDLLLAFGVRFD


 


DRVTGKLEAFASRACIVHIDIDPAEICKNKEAHIPICADLRASLIALNEL


 


LRRDPLPEGAFADWRAAIEAKKQEFPMTFPERDDVIIPQRAIQMLYEETN


 


GEAIISTGVGQHQMWAAQWYQYNEPRRWVTSGGLGSMGFGLPSALGAAAV


 


AYDGTDGRPKKVVVDIDGDGSFLMNCQELATAAVEGLETKIMILNNQHLG


 


MVVQWEDRFYKANRAHTYLGHRANEYHTTLDESHIFPDFVMMAKSCGVPG


 


RRVIKPEELRGAIREMLDTPGPFLLDVMVPHVEHVLPMIPGGGSFKDIIT


 


KGDGRDEY.






In another embodiment, the AHAS of the present invention comprises an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% homologous to the amino acid sequence of SEQ ID NO: 1. Each possibility represents a separate possibility of the invention.
In another embodiment, the AHAS of the present invention comprises an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identical to the amino acid sequence of SEQ ID NO: 1. Each possibility represents a separate possibility of the invention.
In another embodiment, the AHAS as described herein comprises at least a portion of the amino acid shown in SEQ ID NO: 1. In another embodiment, the AHAS as described herein is a variant of SEQ ID NO: 1.
The term “variant” as used herein refers to a protein or a polypeptide which is derived from the sequence through the insertion or deletion of one or more amino acid residues or the substitution of one or more amino acid residues with amino acid residues having similar properties, e.g., the replacement of a polar amino acid residue with another polar amino acid residue, or the replacement of a non-polar amino acid residue with another non-polar amino acid residue. In all cases, variants must have an AHAS function as defined herein.
In another embodiment, the AHAS as described herein further comprises a leader peptide. In another embodiment, the leader peptide allows the polypeptide to be specifically localized or targeted to a target organelle within the cell (e.g. chloroplast and mitochondria). In another embodiment, the AHAS as described herein further comprises a chemical modification such as glycosylation that increases its stability. In another embodiment, the AHAS as described herein further comprises a peptide unrelated to AHAS which increases its stability.
In another embodiment, the present invention provides an isolated AHAS. In another embodiment, the present invention provides that the polypeptide has the function of synthesizing branched-chain amino acid. In another embodiment, the branched-chain amino acid is leucine, isoleucine and valine. Each possibility represents a separate embodiment of the invention.
In another embodiment, the present invention provides an isolated AHAS which catalyzes the synthesis of (2S)-acetolactate (AL) from two molecules of pyruvate. In another embodiment, the present invention provides an isolated AHAS which catalyzes the synthesis of (2S)-2-aceto-2-hydroxybutyrate (AHB) from pyruvate and 2-ketobutyrate. In another embodiment, the present in invention provides an isolated AHAS comprising the amino acid sequence of SEQ ID NO: 1.
In another embodiment, the present invention provides a protein comprising an AHAS activity. In another embodiment, the present invention provides that the protein of the invention is a recombinant AHAS.
In another embodiment, the present invention provides an isolated polynucleotide encoding the protein comprising the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the isolated polynucleotide is an isolated DNA molecule. In another embodiment, the isolated polynucleotide is an isolated cDNA molecule. In another embodiment, the isolated polynucleotide comprises a DNA sequence encoding an AHAS as described herein. In another embodiment, the isolated polynucleotide comprises a DNA sequence encoding a polypeptide comprising an AHAS activity.
In another embodiment, the isolated polynucleotide comprises a DNA sequence comprising the sequence:







(SEQ ID NO: 3)


ATGCAAGGCACTATGCGGCCGACGGCTGGAGCACTGCAGCAGACCGTCGG


 


CTGCTGGCACGTCCCGGCCGGCATCCCCCACGCACAGCAGGCGCTGCGAG


 


GCCGTATACTGCCCGAGGAGCTGAAGCAGCGGTGTTCAGCAACCAAACCT


 


AGGGCGGCCAGGCAGTCAGCGGTGACCGCTGCCAAGCTTGCAGAGGGCAA


 


GGCAGGCACACCATCACGGAGCTTGCGGCAGCAGCCGGCAGCGCCGCAGC


 


AGCAGCAGCAGCAGCAGGATAGCAATGAGCTGGTGGCGTTGCGGGAGGCT


 


GCCAAGGCCTCGCTCTCATCCCCCGCGCCAGCCGAGTGGGTGGATCGGTT


 


TGGGTCGGAGCCCCGCAAGGGCGCGGATATCCTGGTGCAGTGCCTGGAGC


 


GCGAGGGGGCCTTCCGCGTGTTTGCCTACCCCGGCGGCGCCAGCATGGAG


 


ATCCATCAGGCCTTGACGCGCAGCGGCATCATCCGCAACATCCTGTGCCG


 


CCATGAGCAGGGCGAGATCTTTGCGGCGGCGGAGGGCTATGCCAAGTGCA


 


CGGATGTGGGCGTGTGCATCGCGACCAGCGGGCCCGGCGCCACCAACCTC


 


GTGACTGGCCTGGCTGATGCCATGCTGGACAGCGTTCCGCTTGTTGCCAT


 


CACAGGGCAGGTGCCCCGCAAGATGATCGGCACGGACGATGGGGTTCCAG


 


GAGACGCCGATTGTGGAGGTGACGCGGCAGATCACCAAGCACAACTTCCT


 


GGTCACCTGGATGACCTGCCGCGCATCATGAAGGAGGCATTCTACCTGGC


 


ACGGGGGCGCCGGCCGGGCCCGGTGCTGGTGGATGTGCCCAAGGACATCC


 


AGCAGCAGCTGGCGGTGCCGGACTGGGACACGCCCATGGCCATCAGCGGC


 


TACATGTCGCGCCTGCCGGCCCCGCCTAACCCCTCACAGCTGGCCGCGGT


 


CGTGCGCGCGCTGAAGGAGGCTAAGAGGCCGACGCTGTACGTGGGCGGTG


 


GCGCGCTCGACTCGAGCGCTGAGCTGCGCGAGTTTGTGCGGCTGACGGGC


 


CCCGCTATCCCCGTCGCGCAGACGCTTATGGGCCTGGGCACCTTCCCCGA


 


GGAAGAGGCACTGCAGATGCTGGGCATGCACGGTACGGTGTACGCCAACT


 


ACGCCGTCAACGACAGCGACCTGCTGCTGGCGTTTGGCGTGCGCTTCGAC


 


GACCGCGTGACTGGCAAGCTGGAGGCGTTTGCTTCGCGCGCGTGCATCGT


 


GCACATCGACATCGACCCTGCGGAGATCTGCAAGAACAAGGAGGCCCACA


 


TACCCATCTGTGCAGACCTGCGTGCGTCGTTGATAGCGCTGAATGAGCTG


 


TTGCGGCGAGACCCTCTGCCAGAGGGCGCCTTTGCGGACTGGCGGGCGGC


 


CATTGAGGCCAAGAAGCAGGAGTTCCCCATGACCTTCCCGGAGCGGGACG


 


ACGTGATCATCCCACAGCGTGCCATCCAGATGCTGTATGAGGAGACAAAC


 


GGCGAGGCCATCATCAGCACCGGCGTGGGCCAGCACCAGATGTGGGCGGC


 


GCAGTGGTACCAGTACAACGAGCCGCGGAGATGGGTCACGTCCGGCGGCC


 


TCGGTACGACGGCACTCCATGGGCTTTGGCCTGCCATCCGCCCTCGGCGC


 


GGCGGTGGCCCGGACGGTCGGCCCAAGAAGGTGGTGGTGGACATCGACGG


 


TTCCTGATGAACTGCCAGGAGCTGGCGACGGCGGCGGTGGAGGCGACGGC


 


AGTGCCTGGAGACCAAGATCATGATCCTCAACAACCAGCACCTGGGCATG


 


GTGGTCCAGTGGGAGGACCGCTTCTACAAGGCCAACCGCGCACACACCTA


 


CCTCGGCCACCGGGCCAATGAGTACCACACGACGCTGGACGAGAGCCACA


 


TCTTCCCCGACTTTGTCATGATGGCCAAGTCGTGCGGCGTCCCAGGCCGG


 


CGCGTCATCAAGCCCGAGGAGCTGCGCGGGGCCATCAGGGAGATGCTGGA


 


CACGCCCGGCCCCTTCCTGCTGGACGTGATGGTGCCGCATGTGGAGCACG


 


TGCTGCCCATGATCCCGGGCGGCGGCTCCTTCAAGGACATCATCACCAAG


 


GGCGACGGCCGCGACGAGTACTAA






In another embodiment, the isolated polynucleotide comprises a DNA sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% homologous to the amino acid sequence of SEQ ID NO: 3. Each possibility represents a separate possibility of the invention.
In another embodiment, the isolated polynucleotide comprises a DNA sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identical to the amino acid sequence of SEQ ID NO: 3. Each possibility represents a separate possibility of the invention.
In another embodiment, the present invention comprises a AHAS or a nucleic acid encoding same combined with additional proteins and/or enzymes and/or substrates that are involved in the biosynthesis of BCCA. In another embodiment, the present invention comprises a composition comprising a AHAS or a nucleic acid molecule encoding same combined with additional proteins and/or enzymes and/or substrates that are involved in the biosynthesis of BCCA. In another embodiment, the present invention comprises a transformed or transfected cell comprising AHAS or a nucleic acid molecule encoding same combined with additional proteins and/or enzymes and/or substrates that are involved in the biosynthesis of BCCA. In another embodiment, the present invention comprises a transgenic alga comprising AHAS or a nucleic acid molecule encoding same combined with additional proteins and/or enzymes and/or substrates that are involved in the biosynthesis of BCCA.
In another embodiment, the present invention provides an algal AHAS or a nucleic acid molecule encoding same combined with additional algal proteins and/or enzymes and/or substrates that are involved in the biosynthesis of BCCA. In another embodiment, the alga is microalgae. In another embodiment, the present invention provides a microalgae AHAS or a nucleic acid molecule encoding same combined with additional microalgae proteins and/or enzymes and/or substrates that are involved in the biosynthesis of BCCA. In another embodiment, the microalgae is Parietochloris incisa. In another embodiment, the present invention provides a Parietochloris incisa AHAS or a nucleic acid molecule encoding same combined with additional Parietochloris incisa proteins and/or enzymes and/or substrates that are involved in the biosynthesis of BCCA.
In another embodiment, the present invention provides a combination of AHAS and additional proteins and/or enzymes and/or substrates involved in the biosynthesis of BCCA. In another embodiment, the present invention provides a combination of AHAS and Threonine deaminase (TD), ketol-acid Reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD); transaminase (TA), 2-isopropylmalate synthase (2-IPMS), 3-isopropylmalate dehydratase (3-IPMD) and 3-isopropylmalate dehydrogenase (3-IPMDH). Each possibility represents a separate embodiment of the invention.
In another embodiment, the present invention provides a composition comprising the protein as described herein to be used in foodstuffs, dietary supplements or pharmaceutical compositions. In another embodiment, the present invention provides a composition comprising the protein described herein to be used in industrial applications for the manufacturing of BCCA. In another embodiment, the present invention provides composition comprising the BCCA. In another embodiment, a composition comprising BCCA is used in foodstuffs, dietary supplements or pharmaceutical compositions.
In another embodiment, an algal AHAS gene and AHAS protein of the present invention is superior when compared to its homologues with respect to efficient production of BCCA. In another embodiment, transforming a first alga with an algal AHAS derived from a second alga such as described herein is more efficient in the production of BCCA in comparison to transforming with a fungal AHAS. In another embodiment, transforming a first alga with an algal AHAS gene derived from a second alga such as described herein in combination with additional genes that encode proteins and/or enzymes involved in the biosynthesis of BCCA, is more efficient in the production of BCCA in comparison to transforming with AHAS fungal genes in combination with additional fungal genes. In another embodiment, the first alga is P. incisa. In another embodiment, P. incisa is the second alga. In another embodiment, P. incisa is the source of the additional genes that are involved in the biosynthesis of BCCA. In another embodiment, the source of the additional genes that are involved in the biosynthesis of BCCA is any organism comprising such genes.
In another embodiment, the present invention provides a method for producing branched-chain amino acid (BCCA) in a cell, comprising the step of transforming the cell with the polynucleotide as described herein. In another embodiment, the present invention provides a method for substantially enhancing the production of BCCA in a cell, comprising the step of transforming a cell with said polynucleotide as described herein.
In another embodiment, the present invention provides a method for producing branched-chain amino acid (BCCA) in a cell, comprising the step of transforming the cell with the polynucleotide as described herein, with an exogenous polynucleotide as described herein, with a combination of exogenous polynucleotides as described herein or with a vector comprising an exogenous polynucleotide as described herein, with a vector comprising a combination of exogenous polynucleotides as described herein, thereby producing BCCA in a cell, wherein each possibility represents a separate embodiment of the invention.
In another aspect, the present invention provides a protein comprising an amino acid sequence set forth in SEQ ID NO: 2. In one embodiment the present invention provides that the protein is a polypeptide. In another embodiment, the present invention provides that the protein is an enzyme. In another embodiment, the present invention provides a protein comprising an amino acid sequence set forth in SEQ ID NO: 1, wherein said sequence comprises at least one amino acid substitution. In another embodiment, the at least one amino acid substitution is at residue Trp 605. In another embodiment, the at least one amino acid substitution is W605S.
In another embodiment, the present invention provides a protein comprising the amino acid sequence:







(SEQ ID NO: 2)


MQGTMRPTAGALQQTVGCWHVPAGIPHAQQALRGRILPEELKQRCSATKP


 


RAARQSAVTAAKLAEGKAGTPSRSLRQQPAAPQQQQQQQDSNELVALREA


 


AKASLSSPAPAEWVDRFGSEPRKGADILVQCLEREGAFRVFAYPGGASME


 


IHQALTRSGIIRNILCRHEQGEIFAAEGYAKCTGDVGVCIATSGPGATNL


 


VTGLADAMLDSVPLVAITGQVPRKMIGTDGFQETPIVEVTRQITKHNFLV


 


MDLDDLPRIMKEAFYLARTGRPGPVLVDVPKDIQQQLAVPDWDTPMAISG


 


YMSRLPAPPNPSQLAAVVRALKEAKRPTLYVGGGALDSSAELREFVRLTG


 


IPVAQTLMGLGTFPEEDPLALQMLGMHGTVYANYAVNDSDLLLAFGVRFD


 


DRVTGKLEAFASRACIVHIDIDPAEICKNKEAHIPICADLRASLIALNEL


 


LRRDPLPEGAFADWRAAIEAKKQEFPMTFPERDDVIIPQRAIQMLYEETN


 


GEAIISTGVGQHQMWAAQWYQYNEPRRWVTSGGLGSMGFGLPSALGAAAV


 


AYDGTDGRPKKVVVDIDGDGSFLMNCQELATAAVEGLETKIMILNNQHLG


 


MVVQSEDRFYKANRAHTYLGHRANEYHTTLDESHIFPDFVMMAKSCGVPG


 


RRVIKPEELRGAIREMLDTPGPFLLDVMVPHVEHVLPMIPGGGSFKDIIT


 


KGDGRDEY.






In another embodiment, the protein comprises an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% homologous to the amino acid sequence of SEQ ID NO: 2. Each possibility represents a separate possibility of the invention.
In another embodiment, the protein comprises an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identical to the amino acid sequence of SEQ ID NO: 2. Each possibility represents a separate possibility of the invention.
In another embodiment, the protein as described herein, comprises at least a portion of the amino acid shown in SEQ ID NO: 2. In another embodiment, the protein as described herein is a variant of SEQ ID NO: 2.
In another embodiment, the present invention provides a polynucleotide encoding a protein comprising the amino acid set forth in SEQ ID NO: 2. In another embodiment, the polynucleotide is a DNA molecule. In another embodiment, the polynucleotide is a cDNA molecule.
In another embodiment, the polynucleotide comprises a DNA sequence comprising the sequence:







(SEQ ID NO: 4)


ATGCAAGGCACTATGCGGCCGACGGCTGGAGCACTGCAGCAGACCGTCGG


 


CTGCTGGCACGTCCCGGCCGGCATCCCCCACGCACAGCAGGCGCTGCGAG


 


GCCGTATACTGCCCGAGGAGCTGAAGCAGCGGTGTTCAGCAACCAAACCT


 


AGGGCGGCCAGGCAGTCAGCGGTGACCGCTGCCAAGCTTGCAGAGGGCAA


 


GGCAGGCACACCATCACGGAGCTTGCGGCAGCAGCCGGCAGCGCCGCAGC


 


AGCAGCAGCAGCAGCAGGATAGCAATGAGCTGGTGGCGTTGCGGGAGGCT


 


GCCAAGGCCTCGCTCTCATCCCCCGCGCCAGCCGAGTGGGTGGATCGGTT


 


TGGGTCGGAGCCCCGCAAGGGCGCGGATATCCTGGTGCAGTGCCTGGAGC


 


GCGAGGGGGCCTTCCGCGTGTTTGCCTACCCCGGCGGCGCCAGCATGGAG


 


ATCCATCAGGCCTTGACGCGCAGCGGCATCATCCGCAACATCCTGTGCCG


 


CCATGAGCAGGGCGAGATCTTTGCGGCGGAGGGCTATGCCAAGTGCACGG


 


GCGATGTGGGCGTGTGCATCGCGACCAGCGGGCCCGGCGCCACCAACCTC


 


GTGACTGGCCTGGCTGATGCCATGCTGGACAGCGTTCCGCTTGTTGCCAT


 


CACAGGGCAGGTGCCCCGCAAGATGATCGGCACGGACGGGTTCCAGGAGA


 


CGCCGATTGTGGAGGTGACGCGGCAGATCACCAAGCACAACTTCCTGGTC


 


ATGGACCTGGATGACCTGCCGCGCATCATGAAGGAGGCATTCTACCTGGC


 


GCGCACGGGGCGGCCGGGCCCGGTGCTGGTGGATGTGCCCAAGGACATCC


 


AGCAGCAGCTGGCGGTGCCGGACTGGGACACGCCCATGGCCATCAGCGGC


 


TACATGTCGCGCCTGCCGGCCCCGCCTAACCCCTCACAGCTGGCCGCGGT


 


CGTGCGCGCGCTGAAGGAGGCTAAGAGGCCGACGCTGTACGTGGGCGGTG


 


GCGCGCTCGACTCGAGCGCTGAGCTGCGCGAGTTTGTGCGGCTGACGGGC


 


ATCCCCGTCGCGCAGACGCTTATGGGCCTGGGCACCTTCCCCGAGGAAGA


 


CCCGCTGGCACTGCAGATGCTGGGCATGCACGGTACGGTGTACGCCAACT


 


ACGCCGTCAACGACAGCGACCTGCTGCTGGCGTTTGGCGTGCGCTTCGAC


 


GACCGCGTGACTGGCAAGCTGGAGGCGTTTGCTTCGCGCGCGTGCATCGT


 


GCACATCGACATCGACCCTGCGGAGATCTGCAAGAACAAGGAGGCCCACA


 


TACCCATCTGTGCAGACCTGCGTGCGTCGTTGATAGCGCTGAATGAGCTG


 


TTGCGGCGAGACCCTCTGCCAGAGGGCGCCTTTGCGGACTGGCGGGCGGC


 


CATTGAGGCCAAGAAGCAGGAGTTCCCCATGACCTTCCCGGAGCGGGACG


 


ACGTGATCATCCCACAGCGTGCCATCCAGATGCTGTATGAGGAGACAAAC


 


GGCGAGGCCATCATCAGCACCGGCGTGGGCCAGCACCAGATGTGGGCGGC


 


GCAGTGGTACCAGTACAACGAGCCGCGGAGATGGGTCACGTCCGGCGGCC


 


TCGGCTCCATGGGCTTTGGCCTGCCATCCGCCCTCGGCGCGGCGGTGGCC


 


TACGACGGCACGGACGGTCGGCCCAAGAAGGTGGTGGTGGACATCGACGG


 


CGACGGCAGTTTCCTGATGAACTGCCAGGAGCTGGCGACGGCGGCGGTGG


 


AGGGCCTGGAGACCAAGATCATGATCCTCAACAACCAGCACCTGGGCATG


 


GTGGTCCAGTCGGAGGACCGCTTCTACAAGGCCAACCGCGCACACACCTA


 


CCTCGGCCACCGGGCCAATGAGTACCACACGACGCTGGACGAGAGCCACA


 


TCTTCCCCGACTTTGTCATGATGGCCAAGTCGTGCGGCGTCCCAGGCCGG


 


CGCGTCATCAAGCCCGAGGAGCTGCGCGGGGCCATCAGGGAGATGCTGGA


 


CACGCCCGGCCCCTTCCTGCTGGACGTGATGGTGCCGCATGTGGAGCACG


 


TGCTGCCCATGATCCCGGGCGGCGGCTCCTTCAAGGACATCATCACCAAG


 


GGCGACGGCCGCGACGAGTACTAA






In another embodiment, the polynucleotide comprises a DNA sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% homologous to the amino acid sequence of SEQ ID NO: 4. Each possibility represents a separate possibility of the invention.
In another embodiment, the polynucleotide comprises a DNA sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identical to the amino acid sequence of SEQ ID NO: 4. Each possibility represents a separate possibility of the invention.
In another embodiment, the present invention comprises a composition comprising a protein comprising or consisting the sequence set forth in SEQ ID NO: 2 or a nucleic acid molecule encoding same. In another embodiment, the present invention comprises a transformed or transfected cell comprising the protein comprising the sequence set forth in SEQ ID NO: 2 or a nucleic acid molecule encoding same. In another embodiment, the present invention comprises a transgenic alga comprising the protein comprising the sequence set forth in SEQ ID NO: 2 or a nucleic acid molecule encoding same.
In another embodiment, the present invention provides a protein isolated from alga and further mutated, the protein comprising the sequence set forth in SEQ ID NO: 2 or a nucleic acid molecule encoding same. In another embodiment, the alga is microalgae. In another embodiment, the present invention provides a protein isolated from microalgae and further mutated, the protein comprising the sequence set forth in SEQ ID NO: 2 or a nucleic acid molecule encoding same. In another embodiment, the microalgae is Parietochloris incisa. In another embodiment, the present invention provides a protein isolated from Parietochloris incisa and further mutated, the protein comprising the sequence set forth in SEQ ID NO: 2 or a nucleic acid molecule encoding same.
In another embodiment, the present invention provides a combination of the protein comprising the sequence set forth in SEQ ID NO: 2 and additional proteins conferring resistance to herbicides. In another embodiment, the present invention provides a combination of the protein comprising the sequence set forth in SEQ ID NO: 2 and proteins having target-site mutations which confer resistance to herbicides. In another embodiment, proteins having target-site mutations which confer resistance to herbicides include but are not limited to phytoene desaturase (PDS) and tubulin.
In another embodiment, the present invention provides a method for conferring herbicide resistance in a cell comprising the step of transforming the cell with a polynucleotide encoding the protein comprising the sequence set forth in SEQ ID NO: 2. In another embodiment, the present invention provides a method for conferring herbicide resistance in a cell comprising the step of transforming the cell with a polynucleotide comprising the sequence set forth in SEQ ID NO: 4.
In another embodiment, the present invention provides a method for conferring resistance to herbicides in a cell, comprising the step of transforming the cell with a polynucleotide comprising the sequence set forth in SEQ ID NO: 4, wherein the herbicides include but are not limited to sulfonylureas, imidazolinones and triazolopyrimidines. In another embodiment, sulfonylurea herbicides include but are not limited to pyrimidinylsulfonylurea herbicides and triazinylsulfonylurea herbicides. In another embodiment, pyrimidinylsulfonylurea herbicides include but are not limited to: amidosulfuron, azimsulfuron, bensulfuron, chlorimuron, cyclosulfamuron, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, foramsulfuron, halo sulfuron, imazosulfuron, me s o sulfuron, metazosulfuron, methiopyrisulfuron, monosulfuron, nicosulfuron, orthosulfamuron, oxasulfuron, primisulfuron propyrisulfuron, pyrazosulfuron, rimsulfuron, sulfometuron, sulfometuron methyl, sulfosulfuron, trifloxysulfuron and zuomihuanglong. In another embodiment, triazinylsulfonylurea herbicides include but are not limited to chlorsulfuron, cinosulfuron, ethametsulfuron, iodosulfuron, iofensulfuron, metsulfuron, prosulfuron, thifensulfuron, triasulfuron, tribenuron, triflusulfuron and tritosulfuron.
In another embodiment, the present invention provides a method for conferring herbicide resistance in a cell, comprising the step of transforming the cell with the polynucleotide described herein, wherein the cell produces long-chain polyunsaturated fatty acid (LC-PUFA), arachidonic acid (ARA) and rich triacylglycerols (TAG). In another embodiment, the present invention provides a method for conferring herbicide resistance in an alga, comprising the step of transforming the alga with the polynucleotide described herein, wherein the alga produces long-chain polyunsaturated fatty acid (LC-PUFA), arachidonic acid (ARA) and rich triacylglycerols (TAG). In another embodiment, the present invention provides a method for conferring herbicide resistance in Parietochloris incisa, comprising the step of transforming e Parietochloris incisa with the polynucleotide described herein, wherein Parietochloris incisa produces long-chain polyunsaturated fatty acid (LC-PUFA), arachidonic acid (ARA) and rich triacylglycerols (TAG).
In another embodiment, the method of the present invention further comprises the step of transforming a cell with at least one polynucleotide encoding an enzyme involved in the biosynthesis of ω-3 LC-PUFA, thereby producing a cell having herbicide resistance and at least one enzyme involved in the biosynthesis of ω-3 LC-PUFA. In another embodiment, the method of the present invention further comprises the step of transforming a cell with ω-3 desaturase, C20 PUFA elongase, Δ4 desaturase or combinations thereof, thereby producing a cell having herbicide resistance and at least one enzyme involved in the biosynthesis of ω-3 LC-PUFA.
In another embodiment, the expression and/or transcription of the enzyme involved in the biosynthesis of ω-3 LC-PUFA as described herein is up-regulated during nitrogen starvation. In another embodiment, the expression and/or transcription of ω-3 desaturase, C20 PUFA elongase and Δ4 desaturase as described herein, is up-regulated during nitrogen starvation.
In another embodiment, the protein comprising the sequence set forth in SEQ ID NO: 2 is used indirectly or directly for conferring herbicide resistance in cells and capable of producing polyunsaturated fatty acids. In another embodiment, “Directly” is meant to encompass the situation where the enzyme directly enhances herbicide resistance in a cell. In another embodiment, “Indirectly” is meant to encompass the situation where a protein, other than the protein of the invention, is modified to be herbicide resistant by the protein of the invention, described herein. In another embodiment, a very long-chain polyunsaturated fatty acid produced by cells having herbicide resistance, either directly or indirectly is added to a nutritional composition, pharmaceutical compositions, cosmetics, and animal feeds, all of which are encompassed by the present invention.
In another embodiment, a long-chain polyunsaturated fatty acid produced by a protein or a combination of proteins in a cell or alga cell having herbicide resistance produced by methods described herein, is utilized in an infant formula. In another embodiment, a long-chain polyunsaturated fatty acid produced by a protein or a combination of proteins in a cell or alga cell having herbicide resistance produced by methods described herein, is administered to a subject having a deficiency in very long-chain polyunsaturated fatty acid.
In another embodiment, a cell is a eukaryotic cell. In another embodiment, a cell is a prokaryotic cell. In another embodiment, a cell is a plant cell. In another embodiment, a cell is an algal cell. In another embodiment, a cell is a microalga cell. In another embodiment, a cell is a Parietochloris incisa cell. In another embodiment, a cell is a transfected cell. In another embodiment, a cell is transiently transfected with a polynucleotide or a combination of polynucleotides as described herein. In another embodiment, a cell is stably transfected cell with a polynucleotide or a combination of polynucleotides as described herein.
In another embodiment, the present invention provides a composition comprising a polypeptide as described herein. In another embodiment, the present invention provides a composition comprising a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Each possibility represents a separate embodiment of the invention.
In another embodiment, the present invention provides a composition comprising the polynucleotide as described herein. In another embodiment, the present invention provides a composition comprising the polynucleotide encoding the protein comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Each possibility represents a separate embodiment of the invention.
In another embodiment, the present invention provides a composition comprising the polynucleotide comprising the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4. Each possibility represents a separate embodiment of the invention.
In another embodiment, the present invention provides a composition comprising a vector comprising a polynucleotide as described herein. In another embodiment, the present invention provides composition comprising a combination of vectors which comprise polynucleotides as described herein. In another embodiment, a composition such as described herein, comprises an excipient. In another embodiment, a composition such as described herein, comprises a carrier. In another embodiment, a carrier stabilizes a protein or a nucleic acid molecule of the invention. In another embodiment, one of skill in the art will readily identify a known suitable carrier to be used with the composition as described herein. In another embodiment, one of skill in the art is able to prepare a composition comprising AHAS as described herein, or a polynucleotide as described herein, or a combination of polynucleotides and vectors as described herein. Each possibility represents a separate embodiment of the invention.
In another embodiment, algae as described herein are eukaryotic organisms. In another embodiment, algae as described herein are photoautotrophic. In another embodiment, algae as described herein are mixotrophic. In another embodiment, algae as described herein are unicellular. In another embodiment, algae as described herein are multicellular. In another embodiment, algae as described herein are Excavata algae. In another embodiment, algae as described herein are Rhizaria algae. In another embodiment, algae as described herein are Chromista algae. In another embodiment, algae as described herein are Alveolata algae.
Polypeptides and Polynucleotides
In some embodiments, the terms “protein” or “polypeptide” are used herein interchangeably and encompasses native polypeptides (either degradation products, synthetically synthesized polypeptides or recombinant polypeptides) and peptidomimetics (typically, synthetically synthesized polypeptides), as well as peptoids and semipeptoids which are polypeptide analogs, which have, in some embodiments, modifications rendering the polypeptides/proteins even more stable while in a body or more capable of penetrating into cells.
In some embodiments, modifications include, but are not limited to N terminus modification, C terminus modification, polypeptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein.
In some embodiments, polypeptide bonds (—CO—NH—) within the polypeptide are substituted. In some embodiments, the polypeptide bonds are substituted by N-methylated bonds (—N(CH3)—CO—), by ester bonds (—C(R)H—C—O—O—C(R)—N—), by ketomethylene bonds (—CO—CH2-) or α-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carbo bonds (—CH2-NH—). Each possibility represents a separate embodiment of the invention. In some embodiments, the polypeptide bonds are substituted by hydroxyethylene bonds (—CH(OH)—CH2-), by thioamide bonds (—CS—NH—), by olefinic double bonds (—CH═CH—) or by retro amide bonds (—NH—CO—), by polypeptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturally presented on the carbon atom. In some embodiments, these modifications occur at any of the bonds along the polypeptide chain and even at several (2-3 bonds) at the same time.
In some embodiments, natural aromatic amino acids of the polypeptide such as Trp, Tyr and Phe, be substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. In some embodiments, the polypeptides of the present invention include one or more modified amino acid or one or more non-amino acid monomers (e.g., fatty acid, complex carbohydrates, etc.).
In one embodiment, “amino acid” or “amino acids” is understood to include the 20 naturally occurring amino acid; those amino acid often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acid including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. In one embodiment, “amino acid” includes both D- and L-amino acid.
In some embodiments, the polypeptides or proteins of the present invention are utilized in a soluble form. In some embodiments, the polypeptides or proteins of the present invention include one or more non-natural or natural polar amino acid, including but not limited to serine and threonine which are capable of increasing polypeptide or protein solubility due to their hydroxyl-containing side chain.
In some embodiments, the polypeptides or proteins of the present invention are utilized in a linear form, although it will be appreciated by one skilled in the art that in cases where cyclization does not severely interfere with polypeptide characteristics, cyclic forms of the polypeptide can also be utilized.
In some embodiments, the polypeptides or proteins of present invention are biochemically synthesized such as by using standard solid phase techniques. In some embodiments, these biochemical methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. In some embodiments, these methods are used when the polypeptide is relatively short (about 5-15 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
In some embodiments, solid phase polypeptide or protein synthesis procedures are well known to one skilled in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). In some embodiments, synthetic polypeptides or proteins are purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.], and the composition of which can be confirmed via amino acid sequencing by methods known to one skilled in the art.
In some embodiments, recombinant protein techniques are used to generate the polypeptides of the present invention. In some embodiments, recombinant protein techniques are used for generation of relatively long polypeptides (e.g., longer than 18-25 amino acid). In some embodiments, recombinant protein techniques are used for the generation of large amounts of the polypeptide of the present invention. In some embodiments, recombinant techniques are described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al, (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.
In one embodiment, a polypeptide or protein of the present invention is synthesized using a polynucleotide encoding a polypeptide or protein of the present invention. In some embodiments, the polynucleotide encoding a polypeptide of the present invention is ligated into an expression vector, comprising a transcriptional control of a cis-regulatory sequence (e.g., promoter sequence). In some embodiments, the cis-regulatory sequence is suitable for directing constitutive expression of the polypeptide of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing tissue specific expression of the polypeptide of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing inducible expression of the polypeptide of the present invention.
In one embodiment, the phrase “a polynucleotide” refers to a single or double stranded nucleic acid sequence which may be isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above). Each possibility represents a separate embodiment of the invention. In one embodiment, “genomic polynucleotide sequence” refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
In one embodiment, “composite polynucleotide sequence” refers to a sequence, which is at least partially complementary and at least partially genomic. In one embodiment, a composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing there between. In one embodiment, the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. In one embodiment, intronic sequences include cis acting expression regulatory elements.
In one embodiment, the polynucleotides of the present invention further comprise a signal sequence encoding a signal peptide for the secretion of the polypeptides of the present invention. In one embodiment, following expression, the signal peptides are cleaved from the precursor proteins resulting in the mature proteins.
In some embodiments, polynucleotides of the present invention are prepared using PCR techniques or any other method or procedure known to one skilled in the art. In some embodiments, the procedure involves the legation of two different DNA sequences (See, for example, “Current Protocols in Molecular Biology”, eds. Ausubel et al., John Wiley & Sons, 1992).
In one embodiment, polypeptides or proteins of the present invention are purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
In one embodiment, the polypeptide or protein of the present invention is retrieved in “substantially pure” form. In one embodiment, the phrase “substantially pure” refers to a purity that allows for the effective use of the protein in the applications described herein.
In one embodiment, the polypeptide or protein of the present invention can also be synthesized using in vitro expression systems. In one embodiment, in vitro synthesis methods are well known in the art and the components of the system are commercially available.
Expression and Transformation Systems
In another embodiment, the terms “transformation”, “transduction”, “transfection” and “conjugation” are used herein interchangeably and refer to the insertion of new genetic material into nonbacterial cells including animal and plant cells, applied to eukaryotic and prokaryotic cells.
In another embodiment, the present invention provides an engineered organism such as a transgenic plant, a transgenic seed, a transgenic alga and a transgenic animal. Each possibility represents a separate embodiment of the invention. In another embodiment, an engineered organism is engineered to express a protein, a polypeptide, a combination of polypeptides, a polynucleotide and a combination of polynucleotides as described herein. Each possibility represents a separate embodiment of the invention.
In one embodiment, polynucleotides of the present invention are inserted into expression vectors (i.e., a nucleic acid construct) to enable expression of the recombinant polypeptide. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention includes a shuttle vector which renders this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, cloning vectors comprise transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals).
In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the polypeptide coding sequence.
In some embodiments, non-bacterial expression systems are used (e.g., plant expression systems) to express the polypeptide of the present invention. In one embodiment, yeast expression systems are used. In one embodiment, algae expression systems are used. In one embodiment, plant expression systems are used. In one embodiment, a number of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Pat. No: 5,932,447 which is hereby incorporated in its entirety by reference. In another embodiment, vectors which promote integration of foreign DNA sequences into the yeast chromosome are used.
In another embodiment, expression in a host cell can be accomplished in a transient or a stable fashion. In another embodiment, a host cell is a cell as described herein. In another embodiment, transient expression is from introduced constructs which contain expression signals functional in the host cell, but which constructs do not replicate and rarely integrate in the host cell, or where the host cell is not proliferating. In another embodiment, transient expression also can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest.
In another embodiment, stable expression is achieved by introduction of a construct that integrates into the host genome. In another embodiment, stable expression comprises autonomously replication within the host cell. In another embodiment, stable expression of the polynucleotide of the invention is selected for the use of a selectable marker located on or transfected with the expression construct, followed by selection for cells expressing the marker. In another embodiment, the site of the construct's integration can occur randomly within the host genome or can be targeted through constructs containing regions of homology with the host genome sufficient to target recombination with the host locus. In another embodiment, constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.
In another embodiment, a plant or plant tissue is utilized as a host or host cell, respectively, for expression of the protein/s disclosed herein, used in turn, for the production of BCCA In another embodiment, a vector which comprises a DNA sequence encoding the protein/s as described herein is linked to a promoter, and is introduced into the plant tissue or plant for a time and under conditions sufficient for expression of the protein.
In another embodiment, the regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (for example: Weissbach and Weissbach, In: Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc. San Diego, Calif., (1988)). In another embodiment, regeneration and growth process comprises the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. In another embodiment, transgenic embryos and seeds are similarly regenerated. In another embodiment, resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
In another embodiment, regeneration and growth process of algae are known to one of skill in the art. In another embodiment, identification, selection, of transgenic algae are known to one of skill in the art.
In another embodiment, development or regeneration of plants containing an exogenous polynucleotide as described herein encodes a protein as described herein and is well known in the art. In another embodiment, development or regeneration of algae containing an exogenous polynucleotide as described herein encodes a protein as described herein and is well known in the art. In another embodiment, the regenerated plants are self-pollinated to provide homozygous transgenic plants. In another embodiment, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. In another embodiment, pollen from plants of these important lines is used to pollinate regenerated plants. In another embodiment, a transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
In another embodiment, a variety of methods can be utilized for the regeneration of plants from plant tissue. In another embodiment, the method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. In another embodiment, methods for transforming dicots, primarily by use of Agrobacterium tumefaciens, and obtaining transgenic plants are known in the art McCabe et al., Biol. Technology 6:923 (1988), Christou et al., Plant Physiol. 87:671-674 (1988)); Cheng et al., Plant Cell Rep. 15:653657 (1996), McKently et al., Plant Cell Rep. 14:699-703 (1995)); Grant et al., Plant Cell Rep. 15:254-258, (1995).
In another embodiment, transformation of monocotyledons using electroporation, particle bombardment, and Agrobacterium are known in the art. In another embodiment, transformation and plant regeneration are well established in the art. In another embodiment, assays for gene expression based on the transient expression of cloned nucleic acid constructs have been developed by introducing the nucleic acid molecules into plant cells by polyethylene glycol treatment, electroporation, or particle bombardment (Marcotte et al., Nature 335:454-457 (1988); Marcotte et al., Plant Cell 1:523-532 (1989); McCarty et al., Cell 66:895-905 (1991); Hattori et al., Genes Dev. 6:609-618 (1992); Goff et al., EMBO J. 9:2517-2522 (1990)).
In another embodiment, transient expression systems are used to functionally dissect the oligonucleotides constructs. In another embodiment, practitioners are familiar with the standard resource materials which describe specific conditions and procedures for the construction, manipulation and isolation of macromolecules (e.g., DNA molecules, plasmids, etc.), generation of recombinant organisms and the screening and isolating of clones, (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989); Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995); Birren et al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor, N.Y. (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, Cold Spring Harbor, N.Y. (1998); Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer, N.Y. (1997)).
In one embodiment, the expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. In some embodiments, recombinant viral vectors are useful for in vivo expression of the polypeptides of the present invention since they offer advantages such as lateral infection and targeting specificity. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus, and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.
It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide or protein), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide or protein.
In some embodiments, transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide or protein. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide or protein of the present invention. In some embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art.
In some embodiments, depending on the vector and host system used for production, resultant polypeptides or proteins of the present invention either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, or retained on the outer surface of a cell or viral membrane.
Nutritional Compositions
In another embodiment, the described compositions are utilized in connection with animals (i.e., domestic or non-domestic), as well as humans, as animals experience many of the same needs and conditions as humans.
In another embodiment, nutritional compositions include any food or preparation for human or animal consumption including for enteral or parenteral consumption, which when taken into the body (a) serve to nourish or build up tissues or supply energy and/or (b) maintain, restore or support adequate nutritional status or metabolic functions. In another embodiment, the composition includes edible macronutrients, vitamins and minerals in amounts desired for a particular use. In another embodiment, the amount of such ingredients will vary depending on whether the composition is intended for use with normal, healthy infants, children or adults having specialized needs such as those which accompany certain metabolic conditions (e.g., metabolic disorders).
In another embodiment, the macronutrients include edible fats, carbohydrates and proteins. In another embodiment, edible fats include but are not limited to coconut oil, soy oil, and mono- and diglycerides. In another embodiment, carbohydrates include but are not limited to glucose, edible lactose and hydrolyzed search. In another embodiment, proteins which are utilized in the nutritional composition of the invention include but are not limited to soy proteins, electrodialysed whey, electrodialysed skim milk, milk whey, or the hydrolysates of these proteins.
In another embodiment, vitamins and minerals are added to the nutritional compositions of the present invention and include but are not limited to: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other such vitamins and minerals may also be added.
In another embodiment, components utilized in the nutritional compositions of the present invention will be of semi-purified or purified origin. By semi-purified or purified is meant a material which has been prepared by purification of a natural material or by synthesis. In another embodiment, nutritional compositions of the present invention include but are not limited to infant formulas, dietary supplements, dietary substitutes, and rehydration compositions. In another embodiment, a nutritional composition of the present invention may also be added to food even when supplementation of the diet is not required. In another embodiment, a composition is added to food of any type including but not limited to margarines, modified butters, cheeses, milk, yogurt, chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats, fish and beverages.
In another embodiment, a nutritional composition is an enteral nutritional product. In another embodiment, a nutritional composition is an adult or pediatric enteral nutritional product. In another embodiment, a composition is administered to adults or children experiencing stress or having specialized needs due to chronic or acute disease states. In another embodiment, a composition comprises, in addition to polyunsaturated fatty acids produced in accordance with the present invention, macronutrients, vitamins and minerals as described above. In another embodiment, the macronutrients may be present in amounts equivalent to those present in human milk or on an energy basis, i.e., on a per calorie basis.
In another embodiment, the present invention includes an enteral formula comprising polyunsaturated fatty acids produced in accordance with the teachings of the present invention. In another embodiment, an enteral formula is sterilized and subsequently utilized on a ready-to-feed basis or stored in a concentrated liquid or powder. In another embodiment, a powder is prepared by spray drying the formula prepared as indicated above, and reconstituting it by rehydrating the concentrate. In another embodiment, the present invention includes an adult and pediatric nutritional formulas. In another embodiment, adult and pediatric nutritional formulas are known in the art and are commercially available (e.g., Similac®, Ensure®, Jevity® and Alimentum® from Ross Products Division, Abbott Laboratories).
In another embodiment, PUFAs produced in accordance with the present invention, or derivatives thereof, are added to a dietary substitute or supplement, particularly an infant formula, for patients undergoing intravenous feeding or for preventing or treating malnutrition or other conditions or disease states. In another embodiment, PUFAs produced in accordance with the present invention are used to alter, the composition of infant formulas in order to better replicate the PUFA content of human breast milk or to alter the presence of PUFAs normally found in a non-human mammal's milk.
Pharmaceutical Composition
In one embodiment, the polypeptides or proteins of the present invention can be provided to the individual as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. In one embodiment, a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. In one embodiment, “active ingredient” refers to the polypeptide or protein sequence of interest.
In one embodiment, the phrase “physiologically acceptable carrier” refers to a carrier or a diluent that does not cause significant irritation to a tissue such as a plant tissue or a cell such as a plant cell; and does not abrogate the biological activity and properties of the protein or polynucleotide of the invention. An adjuvant is included under these phrases. In one embodiment, one of the ingredients included in the physiologically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).
In one embodiment, “excipient” refers to an inert substance added to the composition to further facilitate administration of an active ingredient. In one embodiment, excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
In another embodiment, techniques for formulation and administration of peptide to plants or in-vitro are known to one of skill in the art. In one embodiment, compositions of the present invention are manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, or lyophilizing processes. In one embodiment, compositions for use in accordance with the present invention is formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the proteins/polynucleotides into preparations. In one embodiment, formulation is dependent upon the method of administration chosen.
The compositions also comprise, in some embodiments, preservatives, such as benzalkonium chloride and thimerosal and the like; chelating agents, such as edetate sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfote and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acid and bases to adjust the pH of these aqueous compositions as needed. The compositions also comprise, in some embodiments, local anesthetics or other actives. The compositions can be used as sprays, mists, drops, and the like.
Additionally, suspensions of the active ingredients, in some embodiments, are prepared as appropriate oily or water based suspensions. Suitable lipophilic solvents or vehicles include, in some embodiments, fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions contain, in some embodiments, substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. In another embodiment, the suspension also contains suitable stabilizers or agents, which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
In another embodiment, the proteins as described herein can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid). In some embodiments, the protein as described herein is in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use. In another embodiment, compositions are contained in a container with attached atomizing means.
In some embodiments, compositions suitable for use in context of the present invention include compositions wherein the proteins or oligonucleotides are contained in an amount effective to achieve the intended purpose. In one embodiment, determination of the effective amount is well within the capability of those skilled in the art.
Some examples of substances which can serve as carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TweenTM brand emulsifiers; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions. The choice of a pharmaceutically-acceptable carrier to be used in conjunction with the compound is basically determined by the way the compound is to be administered. If the subject compound is to be injected, in one embodiment, the pharmaceutically-acceptable carrier is sterile, physiological saline, with a blood-compatible suspending agent, the pH of which has been adjusted to about 7.4.
In addition, the compositions further comprise binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCl., acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents(e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.
The compositions also include incorporation of the proteins or oligonucleotides of the invention into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.
In some embodiments, the proteins or oligonucleotides of the invention modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. In another embodiment, the modified proteins or oligonucleotides of the invention exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds. In one embodiment, modifications also increase the proteins or oligonucleotides solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. In another embodiment, the desired in vivo biological activity is achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound.
The following examples are presented to provide a more complete understanding of the invention. The specific techniques, conditions, materials, proportions and reported data set forth to illustrate the principles of the invention are exemplary and should not be construed as limiting the scope of the invention.
EXAMPLES
Experimental Procedures
Algal Growth Conditions
P. incisa was isolated and maintained (Watanabe S et al. Parietochloris incisa comb. nov. (Trebouxiophyceae, Chlorophyta). Phycol. Res. 1996, 44, 107-108) and subsequently axenic cultures of P. incisa were cultivated on BG-11 nutrient medium (Stanier R. et al. Purification and properties of unicellular blue-green algae (order Chroococcales). Microbiology and Molecular Biology Reviews 1971, 35, 171-205) in 250-ml Erlenmeyer glass flasks in an incubator shaker at a speed of 170 rpm, under an air/CO2 atmosphere (99:1, v/v), controlled temperature (25° C.) and illumination (115 μmol quanta m−2 S−1) (Bigogno C et al. Biosynthesis of arachidonic acid in the oleaginous microalga Parietochloris incisa (Chlorophyceae): radiolabeling studies. Lipids 2002, 37, 209-216).
Construction of a P. incisa cDNA Library
One microgram of total RNA was reverse-transcribed into cDNA using a VersoTM cDNA kit (ABgene, Surrey, UK), according to the manufacturer's instructions. Each 20 μl reaction mix contained 1 μg of total RNA, 300 ng of random hexamers and 125 ng of anchored oligo-dT, dNTP mix (500 μM each), cDNA synthesis buffer, RT enhancer, and Verso enzyme mix. Following cDNA synthesis at 42° C. for 1 h, reactions were stopped by heating at 95° C. for 2 min and cDNA was diluted tenfold with PCR grade water.
Cloning of the PiAHAS cDNA
Several known amino acid and nucleotide sequences of the AHAS gene in related green algae and other higher plants were aligned, using ClustalW, retrieved from the Internet to identify conserved motifs in the enzyme. The conserved “blocks” were used to design two oligonucleotide primer sets, AHAS01 and AHAS02, for cloning partial PiAHAS sequences. All the primers used for PCR and sequencing are listed in Table 1. All the primers were designed by Primer3, version 0.4.0, retrieved from the Internet, software and checked with NetPrimer retrieved from the Internet. PCT amplifications were carried out using the first strand cDNA as a template, primers and 2xPCR ReddyMixTM Master Mix (ABgene), PCR amplification was as follows: denaturation at 94° C. for 3 min. followed by 32 cycles of 94° C. for 30 s, 60° C. for 90 s and 72° C. for 1 min, and a final extension cycle of 72° C. for 10 min. All amplified products were cloned into Pgem T-easy plasmid (Promega, WI, USA) and sequenced. The cloned fragments were analyzed by BLASTX.
The amplified fragments were used for the design of a third primer set, AHAS03. PCR amplifications were carried out using the first strand cDNA as a template, primers and 2xPCR ReddyMixTM Master Mix. Touch-Down PCR (TD-PCR) amplification was as follows: denaturation at 94° C. for 2 min, followed by 30 cycles of 94 ° C. for 30 s, 55° C. for 60 s and 68° C. for 1.5 min, and a final extension cycle of 68° C. for 10 min. The complete internal fragment was used for gene-specific primers (GSPs) design to clone the full-length cDNA of the PiAHAS gene, employing the 3′ and 5′ rapid amplification of the cDNA ends (RACE) method, using a BD smart RACE cDNA Amplification Kit (BD Biosciences, Clontech, Calif., USA) according to the manufacturer's manual. Two sets of RACE-cDNAs were synthesized for 3′ end and 5′ end amplification. The synthesized cDNA was used for PCR amplification of the 3′ cDNA end using the Universal Primer A Mix (UPM) and the 3′AHAS GSP. The PCR amplification was carried out using the BD AdvantageTM 2 PCR Kit (BD Biosciences). PCR amplification was as follows: denaturation at 94° C. for 2 min, followed by 30 cycles of 94° C. for 30 s, 63° C. for 30 s and 72° C. for 2 min, and a final extension cycle of 72° C. for 10 min; the reaction was terminated at 10° C. For the synthesis of the 5′ cDNA end of AHAS, 5′AHAS GSP was used as a reverse primer and UPM as a forward primer and TD-PCR was employed: denaturation at 94° C. for 2 min, followed by 5 cycles of 94° C. for 30 s, 70° C. for 40 s and 72° C. for 2.5 min, followed by 5 cycles of 94° C. for 30s, 68° C. for 40s and 72° C. for 2.5 min, and finally 25 cycles of 94° C. for 30 s, 64° C. for 40s and 72° C. for 2.5 min.
An additional set of primers, PiAHAS-full, was designed based on the cDNA ends. The primers contained the start and stop codons and had SalI restriction sites for future ligations. PCR amplifications were carried out using the first strand cDNA as a template, primers and the PfuUltra DNA polymerase (Stratagene). PCR amplification was as follows: denaturation at 94° C. for 3 min, followed by 30 cycles of 94° C. for 30 s, 65° C. for 60 s and 72° C. for 3 min, and a final extension cycle of 72° C. for 10 min. The full gene was ligated and cloned into pGEM T-easy plasmid.







TABLE 1







Primers used in cDNA cloning













SEQ




Tm
ID


Oligo
Sequence
[C. °]
NO.













AHAS01-F
AACCGGCGCACACGTAC
67
5



CTGG




 


AHAS01-R
CGGGGATCATGGGCAGCACG
67
6


 


AHAS02-F
GGCACCGAIGCITTICAIGAIAC
68
7


 


AHAS02-R
ACGGTGCCITGCATICCIAICAT
69
8


 


AHAS03-F
AGATCACCAAGCACAACTTCCT
60
9


 


AHAS03-R
ATGACAAAGTCG
60
10



GGGAAGATGT




 


3′AHAS GSP
ACGCTGGACGAGAGCCACATCT
69
11



TC




 


5′AHAS GSP
CCCTGTGATGGCAACAAGCGGA
69
12



AC




 


PiAHAS-full
ATAGTCGACAGCATGCAAGGCA
68
13


F
CTATG




 


PiAHAS-full
AATGTCGACCTGCGCCTTAGTA
69
14


R
CTCG




 


AHAS-Mut-F
GCATGGTGGTCCAGTcGGAGGA
79
15



CCGCTTCTACA




 


AHAS-Mut-R
TGTAGAAGCGGTCCTCCgACTG
79
16



GACCACCATGC




 


pAH29-
TATAGTCGACGCGCAAAAGGAA
65
17


empty-F
TATAAAAA




 


pAH29-
TATAGGATCCCATAGTTAGTTCC
70
18


empty-R
CCGTCC




 


PiAHAS-
GGATCCATGCAAGGCACTATG
63
19


ORF





 


PiAHAS-
GGATCCAATGAGCTGGTGGC
61
20


trunc





 


PiAHAS-R
GTCGACCTGCGCCTTAGTACTC
66
21









Site-directed Mutagenesis of the PiAHAS Gene

A specific point mutation was designed for substitution of the PiAHAS Trp605 with Serine. Mutagenesis of the PiAHAS wild type cDNA was performed with the QuikChange site-directed mutagenesis kit (Stratagene, Calif., USA). A complementary oligonucleotide primer set, AHAS-Mut, was designed with the intended point mutation. PCR amplification was carried out using the pGEM T-easy plasmid with the AHAS gene as a template, primers and the proofreading PfuUltra DNA polymerase (Stratagene). PCR amplification was as follows: denaturation at 94° C. for 1 min, followed by 12 cycles of 94° C. for 30 s, 55° C. for 60 s and 72° C. for 5 min, and a final extension cycle of 72° C. for 10 min. After the PCR reaction, the parental DNA template was digested with DpnI restriction enzyme. The PCR amplified plasmid was separated on agarose gel, extracted and inserted into E. coli competent cells. Ampicillin resistant colonies were selected from which the plasmid was extracted. The extracted plasmid was sequenced for confirmation of the desired point mutation.
PiAHAS Functional Expression in Bacteria
E. coli K12 strain BUM1 is a recA mutant of strain CU9090 which does not express any AHAS enzymes and cannot grow on minimal medium lacking isoleucine or valine (Ibdah M, et al. Homology modeling of the structure of bacterial acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis. Biochemistry (N.Y.) 1996, 35, 16282-16291). This strain also requires proline and thiamin, regardless of AHAS expression. Transformation of BUM1 cells was achieved using a standard heat shock protocol. BUM1 cells were grown in LB or in M9 minimal medium (7 mg/ml Na2HPO4; 3 mg/ml KH2PO4; 0.5 mg/ml NaCl; 1 mg/ml NH4Cl; 0.12 mg/ml MgSO4; 0.35 mg/ml thiamin HCl; 2 mg/ml glucose) supplemented with 200 μg/ml proline. Where appropriate, the M9 medium was supplemented with the BCAAs valine, leucine, and isoleucine (150 μg/ml of each) or with 50 μM SMM. BUM1 transformants were propagated and kept on LB medium supplemented with 100 μg/ml Ampicillin. For functional expression assays, the cells were washed and plated on M9 agar plates and incubated for 48 hrs at 37° C.
The expression vector pAH29 (Lawther R et al. Molecular basis of valine resistance in Escherichia coli K-12. Proc. Natl. Acad. Sci. U. S. A. 1981, 78, 922-925) contains the ilvGM genes (i.e., the entire coding region of E. coli AHASII large and small subunits) under the native bacterial promoter of the ilvGMEDA operon, ilvEp (Lopes J et al. Analysis and comparison of the internal promoter, pE, of the ilvGMEDA operons from Escherichia coli K-12 and Salmonella typhimurium. Nucleic Acids Res. 1986, 14, 2779-2798). The pAH29 plasmid was used as a template for amplification of the plasmid back-bone with pAH29-empty primer set, without the ilvGM coding region. To clone the PiAHAS cDNA, three primers were designed. PiAHAS wild type and mutant genes were amplified with the forward primers, PiAHAS-ORF and PiAHAS-trunc, designed to include and exclude the estimated 90 amino-acids chloroplast targeting peptide, respectively. The forward primers were also designed to include BamHI restriction site. The reverse primer PiAHAS-R included a SalI restriction site and was designed based on the 3′ end of the gene.
PiAHAS in vitro Enzyme Assay
To overexpress the hexahistidine (6xHis)-tagged PiAHAS genes in E. coli strain JM109, plasmids pQE-WT and pQE-MUT were used. These plasmids were constructed by inserting the BamHI-SalI fragment containing the cloned PiAHAS wt and mutant genes into plasmid pQE30 (Qiagen, Hilden, Germany). These plasmids express PiAHAS fused at its N-terminus to the pQE30 6xHis leader. pQE-WT and pQE-MUT were transformed into E. coli for expression. The transformed cells were grown, IPTG induced, and harvested as described previously (Hill C et al. Purification of Escherichia coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits. Biochem. J. 1997, 327, 891-898). Bacterial cells were disrupted by sonication in a binding buffer with 20 mM imidazol, 0.5 M sodium chloride, 50 mM sodium dihydrogen phosphate (pH 8.0), 20 μM FAD and 50 μl/g cells of protease inhibitor cocktail (Sigma-Aldrich, Israel). After 30 min of centrifugation at 20,000×g, the supernatant was loaded on a 1.5×8 cm column of Ni2+-nitrilotriacetatic acid-agarose (Qiagen) previously washed with the binding buffer. The column was then washed with 80 ml of the binding buffer, and the His-AHAS protein eluted with 0.4 M imidazol in the binding buffer. The fractions were dialyzed against 50 mM potassium phosphate buffer (pH 7.6), containing 20 μM FAD. The protein was concentrated for storage at −20oC by dialysis against same buffer, containing 50% glycerol. The AHAS catalytic activity was determined as previously described (Bar-Ilan A et al. Binding and activation of thiamin diphosphate in acetohydroxyacid synthase. Biochemistry (N.Y.) 2001, 40, 11946-11954). The reactions were carried for 20 min at 37oC in 0.1 M potassium phosphate buffer (pH 7.6), containing 10 mM magnesium chloride, 0.1 mM ThDP, 75 μM FAD, 5 mM EDTA, 1 mM DTT with 100 mM pyruvate as substrate, except where otherwise indicated. The activity is expressed in units (U) (1 U=1 μmol of acetolactate formed min−1). Km for pyruvate and Ki for SMM were determined by varying the concentration of the factor in question. Protein concentration was determined by the dye-binding method of Bradford (Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254), with bovine serum albumin as standard. Km calculations were fit with the program “Sigma-Plot” to Michaelis-Menten equation. For Ki calculations, the lines were fit to equations:

V=Vo×Ki/(Ki+[SMM]), for wild-type;

V=Vf+(Vo−Vf)×Ki/(Ki+[SMM]), for mutant.

V is the rate of acetolactate formation by AHAS;
Vo and Vf are beginning and final rates of acetolactate formation;
[SMM] is concentration of SMM;
Example 1
Cloning of PiAHAS cDNA of P. Incisa 
Multiple sequence alignment, by ClustalW, of the AHAS protein sequences of the green algae Chlorella variabilis (EFN51096.1), Chlamydomonas reinhardtii (AAB88292.1), and Volvox carteri (AAC04854.1), the moss Physcomitrella patens subsp. (XP—001759950.1) and the higher plant Brassica napus (AAA62705.1) showed 4 conserved sequence blocks, designated A-D (FIG. 1). The first set of primers, AHAS01, amplified a 250 by region of the C-terminal part of the gene between Block C and Block D. The second set of primers, AHAS02, was of a degenerate nature in which inosine was introduced at positions of non-conserved nucleotides, amplified a 500 by region of the N-terminal part of the gene between Block A and Block B. The amplified products were found to contain partial AHAS coding sequences. Based on these cloned sequences we designed a third primer pair, AHAS03, which was used to amplify a single band of about 1200 by in size, extending from Block A to Block D. This complete 1200 by fragment was cloned and sequenced and found to encode the PiAHAS gene. The 5′ and 3′-ends of the PiAHAS cDNA were amplified from the RACE Ready cDNA. RACE-PCR enabled amplification and cloning of the gene ends, including 800 by of the 3′ untranslated region (UTR) and 80 by of the 5′ UTR. The clones were sequenced and PiAHAS-full primer set was designed and used to amplify the complete 2100 by AHAS coding sequence.
Example 2
Prediction of PiAHAS Chloroplast-Targeting Signal
The putative PiAHAS protein was found to be 70-75% identical to the AHAS proteins of the green algae C. variabilis, C. reinhardtii, and of V. carteri. When these proteins were aligned by ClustalW (FIG. 2), we found low conservation in the N-terminus between these different green algae. The first  ̃100 amino-acids of the PiAHAS N-terminus showed poor similarity to the other proteins, particularly by the presence of a unique polyQ repeat. We analyzed the encoded protein for the presence of possible chloroplast transit peptides using ChloroP and TargetP softwares. Neither predicts plastidial localization for PiAHS.
Example 3
PiAHAS Site-Directed Mutagenesis
pGEM-T Easy vector, harboring PiAHAS cDNA, was used as a template for site-directed mutagenesis by PCR amplification with AHAS-Mut primers, carrying the desired W605S mutation. The reaction resulted in pGEM-T Easy, containing the mutagenized PiAHAS insert (PimAHAS). The mutation was verified by DNA sequencing.
Example 4
Growth Complementation of AHAS-Deficient E. coli by PiAHAS and PimAHAS
The expression vector pEp-empty, an empty plasmid that retains the native ilvEp promoter and ATG start codon, was created by amplification of pAH29 plasmid with the pAH29-empty primer set, and included BamHI and SalI restriction sites for future fusion with the algal genes. Three forms of PiAHAS genes were used for this experiment: PiAHAS full open reading frame (ORF), truncated PiAHAS without the estimated chloroplast targeting peptide (from Serine91), and PimAHAS containing the point mutation (W605S). The three forms were amplified and digested with BamHI and SalI. Each PiAHAS form was inserted into the pEp-empty, under the native bacterial promoter to form three constructs: pEp-ORF, pEp-truncated and pEp-mut. A vector containing only the Ep, pEp-empty, was used as a negative control. The four constructs, together with pAH29 as a positive control, were transformed into E. coli BUM1 competent cells. The transformed cell lines were plated on three types of agar plates: M9, M9 supplemented with BCAAs and M9 supplemented with 50 μM SMM. The plates were incubated for 48 hrs (FIG. 3) and bacterial growth was determined. All the cell lines were able to grow on the BCAAs supplemented medium. The M9 plates, without BCAAs, were used to select for bacteria with functional AHAS activity. The host strain, BUM1, transformed with the pEp-empty vector was unable to grow on these plates. In contrast, bacteria transformed with the different PiAHAS genes were able to grow on the selective medium, as were cells transformed with pAH29. This result indicates that the algal PiAHAS gene can functionally complement the bacterial mutation, and that the first 90 amino-acids are not required for enzymatic activity. To test whether PimAHAS conferred SMM resistance, we added 50 μAM SMM to the M9 plates. Growth was totally inhibited in the bacteria transformed with the wild type PiAHAS genes, whereas those carrying the mutant gene were not affected by the herbicide. Bacteria transformed with pAH29 were able to grow slowly in the presence of 50 μM SMM, probably due to higher level of SMM resistance of the bacterial AHASII enzyme (Steinmetz, A et al. Valine 375 and Phenylalanine 109 Confer Affinity and Specificity for Pyruvate as Donor Substrate in Acetohydroxy Acid Synthase Isozyme II from Escherichia coli. Biochemistry (N.Y.) 2010, 49, 5188-5199).
Example 5
Biochemical Assay of PiAHAS Activity in Vitro
P. incisa genes encoding the wild-type and mutant forms of AHAS were cloned into the pQE30 expression plasmid and expressed in E. coli JM109 cells as N-terminal hexahistidine-tagged proteins. The molecular weights of the AHAS forms are shown on a Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (FIG. 4). Total protein samples of the transformed bacteria, before and after induction, showed enhancement of a band of about 75 kDa, the expected size of the large subunit of the putative PiAHAS. The proteins were expressed in soluble form and were purified using Ni+-chelating column chromatography. Enzymatic parameters for interaction of the enzyme with its substrate and with the herbicide inhibitor were determined and presented in Table 2.







TABLE 2







Kinetic parameters for wild-type and mutant AHASa.











Parameter
Wild-type
Mutant







Specific activity, U × mg−1
3.00 ± 0.05
1.46 ± 0.03



Km for pyruvate, mM
49.4 ± 2.1 
71.7 ± 2.9 



kcat/Km, M−1 × s−1
83
25



Ki for SMM, μMb
0.15 ± 0.01
 >30 × 106 







aThe kinetic parameters were determined as described under Materials and Methods at 37° C. and pH 7.6.



bThe concentration of pyruvate was 100 mM.






Enzymatic activity of the wild type and mutant forms of AHAS in the presence of different Pyruvate substrate concentrations was assayed with 6.9 μg/ml of PiAHAS wild-type protein and 11.5 μg/ml of mutant protein (FIG. 5) and the specific activity and Km were calculated. The specific activity of the purified PiAHAS was 3 U/mg, whereas the tested mutant W605S showed a 2-fold decrease.
The inhibition of the P. incisa wild type and mutant enzymes, by different SMM concentrations, was also assayed with 8.3 μg/ml of PiAHAS wild-type protein and 23 μg/ml of mutant protein (FIG. 6). The wild type form was strongly inhibited by very low concentration of SMM and its Ki was determined to be 0.15 μM. W605S substitution on the other hand, showed strong resistance to this herbicide, even at high concentrations of 200 μM. The Ki was determined to be >30×106, 7 orders of magnitude higher than that of the wild type.
In summary, the gene encoding PiAHAS was successfully isolated and cloned. The gene was further mutated at a Trp residue. The wild type and mutant genes were functionally expressed in AHAS-deficient bacteria. The genes were shown to complement AHAS activity in vivo and the mutant form was shown to confer SMM resistance. In green algae and higher plants AHAS is encoded in the nucleus and typically, the protein is targeted to the chloroplast by N-terminal chloroplast-targeting signal. Commonly used softwares, like ChloroP and TargetP, did not identify the protein as plastidial targeted, suggesting a unique, species-specific, chloroplast targeting peptide sequence. It was shown that a truncated protein, lacking the first 90 amino-acids of the N-terminus, retained its enzymatic activity in-vivo (FIG. 3). The wild type and mutant proteins were cloned into pQE30 vector, expressed as his-tagged proteins, isolated and characterized in-vitro. The molecular weight of the protein was determined to be 75 kDa by SDS-PAGE (FIG. 4). The W605S mutation caused a 2-fold decrease in enzymatic activity and in the affinity to the Pyruvate substrate, compared to that of the wild type. The mutant protein showed 7 orders of magnitude higher resistance to the SMM herbicide than that of the wild type.
Example 6
Herbicide Sulfometuron Methyl (SMM) Resistance to the Endogenous Copy of AHAS Gene
Combination of native regulatory elements and natural codon-usage was shown to drive efficient and stable expression of the endogenous marker-gene in algal cells as compared with antibiotics resistant genes. To facilitate stable nuclear transformation of P. incisa cells, in this experiment the same mutation, responsible for resistance to herbicide sulfometuron methyl (SMM), was introduced into the endogenous copy of AHAS gene: gDNA sequence of the gene was retrieved by blasting of cDNA to P. incisa genome sequence (giavap-genomes.ibpc.fr).
For amplification of an AHAS gene, utilizing its endogenous regulatory elements, 5′ forward primer in 1 kb upstream to ATG translation initiation codon and 3′ primer in 0.15 kb downstream after TAA translation Stop-codon, were used. Due to the large size of the whole amplicon, (7.2 kb), internal primers for amplification of two overlapping fragments of AHAS genomic loci were designed. After amplification, fragments with 5′ and 3′ parts of AHAS gene were cloned into pJET 2.1 (Fermentas) cloning vector. Then, 3′ fragment was mutagenized according to adapted QuickChange (Stratagene) protocol and the whole chromosomal fragment was assembled by insertion of NheI-NotI fragment of 3′ part of AHAS gene into NheI-NotI restriction gate in plasmid with 5′ of AHAS gene (FIG. 8. P. incisa gAHAS cloning). For amplification of 5′ and 3′ parts of AHAS genomic loci following pair of primers were used: AGAGGGGCATCAAAACCAGG (SEQ ID NO: 22) CGTAGTTGGCGTACACCGTA (SEQ ID NO: 23) and CCTCAGCGACGATCCTCTTC (SEQ ID NO: 24) CCATACCAGGCCCATTGTGT (SEQ ID NO: 25). Point mutation was introduced with primers ctgggcatggtggtccagtCggaggaccgcttctacaag (SEQ ID NO: 26) and cttgtagaagcggtcctccGactggaccaccatgcccag (SEQ ID NO: 27).
The actual sequence encoding the genomic fragment of P. incisa, containing AHAS gene is:








(SEQ ID NO: 28)



AGAGGGGCATCAAAACCAGGgaaacagcctcgcgcaaaccagcaacaattgtgagcagcg



 


ctgtttgcggctgtgtgagtgctcggtcgctctgtggggcaggtgcttccactatcacattatcatgagattgccctgccatccc


 


tgctcctctctgccacgctgggcgagaaggagctgtcgaaatcgcctccaggagtggccgtgactgcgatcgagttgacgt


 


tagcacctgaatagtggtactggcacttcagagatagctgcaggtcagcatgcaaggcaccatgcggccgacggctggag


 


cactgcagcagaccgtcggctgctggcacgtcccggccggcatcccccacgcacagcaggtgtttcaagttgttaggtggt


 


gataagtgcctactgaagattcctttatgcagctgaacgaggcagcgtcccttgcttcccagatcaacagccccttgtcactgg


 


tctgacaaaacgtgtgtgacaccctgacctgcgcatgcaggcgctgcgaggccgtatactgcccgaggagctgaagcagc


 


ggtgttcagcaaccaaacctaggtacaattgcagcagtagctcgacatgcatacacgcttaacgcagctgctggcagcagtc


 


tcgtgcctactgcttgccgccgcagcatgggctggctaccactacccttgaaacacacacaaggctcacaaaggtcctccct


 


ttcgtcccttgaggcagatgtgacgcctgctttgaccctgtgcacagggcggccaggcagtcagcggtgaccgctgccaag


 


cttgcagagggcaaggcaggcacaccatcacggagcttgcggcagcagccggcagcgccgcagcagcagcagcagca


 


gcannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnaggtcgtctggcacgggtggcaggtggcacgctcttcag


 


gcacttggtttggacaccctagcagtatgtcgtcgagttcagATGGAAGTCCTAGTACAGTGTGAGgt


 


cagcgttggcaagcctgccttgtcaatgtggcacactgatgcctttgcgcagcacatgcggatggggtggagtgtcgcatga


 


gctggcatggtccagttggaccctgctggggatgctgctagtatgggttgctggaccagatgaccctggtcttgagtggtga


 


ccatggcgccagtgcatccaactgctggtgtggtttgcttctgccccagctgtcaagctactgcaggccgggcggtgcagg


 


caaaacaccaggcagtacatgtagcagcttgacggcagttgtagtagctcaccgccaccaatagtttgacgcactggcaga


 


ctacctcagggcatgcctgcagctgctgtggcaagagggcatgagcctatccaatgcagctgctgtgaccacacatacctg


 


gcctgtctgtgcagCTGGTGGCGTTGCGGGAGGCTGCCAAGGCCTCGCTCTCATCC


 


CCCGCGCCAGCCGAGTGGGTGGATCGGTTTGGGTCGGAGCCCCGCAAGGG


 


CGCGGATATCCTGGTGCAGTGCCTGGAGCGCGAGGGGGCCTTCCGCGTGTT


 


TGCCTACCCCGGCGGCGCCAGCATGGAGATCCATCAGGCCTTGACGCGCA


 


GCGGCATCATCCGCAACATCCTGTGCCGCCATGAGCAGgtggggatggaccagactg


 


gcctggtgtggggcgaacaagtaggaaccagtgggccacggcacaggcggtacacatgcgtatggcatgggaggctgat


 


agtgctcgcatgtagggatggcattttgcctgccaaagggcttggctatgcttgtgatgcacagtgggcctgcatctgagcac


 


tgaagccagtcagtcattggctgattgatgaatgccgcttcacccagtgacagatgcattggcctcgggtgtggttcaaagct


 


acagcaactgaaggacgctgagggcatgtgcatgtccaccaccttcacttactgtcaagtcttgcctacccatgcctgatcca


 


gcatgaacgccatgtcgcagGGCGAGATCTTTGCGGCGGAGGGCTATGCCAAGTGCAC


 


GGGCGATGTGGGCGTGTGCATCGCGACCAGCGGGCCCGGCGCCACCAACC


 


TCGTGACTGGCCTGGCTGATGCCATGCTGGACAGCGTTCCGCTTGTTGCCA


 


TCACAGGGCAGgtgtgcacgcaagtcatcctgttgtttctcggcctgcctgtatggtctgtagcctgtttcaggtgct


 


ctgctcagcatcgcagatgcctcactgctttctgttgtggcgggggtcactaacctgcataaagtttccaccttgagcagtcgtt


 


caagcactactacttgtactgacagtcaacaatgagtccactgatcaggctacgaatgcaatgtggcggctgctcgaccacat


 


gtcagcgctctggcagtaaaccggggttccgcatgcaggTGCCCCGCAAGATGATCGGCACGGAC


 


GGGTTCCAGGAGACGCCGATTGTGGAGGTGACGCGGCAGATCACCAAGCA


 


CAACTTCCTGGTCATGGACCTGGATGACCTGCCGCGCATCATGAAGGAGGt


 


aggtgtggttcacctgcaacagaagcaaggcaatgtgcttgcacagcctgctcttctgcgcacagcagtccctgctgattgct


 


tgtttggaagctaggcaacagctgctgcagacgcacaagcaacatcacatgcagaggcatcacacgcgattttggcaacct


 


gccggctaggtggcctaatcctgactctctatcaccctgtctgtttttgcaggCATTCTACCTGGCGCGCAC


 


GGGGCGGCCGGGCCCGGTGCTGGTGGATGTGCCCAAGGACATCCAGCAGC


 


AGCTGGCGGtgccggactgggacacgcccatggccatcagcggctacatgtcgcgcctgccnnnnnnnnnnn


 


nnnnnnnnnnnnnnnnnnnnnncccgcctaacccctcacagctggccgcggtcgtgcgcgcgctgaaggaggtacg


 


catgggtgatgcgcagcctcggtcgcctttctgaagtagacagcacgacctcagcgacgatcctcttcagttgagccgtgcc


 


catgcatgcatgtaagtgcatggatgcaagcatgctgaatcgctttctgagacagactgtgcatgctatgggcaaggtacatc


 


attaatggaaagttgctgctagctgcaagcctgcgaaccggccccgctttcaccagcgtttggtcgccttcagcacagtgcac


 


ctgtccatacggaacgcttctttcccacatcatgcacggtgcatcatgcacggtacaatcaagctgtctggcgtgataaggag


 


ccgtggctgacatctcggcaggaaacttatgctttccccacttcctgtaggCTAAGAGGCCGACGCTGTAC


 


GTGGGCGGTGGCGCGCTCGACTCGAGCGCTGAGCTGCGCGAGTTTGTGCG


 


GCTGACGGGCATCCCCGTCGCGCAGACGCTTATGGGCCTGGGCACCTTCCC


 


CGAGGAAGACCCGCTGGCACTGCAGgtaggcttccttgctggcgggagactggtaacaaaggagg


 


gagcagtttgtgagcctatgggtgcgggacagtgcgagatctggttgaatgatgtgttgtcatgcggcttgtgacacgttgtg


 


gcacctgcggcagctggatgccatcttatgggcagccatggcaccagcgcgtggggctatgggtcatgctgacaaggcca


 


gtgccattgttttggatgcagATGCTGGGCATGCACGGTACGGTGTACGCCAACTACGC


 


CGTCAACGACAGCGACCTGCTGCTGGCGTTTGGCGTGCGCTTCGACGACCG


 


CGTGACTGGCAAGCTGGAGGCGTTTGCTTCGCGCGCGCGCATCGTGCACAT


 


CGACATCGACCCTGCGGAGATCTGCAAGAACAAGGAGGCCCACATACCCA


 


TCTGTGCAGgttggttcgtatagagcagcacccattttgttttcgatgtcagtgcaaatgctagaaagccagttttgaca


 


gccagcacctcatgcagggctatgtatacttgcatactcagggcccgactgggcatgttgactgccaggttccttactgctcat


 


gttggctgcagACCTGCGTGCGTCGTTGATAGCGCTGAATGAGCTGTTGCGGCGA


 


GACCCTCTGCCAGAGGGCGCCTTTGCGGACTGGCGGGCGGCCATTGAGGC


 


CAAGAAGCAGGAGTTCCCCATGACCTTCCCGGAGCGGGACGACGTGATCA


 


TCCCACAGCGTGCCATCCAGGTGAGAGGCGATGTCATGGATTCAgtgagacagc


 


cgcaagacatgttggcatgacatgttttcctctctgttctcttgctgggaatgtcattgtttgcgagcaggccacatctgcaagca


 


aaaccgtcttgcttgcctcgtgcagATGCTGTATGAGGAGACAAACGGCGAGGCCATCAT


 


CAGCACCGGCGTGGGCCAGCACCAGATGTGGGCGGCGCAGTGGTACCAGT


 


ACAACGAGCCGCGGAGATGGGTCACGTCCGGCGGCCTCGGCTCCATGGGC


 


TTTGGCCTGCCATCCGCCCTCGGCGCGGCGGTGGCCTACGACGGCACGGAC


 


GGTCGGCCCAAGAAGGTatccgtcatatggaatcctctggctgaatggcatgcctttttttaaactctgcctggt


 


atctgggtgaaagcgggtgaaagcgatgtctggccctacaaggagttggcgtgccactagaaatgcctgtgatagcaccaa


 


tctctgtttggagagagttcattgttgtatgaaggatacggggcaacccttgatccatgtacatacatgtctgtatgtgccctcgc


 


tgtttgaccggtcaggtgagcgtgcatggtgctgcactgggctacccagtgcagagggatgtaggctgccatctgtgttcgtc


 


tagccgtgacgcgttttgtcaatgtatcatcagctggcgggtatggcaaccaaagatggtaagcttggatgatgagggtgacc


 


tgttacctgacccagcgctctggcaaggcccttcatcatccagcgttactctactggctacattggttcagtggcggacggtgt


 


atctgctaggttgtgcacaccagtcagccagatgctggtagggcaattggttgtcctcctatgagccaacgacgacctttgct


 


gatcgggtgtcaccctgacgcaggcttgaccatgaagtctaagtagacgatgctgcttttcgcacacctctgctcaataaaata


 


tgtggttggcatatgtgcatggccaagtgtgcggcatgaccagctgtctataaggccccgacatagctggcctttgttaggct


 


gacgcctcatgaggttgctgccatcatcttgaccgctcacatggtatgacgacggttatgacgacggttatgacgacggttat


 


ggcacgtggttgagcgtggtcaggtcctcgcatcacactacaatctttacatgcagatgctgtgtgcctatttgtgtgttatgga


 


gatagcatcgacggcaggccagcagcctacaaggcagcggggactaaagcagtggatctggtgaccttgcttcgtagttg


 


ccactaccaggcaatagcaacatggggatcttgcagttgcaaggctcagcttgtgactcaatctcgggcattgcgtccaatct


 


tgtgcaggcgaggtggacgtggacatctaagtgtatgcttgctgacctgctaaaagtgtctgagtagaagcagtgaatatccg


 


acgtggcatcaaggagtgagccgccttgtcgcgcaggtcgtcgtggacatcgatgatggccacagcttctgaagattcgca


 


cacagcatcctgttggaacatgaggcttgcctgcgcaggtGGTGGTGGACATCGACGGCGACGGC


 


AGTTTCCTGATGAACTGCCAGGAGCTGGCGACGGCGGCGGTGGAGGGCCT


 


GGAGACCAAGATCATGATCCTCAACAACCAGCACCTGGGCATGGTGGTCC


 


AGTGGGAGGACCGCTTCTACAAGGCCAACCGCGCACACACCTACCTCGGC


 


CACCGGGtgcgtcccacaggctgctggtcttgccggtttcggtccagctgacttgttgtgacgctgttattgctggtgct


 


gtgtgagactgacatgaagttgctcttgcaagaggttggggcagagtggcagtgaaaaataagttgcaggcttcaaaccac


 


gcaatgcatgcaggCCAATGAGTACCACACGACGCTGGACGAGAGCCACATCTTC


 


CCCGACTTTGTCATGATGGCCAAGTCGTGCGGCGTCCCAGGCCGGCGCGTC


 


ATCAAGCCCGAGGAGCTGCGCGGGGCCATCaggtgggggctgctgccacgggcgcagtgctt


 


gcagcatgcacactgtctgcaacttggtgaaccctggctgtggtgtgtggagatggcacattaagcacgtgcatcgcactgc


 


tgctgccaccctacaggtggagtccctctgctcttgctgcgctcgtcgcactggtggaagctcagcagctctattcctgcagc


 


agctgctgaagtgatgtgtctccactgacaGGGAGATGCTGGACACGCCCGGCCCCTTCCTG


 


CTGGACGTGATGGTGCCGCATGTGGAGCACGTGCTGCCCATGATCCCGGGC


 


GGCGGCTCCTTCAAGGACATCATCACCAAGGGCGACGGCCGCGACGAGTA


 


CTAAggcgcaggtcgcataggttgccatgggcaaggggctgccatggttgacttggtcgtgaccgatggttgtctgtcc


 


ggacgttttcggtaacgtcctgcgctgtcctgctaccaaggtgctgtgctgtaggcACACAATGGGCCTGGT


 


ATGG.






Detailed analysis and description of SEQ ID NO: 28 is further provided in FIG. 7.

## Claims

1. An expression vector comprising a polynucleotide, said polynucleotide comprises: (a) a nucleic acid sequence encoding a protein, said protein comprises the amino acid sequence set forth in SEQ ID NO: 1; or (b) the nucleic acid sequence of SEQ ID NO: 28.

2. A transgenic alga, a transgenic plant, or a transgenic seed, comprising the expression vector of claim 1.

3. The transgenic plant of claim 2, wherein said transgenic plant is a transgenic embryo or a transgenic shoot.

4. A cell transformed by the expression vector of claim 1.

5. The cell of claim 4, wherein the cell is derived from a transgenic alga, a transgenic plant, or a transgenic seed.

6. The cell of claim 5, wherein said transgenic plant is a transgenic embryo or a transgenic shoot.

7. A method for enhancing the production of branched-chain amino acid (BCAA) in a cell comprising the step of transforming a cell with the expression vector of claim 1, thereby increasing the production of BCAA in a cell.

8. The method of claim 7, wherein the cell is an alga cell.

9. The method of claim 7, further comprising the step of transforming said cell with a polynucleotide encoding an enzyme selected from the group consisting of: Threonine deaminase (TD), ketol-acid Reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD); transaminase (TA), 2-isopropylmalate synthase (2-IPMS), 3-isopropylmalate dehydratase (3-IPMD) and 3-isopropylmalate dehydrogenase (3-IPMDH), or a combination thereof.