EP1546314A1 - Bhyd gen - Google Patents

Bhyd gen

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Publication number
EP1546314A1
EP1546314A1 EP03748055A EP03748055A EP1546314A1 EP 1546314 A1 EP1546314 A1 EP 1546314A1 EP 03748055 A EP03748055 A EP 03748055A EP 03748055 A EP03748055 A EP 03748055A EP 1546314 A1 EP1546314 A1 EP 1546314A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
sequence
polynucleotide
polypeptide
carotene hydroxylase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03748055A
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English (en)
French (fr)
Inventor
Tatsuo Hoshino
Kazuyuki Ojima
Yutaka Setoguchi
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DSM IP Assets BV
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DSM IP Assets BV
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Filing date
Publication date
Application filed by DSM IP Assets BV filed Critical DSM IP Assets BV
Priority to EP03748055A priority Critical patent/EP1546314A1/de
Publication of EP1546314A1 publication Critical patent/EP1546314A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)

Definitions

  • the present invention relates to a gene useful in a process to increase the microbial production of carotenoids.
  • the genes encoding ⁇ -carotene hydroxylase which is a biological material useful in the improvement of the production process for ⁇ -cryptoxanthin are provided.
  • This invention involves cloning and determination of the genes encoding ⁇ -carotene hydroxylase from plant species, Arabidopsis thaliana and Glycine max.
  • genes may be modified in a suitable host, such as P. rhodozyma to overproduce thus cloned ⁇ -hydroxylase and the effect on the carotenogenesis can be confirmed by the culti- vation of such a transformant in an appropriate medium under an appropriate cultivation condition.
  • novel DNA fragments encoding the enzymes, from Arabidopsis thaliana and Glycine max.
  • the said DNA means a cDNA which contains only open reading frame flanked between the short fragments in its 5'- and 3'- untranslated region.
  • the present invention relates to a polynucleotide comprising a nucleic acid molecule selected from the group consisting of: (a) nucleic acid molecules encoding at least the mature form of the polypeptide depicted in SEQ ID NO:2 or 4; (b) nucleic acid molecules comprising a coding sequence as depicted in SEQ ID NO:l or 3; (c) nucleic acid molecules whose nucleotide sequence is degenerate as a result of the genetic code to a nucleotide sequence of (a) or (b);
  • nucleic acid molecules encoding a polypeptide derived from the polypeptide encoded by a polynucleotide of (a) to (c) b way of substitution, deletion and/or addition of one or several amino acids of the amino acid sequence of the polypeptide encoded by a polynucleotide of (a) to (c);
  • nucleic acid molecules encoding a polypeptide whose sequence has an identity of 72.8 % or more to the amino acid sequence of the polypeptide encoded by a nucleic acid molecule of (a) or (b);
  • nucleic acid molecules comprising a fragment or a epitope-bearing portion of a polypeptide encoded by a nucleic acid molecule of any one of (a) to (e) and having ⁇ -carotene hydroxylase activity;
  • nucleic acid molecules comprising a polynucleotide having a sequence of a nucleic acid molecule amplified from Arabidopsis or Glycine nucleic acid library using the primers de- picted in SEQ ID NO:5, 6, and 7;
  • nucleic acid molecules encoding a polypeptide having ⁇ -carotene hydroxylase activity, wherein said polypeptide is a fragment of a polypeptide encoded by any one of (a) to (g); (i) nucleic acid molecules comprising at least 15 nucleotides of a polynucleotide of any one of (a) to (h); (j) nucleic acid molecules encoding a polypeptide having ⁇ -carotene hydroxylase activity, wherein said polypeptide is recognized by antibodies that have been raised against a polypeptide encoded by a nucleic acid molecule of any one of (a) to (h); (k) nucleic acid molecules obtainable by screening an appropriate library under stringent conditions with a probe having the sequence of the nucleic acid molecule of any one of (a) • to (j) and having ⁇ -carotene hydroxylase activity;
  • nucleic acid molecules the complementary strand of which hybridizes under stringent conditions with a nucleic acid molecule of any one of (a) to (k) and having ⁇ -carotene hydroxylase activity.
  • nucleic acid molecule(s) refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule.
  • this term includes double- and single- stranded DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analog.
  • the DNA sequence of the invention comprises a coding sequence encoding the above-defined polypeptide.
  • a “coding sequence” is a nucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus.
  • a coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.
  • the gene encoding ⁇ -carotene hydroxylase ⁇ BHYD) gene from A. thaliana and G. max was cloned.
  • the invention further encompasses polynucleotides that differ from one of the nucleotide sequences shown in SEQ ID NO:l and 3 (and portions thereof) due to degeneracy of the genetic code and thus encode a ⁇ -carotene hydroxylase as that encoded by the nucleotide sequences shown in SEQ ID NO:l and 3.
  • the polynucleotide of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2 and 4.
  • the polynucleotide of the invention encodes a full length Arabidopsis thaliana and Glycine max protein which is substantially homologous to an amino acid sequence of SEQ ID NO:2 and 4.
  • BHYD gene and “recombinant BHYD gene” refer to nucleic acid molecules comprising an open reading frame encoding a ⁇ -carotene hydroxylase, preferably a ⁇ -carotene hydroxylase from A. thaliana or G. max.
  • DNA sequence polymorphism that leads to changes in the amino acid sequences of enzymes may exist within a population (e.g., A. thaliana or G. max population) due to natural variation.
  • Such natural variations can typically result in 1-5% variance in the nucleotide sequence of the ⁇ -carotene hydroxylase gene. Any and all such nucleotide variations and resulting amino acid polymorphism in ⁇ -carotene hydroxylase that are the result of natural variation and that do not alter the functional activity of ⁇ -carotene hydroxylase are intended to be within the scope of the invention.
  • Polynucleotides corresponding to natural variants and non-A thaliana or non-G. max homologues of the ⁇ -carotene hydroxylase cDNA of the invention can be isolated based on their homology to A. thaliana or G.
  • a polynucleotide of the invention is at least 15 nucleotides in length. Preferably it hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of the polynucleotide of the present invention, e.g. SEQ ID NO:l or 3. In other embodiments, the nucleic acid is at least 20, 30, 50, 100, 250 or more nucleotides in length.
  • hybridizes under stringent conditions is defined above and is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 75% identical to each other typically remain hybridized to each other.
  • the conditions are such that sequences at least about 80%, more preferably at least about 85% and even more preferably at least about 90% or 95% or more identical to each other typically remains hybridized to each other.
  • polynucleotide of the invention that hybridizes under stringent conditions to a sequence of SEQ ID NO: 1 or 3 corresponds to a naturally occurring nucleic acid molecule.
  • the polynucleotide sequence includes SEQ ID NO:l or 3, and fragments thereof having polynucleotide sequences which hybridize to SEQ ID NO:l or 3 under stringent conditions which are sufficient to identify specific binding to SEQ ID NO:l or 3.
  • stringent conditions which are sufficient to identify specific binding to SEQ ID NO:l or 3.
  • any combination of the following hybridization and wash conditions may be used to achieve the required specific binding: High Stringent Hybridization: 6X SSC; 0.5% SDS; 100 ⁇ g/ml denatured salmon sperm DNA; 50% formamide; incubate overnight with gentle rocking at 42°C.
  • High Stringent Wash 1 wash in 2X SSC, 0.5% SDS at room temperature for 15 minutes, followed by another wash in 0.1X SSC, 0.5% SDS at room temperature for 15 minutes.
  • Low Stringent Hybridization 6X SSC; 0.5% SDS; 100 ⁇ g/ml denatured salmon sperm DNA; 50% formamide; incubate overnight with gentle rocking at 37°C.
  • Low Stringent Wash 1 wash in 0.1X SSC, 0.5% SDS at room remperature for 15 minutes.
  • Moderately stringent conditions may be obtained by varying the temperature at which the hybridization reaction occurs and/or the wash conditions as set forth above.
  • the term "homology” means that the respective nucleic acid molecules or encoded proteins are functionally and/or structurally equivalent.
  • the nucleic acid molecules that are homologous to the nucleic acid molecules described above and that are derivatives of said nucleic acid molecules are, for example, variations of said nucleic acid molecules which represent modifications having the same biological function, in particular encoding proteins with the same or substantially the same biological function. They may be naturally occurring variations, such as sequences from other plant varieties or species, or mutations. These mutations may occur naturally or may be obtained by mutagenesis techniques.
  • the allelic variations may be naturally occurring allelic variants as well as synthetically produced or genetically engineered variants. For example, structurally equivalents can be identified by testing the binding of said polypeptide to antibodies. Structurally equivalent have the similar immunological characteristic, e.g. comprise similar epitopes.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • the polynucleotide encodes a natural A. thaliana or G. max ⁇ -carotene hydroxylase.
  • nucleotide sequence of the polynucleotide encoding ⁇ - carotene hydroxylase can be introduced by mutation into a nucleotide sequence of the polynucleotide encoding ⁇ - carotene hydroxylase, thereby leading to changes in the amino acid sequence of the encoded ⁇ -carotene hydroxylase, without altering the functional ability of the ⁇ -carotene hydroxylase.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in a sequence of the polynucleotide encoding ⁇ -carotene hydroxylase, e.g. SEQ ID NO:l or 3.
  • non- essential amino acid residue is a residue that can be altered from the wild-type sequence of one of the ⁇ - carotene hydroxylase without altering the activity of said ⁇ -carotene hydroxylase, whereas an "essential" amino acid residue is required for ⁇ -carotene hydroxylase activity.
  • Other amino acid residues e.g., those that are not conserved or only semi-conserved in the domain having ⁇ -carotene hydroxylase activity
  • the invention relates to polynucleotides encoding ⁇ -carotene hydroxylase that contain changes in amino acid residues that are not essential for ⁇ -carotene hydroxylase.
  • ⁇ -carotene hydroxylase differs in amino acid sequence from a sequence contained in SEQ ID NO:2 or 4 yet retain the ⁇ -carotene hydroxylase activity described herein.
  • the polynucleotide can comprise a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least about 72.8% identical to an amino acid sequence of SEQ ID NO:2 or 4 and is capable of participation in the synthesis of ⁇ -cryptoxanthin.
  • the protein encoded by the nucleic acid molecule is at least about 75-80% identical to the sequence in SEQ ID NO:l or 3, more preferably at least about 80-85% identical to one of the sequences in SEQ ID NO:l or 3, even more preferably at least about 85-90%, 90-95% homologous to the sequence in SEQ ID NO:l or 3, and most preferably at least about 96%, 97%, 98%, or 99% identical to the sequence in SEQ ID NO:l or 3.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid).
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in one sequence e.g., one of the sequences of SEQ ID NO:l, 2, 3, or 4
  • the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the homology can be determined by computer programs as Blast 2.0.
  • GENETYX-SV/RC software Software Development Co., Ltd., Tokyo, Japan
  • This software uses the Lipman-Pearson method for its analytic algo- rithm.
  • a nucleic acid molecule encoding a ⁇ -carotene hydroxylase homologous to a protein sequence of SEQ ID NO:2 or 4 can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of the polynucleotide of the present invention, in particular of SEQ ID NO:l or 3 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into the sequences of, e.g., SEQ ID NO:l or 3 by standard techniques, such as site-directed mutagenesis and PCR- mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glut- amine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • a predicted nonessential amino acid residue in a ⁇ -carotene hydroxylase is preferably replaced with another amino acid residue from the same family.
  • mutations can be introduced randomly along all or part of a ⁇ -carotene hydroxylase coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a ⁇ -carotene hydroxylase activity described herein to identify mutants that retain ⁇ -carotene hydroxylase activity.
  • the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, "color complementation" described herein.
  • the polynucleotide of the present invention is DNA or RNA.
  • a polynucleotide of the present invention e.g., a nucleic acid molecule having a nucleotide sequence of SEQ ID NO:l or 3, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • ⁇ -carotene hydroxylase cDNA can be isolated from a library using all or portion of one of the sequences of the polynucleotide of the present invention as a hybridization probe and standard hybridization techniques.
  • a polynucleotide encompassing all or a por- tion of one of the sequences of the polynucleotide of the present invention can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence (e.g., a nucleic acid molecule encompassing all or a portion of one of the sequences of polynucleotide of the present invention can be isolated by the polymerase chain reaction using oligonucleotide primers, e.g. of SEQ ID NO: 5, 6, or 7, designed based upon this same sequence of polynucleotide of the present invention.
  • oligonucleotide primers e.g. of SEQ ID NO: 5, 6, or 7, designed based upon this same sequence of polynucleotide of the present invention.
  • mRNA can be isolated from cells, e.g.
  • Arabidopsis or Glycine e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al.
  • cDNA can be prepared using reverse transcript- ase (e.g., Moloney MLV reverse transcriptase or AMV reverse transcriptase available from Promega (Madison, USA)).
  • reverse transcript- ase e.g., Moloney MLV reverse transcriptase or AMV reverse transcriptase available from Promega (Madison, USA
  • Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in SEQ ID NO:l or 3.
  • a polynucleotide of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the polynucleotide so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to a ⁇ -carotene hydroxylase nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • fragment means a truncated sequence of the original sequence referred to.
  • the truncated sequence can vary widely in length; the minimum size being a sequence of sufficient size to provide a sequence with at least a comparable function and/or activity of the original sequence referred to, while the maximum size is not critical. In some applications, the maximum size usually is not substantially greater than that required to provide the desired activity and/or function(s) of the original sequence.
  • the truncated amino acid sequence will range from about 5 to about 60 amino acids in length. More typically, however, the sequence will be a maximum of about 50 amino acids in length, preferably a maximum of about 30 amino acids. It is usually desirable to select sequences of at least about 10, 12 or 15 amino acids, up to maximum of about 20 or 25 amino acids.
  • epitope relates to specific immunoreactive sites within an antigen, also known as antigenic determinates. These epitopes can be a linear array of monomers in a polymeric composition - such as amino acids in a protein - or consist of or comprise a more complex secondary or tertiary structure.
  • immunogens i. e., substances capable of eliciting an immune response
  • antigens are antigens; however, some antigen, such as haptens, are not immunogens but ma be made immunogenic by coupling to a carrier molecule.
  • antigen includes references to a substance to which an antibody can be generated and/or to which the antibody is specifically immunoreactive.
  • one or several amino acids relates to at least one amino acid but not more than that number of amino acids which would result in a homology of below 75% identity.
  • identity is more than 80%, more preferred are 85%, 90% or 95%, even more preferred are 96%, 97%, 98%, or 99% identity.
  • ⁇ -carotene hydroxylase or " ⁇ -carotene hydroxylase activity” relates to enzymatic activities of a polypeptide as described below or which can be determined in enzyme assay method. Furthermore, polypeptides that are inactive in an assay herein but are recognized by an antibody specifically binding to ⁇ -carotene hydroxylase, i.e., having one or more ⁇ -carotene hydroxylase epitopes, are also comprised under the term " ⁇ -carotene hydroxylase”. In these cases activity refers to their immunological activity.
  • polynucleotide and nucleic acid molecule also relate to “isolated” polynucleotides or nucleic acids molecules.
  • An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the ⁇ -carotene hydroxylase polynucleotide can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g., a Arabidopsis or Glycine cell).
  • polynucleotides of the present invention in particular an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the polypeptide of the invention comprises one of the nucleotide sequences shown in SEQ ID NO:l or 3.
  • the sequence of SEQ ID NO:l and 3 corresponds to the Arabidopsis thaliana and Glycine max ⁇ -carotene hydroxylase cDNAs of the invention.
  • the polynucleotide of the invention comprises a nucleic acid molecule which is a complement of one of the nucleotide sequences of above mentioned polynucleotides or a portion thereof.
  • a nucleic acid molecule which is complementary to one of the nucleotide sequences shown in SEQ ID NO: 1 or 3 is one which is sufficiently complementary to one of the nucleotide sequences shown in SEQ ID NO: 1 or 3 such that it can hybridize to one of the nucleotide sequences shown in SEQ ID NO: 1 or 3, thereby forming a stable duplex.
  • the polynucleotide of the invention comprises a nucleotide sequence which is at least about 75%, preferably at least about 75-80%, more preferably at least about 85-90% or 90- 95%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence shown in SEQ ID NO.T or 3, or a portion thereof.
  • the polynucleotide of the invention comprises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions as defined herein, to one of the nucleotide sequences shown in SEQ ID NO:l or 3, or a portion thereof.
  • the polynucleotide of the invention can comprise only a portion of the coding region of one of the sequences in SEQ ID NO:l or 3, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a ⁇ -carotene hydroxylase.
  • the nucleotide sequences determined from the cloning of the ⁇ -carotene hydroxylase gene from A. thaliana and G. max allows for the generation of probes and primers designed for use in identifying and/or cloning ⁇ -carotene hydroxylase homologues in other cell types and organisms.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 15 preferably about 20 or 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand of one of the sequences set forth, e.g., in SEQ ID NO:l or 3, an anti-sense sequence of one of the sequences, e.g., set forth in SEQ ID NO:l or 3, or naturally occurring mutants thereof.
  • Primers based on a nucleotide of invention can be used in PCR reactions to clone ⁇ -carotene hydroxylase homologues.
  • Probes based on the ⁇ - carotene hydroxylase nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe can further comprise a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a genomic marker test kit for identifying cells which express a ⁇ -carotene hydroxylase, such as by measuring a level of a ⁇ -carotene hydroxylase -encoding nucleic acid molecule in a sample of cells, e.g., detecting ⁇ -carotene hydroxylase mRNA levels or determining whether a genomic ⁇ -carotene hydroxylase gene has been mutated or deleted.
  • the polynucleotide of the invention encodes a polypeptide or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of SEQ ID NO:2 or 4 such that the protein or portion thereof maintains the ability to participate in the synthesis of ⁇ -cryptoxanthin, in particular a ⁇ -carotene hydroxylase activity as described in the examples in microorganisms or plants.
  • the language "sufficiently homologous” refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain as an amino acid residue in one of the sequences of the polypeptide of the present invention amino acid residues to an amino acid sequence of SEQ ID NO: 2 or 4 such that the protein or portion thereof is able to participate in the synthesis of ⁇ -cryptoxanthin in microorganisms or plants. Examples of a ⁇ -carotene hydroxylase activity are also described herein.
  • the protein is at least about 75-80%, preferably at least about 80-85%, and more preferably at least about 85-90%, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence of SEQ ID NO:2 or 4.
  • Portions of proteins encoded by the ⁇ -carotene hydroxylase polynucleotide of the inven- tion are preferably biologically active portions of one of the ⁇ -carotene hydroxylase.
  • biologically active portion of ⁇ -carotene hydroxylase is intended to include a portion, e.g., a domain/motif, that participates in the biosynthesis of ⁇ -cryptoxanthin or has an immunological activity such that it is binds to an antibody binding specifically to ⁇ -carotene hydroxylase.
  • an assay of enzymatic activity may be performed. Such assay methods including "color complementation" are well known to those skilled in the art, as detailed in the Examples.
  • Additional nucleic acid fragments encoding biologically active portions of a ⁇ -carotene hydroxylase can be prepared by isolating a portion of one of the sequences in SEQ ID NO:l or 3, expressing the encoded portion of the ⁇ -carotene hydroxylase or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the ⁇ -carotene hydroxylase or peptide.
  • degenerate PCR is a method to clone a gene of interest which has high homology of amino acid sequence to the known enzyme from other species which has a same or similar function.
  • Degenerate primer which is used as a primer in degenerate PCR, was designed by a reverse translation of the amino acid sequence to corresponding nucleotides ("degenerated").
  • a mixed primer which consists any of A, C, G or T, or a primer containing inosine at an ambiguity code is generally used.
  • such the mixed primers were used for degenerate primers to clone above gene.
  • cDNA libraries pur- chased from Stratagene (La Jolla, USA) were used as such a PCR template to clone BHYD genes from Arabidopsis thaliana and Glycine max. cDNA of interest thus obtained was confirmed in its sequence.
  • a sequence of a coding region was used for a cloning of cDNA of corresponding gene.
  • the PCR method was also exploited to clone cDNA fragment.
  • the PCR primers whose sequences were identical to the sequence at the 5'- and 3'- end of the open reading frame (ORF) were synthesized with an addition of an appropriate restriction site, and PCR was performed by using those PCR primers.
  • a cDNA library was used as a template in this PCR cloning of cDNA.
  • the said cDNA library consists of various cDNA species which were synthesized in vitro by the viral reverse transcriptase and Taq polymerase and is available from commercial supplier.
  • the present invention relates to a method for making a recombinant vector comprising inserting a polynucleotide of the invention into a vector.
  • the present invention relates to a recombinant vector containing the polynucleotide of the invention or produced by said method of the invention.
  • vector refers to a nucleic acid molecule capable of transporting a polynucleotide to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA or PNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the present invention also relates to cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering that contain a nucleic acid molecule according to the invention. Methods which are well known to those skilled in the art can be used to construct various plasmids and vectors. Alternatively, the nucleic acid molecules and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
  • control sequences In an other preferred embodiment to present invention relates to a vector in which the polynucleotide of the present invention is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic host cells.
  • control sequences generally include promoter, ribosomal binding site, and terminators.
  • control sequences In eukaryotes, generally control sequences include promoters, terminators and, in some instances, enhancers, transactivators; or transcription factors.
  • control sequence is intended to include, at a minimum, components the pre- sence of which are necessary for expression, and may also include additional advantageous components.
  • operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • the control sequence is a promoter, it is obvious for a skilled person that double- stranded nucleic acid is used.
  • Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by polynucleotides as described herein.
  • the recombinant expression vectors of the invention can be designed for expression of ⁇ -carotene hydroxylase in prokaryotic or eukaryotic cells.
  • genes encoding the polynucleotide of the invention can be expressed in bacterial cells such as E.
  • coli insect cells (using baculovirus expression vectors), yeast and other fungal cells, algae, ciliates of the types: Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpi- dium, Glaucoma, Platyophrya, Potomacus, Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especially of Stylonychia lemnae with vectors following, a transformation method as described in WO 98/01,572 and multicellular plant cells or mammalian cells. Suitable host cells are known to the skilled artisan.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc.), pMAL (New England Biolabs, Beverly, USA) and pRIT5 (Pharmacia, Piscataway, USA) which fuse glu- tathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glu- tathione S-transferase
  • the coding sequence of the polypeptide encoded by the polynucleotide of the present invention is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein.
  • the fusion protein can be purified by affinity chromatography using glutathione-agarose resin, e.g. recombinant ⁇ -carotene hydroxylase unfused to GST can be recovered by cleavage of the fusion protein with hrombin.
  • suitable inducible non-fusion E. coli expression vectors include pTrc and pET lid.
  • Target gene expression from the pTrc vector relies on host RNA polymerase tran- scription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 1 Id vector relies on transcription from a T7 g ⁇ lO-lac fusion promoter mediated by a co- expressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression is to express the protein in host bacteria with an impaired capacity to proteolytically cleave the recombinant protein.
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression, such as E. coli.
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the ⁇ -carotene hydroxylase vector can be a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerevisiae include pYepSecl, pMFa, pJRY88, and pYES2 (Invitrogen, San Diego, USA).
  • Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, are known to the skilled arti- san.
  • the polynucleotide of the invention can be introduced in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series and the pVL series.
  • the polynucleotide of the invention is introduced in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM ⁇ and pMT2PC.
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the recombinant mammalian expression vector can be capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver- specific), lymphoid- specific promoters, in particular promoters of T cell receptors and immunoglobulins, neuron-specific promoters (e.g., the neurofilament promoter), pancreas-specific promoters, and mammary gland-specific promoters (e.g., milk whey promoter; US 4,873, 316 and EP 264,166).
  • Developmentally- regulated promoters are also encompassed, for example the murine hox promoters and the fetoprotein promoter.
  • the reaction mixture (600 ⁇ l) includes 1 mM dithiothreitol, 0.5 mM FeS0 4 , 5 mM ascorbic acid, 0.5 mM 2-oxoglutarate, enzyme, and deoxycholate (0.1 % mass/vol.).
  • the reaction mixture (600 ⁇ l) includes 1 mM dithiothreitol, 0.5 mM FeS0 4 , 5 mM ascorbic acid, 0.5 mM 2-oxoglutarate, enzyme, and deoxycholate (0.1 % mass/vol.).
  • Additions of catal- ase (1 mg/ml) and lipid suspension (200 ⁇ g) are optional.
  • Said lipid suspension is pre- pared as follows: the lipids, L-phosphatidylcholine (Sigma Type II-S, from Soybean
  • soybean lecithin (WAKO, Osaka, Japan) are used for vesicle preparation.
  • 10 mg of lipid is suspended in chloroform or acetone (1 ml) and centrifuged at 12000 g for 5 minutes.
  • the clarified solution is placed in a clear round-bottomed glass tube, when incorporating substrates into the suspension carotenoid 100 ⁇ l (1-2 mg/ml) is also added.
  • the lipid is dried onto the surface of the glass tubes under nitrogen.
  • 100 mM Tris-HCl (pH 8.0) containing 1 mM dithiothreitol (0.5 ml) is placed onto the residue.
  • E.coli extracts 300 ⁇ l 250-400 ⁇ g protein are added, mixed and equilibrated at 30°C for 5 minutes. Reactions are started by adding the ⁇ -carotene in chloroform (0.8-1.0 % by volume). Products and substrates from the in vitro incubations are extracted and separated by HPLC.
  • Codon complementation method is the method frequently used for an identification of carotenogenic gene product by visual screening. Cloned gene can be detected through their ability to confer a specific function or trait onto a host cell upon transforma- tion. In certain cases, the gene complements a missing function which, if essential for the host cell, will enable functional selection and remove the need for screening. This approach is very effective in isolation of genes that confer resistance to an inhibitor that affects the host cells. Functional complementation has been used successfully for cloning genes that encode carotenoid biosynthesis enzymes where the means for screening the gene library was color change in the host cells.
  • a recombinant pACYC184 plasmid is constructed with previously cloned genes from different species in such a way that they are functionally expressed in E. coli.
  • Cells of E. coli that carry this plasmid produce a specific carotenoid which serves as a substrate for the enzyme under investigation.
  • the carotenoid accumulated in the bacteria imparts a characteristic color that can be seen by the eye, to the colony of this strain when grown on petri plates.
  • a cDNA library of the appropriate tissue is constructed in an expression vector, such as ⁇ ZAP II phage (Stratagene). Plasmids that represent the entire mRNA repertoire are excised from the library and transfected into the E. coli cells that contain the recombinant pACYC184 plasmid and produce the colored carotenoid precursor. Following incubation under proper selection conditions, colonies of cells that carry two plasmids emerge on the plates. The screening for the carotenoid gene is based on color visualization of colonies of a size 3 mm in diameter.
  • chromophore of the new carotenoid product is different from that of the precursor carotenoid, colonies of a different color or hue are produced and these can be detected easily after about two days.
  • This method has been used successfully for cloning of crtO, a cDNA from Haematococcus pluvialis that encodes ⁇ -C(4)-oxygenase, and cloning the cDNA for lycopene ⁇ -cyclase ⁇ CrtL-e) and ⁇ -carotene hydroxylase from A thaliana.
  • an expressed protein would be purified and used for raising of the antibody against the purified enzyme.
  • Antibody thus prepared would be used for a characterization of the expression of the corresponding enzyme in a strain improvement study, an optimization study of the culture condition, and the like.
  • the present invention relates to an antibody that binds specifically to the polypeptide of the present invention or parts, i.e. specific fragments or epitopes of such a protein.
  • the antibodies of the invention can be used to identify and isolate other ⁇ -carotene hydroxylase and genes. These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or scFv fragments etc.
  • Monoclonal antibodies can be prepared by known techniques, which,e.g. comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals.
  • antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods known to the skilled artisan.
  • Antibodies can be used, e.g., for the immunoprecipitation and immunolocalization of proteins according to the invention as well as for the monitoring of the synthesis of such proteins, for example, in recombinant organisms, and for the identification of compounds interacting with the protein according to the invention.
  • surface plasmon resonance as employed in the BlAcore system can be used to increase the efficiency of phage antibodies selections, yielding a high increment of affinity from a single library of phage antibodies which bind to an epitope of the protein of the invention.
  • the binding phenomenon of antibodies to antigens is equivalent to other ligand/anti-ligand binding.
  • the gene fragment for ⁇ -carotene hydroxylase was cloned from A. thaliana and G. max with a purpose to express heterologously in P. rhodozyma by genetic method using the cloned gene fragment.
  • the present invention relates to a method of making a recombinant host cell comprising introducing the vector or the polynucleotide of the present invention into a host cell.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection”, conjugation and transduction are intended to refer to a variety of art- recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electropora- tion.
  • Suitable methods for transforming or transfecting host cells including plant cells are known to the skilled artisan.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that en- coding the polypeptide of the present invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by, for example, drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a vector which contains at least a portion of the polynucleotide whose sequence is homologous enough to recom- bine with the host genomic DNA.
  • the heterologous gene whose sequence is not be homologous to the host genomic DNA, other polynucleotide fragment than the objective gene to be expressed heterologously is introduced on a vector.
  • rDNA gene fragment is frequently used. rDNA is a kind of satellite DNA which exist in multicopies on the genome.
  • the objective DNA to be overexpressed on said vector also can exist in multicopies and it is expected that its gene dosage effects can contribute to the overexpression of the objective enzymes.
  • rDNA fragment was conveniently used for this purpose.
  • Further host cells can be produced which contain selection systems which allow for regulated expression of the introduced gene.
  • selection systems which allow for regulated expression of the introduced gene.
  • inclusion of the polynucleotide of the invention on a vector placing it under control of the lac operon permits expression of the polynucleotide only in the presence of IPTG.
  • Such regulatory systems are well known in the art.
  • the introduced nucleic acid molecule is foreign to the host cell.
  • nucleic acid molecule is either heterologous with, respect to the host cell, this means derived from a cell or organism with a different genomic background, or is homologous with respect to the host cell but located in a different genomic environment than the naturally occurring counterpart of said nucleic acid molecule. This means that, if the nucleic acid molecule is homologous with respect to the host cell, it is not located in its natural location in the genome of said host cell, in particular it is surrounded by different genes. In this case the nucleic acid molecule may be either under the control of its own promoter or under the control of a heterologous promoter.
  • the vector or nucleic acid molecule according to the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained in some form extrachrornosornally.
  • the nucleic acid molecule of the invention can be used to restore or create a mutant gene via homologous recombination.
  • the present invention relates to a host cell genetically engineered with the polynucleotide of the invention or the vector of the invention.
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a polynucleotide of the present invention can be introduced in bacterial cells as well as insect cells, fungal cells or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells), algae, ciliates, plant cells, fungi or other microorganims like E. coli.
  • Other suitable host cells are known to those skilled in the art.
  • E. coli, baculovirus, Agrobacterium or fungal cells are, for example, those of the genus Saccharomyces, e.g. those of the species S. cerevisiae or Phaffia rhodozyma ⁇ Xanthophylomyces dendrorhous).
  • the P. rhodozyma mutant strain, ATCC96815 producing ⁇ -carotene predo- minantly was used as such a host strain.
  • the present invention relates to a method for the production of fungal transformants comprising the introduction of the polynucleotide or the vector of the present invention into the genome of said fungal cell.
  • the molecules are placed under the control of regulatory elements which ensure the expression in fungal cells.
  • regulatory elements may be heterologous or homologous with respect to the nucleic acid molecule to be expressed as well with respect to the fungal species to be transformed.
  • such regulatory elements comprise a promoter active in fungal cells.
  • a consti- tutive promoter may be used, such as the glyceraldehyde-3-dehydrogenase ⁇ GAP) gene promoter derived from P. rhodozyma (WO 97/23,633) to obtain constitutive expression in fungal cells.
  • Inducible promoters may be used in order to be able to exact control expression.
  • An example for inducible promoters is the promoter of genes encoding heat shock proteins.
  • an amylase gene promoter which is a candidate for such inducible promoters has been described (EP 1,035,206).
  • the regulatory elements may further comprise transcriptional and/or translational enhancers functional in fungal cells.
  • the regulatory elements may include transcription termination signals, such as a poly-A signal, which lead to the addition of a poly A tail to the transcript which may improve its stability.
  • Methods for the introduction of foreign DNA into fungal cells are also well known in the art. These include, e.g., transformation with LiCl method, the fusion of protoplasts, electroporation, biolistic methods like particle bombardment other methods known in the art. Methods for the preparation of appropriate vectors are known to the skilled artisan.
  • transformation refers to the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for the transfer.
  • the polynucleotide may be transiently or stably introduced into the host cell and may be maintained non- integrated, for example, as a plasmid or as chimeric links, or alternatively, may be integrated into the host genome.
  • the fungi which can be modified according to the invention and which either show overexpression of a protein according to the invention or a reduction of the synthesis of such a protein can be derived from any desired fungal species.
  • the present invention relates to a fungal cell comprising the polynucleotide the vector or obtainable by the method of the present invention.
  • the present invention relates also to transgenic fungal cells which contain (preferably stably integrated into the genome) a polynucleotide according to the invention linked to regulatory elements which allow expression of the polynucleotide in fungal cells and wherein the polynucleotide is foreign to the transformed fungal cell.
  • a polynucleotide according to the invention linked to regulatory elements which allow expression of the polynucleotide in fungal cells and wherein the polynucleotide is foreign to the transformed fungal cell.
  • the presence and expression of the polynucleotide in the transformed fungal cells modulates, preferably increases the synthesis of ⁇ -cryptoxanthin and leads to the increase of the ⁇ -cryptoxanthin production in thus obtained transformed fungal cells, preferably in P. rhodozyma cells.
  • the present invention also relates to transformed fungal cells according to the invention. Accordingly, due to the heterologous expression of ⁇ -carotene hydroxylase, cells' metabolic products are useful for the novel biological production process of ⁇ -cryptoxanthin.
  • production or “productivity” are art-recognized and include the concentration of the fermentation product (for example fatty acids, carotenoids, (poly)saccharides, lipids, vitamins, isoprenoids, wax esters, and/or polymers like polyhydroxyalkanoates and/or its metabolism products or further desired fine chemical as mentioned herein) formed within a given time and a given fermentation volume (e.g., kg product per hour per liter).
  • concentration of the fermentation product for example fatty acids, carotenoids, (poly)saccharides, lipids, vitamins, isoprenoids, wax esters, and/or polymers like polyhydroxyalkanoates and/or its metabolism products or further desired fine chemical as mentioned herein
  • efficiency of production includes the time required for a particular level of production to be achieved (for example, how long it takes for the cell to attain a particular rate of output of a said altered yield, in particular, into carotenoids, (poly)saccharides, vitamins, isoprenoids, lipids etc.).
  • yield or “product/carbon yield” is art-recognized and includes the efficiency of the conversion of the carbon source into the product (i.e. acetyl CoA, fatty acids, lipids, carotenoids, vitamins, isoprenoids etc. and/or further compounds as defined above and which biosynthesis is based on said products). This is generally written as, for example, kg product per kg carbon source.
  • biosynthesis (which is used synonymously for “synthesis” of “biological production” in cells, tissues plants, etc.) or a “biosynthetic pathway” are art-recognized and include the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds in what may be a multistep and highly regulated process.
  • metabolism is art-recognized and includes the totality of the biochemical reactions that take place in an organism.
  • the metabolism of a particular compound e.g., the metabolism of acetyl CoA, a fatty acid, hexose, isoprenoid, vitamin, carotenoid, lipid etc.
  • acetyl CoA a fatty acid, hexose, isoprenoid, vitamin, carotenoid, lipid etc.
  • Such a genetically engineered P. rhodozyma would be cultivated in an appropriate medium and evaluated in its productivity or/ and yield of carotenoids, especially ⁇ -cryptoxanthin.
  • a hyper producer of ⁇ -cryptoxanthin thus selected would be confirmed in view of the rela- tionship between its productivity and the level of gene or protein expression which is introduced by such a genetic engineering method.
  • E. coli HB101 F “ , mcrB, mrr, hsdS20, (rB “ , mB “ ), recA13, leuB6, ara-14, proA2, lacYl, galKl, xyl-5, mtl-1, rps 2Q (Sm r ), supE44, X (Toyobo, Osaka, Japan)
  • F “ , mcrB, mrr, hsdS20, (rB “ , mB “ ) recA13, leuB6, ara-14, proA2, lacYl, galKl, xyl-5, mtl-1, rps 2Q (Sm r ), supE44, X (Toyobo, Osaka, Japan)
  • coli TOP 0 F " , mcrA, Amrr-hsdRMS-mcrBC), ⁇ 80, AlacZ M15, ⁇ Z ⁇ cX74, recAl, deoR, r ⁇ Dl39, ⁇ ara-leu)7697 , gall], galK, rps (Str r ), endAl, nupG (Invitrogen, Carlsbad, USA)
  • P. rhodozyma was maintained routinely in YPD medium (DIFCO, Detroit, U.S.A.). E. coli was maintained in LB medium (10 g Bacto-trypton, 5 g yeast extract (DIFCO) and 5 g NaCl per liter). When an agar medium was prepared, 1.5 % of agar (WAKO) was supplemented.
  • PCR Polymerase chain reaction
  • thermal cycler from Perkin Elmer model 2400. Each PCR condition is described in examples.
  • PCR primers were purchased from a commercial supplier. Fluorescent DNA primers for DNA sequencing were purchased from Pharmacia. DNA sequencing was performed with the automated fluorescent DNA sequencer (ALFred, Pharmacia).
  • Competent cells of HB101 were purchased from Toyobo (Osaka, Japan).
  • An authentic sample for zeaxanthin and ⁇ -carotene was purchased from EXTRASYN- THESE S.A. (Genay Cedex, France) and WAKO (Osaka, Japan), respectively, ⁇ -cryptoxanthin was obtained from Roche Vitamins AG (Basle, Switzerland).
  • Example 1 Cloning of a partial BHYD ( ⁇ -carotene hydroxylase) gene from A. thaliana and G. max
  • Lycopersicon esculentum Y14810 (EMBL/GenBank/DDBJ)
  • thaliana library was named as pATZ1028 #3 and used for further study.
  • the following PCR primers were constructed: AT9 (sense) (SEQ ID NO:8), AT13 (antisense) ' (SEQ ID NO:9), T7 (antisense) (SEQ ID NO:10) and Longrev (sense) (SEQ ID NO:l l).
  • PCR reaction 25 cycles of 94°C for 15 seconds, 55°C for 30 seconds and 72°C for 1 min by using HF polymerase (Clontech) as a DNA polymerase.
  • the cDNA library derived from A. thaliana and G.
  • thaliana library when AT9 and T7 was used as PCR primers had a sequence whose deduced amino acid sequence was identical to the insert fragment in the pATZl028 #3.
  • One of the clones was named as pATZl206 #9T7.
  • the cDNA library derived from G. max as a PCR template
  • one of the clones whose internal sequence was identical to the insert fragment in the pATZl028 #3 was named as pGMZ1206 #9T7.
  • AT13 and Longrev as PCR primers, one clone derived from A.
  • thaliana library and whose internal sequence was identical to the insert fragment in the pATZ1028 #3 was named as pATZ1215 and used for further study.
  • pGMZ1215 whose internal sequence was similar to the insert fragment in the pATZ1028 #3 was obtained from G. max library and selected for further study.
  • Example 2 Cloning of full-length cDNAs for BHYD ( ⁇ -carotene hydroxylase) gene from A. thaliana and G. max
  • ATZ1 sense
  • ATZ18 antisense
  • ATZ21 sense
  • SEQ ID NO: 14 the nucleotide sequence of ATZ17 corresponds to the 5'-end of mature BHYD gene from ⁇ . thaliana and G. max.
  • ATZ21 corresponded to the internal BHYD sequence which covers the common sequence between bacterial and plant ⁇ -carotene hydroxylase genes which probably corresponds to the catalytic domain of ⁇ -carotene hydroxylase.
  • putative BHYD gene from A thaliana and G. max consists of 945 base pairs were determined for those nucleotide sequence.
  • TABLE 4 is a summary of the cloned gene and its derivatives.
  • Plasmid Length Derivation ⁇ ATZal228 full length A. thaliana pATZdl227 catalytic domain A. thaliana pGMZal228 full length G. max pGMZdl228 catalytic domain G. max
  • Example 3 Construction of expression plasmid for BHYD gene obtained from A. thaliana
  • the cDNA fragment which covered the Arabidopsis BHYD gene was amplified by PCR method and then cloned into integration vector in which said gene was transcribed by GAP promoter functioning as a control sequence for constitutive expression of GAP (glyceraldehyde-3-phosphate dehydrogenase) gene in P. rhodozyma.
  • GAP glycosyl-phosphate dehydrogenase
  • Such primers include asymmetrical recognition sequence for restriction enzyme, Sfi ⁇ (GGCCNNNNNGGCC) but their asymmetrical hang-over sequence is designed to be different. This enables a directional cloning into expression vector which has the same asymmetrical sequence at their ligation sequence. The use of such a construction is described in EP 1,158,051.
  • the plasmid pATZd!227 had such asymmetrical recognition sequence and can be cloned directly into such expression vector.
  • GAP promoter was cloned from PCR by using the PCR primers GAP101 (sense) (SEQ ID NO:15) and GAP102 (antisense) (SEQ ID NO:16).
  • the BHYD expression vector, pATZdl 12 thus prepared is transformed into P. rhodozyma mutant strain, ATCC96815 producing ⁇ -carotene predominantly.
  • the protocol for the biolistic transformation is described in EP 1,158,051. Geneticin was dissolved in Phaffia's transformation medium (YPD medium containing 0.75 M D-sorbitol and D-mannitol) at the concentration of 0.1 mg/ml.
  • BHYD recombinants of P. rhodozyma, ATCC96815 thus obtained were cultured in 50 ml of production medium in 500 ml Erlenmeyer flask at 20°C for 7 days by using their seed culture which grew in 7 ml of seed medium in test tubes (21 mm in diameter) at 20°C for 3 days.
  • appropriate volume of culture broth was withdrawn and used for analysis of their growth, productivity of carotenoids.
  • Medium composition is as follows: Seed medium: Glucose 30.0 g/1; NH 4 C1 .83 g/i; KH 2 P0 4 1.0 g/1; MgS0 4 -7H 2 0 0.88 g/1; NaCl 0.06 g/1; CaCl 2 -2H 2 O 0.2 g/1; KH phtalate 20.0 g/1; FeS0 4 -7H 2 0 28 mg/1; Trace element solution 0.3 ml; Vitamin stock solution 1.5 ml; (pH was adjusted at 5.4 - 5.6).
  • the content of trace element solution is as follows: 4N H 2 S0 4 100 ml/1; citric acid-H 2 0 50.0 g/1; ZnS0 4 -7H 2 0 16.7 g/1; CuS0 4 -5H 2 0 2.5 g/1; MnS0 4 -4,5H 2 O 2.0 g/1; H 3 BO 3 2.0 g/1; Na 2 Mo0 4 2.0 g/1; KI 0.5 g/1.
  • vitamin stock solution for seed medium was as follows: 4N- H 2 S0 17.5 ml/1; myo-inositol 40.0 g/1; nicotinic acid 2.0 g/1; Ca-D-pantothenate 2.0 g/1; vitamin Bi (thiamin HCl) 2.0 g/1; p- aminobenzoic acid 1.2 g/1; vitamin B 6 (pyridoxine HCl) 0.2 g/1; biotin stock solution 8.0 ml.
  • Biotin stock solution was prepared by the addition of 4N- H 2 S0 4 to 50 ml of ethanol to a total of 100 ml. 400 mg of D-biotin was added.
  • Main medium glucose 22.0 g/1; KH 2 PO 4 14.25 g/1; MgS0 4 -7H 2 0 2.1 g/1; CaCl 2 -2H 2 0 0.865 g/1; (NH 4 ) 2 S0 4 3.7 g/1; FeS0 4 -7H 2 0 0.28 g/1; trace element solution 4.2 ml; vitamin stock solution 9.35 ml; (pH was adjusted at 5.5).
  • the content of vitamin stock solution for main medium was as follows: 4N H 2 S0 4 17.5 ml/1; nicotinic acid 2.0 g/1; Ca-D-pantothenate 3.0 g/1; vitamin Bi (thiamin HCl) 2.0 g/l; >- aminobenzoic acid 1.2 g/1; vitamin B 6 (pyridoxine HCl) 0.2 g/1; biotin stock solution 30.0 ml.
  • a portion of seed culture broth (2.5 ml) was transferred to 47.5 ml of main culture broth in 500 ml Erlenmeyer flask. Then the cultivation was performed at 20°C and 200 rpm. At the second day of the fermentation, 5 ml of 50 % glucose was added to each flask and then the fermentation was continued. At the forth day of the fermentation, 2ml of cultured broth was withdrawn for analysis and then 5 ml of 50 % glucose was added to each flask for further cultivation for 3 days. At the seventh day, the whole culture was withdrawn and used for analysis on carotenogenesis and cell growth. For analysis of growth, optical density at 660 nm was measured by using UV-1200 photo- meter (Shimadzu Corp., Kyoto, Japan).
  • cells were harvested from 1.0 ml of broth after microcentrifugation and used for the extraction of the carotenoids from cells of P. rhodozyma by disruption with glass beads. After extraction, disrupted cells were removed by centrifugation and the resultant was analyzed for carotenoid content with HPLC.
  • HPLC condition used was as follows: HPLC column: Chrompack Lichrosorb si-60 (4.6 mm, 250 mm), Temperature: room temperature, Eluent: acetone / hexane (18/82) add 1 ml/L of water to eluent, Injection volume: 10 ⁇ l, Flow rate: 2.0 ml/min, Detection: UV at 450 nm.

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