WO2004029234A1

WO2004029234A1 - Bhyd gene

Info

Publication number: WO2004029234A1
Application number: PCT/EP2003/010402
Authority: WO
Inventors: Tatsuo Hoshino; Kazuyuki Ojima; Yutaka Setoguchi
Original assignee: Dsm Ip Assets B.V.
Priority date: 2002-09-27
Filing date: 2003-09-18
Publication date: 2004-04-08
Also published as: AU2003267384A1; EP1546314A1

Abstract

Disclosed are 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. These biological materials are useful for the improvement of the production process of carotenoids, especially ß-cryptoxanthin.

Description

BHYD Gene

The present invention relates to a gene useful in a process to increase the microbial production of carotenoids.

In accordance with this invention, the genes encoding β-carotene hydroxylase, which is a biological material useful in the improvement of the production process for β-cryptoxanthin are provided. This invention involves cloning and determination of the genes encoding β-carotene hydroxylase from plant species, Arabidopsis thaliana and Glycine max.

These genes may be modified in a suitable host, such as P. rhodozyma to overproduce thus cloned β-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.

According to the present invention, there is provided 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.

These biological materials are useful for the improvement of the production process of carotenoids, especially β-cryptoxanthin.

Accordingly, 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);

(d) 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);

(e) 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); (f) 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 β-carotene hydroxylase activity;

(g) 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;

(h) nucleic acid molecules encoding a polypeptide having β-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 β-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 β-carotene hydroxylase activity;

(1) 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 β-carotene hydroxylase activity.

The terms "gene(s)", "polynucleotide", "nucleic acid sequence", "nucleotide sequence", "DNA sequence" or "nucleic acid molecule(s)" as used herein 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.

Thus, 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. Preferably, 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.

According to this invention, the gene encoding β-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 β-carotene hydroxylase as that encoded by the nucleotide sequences shown in SEQ ID NO:l and 3. Further 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. In a still further embodiment, 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.

As used herein, the terms " BHYD gene" and "recombinant BHYD gene" refer to nucleic acid molecules comprising an open reading frame encoding a β-carotene hydroxylase, preferably a β-carotene hydroxylase from A. thaliana or G. max.

DNA sequence polymorphism that leads to changes in the amino acid sequences of enzymes, e.g. β-carotene hydroxylase, 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 β-carotene hydroxylase gene. Any and all such nucleotide variations and resulting amino acid polymorphism in β-carotene hydroxylase that are the result of natural variation and that do not alter the functional activity of β-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 β-carotene hydroxylase cDNA of the invention can be isolated based on their homology to A. thaliana or G. max β-carotene hydroxylase polynucleotides disclosed herein using the polynucleotide of the invention, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Accordingly, in another embodiment, 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. The term "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. Preferably, 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. Preferably, 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.

In the present invention, 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. For example, 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 μg/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 μg/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. In the present invention, it is preferred to use high stringent hybridization and wash conditions to define the antisense activity against β-carotene hydroxylase gene from A. thaliana or G. max.

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.

As used herein, 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). Preferably, the polynucleotide encodes a natural A. thaliana or G. max β-carotene hydroxylase.

In addition to naturally-occurring variants of the β-carotene hydroxylase sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into a nucleotide sequence of the polynucleotide encoding β- carotene hydroxylase, thereby leading to changes in the amino acid sequence of the encoded β-carotene hydroxylase, without altering the functional ability of the β-carotene hydroxylase. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in a sequence of the polynucleotide encoding β-carotene hydroxylase, e.g. SEQ ID NO:l or 3. A "non- essential" amino acid residue is a residue that can be altered from the wild-type sequence of one of the β- carotene hydroxylase without altering the activity of said β-carotene hydroxylase, whereas an "essential" amino acid residue is required for β-carotene hydroxylase activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having β-carotene hydroxylase activity) may not be essential for activity and thus are likely to be amenable to alteration without altering β-carotene hydroxylase activity. Accordingly, the invention relates to polynucleotides encoding β-carotene hydroxylase that contain changes in amino acid residues that are not essential for β-carotene hydroxylase. Such β-carotene hydroxylase differs in amino acid sequence from a sequence contained in SEQ ID NO:2 or 4 yet retain the β-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 β-cryptoxanthin. Preferably, 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.

To determine the percent homology of two amino acid sequences (e.g., one of the sequen- ces of SEQ ID NO:2 or 4 and a mutant form thereof) or of two nucleic acids, the 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). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence (e.g., one of the sequences of SEQ ID NO:l, 2, 3, or 4) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence (e.g., a mutant form of the sequence selected), then 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 percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = numbers of identical positions/total numbers of positions x 100). The homology can be determined by computer programs as Blast 2.0. In this invention, GENETYX-SV/RC software (Software Development Co., Ltd., Tokyo, Japan) is used by using its default algorithm as such a homology analysis software. This software uses the Lipman-Pearson method for its analytic algo- rithm.

A nucleic acid molecule encoding a β-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). Thus, a predicted nonessential amino acid residue in a β-carotene hydroxylase is preferably replaced with another amino acid residue from the same family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a β-carotene hydroxylase coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a β-carotene hydroxylase activity described herein to identify mutants that retain β-carotene hydroxylase activity. Following mutagenesis of one of the sequences of SEQ ID NO:l or 3, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, "color complementation" described herein.

Accordingly, in one preferred embodiment 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. For example, β-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. Moreover, 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. For example, mRNA can be isolated from cells, e.g. Arabidopsis or Glycine (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al.) and cDNA can be prepared using 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. Furthermore, oligonucleotides corresponding to a β-carotene hydroxylase nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

The terms "fragment", "fragment of a sequence" or "part of a sequence" means a truncated sequence of the original sequence referred to. The truncated sequence (nucleic acid or protein 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.

Typically, 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.

The term "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. Those of skill will recognize that all immunogens (i. e., substances capable of eliciting an immune response) are antigens; however, some antigen, such as haptens, are not immunogens but ma be made immunogenic by coupling to a carrier molecule. The term "antigen" includes references to a substance to which an antibody can be generated and/or to which the antibody is specifically immunoreactive. The term "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. Preferably, the identity is more than 80%, more preferred are 85%, 90% or 95%, even more preferred are 96%, 97%, 98%, or 99% identity.

The term "β-carotene hydroxylase " or "β-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 β-carotene hydroxylase, i.e., having one or more β-carotene hydroxylase epitopes, are also comprised under the term "β-carotene hydroxylase". In these cases activity refers to their immunological activity.

The terms "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. Preferably, 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.

For example, in various embodiments, the β-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). Moreover, the 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.

Preferably, 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 β-carotene hydroxylase cDNAs of the invention.

Further, 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.

Moreover, 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 β-carotene hydroxylase. The nucleotide sequences determined from the cloning of the β-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 β-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 β-carotene hydroxylase homologues. Probes based on the β- 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 β-carotene hydroxylase, such as by measuring a level of a β-carotene hydroxylase -encoding nucleic acid molecule in a sample of cells, e.g., detecting β-carotene hydroxylase mRNA levels or determining whether a genomic β-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 β-cryptoxanthin, in particular a β-carotene hydroxylase activity as described in the examples in microorganisms or plants. As used herein, 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 β-cryptoxanthin in microorganisms or plants. Examples of a β-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 β-carotene hydroxylase polynucleotide of the inven- tion are preferably biologically active portions of one of the β-carotene hydroxylase.

As mentioned herein, the term "biologically active portion of β-carotene hydroxylase " is intended to include a portion, e.g., a domain/motif, that participates in the biosynthesis of β-cryptoxanthin or has an immunological activity such that it is binds to an antibody binding specifically to β-carotene hydroxylase. To determine whether a β-carotene hydroxylase or a biologically active portion thereof can participate in the metabolism, 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 β-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 β-carotene hydroxylase or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the β-carotene hydroxylase or peptide.

At first, we cloned a partial gene fragment containing a portion of β-carotene hydroxylase {BHYD) gene by using degenerate PCR method. The said 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"). In such a degenerate primer, a mixed primer which consists any of A, C, G or T, or a primer containing inosine at an ambiguity code is generally used. In this invention, such the mixed primers were used for degenerate primers to clone above gene. In this invention, 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.

In this invention, we used the automated fluorescent DNA sequencer, ALFred system (Pharmacia, Uppsala, Sweden) using an autocycle sequencing protocol in which the Taq DNA polymerase is employed in most cases of sequencing.

After the determination of the genomic 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. In this invention, 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.

In another embodiment, the present invention relates to a method for making a recombinant vector comprising inserting a polynucleotide of the invention into a vector.

Further, the present invention relates to a recombinant vector containing the polynucleotide of the invention or produced by said method of the invention.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting a polynucleotide to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. 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). Other vectors (e.g., non-episomal mammalian 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. Moreover, 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". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, 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.

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. The nature of such control sequences differs depending upon the host organism. In prokaryotes, control sequences generally include promoter, ribosomal binding site, and terminators. In eukaryotes, generally control sequences include promoters, terminators and, in some instances, enhancers, transactivators; or transcription factors.

The term "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.

The term "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. In case 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 β-carotene hydroxylase in prokaryotic or eukaryotic cells. For example, 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. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. 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. Often, in fusion expression vectors, 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. Such 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. In one embodiment, 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 β-carotene hydroxylase unfused to GST can be recovered by cleavage of the fusion protein with hrombin.

Examples of 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 gπlO-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 λ 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.

Further, the β-carotene hydroxylase vector can be a yeast expression vector. Examples of 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.

Alternatively, 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 (e.g., Sf 9 cells) include the pAc series and the pVL series.

Alternatively, the polynucleotide of the invention is introduced in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDMδ and pMT2PC. When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, 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. Non-limiting examples of 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.

Thus expressed BHYD gene can be verified for its activity such as by enzyme assay method or "color complementation" method. Some experimental protocols as an enzymatic assay for β-carotene hydroxylase are described in the literature. The following is the one of the methods which is used for the determination of β-carotene hydroxylase activity: the reaction mixture (600 μl) includes 1 mM dithiothreitol, 0.5 mM FeS0₄, 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 μg) are optional. Said lipid suspension is pre- pared as follows: the lipids, L-phosphatidylcholine (Sigma Type II-S, from Soybean

(Sigma, St. Louis, USA)) and soybean lecithin (WAKO, Osaka, Japan) are used for vesicle preparation. To provide about 200 μg in a 10-20-μl suspension, 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 μl (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. Sonication (3 x 2-s bursts, full power) is adequate to yield suitable lipid suspension. Lipid vesicles are prepared freshly prior to incubations. Enzyme (cell extract) is prepared after cell harvest in which the foreign BHYD gene is expressed in E. coli as follows: Pelleted cells (by 6000 g centrifugation) are resuspended in 100 mM Tris-HCl (pH 8.0) containing 1 mM dithiothreitol, protease inhibitor cocktail (0.1 mM of phenylmethylsulfonyl fluoride, 1 μg/ml of leupeptine and 1 μg/ml of pepstatin) and 5 % (by volume) glycerol. E.coli extracts 300 μl (250-400 μg protein) are added, mixed and equilibrated at 30°C for 5 minutes. Reactions are started by adding the β-carotene in chloroform (0.8-1.0 % by volume). Products and substrates from the in vitro incubations are extracted and separated by HPLC.

The term, "Color 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. In this "color complementation" method, 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.

To clone the cDNA for an enzyme that uses this carotenoid as a precursor, a cDNA library of the appropriate tissue is constructed in an expression vector, such as λZAP 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. If the 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 β-C(4)-oxygenase, and cloning the cDNA for lycopene ε-cyclase {CrtL-e) and β-carotene hydroxylase from A thaliana.

Succeeding to the confirmation of the enzyme activity, 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.

In a further embodiment, 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 β-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.

Furthermore, 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. For example, 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. In many cases, the binding phenomenon of antibodies to antigens is equivalent to other ligand/anti-ligand binding.

In this invention, the gene fragment for β-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.

In one embodiment 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. As used herein, the terms "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.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, 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. Preferred 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).

To create a homologous recombinant microorganism, a vector is prepared which contains at least a portion of the polynucleotide whose sequence is homologous enough to recom- bine with the host genomic DNA. In the case that 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. For that purpose, rDNA gene fragment is frequently used. rDNA is a kind of satellite DNA which exist in multicopies on the genome. By inserting a rDNA fragment as a targeting DNA to the host genome into recombinant vector, 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. In this invention, such 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. For example, 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.

Preferably, the introduced nucleic acid molecule is foreign to the host cell.

By "foreign" it is meant that the 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. In this respect, it is also to be understood that the nucleic acid molecule of the invention can be used to restore or create a mutant gene via homologous recombination.

Accordingly, in another embodiment the present invention relates to a host cell genetically engineered with the polynucleotide of the invention or the vector of the invention.

The terms "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.

For example, 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. Preferred are 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). In this invention, the P. rhodozyma mutant strain, ATCC96815 producing β-carotene predo- minantly was used as such a host strain.

In addition, in one embodiment, 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.

For the expression of the nucleic acid molecules according to the invention in plant cells, the molecules are placed under the control of regulatory elements which ensure the expression in fungal cells. These 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.

In general, 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. Also 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. Furthermore, 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.

The term "transformation" as used herein, 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.

In general, 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.

Further, in one embodiment, the present invention relates to a fungal cell comprising the polynucleotide the vector or obtainable by the method of the present invention.

Thus, 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. For the meaning of foreign; see supra.

The presence and expression of the polynucleotide in the transformed fungal cells modulates, preferably increases the synthesis of β-cryptoxanthin and leads to the increase of the β-cryptoxanthin production in thus obtained transformed fungal cells, preferably in P. rhodozyma cells.

Thus, the present invention also relates to transformed fungal cells according to the invention. Accordingly, due to the heterologous expression of β-carotene hydroxylase, cells' metabolic products are useful for the novel biological production process of β-cryptoxanthin.

The terms "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).

The term "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.).

The term "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. By increasing the yield or production of the compound, the quantity of recovered molecules, or of useful recovered molecules of that compound in a given amount of culture over a given amount of time is increased.

The terms "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.

The language "metabolism" is art-recognized and includes the totality of the biochemical reactions that take place in an organism. The metabolism of a particular compound, then, (e.g., the metabolism of acetyl CoA, a fatty acid, hexose, isoprenoid, vitamin, carotenoid, lipid etc.) comprises the overall biosynthetic, modification, and degradation pathways in the cell related to this compound.

Such a genetically engineered P. rhodozyma would be cultivated in an appropriate medium and evaluated in its productivity or/ and yield of carotenoids, especially β-cryptoxanthin. A hyper producer of β-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.

The present invention is further illustrated with Examples described below.

The following materials and methods were employed in the Examples described below:

Strains

P. rhodozyma ATCC96815 (re-deposited under the accession No. ATCC 74486 on February 18, 1999 pursuant to the Budapest Treaty)

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) E. coli TOP 0: F^", mcrA, Amrr-hsdRMS-mcrBC), φ80, AlacZ M15, ΔZαcX74, recAl, deoR, rαDl39, {ara-leu)7697 , gall], galK, rps (Str^r), endAl, nupG (Invitrogen, Carlsbad, USA)

Vectors pBluescriptll SK- (Stratagene) pCR2.1TOPO (Invitrogen)

Media

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.

Methods

Restriction enzymes and T4 DNA ligase were purchased from Takara Shuzo (Ohtsu,

Japan).

Polymerase chain reaction (PCR) is performed with the 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 β-carotene was purchased from EXTRASYN- THESE S.A. (Genay Cedex, France) and WAKO (Osaka, Japan), respectively, β-cryptoxanthin was obtained from Roche Vitamins AG (Basle, Switzerland). Example 1: Cloning of a partial BHYD (β-carotene hydroxylase) gene from A. thaliana and G. max

To clone a partial BHYD gene from A. thaliana and G. max, a degenerate PCR method was exploited. Species and accession number to database whose sequence for β-carotene hydroxylase were used for multiple alignment analysis are as follows: Alcaligenes sp. D58422 (EMBL)

Agrobacterium aurantiacum D58420 (EMBL) Pantoea ananas D90087 (EMBL)

Erwinia herbicola M87280 (EMBL) Arabidopsis thaliana U58919 (EMBL/GenBank/DDBJ)

Lycopersicon esculentum Y14810 (EMBL/GenBank/DDBJ)

Two mixed primers whose nucleotide sequences were designed and synthesized based on the common sequence of known β-carotene hydroxylase genes from other species: AT6 (sense) (SEQ ID NO:5), AT7 (antisense) (SEQ ID NO:6) and AT8 (antisense) (SEQ ID NO:7) (in the sequences "n" means nucleotides a, c, g or t, "r" means nucleotides a or g, "y" means nucleotides c or t, and "h" means nucleotides a, c or t).

After the PCR reaction of 25 cycles of 94°C for 15 seconds, 44°C for 30 seconds and 72°C for 30 seconds by using Taq polymerase (Sawady, Tokyo, Japan) as a DNA polymerase. The cDNA library derived from A thaliana and G. max (Stratagene) was purchased from Stratagene and heated at 99°C for 5 min before use as a PCR template. The yielded reaction mixture was applied to agarose gel electrophoresis. Each PCR band that had a desired length was recovered from the PCR reaction mixture and purified by QIAquick (QIAGEN) according to the method by the manufacturer and then ligated to pCR2.1-TOPO (Invitrogen). After transformation of competent E. coli TOP 10, 6 white colonies were selected and plasmids were isolated with Automatic DNA isolation system. As a result of sequencing, it was found that 6 clones derived from A. thaliana library when AT6 and AT8 was used as PCR primers had a sequence whose deduced amino acid sequence was similar to known β- carotene hydroxylase genes. In the case of using AT7 and AT8 as PCR primers, one clone whose sequence had high homology to known β-carotene hydroxylase genes was obtained from G. max library. Interestingly, the insert fragment derived from A. thaliana and G. max had identical nucleotide sequence. One of the clones derived from A. thaliana library was named as pATZ1028 #3 and used for further study. To clone the cDNA covering an entire gene for β-carotene hydroxylase, 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). After the PCR reaction of 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. max (Stratagene) was heated at 99°C for 5 min before use as a PCR template. Then, yielded reaction mixture was applied to agarose gel electropho- resis. Each PCR band that had a desired length was recovered from the PCR reaction mixture and purified by QIAquick (QIAGEN) according to the method by the manufacturer and then ligated to pCR2.1-TOPO (Invitrogen). After transformation of competent E. coli TOP 10, 6 white colonies were selected and plasmids were isolated with Automatic DNA isolation system. As a result of sequencing, it was found that all the clones derived from A. 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. In the case of using 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. In the case of using 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. Using the same combination of PCR primers, 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 (β-carotene hydroxylase) gene from A. thaliana and G. max

The following PCR primers were designed and synthesized: ATZ1 (sense) (SEQ ID NO:12), ATZ18 (antisense) (SEQ ID NO:13) and ATZ21 (sense) (SEQ ID NO: 14). Among these primers, the nucleotide sequence of ATZ17 corresponds to the 5'-end of mature BHYD gene from Λ. thaliana and G. max. On the other hand, ATZ21 corresponded to the internal BHYD sequence which covers the common sequence between bacterial and plant β-carotene hydroxylase genes which probably corresponds to the catalytic domain of β-carotene hydroxylase.

After the PCR reaction of 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. max (Stratagene) was heated at 99°C for 5 min before use as a PCR template. Then, yielded reaction mixture was applied to agarose gel electropho- resis. Each PCR band that had a desired length was recovered from the PCR reaction mix- ; - ture and purified by QIAquick (QIAGEN) according to the method by the manufacturer and then ligated to pCR2.1-TOPO (Invitrogen). After transformation of competent E. coli TOP 10, 6 white colonies were selected from each PCR reaction and plasmids were isolated with Automatic DNA isolation system.

As a sequence of these candidates for BHYD gene from A. thaliana and G. max, putative BHYD gene from A thaliana and G. max consists of 945 base pairs were determined for those nucleotide sequence. The following TABLE 4 is a summary of the cloned gene and its derivatives.

TABLE 4 The summary of cloned BHYD gene from A. thaliana and G. max

Plasmid Length Derivation ρATZal228 full length A. thaliana pATZdl227 catalytic domain A. thaliana pGMZal228 full length G. max pGMZdl228 catalytic domain G. max

In comparison of sequence between pATZal228 and pGMZal228, only one base change which does not cause the change of amino acid residue (silent mutation) was observed. In conclusion, the sequence of BHYD genes cloned from A. thaliana was identical to that from G. max. Nucleotide sequence for BHYD gene obtained from A. thaliana is listed in SEQ ID NO:l and the corresponding amino acid sequence is listed in SEQ ID NO:2. On the other hand, nucleotide sequence for BHYD gene obtained from G. max is listed in SEQ ID NO:3 and the corresponding amino acid sequence is listed in SEQ ID NO:4. The nucleotide sequence for BHYD genes and their deduced amino acid sequence thus cloned are different from reported sequence for β-carotene hydroxylase from A. thaliana [Sun et al., J. Biol. Chem. 271:24349-24351 (1996)].

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. Such primers include asymmetrical recognition sequence for restriction enzyme, Sfiϊ (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. As the promoter fragment which can drive the transcription of the Arabidopsis BHYD gene, GAP promoter was cloned from PCR by using the PCR primers GAP101 (sense) (SEQ ID NO:15) and GAP102 (antisense) (SEQ ID NO:16).

After the PCR reaction of 25 cycles of 94°C for 15 seconds, 55°C for 30 seconds and 72°C for 1.5 min by using HF polymerase (Clontech) as a DNA polymerase. The plasmid, pG4l8Sa330 (EP 1,035,206) was used as a PCR template. Then, yielded reaction mixture was applied to agarose gel electrophoresis. The PCR band that had a desired length was recovered from the PCR reaction mixture and purified by QIAquick (QIAGEN) according to the method by the manufacturer and then ligated to pCR2.1-TOPO (Invitrogen). After transformation of competent E. coli TOP10, 6 white colonies were selected and plasmids were isolated with Automatic DNA isolation system. As a result of sequencing, it was found that five clones had a correct sequence and one of which (pUG4181107#5) was selected for further study.

Next, the vector backbone, pF718 (EP 1,158,051) which contained AST terminator fragment, G418 resistant cassette and rDNA fragment in this order on pBluescriptll SK- (Stratagene) was prepared. Then, the 0.4 kb Notl-Sfil fragment from pUG4181107#5 and the 0.55 kb Sfil fragment from pATZdl227 was ligated to Notl-Sfil doubly digested pF718. Finally, competent E. coli HB101 was transformed by this ligation mixture. A restriction analysis for 6 transformants yielded showed that all the transformants had correct structure in which Arabidopsis BHYD gene was inserted between GAP promoter and AST terminator derived from P. rhodozyma. One of the clones (pATZdl 12) was selected for further study.

Example 4: Transformation of P. rhodozyma with BHYD expression vector, pATZdl 12

The BHYD expression vector, pATZdl 12 thus prepared is transformed into P. rhodozyma mutant strain, ATCC96815 producing β-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.

As a result, 7 transformants which were resistant to 0.1 mg/ml geneticin were evaluated for their carotenoid profile produced in shaking flask culture. Example 5: Characterization of Arabidopsis BHYD recombinant of P. rhodozyma

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. For analysis of carotenoid produced, 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; NH₄C1 .83 g/i; KH₂P0₄ 1.0 g/1; MgS0₄-7H₂0 0.88 g/1; NaCl 0.06 g/1; CaCl₂-2H₂O 0.2 g/1; KH phtalate 20.0 g/1; FeS0₄-7H₂0 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 H₂S0₄ 100 ml/1; citric acid-H₂0 50.0 g/1; ZnS0₄-7H₂0 16.7 g/1; CuS0₄-5H₂0 2.5 g/1; MnS0₄-4,5H₂O 2.0 g/1; H₃BO₃ 2.0 g/1; Na₂Mo0₄ 2.0 g/1; KI 0.5 g/1. The content of vitamin stock solution for seed medium was as follows: 4N- H₂S0 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 B₆ (pyridoxine HCl) 0.2 g/1; biotin stock solution 8.0 ml.

Biotin stock solution was prepared by the addition of 4N- H₂S0₄ 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; KH₂PO₄ 14.25 g/1; MgS0₄-7H₂0 2.1 g/1; CaCl₂-2H₂0 0.865 g/1; (NH₄) ₂S0₄ 3.7 g/1; FeS0₄-7H₂0 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 H₂S0₄ 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 B₆ (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). For analysis of content of β-carotene, β-cryptoxanthin and zeaxanthin, 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. The 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 μl, Flow rate: 2.0 ml/min, Detection: UV at 450 nm.

Fermentation profile by the 7 recombinants thus obtained together with its host strain is described in TABLE 6.

TABLE 6: Carotenogenesis and growth profile in shake flask culture

As described in TABLE 6, specific conversion from β-carotene into β-cryptoxanthin was exemplified in all the Phaffia recombinants transformed with Arabidopsis BHYD gene.

Claims

1. An isolated polynucleotide comprising a nucleic acid molecule one or more 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 the 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); (d) nucleic acid molecules encoding a polypeptide derived from the polypeptide encoded by a polynucleotide of (a) to (c) byway of substitution, deletion and/or addition of one or several amino acids of the amino acid sequence of the polypeptide encoded by a nucleotide of (a) to (c);

(e) nucleic acid molecules encoding a polypeptide derived from the 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);

(f) nucleic acid molecules comprising a fragment encoded by a nucleic acid molecule of any one of (a) to (e) and having β-carotene hydroxylase activity;

(g) nucleic acid molecules comprising a polynucleotide having a sequence of a nucleic acid molecule amplified from a Arabidopsis or Glycine nucleic acid library using the primers depicted in SEQ ID NO:5, 6, and 7;

(h) nucleic acid molecules encoding a polypeptide having β-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 β-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 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 β-carotene hydroxylase activity;

(1) nucleic acid molecules whose complementary strand hybridizes under stringent conditions with a nucleic acid molecule of any one of (a) to (k) and having β-carotene hydroxylase activity.

2. The isolated polynucleotide of claim 1, wherein said polynucleotide encodes amino acid sequence which is identified by SEQ ID NO:2 or 4, or has identity of 72.8 % or more with SEQ ID NO:2 or 4.

3. The isolated polynucleotide of claim 1 or 2, wherein said polynucleotide is derived from a strain of Arabidopsis thaliana or Glycine max.

4. A method for making a recombinant vector comprising inserting the polynucleotide of any one of claims 1 to 3 into a vector.

5. A recombinant vector containing the polynucleotide of any one of claims 1 to 3 or produced by the method of claim 4.

6. The vector of claim 5 in which the polynucleotide of any one of claims 1 to 3 is operatively linked to expression control sequences allowing expression in prokaryotic or eukary- otic cells.

7. A method of making a recombinant organism comprising introducing the vector of claim 5 or 6 into a host organism.

8. The method of claim 7, wherein said host organism is selected from Escherichia coli, baculovirus, Saccharomyces cerevisiae, Phaffia rhodozyma, or Xanthophylomγces dendro- rhous.

9. The recombinant organism containing the vector of claims 5 or 6, or produced by the method of claims 7 or 8.

10. A process for producing a polypeptide having β-carotene hydroxylase activity comprising culturing the recombinant organism of claim 9 and recovering the polypeptide from the culture of said recombinant organism.

11. A polypeptide having the amino acid sequence encoded by the polynucleotide of any one of claims 1 to 3 or obtainable by the process of claim 10.

12. An antibody that binds specifically to the polypeptide of claim 11.

13. The recombinant organism of claim 9, which is characterized in that β-carotene hydroxylase gene is heterologously introduced, and is thereby capable of producing β-cryptoxanthin.

14. The recombinant organism according to claim 13, wherein the β-carotene hydroxylase gene is heterologously introduced by means of the recombinant DNA technique.

15. The recombinant organism according to claim 13 or 14, wherein said organism belongs to a strain oiPhafia rhodozyma or Xanthophylomyces denάrorhous.

16. A process for producing β-cryptoxanthin, which comprises cultivating the recombinant organism of claim 13.

17. The process according to claim 16, wherein said organism belongs to a strain of Phaffia rhodozyma or Xanthophylomyces dendrorhous.