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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
  • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
  • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
  • C12N9/0042—NADPH-cytochrome P450 reductase (1.6.2.4)

Definitions

• Yeast basidiomycete Xanthophyllomyces dendrorhous synthesizes carotenoids, mainly xanthophyll astaxanthin, of great commercial interest due to its use as a food supplement in the aquaculture industry.
• the present invention describes the genetic material and the polypeptide it encodes, which has cytochrome P450 reductase activity and participates in the synthesis of astaxanthin from beta-carotene, acting as an electron donor in the reaction catalyzed by the enzyme astaxanthin synthase .
• Carotenes are a group of natural pigments produced mainly by plants, yeasts and microalgae. Particularly xanthophyll astaxanthin is a compound widely found in nature, it has It is of great commercial importance since it is used as a food additive to pigment trout and salmon meat in aquaculture (Johnson et al. 1977). On the other hand • and due to its highly antioxidant activity, its benefits for human health have been observed, for example, in the prevention of cardiovascular diseases, it favors the immune system, bioactivity against Helicobacter pylori and cataract prevention (Higuera-Ciapara and cois. 2006).
• astaxanthin Another way of obtaining astaxanthin is biosynthesis and extraction from microorganisms, in this sense the use of microalgae (Haematococcus pluvials) and yeast ⁇ Xanthophyllomyces dendrorhous) are more important.
• pluvialis has been recognized for many years as an accumulator of the astaxanthin carotenoid, under certain stress conditions, such as high light intensity and / or nitrogen limitation, the cells of this algae form cysts and accumulate high amounts of astaxanthin (> 1% dry weight) (Margalith, 1999), changing its color from green to red. This microalgae has recently received much attention due to its ability to synthesize and accumulate large amounts of astaxanthin during and at the end of its growth phase (Margalith, 1999).
• H. pluvialis as a source of astaxanthin was poor due to the obtaining of pigments of inferior quality than the commercially required, given the high percentage of esterification of the obtained astaxanthin (Sominer et al 1991).
• the astaxanthin produced by the yeast Xanthophyllomyces dendrorhous is 100% free (not esterified) and is composed of the 3R, 3R 'optical isomer and the "trans" geometric isomer, which gives it a great comparative advantage over the produced by the microalgae (Andrewes and Starr, 1976).
• microalgae are the natural source with the highest known astaxanthin content, the interest for commercial exploitation of these organisms has not been sufficient due to the high rates of contamination with protozoa and a lower development of culture technologies , compared to heterotrophic organisms such as yeasts.
• Figure 1 shows the route identified for X. dendrorhous and details those genes that have been isolated, sequenced, characterized and published (Kajiwara et al., 1997, Niklitschek and cois., 2008, Verdoes and cois., 1999a; Alca ⁇ no, 2002, Verdoes and cois., 1999b; León, 2000).
• astaxanthin from beta-carotene requires two Enzymatic activities: Ketolase that incorporates a keto group at positions 4 and 4 'of the beta-carotene and hydroxylase molecule that introduces a hydroxyl group at positions 3 and 3' of beta-carotene (Cunningham and Gantt 1998; Linden 1999) . Unlike other organisms where two independent genes participate in this step, in X. dendrorhous a crtS gene has been isolated that encodes an astaxanthin synthase enzyme which exhibits both activities (EP 1.035.206; 0j ima and cois., 2006; ⁇ lvarez and cois., 2006).
• cytochrome P450 monooxygenase enzymes require an electron donor system that in eukaryotes is a cytochrome P450 Reductase (CPR) protein (van den Brink et al., 1998).
• CPR cytochrome P450 Reductase
• Oj ima and cois (2006) described a work in which Escherichia was transformed coli with 3 plasmids compatible with each other, one of the plasmids contained the genes necessary for the synthesis of beta-carotene, another contained the crtS gene of X.
• the present invention describes the sequence of the crtR gene and the polypeptide it encodes, which possesses Cytochrome P450 Reductase activity of X. dendrorhous.
• This invention provides technical tools that allow transforming microorganisms either by enhancing their natural ability to produce astaxanthin or by creating new metabolic pathways in other non-carotenogenic organisms that allow the production of this pigment in a commercially attractive manner.
• the present invention relates to the genetic material that codes for the cytochrome P450 reductase enzyme (CPR), genetic material being understood as the nucleic acid sequence of the genomic DNA type, complementary DNA, messenger RNA and the respective allelic variants encoding a polypeptide with CPR activity, useful for the production of astaxanthin by transforming host organisms that may or may not be natural producers of said carotenoid.
• CPR cytochrome P450 reductase enzyme
• Figure 1 Main steps of the astaxanthin biosynthesis path in X. dendrorhous.
• the structural genes of the pathway that have been cloned and sequenced are indicated. It shows the color of the colonies that they accumulate intermediate carotenoids in the biosynthesis pathway. The color is an indicator of the type of pigment that is being obtained in the different stages of the synthesis.
• the underlined sequence fragments correspond to the exons of the gene.
• those nucleic acids identified as donor and receptor sites for splicing processes stand out. Restriction sites for the Bam ⁇ l enzyme are indicated.
• FIG. 3 Comparison of nucleotide sequences of NADPH-cytochrome P450 reductase genes from different fungi.
• C_echinulata Cunninghamella echinulata (Zygomycete) GenBank Access No.: AF195660.
• S_cerevisiae Saccharomyces cerevisiae (Ascomycete) GenBank Access No.: D13788.
• R_minuta Rhodotorula minuta (Basidiomycete) GenBank Accession No.: AB055119. Gray shows where some of the splitters described in table 1 align.
• FIG. 5 Amino acid sequence deduced from the coding region of the crtR gene.
• the conserved domains of the protein stand out, in order they are: Transmembrane domain, FMN binding domain, FAD binding domain and NAD (P) H binding domain, identified by the BlastP program.
• Figure 7 Scheme of mutation events of the crtR gene by homologous recombination in wild strains UCD67-385 (385) and CBS6938 (CBS) of X. dendrorhous, where A shows the transformation of strain UCD67-385. and B shows transformation of strain CBS6938.
• B CBS6938 PCR and CBSTr transformant.
• CBSTr transformant PCR Lane: (1): CBSTr + SEQ754-hygSecR DNA, (2): CBSTr + HF + SEQ35 DNA, (3): CBSTr + HygSecF + M13F DNA, (4): CBSTr + M13R + SEQ15 DNA, (5): CBSTr + SEQ20 + SEQ50 and (6) DNA: CBSTr 4- SEQ30 + SEQ50 DNA.
• the present invention relates to a gene and an enzyme that is involved in the last stage of the synthesis of astaxanthin from beta-carotene.
• the present invention provides a genomic DNA sequence (crtR gene) and complementary DNA encoding for an enzyme with cytochrome P450 reductase (CPR) activity responsible for delivering the electrons necessary for the incorporation of keto and hydroxyl groups to the beta-carotene molecule to obtain astaxanthin, this reaction is catalyzed by the enzyme astaxanthin synthase.
• CPR cytochrome P450 reductase
• the present invention relates to the DNA sequences of the crtR gene of X. dendrorhous, in its preferred embodiment genomic DNA and complementary DNA sequences, as well as fragments of said gene, these being semi-degenerated or not.
• the present invention relates to a genomic DNA sequence of the crtR gene, its complementary strand, its allelic variants and sequences represented with addition, insertion, deletion and / or substitution of one or more nucleotides encoding a polypeptide having CPR enzymatic activity. . Because the genetic code is degenerated, this invention also contemplates nucleic acid sequences that hybridize with the crtR gene, under conditions of standard strictness, such as that described by Sambrook and cois 2001. In its preferred embodiment, The DNA sequence corresponds to the SEQ1 and is characterized by coming from X.
• nucleic acids described as donor and receptor sites for splicing processes are identified, said nucleic acids are labeled in black and underlined in the SEQl (figure 2).
• the genomic DNA sequence of said gene is characterized by having 3 segments of the sequence corresponding to the exons, underlined in SEQl.
• This invention also considers a complementary DNA sequence, its complementary strand and the messenger RNA sequence that generates it, its allelic variants and sequences represented with addition, insertion, deletion and / or substitution of one or more nucleotides encoding a polypeptide that have CPR enzyme activity. Because the genetic code is degenerated, this invention also contemplates nucleic acid sequences that hybridize with the complement of the crtR gene, under conditions of standard strictness, such as that described by Sambrook et al. In its preferred embodiment said complementary DNA is synthesized from the messenger RNAs generated from the crtR gene, and refers to SEQ2 ( Figure 4), said complementary DNA represents the coding segment of the crtR gene and has a length of 2,241 base pairs.
• the invention also relates to methods of obtaining DNA sequences from the crtR gene of X. dendr ⁇ rhous.
• said method corresponds to the identification of the genomic DNA of the gene of interest, or part thereof, using semi-degenerated fragments.
• the gene of interest is identified with fragments of the sequence isolated from the previous stage.
• consensus sequences of segments conserved among genes equivalent to those of interest from other species are used.
• the invention comprises the comparison by means of bioinformatics techniques of known sequences of cytochrome P450 reductase genes from other yeasts, the identification of conserved segments and the design, based on them, of short acid fragments. nucleic (between 15 and 30 base pairs), useful for the identification and amplification of DNA sequences.
• nucleic between 15 and 30 base pairs
• the DNA fragments described in Table 1 were designed.
• the splitters from SEQlO to SEQ13 were used to identify clones of genomic DNA and complementary DNA libraries of X. dendrorhous carrying fragments of the crtR gene, using the PCR technique. The rest of the mentioned starters were used for the complete sequencing of the crtR gene. Table 1: DNA fragments useful for the identification and amplification of the crtR gene.
• this invention relates to an amino acid sequence with cytochrome P450 reductase activity, corresponding in its preferred modality to that described in SEQ3 ( Figure 5), and encoded by the crtR gene, messenger RNA or complementary DNA generated from said gene, its complementary strands, allelic variants and sequences represented by SEQl or SEQ2 with addition, insertion, deletion and / or substitution of one or more nucleotides, or nucleic acid sequences that hybridize under conditions of standard strictness with the sequence genomic or complementary DNA (eg conditions detailed by Sambrook et al. 2001).
• the amino acid sequence of the CPR activity polypeptide has 746 amino acids, SEQ3 ( Figure 5), by comparisons using bioinformatic tools, certain conserved domains marked in SEQ3 were identified, and corresponding, in order of appearance to: transmembrane domain, FMN binding domain, FAD binding domain and domain binding to NAD (P) H.
• the present invention is also directed to variants of the SEQ3 polypeptide.
• Said variations refer to the addition, insertion, deletion and / or substitution of one or more amino acids in said sequences, while these derivatives maintain the described activity.
• Such activity can be measured by any essay known in the state of the art or specifically described in this document.
• Such variants can be synthesized by chemical synthesis or by recombination based on the available DNA sequences, using state-of-the-art methods.
• the present invention relates to the use of the CPR polypeptide, whose preferred modality is SEQ3, for obtaining modified organisms in order to increase the production of astaxanthin or generate new metabolic pathways. conducive to its bioproduction in appropriate organisms.
• the DNA sequence coding for the modified SEQ3 sequence is used to suppress the cytochrome P450 reductase function in astaxanthin producing organisms in conjunction with astaxanthin synthase, this use is suggested for research purposes.
• the invention comprises the suppression of the functionality of the crtR gene in yeast X. dendrorhous and the evaluation of transformed strains at phenotypic and genotypic levels.
• the deletion of the crtR- gene is performed by replacing the native gene in wild strains of X. dendrorhous with a module that has a fragment of said gene interfered with by a selection marker, preferably antibiotic resistance genes.
• said replacement of the native gene was performed by homologous recombination using an appropriate vector containing the module described above, particularly, the vectors described in Figure 6 were designed and two strains of native X.
• dendrorhous were transformed with them obtaining two transformed strains (CBSTr and T13) that have a capacity reduced production of astaxanthin compared to the native strains from which they come.
• the phenotypic evaluation of the transformed strains is carried out by visual inspection of the color of the yeast in culture and its comparison with the native strain, in another modality, by identifying the pigments it contains, preferably using the HPLC technique (High Performance Liquid Chromatography).
• this invention contemplates the analysis and comparison of the genomic sequence corresponding to this gene by means of molecular biology techniques present in the state of the art, preferably through its PCR amplification using the fragments described in Table 1, especially the SEQ14, SEQ15, SEQ20, SEQ30, SEQ35, SEQ50, SEQ76 fragments, and those described in Table 3.
• the present invention is also directed to a vector or plasmid comprising the DNA sequences described above and a host cell transformed or transfected with said DNA or a vector or plasmid described above.
• this invention contemplates compositions, products and / or formulations comprising said vectors or plasmids, genomic DNA, complementary DNA and gene fragments described previously. Said compositions, products and / or formulations, as well as the sequences described above, can be used for research, production, analysis or molecular biology techniques existing in the state of the art. Preferably in the processes necessary for the transformation of organisms in order to obtain a modified production of astaxanthin and evaluation of said transformations.
• the present invention contemplates the obtaining of recombinant organisms transformed from the vectors or plasmids and the DNA they contain, and the culture of these organisms transformed under conditions that lead to the production of said polypeptide or to the production of astaxanthin.
• polypeptide contemplated in the present invention and the sequences encoding it are useful for genetically manipulating naturally producing organisms of astaxanthin in order to enhance its biosynthetic abilities or generate the biosynthetic pathway in other organisms, which due to their growth qualities, culture conditions, bioavailability and others can be productively more attractive for obtaining said pigment, as is the case of Saccharomyces cerevisiae.
• Example 1 Obtaining the genomic DNA sequence of the crtR gene from X. dendrorhous.
• oligonucleotides were designed to amplify parts of the crtR gene using the PCR technique (Chain Polymerase Reaction). To this end, nucleotide sequences of CPR enzyme genes from Cunninghamella echinulata, Saccharomyces yeasts were compared.
• the crtR gene was amplified from X. dendrorhous genomic DNA from wild strain UCD 67-385, using a PCR protocol under the following conditions: initial denaturation 95 ° C by 3 minutes, 35 cycles: denaturation at 94 0 C for 30 seconds, alignment at 55 ° C for 30 seconds, extension at 72 0 C for 3 minutes.
• the crtR gene contains BamHI sites in its sequence, therefore it was not possible to obtain a clone containing the complete crtR gene from the library used. However, two clones containing parts of the gene were obtained. One of them with an insert of 12820 bp that was completely sequenced and contains the crtR gene from nucleotide base No. 82 of exon 1 to the end of the gene, the other clone contains an insert of approximately 6500 bp that was completely sequenced and the promoter region of the crtR gene and 88 bp of exon 1.
• the protocol used for these experiments was the following: The genomic DNA of the UCD67-385 strain of X. dendrorhous was digested with different restriction enzymes and a 0.8% agarose gel electrophoresis was performed. The DNA was then denatured and transferred to a nylon membrane and hybridization was performed using a DNA fragment that corresponds to part of the crtR gene as a probe.
• Membrane preparation and hybridization was performed according to standard methods Sambroock et al., 2001. Based on this background, a new partial library was constructed using fragments I left DNA from X. dendrorhous selected in sizes between approximately 6400 and 4400 bp, as a vector, pBluescript SK + and strain DH5 ⁇ of E. coli were used as host, the preparation This new library was made according to the manufacturer's instructions. Using this new library, inserts were amplified by PCR, using the SEQlO and SEQ13 splitter pairs. In this way a clone was obtained that contained the entire crtR gene (SEQl).
• Example 2 Obtaining the complementary DNA sequence of the crtR gene from X. dendrorhous.
• a complementary DNA library of X. dendrorhous was used, from which sequences were amplified by the PCR technique using the SEQlO and SEQ13 splitter pairs that hybridize with gene coding segments, whose size of amplified Expected from complementary DNA of the crtR gene is 338_pb.
• the cDNA library was constructed using Stratagene's "pBluescript II XR cDNA library construction kit" commercial kit, using Escherichia coli strain XLlO-GOLD as a host, according to the manufacturer's instructions.
• the conditions used for the PCR reactions were: initial denaturation 95 0 C for 3 minutes, 35 cycles: denaturation at 94 0 C for 30 seconds, alignment at 55 ° C for 30 seconds, extension at 72 0 C for 3 minutes. Then a final extension at 72 0 C for 10 minutes.
• the amplifications obtained were loaded on a 0.8% agarose gel, identifying mixtures of positive clones for amplification of the complementary DNA of the crtR gene.
• Example 3 Determination of the functional importance of the CPR polypeptide, encoded by the crtR gene, in the carotenogenic pathway of X. dendrorhous.
• mutants were constructed by deletion of the crtR gene in wild strains of X. dendrorhous and the phenotypes and genotypes of the mutants were compared with the original strain.
• a construction vector was inserted into a construct in which a DNA fragment containing the crtR gene was replaced by a module of resistance to the hygromycin B antibiotic. This module is composed of the hygromycin B antibiotic resistance gene. ⁇ hph), under the control of the TEF gene promoter (which encodes the translation elongation factor, EF-IcO and the terminator region of the GPD gene of X.
• the CPRl .3 vector has a DNA fragment of X. dendrorhous, a segment contained between nucleotides 4531 and 9902 of the SEQl, was digested with the restriction endonucleases BsiWI or Ndel to generate a deletion in the crtR gene. The ends of the fragment obtained (containing the crtR deletion), were filled with the Klenow polymerase to enable the binding of the hygromycin resistance module whose ends are blunt. and obtained two clones: CPRl .3 ⁇ BsiWI + hyg and CPRl .3 ⁇ NdeI + hyg.
• Strain UCD67-385 was transformed with a linear DNA fragment containing the deletion of the crtR gene and the hygromycin resistance module. This fragment was obtained from the digestion of the CPR1 .3 ⁇ BsiWI + hyg clone with the Smal and Spel endonucleases. By a double homologous recombination event, an allele of the crtR gene of strain UCD67-385 was replaced by the deletion, obtaining strain T13.
• the strain CBS6938 was transformed with the clone CPRl .3 ⁇ NdeI + hyg (circular DNA) containing the deletion of the crtR gene and the hygromycin resistance module. By a homologous recombination event, the wild crtR gene was replaced with the mutated gene, obtaining the CBSTr strain.
• Cells were harvested and resuspended in 25 ml of BD (50 mM potassium phosphate buffer, pH 7.0, 25 mM ditiotritol [DTT]) buffer and incubated at 22 0 C for 15 minutes.
• the cells are washed twice with 25 ml of STM buffer (270 mM sucrose, 10 mM Tris-HCl, pH 7.5, ImM MgC12) and resuspended in 1 ml of STM buffer.
• STM buffer 270 mM sucrose, 10 mM Tris-HCl, pH 7.5, ImM MgC12
• To transform, 60 ⁇ l of electrocompetent cells are mixed with 10 to 20 ⁇ g of transforming DNA in a volume of 5 ⁇ l.
• Electroporation conditions are: 125 ⁇ iF, 600 ⁇ , 0.45 kV and then the cells resuspended in 1 ml of liquid YM medium and incubated at 22 0 C for 5 hours. Finally, the cells are plated with YM medium and 2% agar supplemented with 10 ug / ml of hygromycin B. Using this selection medium, clones were identified that possess the insert with the hygromycin resistance gene.
• the CBSTr transformant accumulates beta-carotene and is unable to synthesize astaxanthin, unlike the wild strain from which it originates (CBS 6938), which produces astaxanthin and a minimum amount of Beta-carotene.
• the T13 transformant reduced its production of astaxanthin by approximately 75% at the cost of an increase in the production of beta-carotene, to Unlike the wild strain from which it comes (UCD67-385), which produces high amounts of astaxanthin and minimal amounts of Beta-carotene (Figure 9).
• the affected locus was analyzed by amplification of its inserts by PCR, using genomic DNA from the transformants and wild strains as a template.
• PCR reactions were carried out with the following pairs of splitters: gdp rev + SEQ50, SEQ14 + Pef fwd, SEQ14 + SEQ50 and using as temperate the wild strain UCD 67-385, the transformed strain T13 and distilled water as a negative control.
• PCR reactions were carried out with the following pairs of splitters: Pef fwd + SEQ35, gpd rev + SEQ30 and using the wild strain CBS 6938 as temperate, the transformed CBSTr strain and distilled water as a negative control,
• the splitter pairs were used: SEQ75 + hygSecR, HF + SEQ35, HygSecF + M13F, M13R + SEQ15, SEQ20 + SEQ50 and SEQ30 + SEQ50, using the strain as tempered CBSTr transformed.
• the PCR reactions were performed according to the following protocol: initial denaturation 95 0 C for 3 minutes , 35 cycles: denaturation at 94 0 C for 30 seconds, alignment at 55 ° C for 30 seconds, extension at 72 0 C for 3 minutes. Then a final extension at 72 0 C for 10 minutes and they were loaded in 0.8% agarose gels. The results obtained are shown in Figure 10 and the comparison between the sizes of the expected and obtained amplifications are shown in Table 3.
• the hybridization experiments were performed by digesting the genomic DNA of the wild strains (UCD67-385 and CBS6938) and of the transforming strains (T13 and CBSTr) with different restriction endonucleases. Digestions were run on 0.8% agarose gels and then denaturated and transferred to nylon membranes for hybridization. The membranes were hybridized independently with two probes: 1) crtR gene amplified probe with SEQ4 and SEQ8, 2) hygromycin gene probe. Membrane preparation and hybridization was performed according to standard methods Sambroock et al., 2001.
• the crtS gene of Xanthophyllomyces dendrorhous encodes a novel cytochrome-P450 hydroxylase involved in the conversion of beta-carotene into astaxanthin and other xanthophylls. Fungal Genet Biol. 43: 261-72.

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