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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

Definitions

• the present invention relates to the modification of galactomannans present in the green coffee bean by reducing the endogenous level of ⁇ -D-galactosidase activity.
• the present invention pertains to a plant cell with reduced ⁇ -D-galactosidase activity and to a plant harboring such a plant cell.
• cell wall polysaccharides account for approximately 48 % of mature coffee bean dry weight, of which mannans represent approximately half. These polysaccharides are essentially insoluble in purified form and have very low galactose branching (Bradbury and Haliday, J. agric. Food Chem. 38 (1990), 389-392). Mannan polymers are acknowledged to be the main reason for the large losses of original green coffee weight encountered during preparation of soluble coffee drinks. The losses occur either when insoluble material remains as sediments during initial extraction or when precipitates and gels form during storage of coffee liquors. Mannans have also been shown to be the principal component responsible for cloudiness and precipitation on standing of coffee beverages.
• the coffee ⁇ -D-galactosidase cDNA has been cloned (Zhu and Goldstein, Gene 140 (1994), 227-231). According to the information derived therefrom the mature coffee bean ⁇ -D- galactosidase is presumed to be composed of 363 amino acids and is synthesized as a pre- proenzyme of 420 residues. Following biosynthesis, two protease cleavages then remove a secretion signal (38 residues) and another signal peptide (19 residues) to produce the protein exhibiting the N-terminal amino acid sequence characteristic of the active enzyme.
• an object of the present invention is to provide an improved method for preparing soluble coffee while concurrently obviating the drawbacks known when storing coffee liquors.
• the above problem has been solved by providing a coffee plant cell, and a coffee plant, respectively, wherein the galactose branching in the galacto-mannans is increased.
• this objective may be obtained by reducing the endogeneous level of ⁇ -D-galactosidase activity. This may be achieved by conventional methods of mutation and selection using the techniques available in the art. Thus, plant cells may be subjected to mutagenic treatments, such as by exposing them to chemicals or radiation that bring about an alteration of the cell's DNA. The cells thus treated are subsequently screened for the desired property.
• such a reduced endogeneous level of ⁇ -D- galactosidase activity is obtained by introducing a construct into a coffee plant cell, containing a nucleic acid that is transcribed into an antisense copy of the mRNA encoded by the ⁇ -D-galactosidase gene, or to a part thereof.
• the antisense copy of the mRNA encoded by the ⁇ -D-galactosidase gene may be any ribonucleic acid capable to form dimers under physiological conditions, i.e. to hybridize with the mRNA encoded by the ⁇ -D-galactosidase gene under conditions prevailing in the cell.
• the antisense copy does not need to be a 100 % homologue to the corresponding counterpart, but rather needs to provide sufficient binding for forming a dimer. Consequently, antisense copies (and the corresponding nucleic acids from which they are transcribed), that are modified by substitution, deletion and/or insertion of nucleotides are well within the context of the present invention.
• the antisense copy may represent a full counterpart to the mRNA encoded by the ⁇ -D-galactosidase gene, that is, it may provide a RNA molecule having essentially the same length as the mRNA encoding the ⁇ -D-galactosidase polypeptide.
• the antisense copy may only cover a part of the mRNA encoding the ⁇ -D-galactosidase polypeptide.
• the nucleic acid encoding a ribonucleic acid, antisense to the mRNA encoded by the ⁇ -D- galactosidase gene, or to a part thereof, may be under the control of a constitutive or an inducible promoter, so that the level of the antisense RNA may be conveniently controlled.
• the level of the antisense copy should be sufficiently high so as to reduce the number of mRNA copies encoding the ⁇ -D-galactosidase polypeptide accessible for the ribosomes.
• the promoter utilized is the coffee cspl promoter, which gives a sufficiently high transcription rate.
• the present invention therefore provides for a modified coffee plant cell and a coffee plant, respectively, wherein the level of ⁇ -D-galactosidase activity has been reduced such that eventually the galactose branching on galacto-mannans is increased.
• the plant is a transgenic plant, the cells of which harbor a construct capable to provide an antisense copy of the mRNA derived from the ⁇ -D-galactosidase gene or a part thereof.
• the present invention also provides a method for preparing soluble coffee, which comprises the step of using coffee beans derived from a plant exhibiting a reduced ⁇ -D-galactosidase activity.
• the present invention also provides a method for increasing the solubility of coffee by increasing the galactose branching.
• This invention aims to increase in the solubility of coffee galacto-mannans by increasing their galactose branching.
• the strategy adopted is to reduce the endogenous level of ⁇ -D- galactosidase activity, preferably by introducing an antisense copy of its cDNA under the control of the coffee cspl promoter.
• This C5p7-promoter has already been characterized (Marraccini et al, Plant Physiol. Biochem. 37 (1999), 273-282) and controls the expression a gene encoding coffee US storage protein.
• a cassette containing said promoter was constructed and introduced into the T-DNA region of a binary vector of transformation, which derives from the pTiT37 plasmid (Bevan, Nucl. Acids Res. 12 (1984), 8711-8721).
• This recombinant vector was introduced in Agrobacterium tumefaciens, which was used to transform coffee explants. Coffee plants harboring the T-DNA inserted in their genome were regenerated and analyzed for the ⁇ -D- galactosidase activity in their grains.
• Plant material was ground in liquid nitrogen and extracted in ice cold enzyme extraction buffer (glycerol 10 % v/v, sodium metabisulfite 10 mM, EDTA 5 mM, MOPS (NaOH) 40 mM, pH 6.5) at an approximate ratio -of 20 mg per 100 ⁇ l.
• the mixture was stirred on ice for 20 min, subjected to centrifugation (12,000 g x 30 min), aliquoted and stored at -85°C until use.
• ⁇ -D-galactosidase activity was detected spectrophotometrically with the substrate p- nitrophenyl- ⁇ -D-galactopyranoside (pNGP).
• the reaction mixture contained 200 ⁇ l pNGP 100 mM in Mcllvain's buffer (citric acid 100 mM - Na 2 HPO 4 200 mM pH 6.5) up to final volume of 1 ml with enzyme extract.
• Total protein was measured -in samples extracted in aqueous buffers by the method of Bradford (Anal. Biochem., 72 (1976), 248-254). For the expression of activity, each sample was extracted and aliquoted, and assays were performed in triplicate, the results being expressed as averages.
• ⁇ -D-galactosidase activity is extremely low or undetected in the young grain stages, and reaches a peak that coincides with a reddening of color of the pericarp. In later stages the activity declines while the cherries are still red. Activities are also compared in different tissues of coffee plant. The activity in perisperm, roots and leaves is particularly low, and close to the limits of detection. However, high activities are recorded in the endosperm where the activity reaches a peak at approximately 36 WAF but also in germinating grain following imbibition of water.
• a cDNA library from Coffea arabica var. Caturra T2308 was constructed with polyA+ mRNA extracted at 30 weeks after flowering according to Rogers et al. (Plant Physiol. Biochem., 37 (1999), 261-272).
• This plasmid cDNA library (10 ng) was tested by PCR using the primers BETA1 (SEQ ID NO: 2) and BETA3 (SEQ ID NO: 3) directly deduced from the coffee ⁇ -D-galactosidase cDNA sequence (Zhu and Goldstein, 1994).
• the BETA1 primer is located between the nucleotides 177 and 193 in the sequence SEQ ID NO: 1.
• the BET A3 primer is located between the nucleotides 1297 and 1313 in the sequence SEQ ID NO: 1.
• the PCR reaction was performed with Pfu DNA polymerase (Stratagene, 11011 North Torrey Pines Road, La Jolla, California 92037, USA) in appropriate 1 X Buffer, 0.2 mM of each dNTP and 0.25 ⁇ M of each oligonucleotides. Denaturation, annealing and extension temperatures are 94 °C for 30 s, 46 °C for 30s and 72 °C for 3 min, respectively. This cycle was repeated 30 times in Robocycler Statagene (USA).
• PCR products were purified with a Microcon 100 (Millipore SA, BP307 Saint Quentin Yvelines cedex 78054, France) cartridge and ligated in the pCR-Script SK (+) as described by Stratagene (USA). The ligation mixture was then used to transform E.coli strain XL 1 -Blue MRF' and a recombinant vector containing the ⁇ -D-galactosidase fragment was purified and cloned into the Sfrl site of the pCR-Script SK Amp (+). Its sequence is located between the positions 177 and 1313 of the sequence SEQ ID NO: 1.
• the 5' end of the ⁇ -D-galactosidase cDNA contains 198 bp upstream the 5' end of the BETA 1 primer.
• BETA100 SEQ ID NO: 3
• BETA101 SEQ ID NO: 4
• This C. arabica sequence was cloned into the pCR Script Amp SK (+) vector to give the pLPl, and corresponds to SEQ ID NO: 1.
• the cDNA obtained contains an open reading frame of 1263 bp, beginning in position 51 and ending in position 1313 of the sequence SEQ ID NO: 1.
• the translation product corresponds to sequence SEQ ID NO: 2 suggesting that the coffee ⁇ -D-galactosidase is synthesised as a preproenzyme, using the translational start codon ATG in position 51 instead of the ATG in position 126.
• the analysis of the ⁇ -D-galactosidase cDNA cloned from C. canephora showed that its translation product is very homologous (similarity > 99%) to the protein found in C. arabica.
• RNAs of these coffee beans were denatured for 15 min at 65°C in 1 x MOPS buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7) in the presence of formamide (50%) and formaldehyde (0.66 M final). They were then separated by electrophoresis, for 6 hrs at 2.5 N/cm, in the presence of IxMOPS buffer, on a 1.2-% agarose gel containing 2.2 M formaldehyde as final concentration.
• R ⁇ As were stained with ethidium bromide (BET) according to Sambrook et al (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, USA, 1989, chapter 9.31 to 9.51). This makes it possible to standardize the quantities deposited on a gel from the intensities of fluorescence of the 18S and 25S ribosomal R ⁇ As.
• BET ethidium bromide
• the total R ⁇ As were then transferred and fixed on a positively charged Nylon membrane according to the recommendations provided by Boehringer Mannheim (Roche-Boehringer Mannheim GmbH, Biochemica, Postfach 310120, Mannheim 31, DE). The pre-hybridisation and hybridisation were carried out according to the conditions described above.
• Results from Northern-blotting demonstrated a peak of gene expression during the early phase of endosperm development.
• the peak of specific mRNA expression under greenhouse conditions occurred at approximately 26 WAF, and corresponds to the start of increase in enzyme activity.
• the peak period of mRNA expression corresponds to the major period of endosperm expansion and hardening taking place in the maturing grains under these conditions. Peak expression for ⁇ -galactosidase-specific mRNA either coincided or was slightly later than peak expression of the US grain storage protein mRNA.
• the US promoter sequence (Marraccini et al, Plant Physiol. Biochem. 37 (1999), 273-282) from coffee is amplified with the specific primers UP210-1 corresponding to the sequence SEQ ID NO: 7, and BAGUS2, corresponding to the sequence SEQ ID NO: 8.
• the oligonucleotide UP210-1 corresponds to the sequence between the nucleotides 24 and 76 published by Marraccini et al, supra and contains within its 5' end the synthetic sequence CGGGGTACCCCG containing a Kpnl restriction site and corresponding to the sequence SEQ ID NO: 9.
• the BAGUS2 primer contains in its 5' end the synthetic sequence CGCGGATCCGCG corresponding to the sequence SEQ ID NO: 10 which carries a Bam ⁇ l restriction site. This primer also contains the nucleotides 998 to 976 of the sequence published by Marraccini et al. (1999).
• This reaction is carried out in the presence of Pfu DNA polymerase (3 units), with 10 ng of ⁇ CSPP4 (WO 99/02688), in a final volume of 50 ⁇ l containing 10 mM KC1, 6 mM (NH 4 ) 2 SO 4 , 20 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 2 mM MgCl 2 , 10 ⁇ g/ml BSA, 0.2 mM of each dNTP, 0.25 ⁇ M of each oligonucleotides described above.
• the reaction mixture was then incubated for 30 cycles (94°C-60 s, 55°C-60 s, 72°C-3 min) followed by a final extension cycle at 72°C for 7 min.
• the PCR fragment of about 950 bp was purified on a Microcon 100 cartridge (Millipore, France), and ligated in the pCR-Script Amp SK (+) vector in the presence of T4 DNA ligase (Promega Corporation, 2800 Woods Hollow Road, Madison, Wisconsin 53711 USA), according to the recommendations provided by the supplier.
• T4 DNA ligase Promega Corporation, 2800 Woods Hollow Road, Madison, Wisconsin 53711 USA
• the E. coli strain XL 1 -Blue MRF' was transformed with the entire ligation mixture.
• One transformant was selected and its plasmid was purified to sequence the insert in order to determine the orientation of the PCR fragment. This analysis thus made it possible to select the plasmid pLP7.
• a shorter version of the US promoter was also amplified by the same approach except that the primer UP213-1, having the nucleic sequence SEQ ID NO: 11 replaces the primer UP210-1.
• This primer corresponds to the sequence between the nucleotides 754 and 777 published by Marraccini et al, supra, and contains in its 5' end the synthetic sequence SEQ ID NO: 9. This led to the amplification of a 250 bp fragment of the pi IS coffee promoter which was cloned as described previously to give the plasmid pLP8.
• the TNOS terminator is amplified following the protocol described for the amplification of the pl lS promoter excepted that the primers TNOS1, having the nucleic sequence SEQ ID NO: 12 and TNOS2, having the nucleic sequence SEQ ID NO: 13 were used.
• TNOS1 contains the sequence SEQ ID NO: 10 in its 5' end.
• TNOS2 contains the sequence SEQ ID NO: 9 in its 5' end sequence.
• the PCR product was cloned in the pCR- Script Amp SK (+) vector as described before, leading to the recombinant vector called pLP32 and was sequenced to determine its orientation. This vector was then digested by Bam ⁇ I to remove the TNOS sequence that was treated afterwards by the T4 DNA polymerase to provide blunt ends.
• the pLP7 and pLP8 vectors were linearised by EcoR ⁇ and also treated with T4 DNA polymerase to provide blunt ends.
• the TNOS terminator was then cloned in the correct orientation in the pLP7 and pLP8 vectors leading to the vectors pllSTNOS7 and pllSTNOS7+, respectively.
• the ⁇ -galactosidase cDNA was amplified from the previously isolated vector pLPl, using the conditions described above except that the primers BETA100B1, having the nucleic sequence SEQ ID NO: 14 and BETA101B1, having the nucleic sequence SEQ ID NO: 15 were used.
• oligonucleotides correspond to the previously used BETA101 and BETA100 primers in which a Bam ⁇ I restriction site, corresponding to the sequence SEQ ID NO: 10, has been introduced in their 5' ends.
• the PCR product was cloned into the pCR-Script Amp SK (+) vector as described before, leading to the recombinant vector called pLP20.
• This plasmid was digested with BamHl to release the ⁇ -galactosidase cDNA.
• recipient vectors pi 1STNOS7 and pi 1STNOS7+ were digested independently by means of the same restriction enzyme and dephosphorylated by a CIAP treatment according to the furnisher (Promega, USA).
• the ⁇ -galactosidase cDNA was cloned in the antisense orientation, respectively in the pi 1STNOS7 and pi 1STNOS7+ vectors, leading to the vectors designated pALPHAl and pALPHA9.
• a final PCR reaction with the Pfu DNA polymerase was carried out using the primers UPSAL1, having the nucleic sequence SEQ ID NO: 16 and UPSAL2, having the nucleic sequence SEQ ID NO: 17.
• Both oligonucleotides contain a Sail restriction site and recognize DNA sequences of the pCR Script Amp SK (+) vector flanking the US promoter and the NOS terminator (TNOS) DNA regions. In addition this restriction site is absent from the sequence which was intended to be introduced into the T-DNA of the binary plasmid.
• Plasmids obtained were called pALP414 and pALP50, and derive respectively from pALPHAl and pALPHA9.
• the vector was digested with the Sail restriction enzyme, which recognizes a unique site between the uidA and csrl-1 genes, and was dephosphorylated. After this ligation, the vectors pBIA121, pBIA126 and pBIA9 were selected.
• the Sail cassette obtained from ⁇ ALP414 ⁇ is cloned in the orientation [LB] Gus-intron> pi IS (long) antisense ⁇ -galactosidase cDNA > csrl-1 [RB].
• the same cassette is cloned in the reverse orientation in the pBLA126 vector.
• the Sail cassette obtained from pALP50 cloned in the pBIA9 vector is in the following orientation: [LB] Gus-intron> pi IS (short) antisense ⁇ -galactosidase cDNA > csrl-1 [RB].
• the binary vectors of transformation pBIA121, pBIA126 and pBIA9 described above were introduced independently into the disarmed Agrobacterium tumefaciens strain LBA4404 according to the direct transformation method described by An et al (Plant Mol. Biol. Manuel,” Gelvin, Schilperoort and Nerma Eds, Kluwer Academic Publishers Dordrecht, Netherlands, A3 (1993), 1-19).
• the recombinant Agrobacterium tumefaciens clones were selected on LB medium supplemented with kanamycin (50 ⁇ g/ml), stretomycin (lOO ⁇ g/ml) et rifampicin (50 ⁇ g/ml).
• Leaf explants were cultured and subcultured every five weeks for 3 to 5 months until somatic embryos appeared at the edge o the explants. Somatic embryos were harvested at the torpedo stage, wounded with a sterile scalpel and soaked for two hours in a 0.9% NaCl solution 5 containing recombinant Agrobacterium tumefaciens strain LBA4404 at a OD 60 o nm of 0.3 to 0.5. The coculture was performed in the dark on semi-solid MS medium without hormones during three days and then washed in liquid MS medium containing cefotaxim (lgr/1) for 3 to 5 hours under constant but gentle agitation. Embryos were cultivated on semi-solid medium with 5 ⁇ M of BAP, 90 ⁇ M sucrose in presence of cefotaxim (400mg/l) under low-light
• L0 condition (16 hrs photoperiod per day). After a period of 3 to 4 weeks, they were transferred to a selective MS medium supplemented with cefotaxim (400mg/l) and chlorsulfuron (80 mg/1). They were then transferred every month to a new selective medium until the regeneration of calli. Transformed embryos growing around the calli were then cultured on the semi-solid MS medium with Morel vitamins (l ⁇ M BAP and 30 ⁇ M sucrose) to induce
• Somatic embryos were also induced from leaf explants to detect the presence of ⁇ - galactosidase antisense mRNA. 1 IS coffee storage proteins were detected in somatic embryos
• RNAs were then extracted from lOOmg of transformed somatic embryos as described previously and tested by RT-PCR using the kit Access RT-PCR system (Promega, USA). Firstly the presence of US specific mRNA was confirmed by performing a RT-PCR using the primers located in the coding sequence of the US cDNA. This was performed using the primers SOU corresponding to the sequence SEQ ID NO: 18 and SO2-1 corresponding to the sequence SEQ ID NO: 19.
• the SOU primer corresponds to the sequence between the nucleotides 1035 and 1059 of the sequence published by Marraccini et al. , supra.
• the SO2-1 primer corresponds to the last 24 nucleotides of the sequence published.
• the synthesis of the first strand of the cDNA was performed as described by the furnisher (45 min., 48°C). The following parameters were used for the PCR reaction: 45 cycles (60 sec. at 94°C for the denaturation step, 90 sec at 52°C for the annealing step, 4 min at 68 °C for the elongation step) with a final extension at 68°C for 7 min.
• the detection of the ⁇ -galactosidase antisense mRNA was performed using only the primer Bl l during the phase of reverse transcription (condition 2).
• This primer corresponds to the sequence between the nucleotides 50 and 78 of the sequence SEQ ID NO: 1.
• the reaction mixture was treated at 94°c during 1 min to inactivate the MMLN reverse transcriptase.
• the missing oligonucleotide, BI 1 in the condition 1 and B33 in condition 2 was added and the reaction was continued by a PCR: 45 cycles (60 sec.

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