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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)
  • C12N9/2405—Glucanases
  • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
• • 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)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2445—Beta-glucosidase (3.2.1.21)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01021—Beta-glucosidase (3.2.1.21)

Definitions

• the present invention relates to the cloning of nucleic acid sequences coding for a ⁇ -glucosidase of fungal origin weakly inhibited by glucose, and to the use of said sequences for the production of this enzyme by genetic engineering.
• ⁇ -D-glucoside glucohydrolases commonly known as ⁇ -glucosidases, catalyze the hydrolysis of alkyl- and aryl- ⁇ -D-glucosides, as well as that of various oligosaccharides, and cellobiose.
• ⁇ -glucosidases for the recycling of cellulose by enzymatic saccharification.
• cellobiose ⁇ -D-glucose (1 ⁇ 4) glucose
• endo- ⁇ -1, 4-glucanase: EC 3.2.1.4, and exo- cellobiohydrolase: EC 3.2.1.91 inhibits these latter enzymes.
• the hydrolysis of cellobiose by a ⁇ -glucosidase as it is formed therefore makes it possible to avoid its accumulation in the medium, and to increase the rate of saccharification.
• the inventors' team has thus previously produced from filamentous fungi, such as Aspergillus niger and Aspergill us oryzae, a ⁇ -glucosidase very weakly inhibited by glucose.
• This enzyme is the subject of PCT application WO 97/02341, on behalf of the NATIONAL INSTITUTE FOR AGRONOMIC RESEARCH (Inventors: GUNATA et al.), which indicates that it is present in corresponding fractions at a molecular weight estimated at approximately 30,000, obtained by exclusion chromatography from the culture medium of Aspergillus oryzae; the protein obtained by ion exchange chromatography has an isoelectric point of approximately 4.2, and its optimum pH is between 4.5 and 6.0.
• the inhibition constant (Ki) of this enzyme for glucose is of the order of 1M.
• BGII carried out by the Inventors, made it possible to specify its characteristics.
• Its affinity constant (Km) for pNP ⁇ G was evaluated at 0.55 mM, its inhibition constant for glucose at 1.36 M, and its inhibition constant for gluconolactone at 12.5 mM. His activity is stimulated by ethanol. The properties of this enzyme are recalled in more detail in Example 1 below.
• BGII ⁇ -glucosidase is produced in small quantities by Aspergillus. It is stated in the
• the inventors thus isolated the BGII gene, and observed, by analyzing the deduced polypeptide sequence, that this enzyme was related to the yeast exo- ⁇ -1, 3-glucanases.
• This gene will hereinafter be called EXG1.
• the sequence of this gene is represented in the annexed sequence list under the number SEQ ID NO: 1; the polypeptide sequence of its translation product is represented under the number SEQ ID NO: 2.
• the present invention relates to a nucleic acid fragment characterized in that it comprises a sequence coding for a ⁇ -glucohydrolase homologous to the ⁇ -glucosidase BGII from Aspergil l us oryzae.
• ⁇ -glucohydrolase homologous to ⁇ -glucosidase BGII from Aspergill us oryzae means a ⁇ -glucohydrolase whose peptide sequence has at least 60%, preferably at least 70% and advantageously at least 80% identity with the sequence of the mature form of the peptide encoded by the EXG1 gene from Aspergill us oryzae, and represented in the sequence list in the appendix under the number SEQ ID NO: 2.
• said sequence codes for a ⁇ -glucohydrolase which has the spectrum of activity of ⁇ -glucosidase BGII as defined above, (active on ⁇ -1,3-, ⁇ -1,6- ⁇ - 1,4-, ⁇ -1,3, ⁇ -1,4 and ⁇ -1,6- glucosides), and is not very sensitive to inhibition by glucose, i.e. whose inhibition constant ( Ki) for glucose is greater than about 900 mM.
• the present invention also includes nucleic acid fragments coding for a fragment of at least 24 consecutive amino acids of the polypeptide SEQ ID NO: 2, as well as their complement, and nucleic acid fragments which hybridize specifically, under conditions stringent, with the sequence fragment SEQ ID NO: 1, or its complement. They are in particular nucleic acid fragments of at least 18 bp homologous or complementary to any sequence present in the coding regions of said fragment and absent from the coding regions of the known genes of exo- ⁇ -1, 3-glucanases.
• fragments can in particular be used as hybridization probes, and / or amplification primers, to screen libraries of genomic DNA or cDNA, in particular DNA libraries of fungi, and very particularly of ascomycetes. , in order to isolate and clone the sequences coding for ⁇ -glucohydrolases homologous to BGII.
• the present invention also encompasses recombinant vectors comprising at least one insert consisting of a nucleic acid fragment according to the invention.
• the nucleic acid fragments in accordance with the invention can in particular be used to transform eukaryotic or prokaryotic host cells, in particular in order to express therein a ⁇ -glucohydrolase homologous to BGII.
• the present invention also encompasses said cells and said transformed host organisms.
• Host cells and organisms which can be used for this purpose are, for example bacteria such as Escherichia coli, or fungi, such as
• Kluveromyces Pichia, Yarrowia, Hansenula, etc.
• sequence coding for ⁇ -glucohydrolase can be placed under the control of its own transcription regulation sequences, or under the control of heterologous sequences which are active in the host cell.
• a subject of the present invention is also recombinant vectors, characterized in that they result from the insertion of at least one nucleic acid fragment in accordance with the invention, into an appropriate vector.
• These recombinant vectors can be used to transform host cells, in accordance with the invention.
• Control sequences and vectors suitable for the expression of sequences of interest in different types of host cells are known per se.
• X-Glc 5-bromo-4-chloro-3-indolyl ⁇ -D-glucopyranoside
• the gene coding for ⁇ -glucohydrolase is preferably placed under the control of a strong promoter, such as gpdA p , pcbC pr or penDE p from A. nidulans or glaA p from A. Niger.
• a strong promoter such as gpdA p , pcbC pr or penDE p from A. nidulans or glaA p from A. Niger.
• a strong promoter such as those described by RADZIO and KUCK [Process Biochem., 32, pp. 529-539, (1997)]; GOUKA et al. [Appl. Microbiol. Biotechnol., 47, pp. 1-11, (1997)]; GOMI et al. [Agric. Biol. Chem. , 51, pp.
• CHRISTENSEN Aspergill us oryzae as a host for production of industrial enzymes, pp. 251-259; in: KA POWELL, A. RENWICK, and JFPEBERDY (eds.), The genus Aspergillus. Plenum Press, New York, (1994)].
• yeasts such as S. cerevisiae
• expression systems such as those described by BITTER et al. [Methods Enzymol., 153, pp. 516-544, (1989)] or TUITE [Expression of heterologous genes, pp. 169-212. In MF TUITE, and SG OLIVER (ed.), Biotechnology Handbooks, Vol. 4, Saccharomyces. , Plenum Press, New York, (1991)].
• the gene coding for ⁇ -glucohydrolase can be placed under the control of regulatory elements such as the strong constitutive promoters and terminators of the ADH or PGK genes; one can also use a strong promoter induced in the stationary growth phase [RIOU et al. , Yeast, 13, pp. 903-915, (1997)].
• regulatory elements such as the strong constitutive promoters and terminators of the ADH or PGK genes; one can also use a strong promoter induced in the stationary growth phase [RIOU et al. , Yeast, 13, pp. 903-915, (1997)].
• To obtain the secretion of ⁇ -glucohydrolase one can use its own signal sequences, or else heterologous sequences, such as for example the factor ⁇ (MF ⁇ l) of S. cerevisiae [VAN RENSBURG et al, J. Biotechnol., 55, pp. 43-53, (1997)], or else the signal sequence of the EXG1 genes
• the DNA fragments, recombinant vectors and transformed cells obtained in accordance with the present invention can be advantageously used for all applications where it is necessary to have a ⁇ -glucosidase activity which is not very sensitive to inhibition by glucose. , and in particular in all the uses of BGII which are described in PCT Application WO 97/02341 mentioned above.
• transformed cells obtained in accordance with the present invention expressing a ⁇ -glucohydrolase homologous to BGII
• a ⁇ -glucohydrolase homologous to BGII can be used for the industrial production of this enzyme, which can be recovered from the cell culture medium. They can also be used directly, for the synthesis of alkyl glucosides, for the hydrolysis of ⁇ -D-glucosides precursors of flavor in fruit juices, for the de-amerization of citrus juices, or for hydrolysis enzymatic of cellulose or its derivatives.
• ⁇ -glucohydrolases homologous to BGII can be used for the industrial production of this enzyme, which can be recovered from the cell culture medium. They can also be used directly, for the synthesis of alkyl glucosides, for the hydrolysis of ⁇ -D-glucosides precursors of flavor in fruit juices, for the de-amerization of citrus juices, or for hydrolysis enzymatic of cellulose or its derivatives.
• BGII can be advantageously used in the context from the manufacture of fermented drinks such as wine or beer, not only to improve the release of aromas, but also to eliminate glucans present in the fermentative mixture.
• these glucans prevent the natural sedimentation of the particles in the grape must and cause the clogging of the membranes during the stages of filtration of fermented must (VAN RENSBURG et al., 1997); in beer, there are in particular ⁇ - 1, 3-1, 4-glucans, coming from cereals used as raw material, and which cause the same problems.
• the degradation of these glucans into fermentable sugars by the endo- ⁇ -1, 3-glucanases and the exo- ⁇ -1, 3-glucanases of wine and beer yeasts is limited by the spectrum of action of these enzymes.
• This use can be implemented for example by adding a preparation of ⁇ -glucohydrolase homologous to BGII (possibly associated with one or more other enzymes having endo-glucanase activity) to wine or to beer, after alcoholic fermentation, and before filtration; alcoholic fermentation can also be carried out in the presence of at least one transformed microorganism expressing a ⁇ -glucohydrolase homologous to BGII, and optionally one or more other enzymes having endo-glucanase activity: it may for example be a strain of S. cerevisiae expressing the EXG1 gene of A. oryzae, in synergy with the BGL2 gene of S. cerevisiae [MRSA et al. , J.
• ⁇ -glucosidase BGII is prepared from strain 12559 of A. oryzae from the collection of the Centraalbureau Voor Schimmel Cultures (CBS, Netherlands). This strain is stored on PDA solid medium ("Potato Dextrose Agar", DIFCO). For the production of the enzyme, it is cultivated on a base medium containing the following compounds (w / v): 0.2% NaN0 3 , 0.2% KC1, 0.1% KH 2 P0 flick, 0, 1% NH 4 N0 3 , 0.1% (NH 4 ) H 2 P0 4 , 0.05% MgS0 4 , 7H 2 0, 0.05% yeast extract (DIFCO). This medium is sterilized for 20 minutes at 120 ° C. The carbon source used (quercetin) is sterilized separately (20 minutes, 120 ° C), then mixed with the minimum medium to a final concentration of 0.5% (w / v).
• the cultures after sowing with 10 5 spores of A. oryzae / ml, suspended in a solution of TWEEN 80 at 0.15% (w / v), are carried out at 28 ° C, with stirring (125 revolutions / minute). Samples are taken at various times throughout the cultures to monitor the kinetics of production of the enzyme. The samples are centrifuged (10,000 g, 5 minutes). The culture supernatant is used for the assay of ⁇ -glucosidase activity.
• the assay of ⁇ -glucosidase activity is carried out by incubation for 30 minutes at 50 ° C from 1 to 100 ⁇ l of enzymatic sample in a final reaction volume of 1 ml containing 5 mM of p-nitrophenol 10
• ⁇ -glucopyranoside in 0.1 M acetate buffer, pH5.
• the ⁇ -glucosidase activity is determined by estimating the amount of p-nitrophenol (pNP) released after addition of 2 ml of Na 2 C0 3 and reading of the absorbance at 400 nm. It is expressed in units per ml of enzyme solution (U / ml). One unit corresponds to the number of micromoles of pNP released per minute under the above conditions ( ⁇ mol / min).
• the concentrated crude extract is placed on a filtration column (1.6 x 90 cm) packed with ULTROGEL AcA44 (IBF), having an exclusion threshold of 10 to 130 kDa, previously equilibrated with 10 mM acetate buffer, pH 6, containing 50 mM NaCl and 8 mM EDTA, at 4 ° C. Elution is carried out with the same buffer at a constant flow rate of 20 ml / h.
• the recovered eluate is fractionated using an automatic collector (PHARMACIA) set to 2.5 ml / fraction.
• the chromatographic profile reveals 2 distinct activity peaks, corresponding to the BGI and BGII ⁇ -glucosidases described in PCT application WO 97/02341.
• the fractions exhibiting the BGII activity peak are combined, concentrated and desalted by ultrafiltration on a CENTRIPLUS PM 10 membrane (AMICON). 11
• the fractions of the BGII peak, pooled at the end of the filtration chromatography, concentrated and desalted, are injected onto a TSK DEAE-5PW (BECKMAN) column (7.5 x 75 mm) balanced with a 10 mM citrate-phosphate buffer. , pH 6, containing 1 mM EDTA. Chromatography is performed in HPLC (High Pressure Liquid Chromatography). After injection of the sample, the column is washed for 10 minutes with the same buffer, then another 10 minutes with the same buffer containing 0.13 M NaCl. The BGII is then eluted with a linear gradient of 0.13 to 0.25 M NaCl, for 15 minutes.
• the optimum temperature and the optimum pH were determined as described in PCT Application WO 97/02341.
• the BGII activity is maximum for a temperature of 50 ° C., and a pH of 5.
• the activity curves are represented in FIGS. 1A (activity at pH 5, as a function of temperature) and 1B (activity at 50 ° C as a function of pH).
• FIGS. 1A and 1B also respectively represent the stability curves (O) of the purified homogeneous BGII after 4 hours of incubation at pH 5 at temperatures varying from 20 to 80 ° C., and after 24 hours of incubation at 20 ° C at pH varying from 2.5 to 8. These curves show that under these conditions, the purified BGII is stable up to 45 ° C, and still shows 50% activity at 50 ° C, and that it is stable between pH 4 and 13
• the inhibitory effect of glucose or ⁇ -gluconolactone on BGII purified to homogeneity was determined, under optimal conditions of BGII activity (50 ° C, pH 5, 30 minutes), using the same experimental protocol. than that described in PCT Application WO 97/02341.
• WO 97/02341 have been confirmed or supplemented with the preparation of homogeneously purified BGII.
• the hydrolysis of the substrates was estimated by measuring the release of pNP at 400 nm or by measuring the reducing sugars at 540 nm [MILLER, Anal. Chem. 21,
• Aryl-glycosides (5 mM) p-Nitrophenyl- ⁇ -D-glucoside ⁇ GIc 100 p -Nitrophenyl- ⁇ -D-xyloside ⁇ Xyl 50 p -Nitrophenyl- ⁇ -D-galactoside ⁇ Gal 8.2 p -Nitrophenyl- ⁇ -D-glucoside ⁇ GIc 1.6 p -Nitrophenyl- ⁇ -D-cellobioside ⁇ GIc 3.3 p -Nitrophenyl- ⁇ -D-gentiobioside ⁇ GIc 3.2 p -Nitrophenyl- ⁇ -D-maltopyranoside ⁇ GIc 1.8 p -Nitrophenyl- -L-maltopyranoside ⁇ GIen 1.3 p -arabi ⁇ ofuranopyranoside ⁇ Ara 1.5 p -Nitrophenyl- ⁇ -D-glucuronide
• BGII has a wide spectrum of activity since it is capable of efficiently hydrolyzing ⁇ -D-glucosides having bonds of different types, such as laminaribiose ( ⁇ -1,3), gentiobiose ( ⁇ -1,6), and cellooligosaccharides ( ⁇ -1,4).
• BGII hydrolyzes maltose ( ⁇ -1,4) at 68% of the optimal hydrolysis rate of laminaribiose, it nevertheless seems more specific for ⁇ bonds; in particular, it does not hydrolyze pNP ⁇ G. On the other hand, it hydrolyzes pNP- ⁇ -xyloside (pNP ⁇ X), at 50% of the rate 15
• inhibitors such as SDS, NBS,
• the enzymatic activity is not (or only slightly) affected by other substances such as EDTA, pCMB, dicyclohexyl carbodiimide, reagent K from Woodward, DTT, DMSO, and N-acetylimidazole .
• Co 2+ is not necessary for enzymatic activity. However, this is significantly stimulated by
• Mn 2+ the hydrolysis of the substrate pNP ⁇ G is increased by 77%, in the presence of 5 mM of Mn 2+ .
• Ethanol stimulates the activity of BGII; this stimulation is maximum for ethanol concentrations of the same order as those encountered in wine: the hydrolysis of pNP ⁇ G is thus increased by 30% in the presence of 15% (v / v) of ethanol, and by 15% in the presence of
• the sequencing of the BGII protein was started from the preparation of purified protein with homogeneity described in Example 1 above.
• the N-terminal end of the protein being partially blocked, a first sequencing resulted only in a sequence of 7 N-terminal amino acids.
• the BGII protein was digested with endo H (endo-peptidase from S. aureus), which made it possible to sequence 4 additional small peptides.
• oligonucleotide primers were derived from the sequences of 7 N-terminal amino acids of the BGII protein, and 6 C-terminal amino acids of peptide # 35.
• the sequence of the degenerate oligonucleotide derived from the N-terminal peptide sequence of BGII is as follows: 5 'TT (CT) GA (CT) TA (CT) AA (CT) GGC GA (AG) AA 3' its Tm is from 54.8 ° C.
• the sequence of the degenerate oligonucleotide derived from the C-terminal peptide sequence of # 35 is as follows: 5 'AT (CT) CC (CT) GC (CT) AC (CT) GT (CT) TC 3' its Tm is of 54 ° C.
• primers made it possible to amplify by PCR (30 sec 94 ° C, 30 sec 50 ° C, 1 min 72 ° C, 35 cycles), from the total genomic DNA of A. oryzae, a 601 bp DNA fragment.
• the deduced polypeptide sequence obtained in this way contained, in its N-terminal part, 5 amino acids out of 7 of the sequence N-terminal of BGII determined by direct sequencing of the protein. In contrast, the C-terminal part of the deduced polypeptide sequence did not correspond to that of peptide # 35.
• the inventors searched in the nucleic acid sequence for the presence of splicing sequences and introns, and thus located an entire intron of 64 bp inside the amplified fragment. After elimination of the intron sequence, the deduced C-terminal polypeptide sequence contained 19 of the 21 amino acids of peptide sequence # 35.
• the protein has a molecular mass of 43 kDa, and that the genes already isolated from Aspergillus oryzae present on average 4 to 6 introns of 50 to 80 bp [UNKLES, In: Applied Molecular Genetics of Filamentous Fungi. J.R. Kinghorn and G. Turner (Eds), Blackie Académie and Professional, Glasgow, pp. 28-53, (1992)], the total size of the gene corresponding to BGII has been estimated at approximately 1500 bp. To clone the complete gene, the total genomic DNA of A. oryzae was digested with the restriction enzyme BamHI; the EXG1 gene was recovered by reverse PCR, from the fragments obtained, using primers chosen from the partial sequence obtained as indicated above.
• BamHI restriction enzyme
• the putative signal peptide, of 14 amino acids is framed on the polypeptide sequence of Figure 2. Analysis of the polypeptide sequence further shows the absence of glycosylation sites.
• the inventors compared the polypeptide sequence with those present in the databases, using the BLASTP software [ALTSCHUL et al. Nucleic Acids Res. 25: 3389-3402, (1997)].
• FIG. 3 represents the alignment of peptide sequences obtained using the software
• PBU26160 precursor of the glycoprotein gp43 of
• EXG_CANAL exo-1, 3- ⁇ -glucanase from Candida albicans
• EXG_YARLI exo-1, 3- ⁇ -glucanase from Yarrowia lipolytica
• KLEXG1 exo-1,3- ⁇ -glucanase from Kluyveromyces lactis
• HPEXG1 exo-1, 3- ⁇ -glucanase from Hansenula polymorpha
• ABEXG1G2 exo-1, 3- ⁇ -glucanase from Agaricus bisporus;
• SPR1_YEAST exo-1, 3- ⁇ -glucanase from Saccharomyces cerevisiae, specific for sporulation
• SOEXG1 exo-1, 3- ⁇ -glucanase from Schwanniomyces occidentalis;
• EXG1_YEAST exo-1 3- ⁇ -glucanase 1 from Sa ccharomyces cerevisiae
• EXG2_YEAST exo-1 3- ⁇ -glucanase 2 from Sa ccharomyces cerevisiae.
• RNAs of A. oryzae grown on quercetin were isolated and the messenger RNA (mRNA) purified.
• the cDNAs are prepared by reverse transcription, and the specific cDNA corresponding to the gene sought is amplified using oligonucleotide primers derived from the 601 bp fragment of the BGII gene. The thus amplified cDNA is cloned into the vector pAMP10 (GIBCO-BRL) and sequenced. 21
• the cDNA sequence compared to the genomic ADB sequence made it possible to demonstrate the presence of a single 64 bp intron (FIG. 2 a) and to locate the polyadenylation site (TCA in bold, FIG. 2 b). 200 bases downstream from the STOP codon ( Figure 2 b).
• the coding sequence thus obtained can be expressed in microorganisms, such as E. coli and S. cerevisiae.

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