NUCLEIC ACID CODING FOR AN ALPHA-ACETOLACTATE SYNTHASE FROM LACTOCOCCUS AND ITS APPLICATIONS

Abstract: The invention relates to the cloning and sequencing of the nucleic acid coding for (alpha)-acetolactate synthase of (L. lactis) subsp. (lactis), as well as to the polypeptide sequence encoded by this nucleic acid. The invention also relates to the use of the above-mentioned nucleic acid for the transformation of host cells and the production of diacetyl.

Description: NUCLEIC ACID CODING FOR AN ALPHA-ACETOLACTATE SYNTHASE FROM LACTOCOCCUS AND ITS APPLICATIONS The invention relates to a nucleic acid coding for an α-acetolactate synthase, as well as to vectors contain- ing this nucleic acid and to the use of these vectors for transforming microorganisms in which the production of α-acetolactate and diacetyl will be promoted. Diacetyl is a flavouring sought in the production of food products such as butter, creme fraϊche and some cheeses. Conversely, this flavouring is undesirable in the production of other food products such as beer. A limited number of bacteria, such as Lactococcus lactis subsp. lactis biovar diacetylactis as well as bacteria of the genera Leuconostoc Pediococcus, Lactobacillus, naturally produce diacetyl; this production takes place from a substrate present in milk in small amounts, citrate; these bacterial strains are capable of metabolizing citrate to pyruvate, which is then converted to acetoin and to diacetyl. α-Acetolactate is also an intermediate of the pathway of synthesis of the branched amino acids, which are leucine, isoleucine and valine. This pathway has been very widely studied in bacteria, fungi and plants (1, 2, 3, 4). However, the sequences of the assembly of genes involved in this pathway in one and the same microorganism have never been reported. The regulation of the expression of the genes involved in the pathway of synthesis of the branched amino acids is complex on account of the steps common to the synthesis of these three amino acids, and this pathway is often presented as a model for the study of organization and regulation. The organization of these genes has been charac¬ terized in Escherichia coli (3) , Salmonella typhinium (3) , Bacillus subtilis (5, 6, 7), Corynebacterium gluamicum (8) and Staphylococcus aureus (9) . On the E. coli chromosome, the genes are localized in three groups (10) . The largest, located at 85 minutes, is organized in one large transcription unit and two small SUBSTITUTE SHEET transcription units, comprising the ilvGMEDA, ilvY and ilvc genes, respectively (11, 12); another group, located at 2 minutes, is composed of two transcription units comprising the ilvH and leuACBD genes (13, 14), and the last group, at 82 min, groups together the ilvBN genes in a single transcription unit. A similar organization is found in other Entero- bacteriaceae. In B. subtilis, the ilvBN and leuACBD genes are encountered in one region of the chromosome (5) and the ilvAD genes in another region. Three cloned nonadjacent chromosomal fragments of C. σlutamicum carry five genes of the pathway of synthesis of the branched amino acids, ilvCBN, ilvA and ilvE (9). In S. aureus, genetic mapping shows that eight genes are grouped together in the following order: ilvAB- CO, leuABCD. The synthesis of the branched amino acids leucine, isoleucine and valine in L. lactis subsp. lactis is a complex pathway involving eight enzymes, four of which are common to the synthesis of the above three amino acids. The synthesis of valine involves only these four, whereas the synthesis of isoleucine and of leucine involves, respectively, one and four additional specific enzymes. In addition, this system is complicated by the fact that the excess of one of the three amino acids interferes in the synthesis of the other amino acids. The present invention relates to the cloning, characterization and sequencing of the genes involved in the pathway of synthesis of the branched amino acids in L. lactis subsp. lactis. This work was carried out using the strain NCD02118 (AFRC, Institute of Food Research Reading Laboratory, Shinfield, Reading, Berks, United Kingdom) . The genes were characterized by cloning, comple- mentation in E. coli and B. subtilis and sequence analy¬ sis. Nine structural genes are assembled on a 12-kb DNA fragment in the following order: leuABCDilvDBNCA. Among these genes, the invention relates to a nucleic acid sequence coding for two polypeptides forming SUBSTITUTE SHEET the subunits of a protein possessing α-acetolactate synthase activity; this enzyme is of very special interest since it is responsible, in L. lactis subsp. lactis, for the direct conversion of pyruvate to α-acetolactate independently of the presence of citrate in the medium. The techniques used for cloning and sequencing the nucleic acid of the invention will be described more particularly in the detailed description of the invention. Reference will be made in what follows to the tables and figures, wherein: - Table I shows the bacterial strains, plasmids and phages used for cloning the L. lactis subsp. lactis genes involved in the pathway of synthesis of the branched amino acids; - Table II shows a comparison of proteins involved in the pathway of synthesis of the branched amino acids between L. lactis subsp. lactis and different microorgan¬ isms; - Figure 1 shows the structure of the region of L. lactis subsp. lactis DNA carrying the genes involved in the pathway of synthesis of the branched amino acids; Figure 2 shows the nucleotide sequence, together with the polypeptide sequence deduced from this nucleotide sequence, of the region of L. lactis subsp. lactis strain NCD02118 DNA carrying the genes involved in the pathway of synthesis of the branched amino acids; - Figure 3 shows the alignment of 6 ATP-binding proteins with ORF2 of L. lactis; - Figure 4 shows the secondary structures of the major transcript serving as a transcription attenuator. Research work carried out on the DNA of Figure 2, and also on the polypeptide deduced from this DNA, leads to the following observations: Ten genes are present in the region of L. lactis subsp. lactis DNA shown in Figure 2: the leuA gene of 1539 nucleotides, located between nucleotides 450 and 1988 and coding for a polypeptide of 513 amino acids; the leuB gene of 1041 nucleotides, located SUBSTITUTE SHEET between nucleotides 2003 and 3037 and coding for a polypeptide of 347 amino acids; the leuC gene of 1380 nucleotides, located between nucleotides 3403 and 4782 and coding for a polypeptide of 460 amino acids; • the leuD gene of 573 nucleotides, located between nucleotides 4805 and 5377 and coding for a polypeptide of 191 amino acids; ■ the orf2 gene of 606 nucleotides, located between nucleotides 5394 and 6170 and coding for a polypeptide of 202 amino acids; the ilvD gene of 1710 nucleotides, located between nucleotides 6295 and 8004 and coding for a polypeptide of 570 amino acids; • the ilvB gene of 1725 nucleotides, located between nucleotides 8018 and 9742 and coding for a polypeptide of 575 amino acids; • the ilvN gene of 474 nucleotides, located between nucleotides 9738 and 10211 and coding for a polypeptide of 158 amino acids; the ilvC gene of 1032 nucleotides, located between nucleotides 10260 and 11291 and coding for a polypeptide of 344 amino acids; the ilvA gene of 1323 nucleotides, located between nucleotides 11337 and 12659 and coding for a polypeptide of 441 amino acids. The invention relates to any nucleic acid com¬ prising all or part of the DNA sequence bounded by the nucleotides located at positions 8018 and 10211 of Fig- ure 2, and coding for one or both subunits of a protein capable of converting pyruvate to α-acetolactate; and for any other novel polynucleotide, peptide or protein dis¬ closed herein. α-Acetolactate synthase of L. lactis subsp. lactis is a protein consisting of two subunits; the first subunit is a polypeptide of 575 amino acids encoded by the frag¬ ment bounded by the nucleotides located at positions 8018 and 9742 of Figure 2, and corresponding to the ilvB gene; the second subunit is a polypeptide of 474 amino acids SUBSTITUTE SHEET encoded by the fragment bounded by the nucleotides located at positions 9738 and 10211 of Figure 2, and corresponding to the ilvN gene. Consequently, the α-acetolactate synthase activity of L. lactis subsp. lactis is possible only if both of these genes are expressed. The subject of the invention is, more especially, the fragments of the above nucleic acid coding for one or both subunits of a protein capable of possessing the enzymatic properties of α-acetolactate synthase of L. lactis subsp. lactis. More specifically, the invention relates to nucleic acids comprising one or other or both of the following segments: - the first segment corresponding to the ilvB gene, bounded by the nucleotides located at positions 8018 and 9742 of Figure 2, and coding for the polypeptide of 1725 amino acids corresponding to one of the two subunits of α-acetolactate synthase of L. lactis subsp. lactis; - the second segment corresponding to the ilvN gene, bounded by the nucleotides located at positions 9738 and 10211 of Figure 2, and coding for the polypeptide of 474 amino acids corresponding to the other subunit of α-acetolactate synthase of L. lactis subsp. lactis. The invention also relates to the above nucleic acids in which the sequences are modified, provided the polypeptides encoded by these nucleic acids retain their enzymatic properties of the α-acetolactate synthase type. Such modifications, without implied limitation, lead, for example, to variant nucleic acids which differ from the nucleic acid of the invention by the addition and/or deletion of one or more nucleotides and/or modifi¬ cation of one or more nucleotides. Thus, the invention further relates to DNA sequences coding for α-acetolactate synthase of the pathway of synthesis of the branched amino acids of bacteria of the genus Lactococcus, such as L. plantarum, L. rafinolactis and L. lactis. for instance L. lactis subsp. cremoris; which DNA sequences display a strong homology with the nucleic acid sequence of ilvBN. Advantageously, these DNA sequences are identified, SUBSTITUTE SHEET and where appropriate selected, by genetic hybridization with one or more nucleic acid probes originating from the above nucleic acid sequences, under moderately stringent conditions. As an example, a method for the identification of such a DNA sequence comprises the following steps: - extraction of the DNA from the bacterium; - digestion of this DNA with one or more restric¬ tion enzymes such as EcoRI; - transfer of the DNA fragments obtained onto a nitrocellulose membrane; - hybridization with the complete ilvBN sequence in a hybridization solution whose composition is: 6 x SSC, approximately 10% formamide, 5 x Denhardt, 100 ml of phosphate buffer pH 7. The invention also relates to polypeptide sequences comprising all or part of the polypeptide sequences encoded, on the one hand by the DNA sequence bounded by the nucleotides located at positions 8018 and 9742 of Figure 2, and on the other hand by the DNA sequence bounded by the nucleotides located at positions 9738 and 10211 of Figure 2; these two polypeptides constituting the subunits of a protein possessing the enzymatic properties of α-acetolactate synthase of L. lactis subsp. lactis. The subject of the invention is also any polypeptide sequence originating from each of the above polypeptide sequences, provided the protein formed from these polypeptides possesses enzymatic properties of the type possessed by α-acetolactate synthase. The above polypeptides may be modified provided they retain the enzymatic properties defined above. For example, without implied limitation, polypeptides lying within the scope of the invention can differ from those defined above by the addition and/or deletion of one or more amino acids and/or modification of one or more amino acids. A person skilled in the art has at his disposal means enabling him to identify those polypeptides of shorter sequences which lie within the field of the SUBSTITUTE SHEET invention. A general means enabling him to undertake this identification consists in treating each of the above polypeptides with a protease that cleaves the polypeptides at a specific site, and then separating the fragments obtained and testing them for their enzymatic activity with respect to pyruvate. Another means enabling regions of the polypeptides which are essential to the α-acetolactate synthase activ¬ ity to be identified consists in cleaving the nucleic acids corresponding to the ilvB and ilvN genes, for example using one or more restriction enzymes, before introducing them into the cell host or the expression vector used for the production of a protein displaying α-acetolactate synthase activity. The truncated nucleic acid may thus be tested for its capacity to express effectively a product possessing α-acetolactate synthase activity or, on the contrary, no longer possessing it. The subject of the invention is also any recom¬ binant nucleic acid comprising at least one nucleotide sequence of the abovementioned type coding for α-aceto¬ lactate synthase, combined with at least one promoter and/or one transcription terminator recognized by the polymerases of the host into which the said recombinant nucleic acid is introduced. The introduction of the recombinant nucleic acid may be carried out using vectors of the plasmid type capable of replicating in the host microorganism and of permitting the expression therein of the sequence coding for the enzyme, or alternatively the recombinant nucleic acid may be introduced directly into the genome by genetic engineering techniques known to a person skilled in the art. The host microorganisms into which the recombinant nucleic acid is capable of being introduced are preferably prokaryotic cells such as Lactococcus lactis. Leuconostoc lactis, Lactobacillus lactis. Lactobacillus plantarum. but may also be eukaryotes such as yeasts. More especially, the invention relates to all microorganisms capable of being used as leaven in an agri- SUBSTITUTE SHEET foodstuffs process, in which microorganisms the recombi¬ nant nucleic acid of the invention is introduced for the purpose of obtaining an overproduction of diacetyl in an agri-foodstuffs product such as dairy product derivatives, fermented wheys, soft white cheeses, and the like. Advantageously, the introduction of the recombinant nucleic acid of the invention into L. lactis bacteria is combined with an inhibition of the enzyme responsible in these bacteria for the conversion of acetolactate to acetoin (α-acetolactate decarboxylase) and/or of the enzyme involved in the conversion of pyruvate to lactate (lactate dehydrogenase) , in order to increase the acetola¬ ctate pool leading to diacetyl. The inhibition of these enzymes may be achieved by all chemical or biological means that enable the metabolic flux towards diacetyl production to be increased; a first biological means consists in deleting all or part of the wild-type genes coding for these enzymes; a second bio¬ logical means consists in replacing the wild-type genes coding for these enzymes by mutated genes incapable of expressing the enzymes in question. The recombinant nucleic acid of the invention may also be introduced into other microorganisms, such as Bacillus, for the production of diacetyl for non-agri- foodstuffs purposes, for example in cosmetology and as an antibacterial. Other features of the invention will become appar¬ ent in the description which follows, which relates on the one hand to the cloning, characterization and sequencing of the region of L. lactis subsp. lactis DNA carrying the genes involved in the pathway of synthesis of the branched amino acids, and on the other hand to the introduction of a nucleic acid according to the invention, coding for a polypeptide displaying α-acetolactate synthase activity, into a cell host. I - CLONING OF THE GENES INVOLVED IN THE SYNTHESIS OF AMINO ACIDS IN L. LACTIS SUBSP. LACTIS 1) Materials and methods a) Bacterial strains, plasmids and culture media SUBSTITUTE SHEET The bacterial strains, plasmids and phages used for gene cloning are listed in Table I below. SUBSTITUTE SHEET Table I SUBSTITUTE SHEET Table I ( continued) L. lactis subsp. lactis strain IL1403 was cultured at 37°C on M17 medium (25) in which lactose was replaced by glucose. Escherichia coli was cultured on Luria-Bertani (LB) medium or in M9 minimum medium (26) at 37°C. Bacillus subtilis was cultured on a medium at 37°C, on LB medium or in Spizizen-salt minimum medium supplemented with the appropriate amino acids. If necessary, erythromycin (5 μg/ml for L. lactis subsp. lactis or 0.3 μg/ml for B. subtilis) , ampicillin (50 μg/ml for E. coli) or tetrac- ycline (10 μg/ml for E. coli) is added to the medium, b) Molecular cloning and DNA manipulation The plasmids and the chromosomal DNA were prepared according to known techniques of the prior art (26, 27, 28, 29). Transformation of E. coli cells was carried out either by a standard CaCl 2 procedure (36) or by electropo- SUBSTITUTE SHEET ration (30) . Induction of competence and transformation of B. subtilis cells were carried out as described by Anagno- topoulos and Spizizen (31), with a few modifications (32). Southern blotting and DNA hybridizations were carried out as described by Maniatis et al. (26) . Nucleic acid probes were prepared using [α- 32 P]-dCTP and a nick translation kit in accordance with the supplier's recommendation. The other molecular techniques were carried out as described by Maniatis et al. (26) . c) Analysis of the DNA sequence The E. coli clones used for sequencing were obtained by subcloning DNA fragments into plasmids of the pBluescript family and using exonuclease III and mung bean nuclease from the company Stratagene in order to generate a series of clones containing overlapping DNA fragments. The DNA was then sequenced using the "Taq DyeDeoxy Terminator Cycle Sequencing" kit and the "Sequencer 370A" marketed by the company Applied Biosystems. Nucleotide and polypeptide sequences were analysed with the University of Wisconsin's "BISANCE" and "GCG" softwares. Protein sequences were aligned with a "MULTALIN" software (33) . The nucleic acid sequences were determined on both strands. Restrictively, B. subtilis strains mutated in the ilvA gene were prepared by transforming competent MT119 cells with pHV438 (34) to Cm R . Integration of this plasmid in the chromosome by double crossing-over replaces the ilvA gene by the cm R gene (24) . A representative ilvA leuB6 r " /m " Cm R clone is desig¬ nated IL2685. An ilvD4 leuB6 double mutant of B. subtilis was constructed by transformation, using GSY276 DNA to trans¬ form competent 1012 cells to cells which were prototrophic for methionine. The transformed cells were then tested for their requirements for isoleucine and leucine and for the absence of restriction by titration of the rho phage. An ilvD4 leuB6 r " /m + clone was designated IL3151. 2) Gene cloning Total DNA of L. lactis subsp. lactis NCD02118 was partially digested with the endonuclease Sau3Al. 20 μg of SUBSTITUTE SHEET the DNA segments of size larger than 10 kb were ligated with 10 μg of DNA of plasmid vector pIL253 cleaved with BamHI, at a final concentration of 500 μg/ml. The ligated mixture was used to transform competent cells of the mutant B. subtilis strain leuB6 ilV4, IL3151 to isoleucine-independent cells. Four transformants were also Em R , and contained 18-, 14.2-, 13.5- or 9.5-kb plasmids designated pIL384, pIL371, pIL374 and pIL373, respectively. According to a similar experiment, L. lactis subsp. lactis DNA cleaved with Xbal was cloned into pIL253 using the mutant B. subtilis ilvA strain IL2685 as recipient. The transformants were selected on a minimum medium enriched with leucine and tryptophan but lacking isoleucine. Two Em R Ile + clones, containing an apparently identical 23.5-kb plasmid, designated pIL500, were obtained. 3) Complementation experiments The cloned DNA segments were used to complement, in B. subtilis, leu and ilv mutants, and in E. coli after subcloning into pBluescript. The results relating to these studies are reported in Figure l. Figure 1 shows the structure of the DNA region carrying the genes for the enzymes involved in the pathway of synthesis of the branched amino acids in L. lactis. In this figure, the open bars represent the segments used for the complementation experiments in B. subtilis and E. coli. These segments are carried by the plasmids indicated in the figure, which were constructed as described in Table I above. The results of the complementation experiments with the mutants listed in Table I are represented by (+) and (-) , referring to growth or the absence of growth on the culture medium, lacking the corresponding amino acids. The organization of the DNA region carrying the genes involved in the pathway of synthesis of the branched amino acids (orfl, leuA, leuB, leuC, leuD, orf2, ilvD, ilvB, ilvN, ilvc, ilvA) is also shown at the bottom of Figure I; pi and p2 indicate the transcription promoters and tl the transcription SUBSTITUTESHEET terminator. The open reading frame between pi and tl represents the leader peptide. In these complementation experiments, the E. coli nomenclature has been used; the leuB and leuC genes of E. coli correspond to the leuC and leuB genes, respect¬ ively, of B. subtilis, and the three isoenzymes of acetolactate synthase and of acetohydroxide synthase encoded by ilvBN, ilvIH and ilvGN in E. coli correspond to a single enzyme encoded by ilvBN in B. subtilis. The leuA, leuB, leuC, ilvD and ilvA genes complement the correspon¬ ding mutations in B. subtilis; the leuA, leuB, leuC, leuD and ilvc genes complement the corresponding mutations in E. coli. These results are sufficient to identify nine genes involved in the pathway of biosynthesis of the branched amino acids in L. lactis subsp. lactis. 4) Nucleotide sequences of the genes The nucleotide sequence of a region of 12,720 bp was determined. This sequence is shown in Figure 2. A computer analysis according to the method of Griskov et al. (35) discloses ten open reading frames (ORF) of size larger than 200 bp. Each ORF is preceded by a ribosome binding site, complementary to the 3' end of rRNA 163 of L. lactis subsp. lactis (36) . All the ORFs begin with the codon ATG, except for the second which begins with the codon TTG; furthermore, four 72-bp repeat units are present between the second and the third ORF. Figure 2 shows the nucleotide sequence, and the polypeptide sequence deduced from this nucleotide sequence, of the DNA region coding for the genes involved in the pathway of synthesis of the branched amino acids of L. lactis strain NCD02118; the numbers at the right indicate the position of the oligonucleotides; the names of the genes are indicated at the beginning of each amino acid sequence; the transcription stop codons and also the ribosome binding sites are indicated respectively by (*) and RBS; the consensus sequences "-10" and "-35" of the promoters are underlined; inverted repeat units, which can constitute "preamptors" and "terminators", are numbered SUBSTITUTE SHEET (1, 2) and (5, 6), respectively, and those capable of forming "antiterminators" are numbered (3 and 5), (see Figure 4 below); the arrow ( >) indicates the 72-bp direct repeat unit, and the bold characters indicate the 6-bp inverted repeat units which it contains. 5) Assignment of the ORFs The proteins deduced from the ten ORFs were com¬ pared with the GENPRO and NBRF protein databases. Signifi¬ cant homologies were identified for ten ORFs; nine of them correspond to the genes involved in the pathway of syn¬ thesis of the branched amino acids, as seen in Table II below. SUBSTITUTE SHEET Table II CO c CD 0) X m I en 03 Ό •H o SUBSTITUTE SHEET In Table II : - C) indicates that the percentage was calculated using a software (52) from the percentage identity divided by the size of the smallest protein; - ( b ) indicates sequences not available; - ( c ) indicates the comparison with IlvB nucleic acid; - ( d ) indicates the comparison with IlvN nucleic acid; - ( c ) indicates the comparison with Ilvl nucleic acid; - ( f ) indicates the comparison with IlvH nucleic acid; - ( κ ) indicates the comparison with live nucleic acid; - ( h ) indicates the comparison with IlvM nucleic acid; - (') N-terminal end of Leul; - ( j ) C-terminal end of Leul. In addition, two genes detected by complementation, the ilvB gene (homologous to ilvB, ilvl and ilvG of E. coli) and the ilvN gene (homologous to ilvH of E. coli) , for which complementation data were not obtained, were identified. Most of the proteins of L. lactis were similar in size to their homologues. However, three exceptions were observed. Relative to the proteins of E. coli, the lactococcal proteins IlvA and live lack the C-terminal amino acids 73 and 147, respectively, and IlvD displays a deletion of 36 amino acids, one of the ORFs, designated ORF2, codes for a protein not displaying any homology with the enzymes of the pathway of synthesis of the branched amino acids, but carries two regions that are conserved in a superfamily of ATP-binding proteins (53). Figure 3 shows the alignment of 6 ATP-binding proteins with the ORF2 sequence of L. lactis. Needleman's software was used to obtain these results (54). In Fig¬ ure 3: - the binding domains (53) are represented by NBl SUBSTITUTE SHEET and NB2 ; - a short sequence shared by all the members of the ATP-dependent family is indicated by the symbols (#) ; - the symbols ( λ ) indicate amino acids common to 5 all the proteins; - the symbols (.) indicate conserved substitutions; - the amino acids shown in bold characters are present in at least five proteins; - the figures at the left indicate the position of 10 the amino acids, and the figures in brackets indicate the size of the proteins; - the functions of the different proteins are shown in the following manner: (') , nodulation in Rhizobium leguminosarum (55); 15 ( 2 ) , maltose transport in E. coli (56); ( 3 ) , glutamine transport in E. coli (57); ( 4 ) . glyoine, betaine and proline transport in E. coli (58); ι ( s ) , haemolysin secretion in E. coli (59); 20 ( 6 ) , cyclolysine secretion in Bordetella pertussis (60). The organization of the sequenced segments is presented in Figure 1. All the genes, except for the first one, upstream of LeuA (ORF1) , are transcribed in the same 25 direction. The leu and ilv genes are grouped together, and the two groups are separated by 121 bp. The leu genes are separated by less than 19 bp, except for leuB and leuC which are separated by four 72-bp direct repeats. The distance between the ilv genes is between approximately 30 10 and 42 bp, except for ilvB and ilvN which are separated by 9 bp. 6) Transcription signals The sequences conforming to the consensus units of the lactococcus promoters were identified upstream of the 35 assemblies of leu and ilv genes (see the positions of pi and p2 in Figure 1) . The region between pi and leuA strongly resembles the regulatory regions of the amino acid biosynthesis operons controlled by attenuation (61). The transcript initiated at pi can follow two paths. One SUBSTITUTE SHEET leads to the formation of a rho-independent transcription "terminator" (as shown in Figure 4A) . In addition, the transcript carries a 51-bp message, beginning with an ATG codon and ending with a TAG codon, which determines a leader peptide of 16 amino acids, four of which are a succession of leucine and isoleucine (as shown in Figure 4B) . The ribosome sited at the Leu and lie codons prevents formation of the termination signal and leads to tran¬ scription of the leu genes downstream. Independent tran- scription "terminators" were not found between the two assemblies of genes or downstream of the ilvA gene. 7) Organization of the ilv operon The genes involved in the pathway of synthesis of the branched amino acids in L. lactis subsp. lactis are organized in a large assembly divided into two units grouping together the leu and ilv genes. The two units are necessary for the synthesis of leucine, whereas only the second is required for the synthesis of isoleucine and valine. A transaminase, involved in the last step of the pathway of synthesis of the branched amino acids (product of the ilvE gene in E. coli) , is not encoded by the assembly. This implies that this reaction is implemented by a non-specific transaminase, or alternatively that the corresponding gene in L. lactis subsp. lactis occurs elsewhere on the chromosome. The leu and ilv genes are both preceded by a promoter; however, they are not separated by an indepen¬ dent transcription "terminator", suggesting that they form a single operon. This operon extends beyond the last biosynthesis gene, ilvA. 8) Regulation of the ilv operon Sequence analysis strongly suggests that the operon is regulated by an attenuation mechanism, the mediator of which is a leucine-rich leader peptide. This peptide very closely resembles the leader peptide of the leu operon of E. coli and of S. typhimurium (62, 63), but differs from that of the ilvBN and ilvGMEDA operons of E. coli. Figure 4 shows the secondary structures of the leader transcript capable of participating as a mediator SUBSTITUTE SHEET in transcription attenuation: - at A, the termination topology is shown; units (1) and (2) are paired, and units (5) and (6) form a transcription terminator; - at B, the antitermination topology is shown; units (3) and (4) are paired, and mask units (5) and (6). In Figure 4, the nucleotides in bold characters indicate the sequence coding for the leader peptide, and the nucleotides in bold italics indicate the ribosome binding sites of the leader peptide and the leuA gene. Stalling of the ribosome at the successive Leu and lie residues could mask unit (1) and favour this topology. Current models propose that the strength of the attenuation is dependent on the tRNA leu load during trans- lation of the leader peptide. The presence of rare codons increases the response to leucine starvation by increasing the duration of ribosome stalling. In E. coli, the codon corresponding to the four leucine residues present in the leader peptide is CUA, which corresponds to only 2% of the leucine codons used for the proteins of this organism (64) . In contrast, the leucine codon UUC, encountered three times in the leader transcript of the pathway of synthesis of the branched amino acids in L. lactis, corresponds to 24% of the leucine codons in the proteins of L. lactis (65) . The isoleucine codon is also present in the leader peptide of L. lactis, following very closely the Leu codons, which is not the case in E. coli, and may hence affect the response of the operon to an arrest of the pathway of synthesis of the branched amino acids. Other studies are necessary to determine whether the model proposed for regulation in E. coli may be applied directly to L. lactis. 9) The open reading frame ORF2 ORF2, inasmuch as it corresponds to a product belonging to the superfa ily of ATP-binding proteins, is not a biosynthesis gene in the pathway of synthesis of the branched amino acids. The proteins of this superfamily are encountered both in prokaryotes and in eukaryotes, and share a similar base organization (66, 67) . In SUBSTITUTE SHEET prokaryotes, most of the members of this superfamily are components of the transport systems involving periplasmic binding proteins. These genes are generally cotranscribed with other genes (67), and have never been found in the biosynthesis operon. Other studies are necessary to establish the function of ORF2, but the latter is doubt¬ less involved in the transport of the chain of synthesis of the branched amino acids, or in regulation of the genes of this pathway. Isoleucine, leucine and valine represent 20% of the amino acids of L. lactis (68), whereas tryptophan and histidine, two other amino acids whose pathways of biosynthesis in L. lactis have been studied, represent less than 2%. This suggests that a fine regulation of the expression of the genes of the pathway of synthesis of the branched amino acids is necessary. II - INTRODUCTION OF A NUCLEIC ACID CODING FOR AN α-ACETOLACTATE SYNTHASE INTO A HOST The nucleic acid consisting of the DNA sequence bounded by the nucleotides located at positions 7977 and 10246 of Figure 2, and coding for the IlvB and IlvN polypeptides, is amplified by the Taq polymerase chain reaction from the DNA of Figure 2, and using two oligo¬ nucleotides located upstream and downstream of the nucleic acid sequence coding for IlvBN. The following two oligonucleotides were used: CTAGTGAAGGTTGCGTTACA (SEQ ID No 15) TGCCATTTTTGTTTCCTCTA (SEQ ID No 16) After the polymerase chain reaction, the product of the reaction is cloned into an expression vector, such as pIL252, pIL253, pWVOl, containing a promoter such as the promoter of the lactose operon or of the tryptophan operon, and where appropriate a terminator. After cloning of the amplified segment in the appropriate orientation, the plasmid obtained is intro¬ duced by transformation into a strain of L. lactis. such as IL1403 or MG1363, which expresses IlvBN at a high level. This results in diversion of a part of the pyruvate pool obtained after glycolysis towards α-acetolactate, a SUBSTITUTE SHEET precursor of diacetyl. SUBSTITUTE SHEET REFERENCES 1. Calvo, J.M. (1983) p. 267-284. In Herrman, K.M. Somerville, R.L. (eds.) Amino acids, biosynthesis and genetic regulation, Addison-Wesley, Reading, Mass. 2. Kohlhaw, G.B. (1983) p. 285-299. In Herrman, K.M. and Somerville, R.L. (eds.) Amino acids, biosynthesis and genetic regulation, Addison- Wesley, Reading, Mass. 3. Umbarger, H.E. (1983) p. 245-266. In Herman, K.M. and Somerville, R.L. (eds.) Amino acids, biosynthesis and genetic regulation, Addison- Wesley, Reading, Mass. 4. Umbarger, H.E. (1987) p. 353-367. In F.C. Neidhardt, J.L. Ingraham, K.B. Low, B. Magasanik, M. Schaechter and H.E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: Cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C. 5. Mackey, C.J., R.J. Warburg, H.o. Halvorson and S.A. Zahler. Gene 32, 49-56. 6. Vandeyar, M.A. , C.J. Mackey, R.H. Lipsky and Z.A. Zahler (1986) p. 295-305. In Ganesan, A.T. and Hoch, J.A. (eds.), Bacillus molecular genetics and biotechnology applications. Academic Press Inc. 7. Ward, J.B., and S.A. Zahler (1973) J. Bacteriol. 116, 719-726. 8. Cordes, C, L. Eggeling and H. Sahm (1990) p. 339- 351. In Heshlot, H., Davies, J., Florent, J., Bobichon, L., Durand, G., Penasse, L. (eds.), Societe Franς:aise de microbiologie. 9. Pattee, P.A. (1976) J. Bacteriol. 127, 1167-1172. 10. Bachmann, B.J. (1987) p. 807-877. in F.C. Neidhardt, J.L. Ingraham, K.B. Low, B. Magasanik, M. Schaechter and H.E. Umbarger (ed.), Escherichia Coli and Salmonella typhymurium: Cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C. SUBSTITUTE SHEET 11. Lawther, R.P., R.C. Wek, J.M. Lopes, R. Pereira, B.E. Taillon, and G.W. Hatfield (1987) Nucleic Acids Res. 15, 2137-2155. 12. Wek, R.C. and G.W. Hatfield (1986) J. Bioch. Chem. 5 261, 2441-2450. 13. Friedberg, D., E.R. Rosenthal, J.W. Jones and J.M. Calvo (1985) Mol. Gen. Genet. 199, 486-494. 14. Squires, C.H., M. DeFelice, S.R. Wessler and J.M. Calvo (1981) J. Bacteriol. 147, 797-804. 10 15. Mackey, C.J. and S.A. Zahler (1982) J. Bacteriol. 151, 1222-1229. 16. Tanaka, T. (1979) Mol. Gen. Genet. 175, 235-237. 17. Barat, M.C. Anagnostopoulos, and A.M. Schneider (1965) J. Bacteriol. 90, 357-369. 15 18. Ikawa, S., T. Shibata, T. Ando, and H. Saito (1980) Mol. Gen. Genet. 177, 359-368. 19. Somers, J.M., A. Amzallag, and R.B. Middleton (1973) J. Bacteriol. 113, 1268-1272. 20. Marsch, N.J., and D.E. Duggan (1972) J. Bacteriol. 20 109, 730-740. 21. Butlin, J.D., G.B. Cox and F. Gibson (1971) Biochem. J. 124, 75-81. 23. Simon, D., and A. Chopin (1988) Biochimie 70, 559- 566. 25 24. Niaudet, B., L. Janniere, and S.D. Ehrlich (1985) J. Bacteriol. 163, 111-120. 25. Terzaghi, B. and W.E. Sandine (1975) Appl. Microbiol. 29, 807-813. 26. Cold Spring Harbor Laboratory, Cold Spring Harbor, 30 N.Y. Maniatis, T., E.F. Fritsh, J. Sambrook (1982) Molecular cloning: a laboratory manual. 27. Loureire des Santos, A.L., A. Chopin (1987) FEMS Microbiol. Lett. 42, 209-212. 28. Simon, D., A. Rouault, M.C. Chopin (1985) FEMS 35 Microbiol. Lett. 26, 239-241. 29. te Riele, H., B. Michel, S.D. Ehrlich (1986) Proc. Natl. Acad. Sci. USA 83, 2541-2545. 30. Dower, W.J., J.F. Miller, C.W. Ragsdale (1988) Nuclei. Acid. Res. 16, 6127-6145. SUBSTITUTE SHEET 31. Anagnostopoulos, C. J. Spizizen (1961) J. Bacteriol. 81, 741-746. 32. Bron, S. (1990) Plasmids; in "Molecular Biology for Bacillus, CR. Harwood, S.M. Cutting, pp. 148-149, John Wiley and Sons Ltd. 33. Corpet, F. (1988) Nuclei. Acid. Res. 16, 10881- 10890. 34. Niaudel, B., and S.D. Ehrlich (1982) p. 201-209. in M. Polsinelli and G. Mazza (eds.), Transformation- 80. Cotswold Press, Oxford UK. 35. Gribskov, G., J. Devereux, and R.R. Burgess (1984) Nucleic Acids Res. 12, 539-549. 36. de Vos, W.M. (1987) FEMS Microbiol. Rev. 46, 281- 295. 37. Wek, R.C, CA. Hauser and G.W. Hatfield (1985) Nucleic Acid Res. 13, 2995-4010. 38. Squires, CH., M. Defelice, J. Devereux and J.M. Calvo (1983) Nucleic Acids Res. 11(52), 5299-5313. 39. Ricca, E. and J.M. Calvo (1990) Nucleic Acid Res. 18, 1290. 40. Rosenthal, E.R. and J.M. Calvo. Nucleic Acid Res. 18, 3072. 41. Imai, R., T. Sekiguchi, Y. Nosoh and K. Tsuda (1987) Nucleic Acids Res. 15, 4988. 42. Sekiguchi, T., Ortega-Cesena, Y. Nosoh, S. Ohashi, K. Tsuda and S. Kanaya (1986) Biochimmica and Biophysica Acta. 867, 36-44. 43. Kagawa, Y., H. Nojim, N. Nukiwa, M. Ishizuka, T. Nakajima, T. Yasuhara, T. Tanaka and T. oshima. (1984) J. Biol. Chem. 259, 2956-2960. 44. Andreadis, A., Y.-P. Hsu, G.B. Kohlaw and P. Schimmel (1982) Cell. 31, 319-325. 45. Beltzer, J.P., L.-F. L. Chang, A.E. Hinkkanen and G.B. Kohlaw (1986) J. Biol. Chem. 261, 5160-5167. 46. Falco, S.C, K.S. Dumas and K.J. Livak (1985) Nucleic Acid Res. 13, 11. 47. Kielland-Brandt M.C. , s. Holmbertg, J.G.L. Petersen and T. Nilssen-Tillgren (1984) carlsberg Res. Commun. 49, 567-575. SUBSTITUTE SHEET 48. Petersen J.G.L. and S. Holmberg (1986) Nucleic Acids Res. 14, 9631-9651. 49. Skala J., E. Capieaux, E. Balzl, W. Chen and A. Goffeau (1991) Yeast 7, 281-285. 5 50. Iturriaga, E.A., J.M. Diaz-Minguez, Z.P. Benito, M.I. Alvarez, and A.T. Eslava (1990) Nucleic Acid Res. 18, 4612. 51. Roncero, M.I.G., L.P. Jepsen, P. Stroman and R. van Heeswijck (1989) Gene 84, 335-343. 10 52. Wilbur, W.J. and D. Lipman (1983) Proc. Natl. Acad. Sci. USA 80, 726-730. 53. Higgins, CF., M.L. Gallagher, M.L. Mimmack and S.R. Pearce (1988) Bioassays 8, 111-116. 54. Needleman, S.B., and CD. Wunsch (1970) J. Mol. 15 Biol. 48, 443-453. 55. Evans, I.J. and J.A. Downie (1986) Gene 43, 95-101. 56. Gilson, E., H. Nikaido and M. Hofnung (1982) Nucleic Acids Res. 10, 7449-7458. 57. Nohno, T., T. Saito and J.-S. Hong (1986) Mol. Gen. 20 Genet. 205, 260-269. 58. Gowrishankar, J. (1989) J. Bacteriol. 171, 1923- 1931. 59. Felmlee, T., S. Pellett and R.A. Welch (1985) J. Bacteriol. 163, 94-105. 25 60. Glaser, P., H. Sakamoto, J. Bellalou, A. Ull ann and A. Danchin (1988) EMBQ J. 7, 3997-4004. 61. Kolter, R. and C Yanofski (1982) Ann. Rev. Genet. 16, 113-134. 62. Frieden, P., T. Newman and M. Freundlich (1982) 30 Proc. Natl. Acad. Sci. USA 79, 6156-6160. 63. Wessler, S.R. and J.M. Calvo (1981) J. Mol. Biol. 149, 579-597. 64. Sharp, P.M., E. Cowe, D.G. Higgins, D.C. Shields, K.H. Wolfe, and F. Wright (1988) Nucleic Acid Res. 35 16, 8207-8211. 65. Van de Cuchte, M., J. Kok and G. Venema (1982) FEMS Microbiol. Rev. 88, 73-92. 66. Higgins, C.F., I.D. Hilce, G.P.C Salmond, D.R. Gill, J.A. Downie, I.J. Evans, I.B. Holland, L. SUBSTITUTESHEET Cray, S.D. Bucked, A.W. Bell and M.A. Hermodson. (1986) Nature 323, 448-450. 67. Higgins, C.F., I.D. Hiles, K. Whalley and D.J. Jamieson (1985) EMBO J. 4, 113-1040. 68. Thomas, T.D. and G.G. Prichard (1987) FEMS Microbiology Rev. 46, 245-268. SUBSTITUTE SHEET - 28a - The following SEQ ID Nos correspond to the sequence indicated . CDS 450-1991; leu A CDS 2003-3040; leu B CDS 3403-4785; leu C CDS 4805-5380; leu D CDS 5394-6173; ORF2 CDS 6295-8007; ilvD CDS 8018-9745; ilvB CDS 9738-10214; ilvN CDS 10260-11294; ilvC CDS 11337-12662; ilvA leu-ilv DNA sequence ilvB DNA sequence ilvN DNA sequence ilvBN DNA sequence CTAGTGAAGGTTGCGTTACA TGCCATTTTTGTTTCCTCTA SUBSTITUTESHEET - 29 - SEQUENCE LISTING LOCUS LACLEUILV 12720 bp ds-DNA BCT 02-NOV-1992 DEFINITION Lactococcus lactis operon (leuA, leuB, leuC, leuD, ilvD, ilvB, ilvN, ilvC, ilvA) genes complete eds; complete ORF2. ACCESSION M90761 KEYWORDS ilvA gene; ilvB gene; ilvC gene; ilvD gene; ilvN gene; leuA gene; leuB gene; leuC gene; leuD gene; operon. SOURCE Lactococcus lactis (library: NCD02118) DNA. ORGANISM Lactococcus lactis Prokaryotae; Firmicutes; Firmibacteria; Gram-positive cocci; Streptococcaceae. REFERENCE 1 (bases 1 to 12720) AUTHORS Godon,J.-J.J.-J. , Chopin,M.-CM. and Ehrlich,D.S. TITLE Branched-chain amino acid biosythesis genes in Lactococcus lactis subup. lactis. JOURNAL J. Bacteriol. 174, 6580-6589 (1992) STANDARD full automatic FEATURES Location/Qualifiers attenuator 260. .450 /note="putative" repeat_region 3039. .3327 /function="unknown" /rpt_type=DIRECT /note="putative" CDS 450. .1991 /EC_number="4.1.3.12" /gene="leuA" /citation=[l] /note="putative" /codon_start=l / t r a n s l a - tion="MRKIEFFDTSLRDGEQTPGVSFSISEKVTIAKQLEKWRISVIEA GFSAASPDSFEAVKQIADSLNDTAVTALARCVISDIDKAVEAVKGAKYPQIHVFIATS PIHMKYKLKISPEEVLKNIDKCVRYARERVEWEFSPEDATRTELNFLLEAVQTAVDA GATYINIPDTVGYTTPEEYGKIFKFLIDNTKSDREIIFSPHCHDDLGMAVANSLAAIK AGAGRVEGTVNGIGERAGNAALEEIAVALHIRKDFYQAQSPLKLSETAATAELISQFS GIAIPKNKAIVGANAFAHESGIHQDGVLKNAETYEIITPELVGIKHNSLPLGKLSGRH AFSEKLTELNIAYDDESLAILFEKFKKLADKKKEITDADIHALFTGETVKNLAGFILD NVQIDGHKALVQLKNQEEEIYVSQGEGSGSVDAIFKAIDKVFNHQLKLISYSVDAVTD GIDAQATTLVSVENLSTGTIFNAKGVDYDVLKGSAIAYMNANVLVQKENLQGKVEQIS AHDGI" CDS 2003. .3040 /gene="leuB" SUBSTITUTE SHEET - 30 - /note="putative" /codon_start=l /translation="LSKKIVTLAGDGIGPEIMSAGLSVLKAVSKKIDFEYELEAKDFG GIAIDKHGHPLPEETLQAVKNADAILLAAIGHPKYNNAKVRPEQGLLALRKELGLYAN VRPLKIYPALKKLSPIRNVENVDFLVIRELTGGIYFGQHELADDKARDVNDYSADEIR RILHFAFKSAQSRPRKLLTSVDKQNVLATSKLWRKMADEIADEYPDVRLEHQLVDSCA MLLITNPQQFDVIVTENLFGDILSDEASSLAGSLGVMPSSSHGFNGLALYEPIHGSAP DIAGKGIANPVSMILSIAMMLRESFGQEDGAAMIEKAVTQTFTDGILTKDLGGTATTK EMTEAILKNCQ" CDS 3403. .4785 /gene="leuC" /note="putative" /codon_start=l /translation="MSGKTIFDKLWDQHVIAGNEGEPQLLYIDLHVIHEVTSPQAFQG LREAGRRVRRKDLTYGTLDHNVPTQNIFNIQDLISKKQIDTFTKNVKEFDVPAETHGG KGQGIVHMVAPESGRTQPGKTIVCGDSHTATNGAFGAIAFGIGTSEVEHVLATQTIWQ VKPKRMKIEFQGHPQKGIYSKDFILALIAKYGVDAGVGYAVEYSGDAISDLSMEERMT ICNMSIEFGAKIGLMNPDEKTYDYVKGREHAPKNFDEAVSKWEKLVSDSDAQYDKILS LDVSQLKPMVTWGTNPGMGLEFGEKFPEINNDLNYERAYQYMDLKPGQTASDIDLGYI FIGSCTNARLGDLEEAAKIIGDRHIADGLTGIWPGSRPVKEAAEAQGLDKIFKEAGF EWREPGCSACLGMNPDQIPEYVHCASTSNRNFEGRQGHNARTHLCSPAMAAAAAIAGK FVDVRMLVTD" CDS 4805. .5380 /gene="leuD" /note="putative" /codon_start=l /translation="MEKFTIYKGTSVPVMNDNIDTDQIIPKQFLKAIDKKGFGKNLFY EWRYLKDYDENPDFILNAPKYKKASLLISGDNFGSGSSREHAAWALSDYGFRAIIAGS YSDIFYNNALKNGLLPIKQPREVLNQLTKLSSQEEITIDLPHQLIITSLGDFHFEIDP IWKDKLINGLDDIGITLQYEEAISAYEQKNQ" CDS 5394. .6173 /function="unknown" /note="ORF2; putative" /codon_start=l /translation="MTIINLKNVNLTRNKKEILKDITWKVNPGENWVILGLNGSGKSS LLKLILAEEWKTSGEITVLNTQFRNGEIPKLRKRISWGSFIAERFQPNIKAENLVYT GKFNSSMLYKPYTDQELDEARQLLRQMGAKSLIGRNYASLSQGEKQVLLIARSLILKP SUBSTITUTESHEET - 31 - ELLILDEATNGLDLFAKEKLLKQLQQINQLKTAPTLIYISHHPDEITDIFTHLLLLRE GKVIQSGKKENLLNEKILTDFYQEKVEVHRFEQKYFVIPAN" CDS 6295. .8007 /gene="ilvD" /note="putative" /codon_start=l /translation="MEFKYNGKVESVELNKYSKTLTPRSTQPATQAMYYGIGFKDEDF KKAQVGIVSMDWDGNPCNMHLGTLGSKIKSSVNQTDGLIGLQFHTIGVSDGIANGKLG MRYSLVSREVIADSIETNAGAEYYDAIVAIPGCDKNMPGSIIGMARLNRPSIMVYGGT IEHGEYKGEKLNIVSAFEALGQKITGNISDEDYHGVICNAIPGQGACGGMYTANTLAA AIETLGMSLPYSSSNPAVSQEKQEECDDIGLAIKNLLEKDIKPSDIMTKEAFENAITI VMVLGGSTNAVLHIIAMANAIGVEITQDDFQRISDIIPVLGDFKPSGKYMMEDLHKIG GLPAVLKYLLKEGKLHGDCLTVTGKTLAENVETALDLDFDSQDIMRPLKNPIKATGHL QILYGNLAQGGSVAKISGKEGEFFKGTARVFDGEQHFIDGIESGRLHAGDVAVIRNIG PVGGPGMPEMLKPTSALIGAGLGKSCALITDGRFSGGTHGFWGHIVPEAVEGGLIGL VEDDDIIEIDAVNNSISLKVSNEEIAKRRANYQKPTPKATRGVLAKFAKLTRPASEGC VTDL" CDS 8018. .9745 /gene="ilvB" /note="putative" /codon_start=l /translation="MKKIKLEKPTSGSQLVLQTLKELGVEIIFGYPGGAMLPLYDAIH NFEGIQHILARHEQGATHEAEGYAKSSGKVGWWTSGPGATNAVTGIADAYLDSVPL LVFTGQVGRQSIGKDAFQEADTVGITAPITKYNYQIRETADIPRIVTEAYYLARTGRP GPVEIDLPKDVSTLEVTEINDPSLNLPHYHESEKATDEQLQELLTELSVSKKPVIIAG GGINYSGSVDIFRAFVEKYQIPWSTLLGLGTLPISHELQLGMAGMHGSYAANMALVE ADYIINLGSRFDDRWSNPAKFAKNAWAHIDIDAAELGKIVKTDIPILSDLKAALSR LLQLNKVRTDFNDWIKTVIENKEKAPFTYEPQNHDIRPQETIKLIGEYTQGDAIIVTD VGQHQMWVAQYYPYKNARQLITSGGMGTMGFGIPAAIGAKLAQPNKNVIVFVGDGGFQ MTNQELALLNGYGIAIKWLINNHSLGMVRQWQESFYEERRSQSVFDVEPNFQLLAEA YGIKHVKLDNPKTLADDLKIITEDEPMLIEVLISKSEHVLPMIPAGLHNDEMIGLHFT DKNEEIDNA" CDS 9738. .10214 /gene="ilvN" /note= M putative" /codon_start=l SUBSTITUTESHEET - 32 - /translation="MRRMIIAKLHNVTGIMNRFTAVLNRRQVNILSITAGVTESQDLT HTTFVIEVDHLDEVEQIIKQLNRLIDVIEVADITDFPHVEREWLIKVSAPPTIRAEI FTMIEPFRVNWDVNLENVTIQLTGDSAKIEALIEWSPYGILNMARTGSAGFERG" CDS 10260. .11294 /gene="ilvC" /note="putative" /codon_start=l /translation="MAVTMYYEDDVEVSALAGKQIAVIGYGSQGHAHAQNLRDSGHNV IIGVRHGKSFDKAKEDGFETFEVGEAVAKADVIMVLAPDELQQSIYEEDIKPNLKAGS ALGFAHGFNIHFGYIKVPEDVDVFMVAPKAPGHLVRRTYTEGFGTPALFVSHQNASGH AREIAMDWAKGIGCARVGIIETTFKEETEEDLFGEQAVLCGGLTALVEAGFETLTEAG YAGELAYFEVLHEMKLIVDLMYEGGFTKMRQSISNTAEFGDYVTGPRIITDEVKKNMK LVLADIQSGKFAQDFVDDFKAGRPKLIAYREAAKNLEIEKIGAEHVKQCHSHNLVMTM PLKSISNFSY" CDS 11337. .12662 /gene="ilvA" /note="putative" /codon_start=l /translation="MISAKEVEDAYDLLKAWTKTPLQLDPYLSNKYQANIYLKEWT KTPLQLDPYLSNKYQANIYLKEENLQKVRSFKLRGAYYSISKLSDEQRSKGWCASAG NHAQGVAFAANQLNISATIFMPVTTPNQKISQVKFFGESHVTIRLIGDTFDESARAAK AFSQDNDKPFIDPFDDENVIAGQGTVALEIFAQAKKQGISLDKIFVQIGGGGLIAGIT AYSKERYPQTEIIGVEAKGATSMKAAYSAGQPVTLEHIDKFADGIAVATVGQKTYQLI NDKVKQLLAVDEGLISQTILELYSKLGIVAEPAGATSVAALELIKDEIKGKNIVCIIS GGNNDISRMQEIEERALVYEGLKHYFVINFPQRPGSLRTFVSDILGPNDDITRFEYIK RADKGKGPCLVGILLSDASDYDSLINRIERFDNRYVNLRGNDSLYELLV" BASE COUNT 4254 a 2046 c 2621 g 3799 t ORIGIN M90761 Length: 12720 April 6, 1993 18:44 Type: N Check: 7819 1 TAAAACTCGA TAATCTTGAG TCATAATTTC TCCTTAATCT TATTAGTACA 51 TTAGAATCCA TTATAATTTA ATCATTTTAT GTCTACCTAA AGCAACAAAA 101 TTGCTTGTAT ATTTTCTAAC AAGCTTAATT ATGTGGATTT AATTGAATAT 151 TAAAGGGAGA AGTTGTAATC TATTTGTTGT TAAATTCTTG SUBSTITUTESHEET - 33 - TTAATACAAA 201 TAAATTTATT AAATATTATT ATTTTATTGA CAATTTAAAA TATTAAGAGT 251 ATTATAATGT AAATTAACAA AAAAAAGAGG AACTTGAAAT GACATACACA 301 CAATTTTCAT TGTTGTTGAT CAAGGTGGAC CTACATTAGC TTTTTTGGCT 351 AAAATATGTG GGTCCTGTTT GGCGATAGTC ATTTCGAGGA CCGAGAGACG 401 TCCTCACGGG CGTCTTTTTT GTTTCTTAAT AAAAAATAGA GGTAATATTA 451 TGCGAAAAAT TGAATTCTTT GACACAAGTT TGAGAGATGG CGAACAGACA 501 CCGGGCGTTA GTTTCTCCAT TTCAGAAAAA GTAACGATTG CTAAACAACT 551 GGAAAAATGG AGGATTTCTG TCATAGAGGC TGGTTTTTCT GCGGCAAGTC 601 CAGATAGTTT TGAAGCAGTA AAGCAAATTG CTGATTCTTT GAATGATACG 651 GCTGTCACTG CATTAGCTCG CTGTGTTATT TCAGATATCG ATAAAGCGGT 701 TGAAGCGGTA AAGGGGGCTA AATATCCGCA AATTCATGTT TTCATTGCAA 751 CTTCACCTAT TCACATGAAA TATAAACTTA AAATCAGTCC CGAAGAAGTT 801 TTGAAAAATA TTGATAAGTG TGTGAGATAC GCACGTGAAC GGGTCGAGGT 851 TGTTGAGTTT TCTCCAGAGG ATGCAACAAG AACGGAGTTG AATTTTCTTT 901 TAGAGGCTGT TCAAACGGCT GTCGATGCTG GAGCAACTTA TATTAATATT 951 CCTGACACTG TCGGTTATAC GACACCAGAA GAATATGGAA AAATTTTTAA 1001 ATTTTTGATT GATAATACTA AGTCTGACCG AGAAATTATT TTTAGTCCAC 1051 ATTGTCATGA TGATTTAGGA ATGGCTGTAG CTAATTCATT AGCTGCAATT 1101 AAAGCTGGGG CTGGGAGAGT TGAAGGAACT GTCAATGGTA TTGGAGAGCG SUBSTITUTE SHEET - 34 - 1151 AGCTGGGAAT GCTGCTCTTG AAGAAATTGC TGTGGCACTA CATATTCGTA 1201 AAGATTTTTA TCAGGCACAA AGTCCTTTAA AACTTTCAGA AACTGCTGCA 1251 ACGGCAGAAC TAATTTCACA ATTTTCAGGA ATTGCTATTC CAAAAAATAA 1301 AGCAATTGTT GGTGCTAATG CTTTTGCACA CGAATCAGGA ATTCATCAAG 1351 ATGGTGTCCT TAAAAATGCT GAAACTTATG AAATTATTAC ACCAGAACTT 1401 GTCGGAATAA AGCATAATTC GTTGCCTTTA GGTAAACTTT CTGGTCGTCA 1451 TGCTTTTAGT GAAAAATTGA CGGAACTTAA TATTGCTTAT GACGATGAAA 1501 GTCTTGCAAT TTTATTTGAA AAATTTAAAA AATTAGCTGA CAAGAAAAAA 1551 GAAATTACTG ACGCAGATAT TCATGCCTTG TTTACAGGAG AAACGGTAAA 1601 AAATCTAGCT GGATTTATAC TTGATAATGT TCAAATTGAT GGGCACAAGG 1651 CATTGGTGCA ACTAAAAAAT CAAGAAGAGG AAATTTATGT TAGCCAAGGA 1701 GAGGGGTCAG GTTCAGTGGA TGCAATTTTT AAAGCTATTG ATAAAGTCTT 1751 TAATCATCAA CTAAAATTAA TTTCCTATTC AGTTGATGCT GTAACTGATG 1801 GAATTGATGC ACAAGCAACG ACTTTGGTTT CTGTTGAAAA TCTATCTACA 1851 GGCACTATAT TTAATGCTAA AGGTGTTGAT TATGATGTAT TGAAAGGAAG 1901 CGCCATTGCT TACATGAACG CTAATGTTTT AGTTCAAAAA GAAAATTTAC 1951 AAGGAAAGGT TGAACAAATT TCAGCTCATG ATGGAATTTA AGGTGAAAAA 2001 TATTGTCTAA AAAAATTGTG ACACTTGCGG GAGATGGAAT TGGGCCAGAA 2051 ATTATGTCAG CTGGTTTAAG TGTTTTAAAA GCTGTCAGTA AAAAAATTGA 2101 TTTTGAGTAT GAATTAGAAG CTAAAGATTT TGGAGGAATT SUBSTITUTE SHEET - 35 - GCAATTGATA 2151 AGCATGGTCA TCCTTTACCA GAAGAAACTT TGCAAGCAGT TAAAAATGCT 2201 GACGCAATCT TGCTCGCTGC AATTGGTCAT CCTAAATACA ACAATGCAAA 2251 AGTTAGACCA GAACAAGGGC TACTTGCTTT ACGAAAAGAA TTAGGACTGT 2301 ATGCTAATGT TCGTCCATTA AAAATTTATC CGGCTCTAAA AAAACTTTCT 2351 CCCATACGAA ATGTTGAAAA TGTTGATTTC CTAGTGATTC GCGAACTTAC 2401 AGGGGGAATC TATTTCGGTC AGCATGAATT GGCAGATGAT AAAGCACGAG 2451 ATGTCAATGA TTATTCTGCT GATGAAATAA GGAGAATTCT TCATTTTGCT 2501 TTCAAAAGTG CTCAAAGTCG GCCCAGAAAA TTACTGACTT CGGTTGATAA 2551 ACAAAATGTT CTTGCAACTT CTAAATTATG GCGAAAAATG GCTGATGAAA 2601 TTGCTGACGA ATATCCTGAT GTACGATTAG AGCACCAATT GGTCGATTCT 2651 TGTGCGATGT TACTGATTAC TAATCCGCAA CAATTTGATG TGATAGTCAC 2701 TGAAAATCTA TTTGGTGATA TTCTCTCTGA TGAAGCAAGT AGTTTGGCCG 2751 GTAGCTTAGG AGTGATGCCT TCGAGTTCGC ATGGATTTAA CGGTTTAGCA 2801 CTCTATGAGC CAATTCATGG TTCGGCACCA GATATTGCAG GAAAAGGAAT 2851 TGCGAACCCT GTTTCGATGA TTCTATCAAT TGCCATGATG CTAAGAGAAT 2901 CTTTTGGGCA AGAAGATGGG GCTGCGATGA TTGAAAAAGC CGTAACCCAA 2951 ACTTTTACTG ACGGAATTTT GACTAAAGAT TTAGGTGGGA CTGCAACAAC 3001 TAAAGAAATG ACAGAAGCAA TCCTGAAAAA TTGTCAGTAA AATGCGATTG 3051 AATAGTGAGC ATTTTAGTTG TAGATAAAAG AACCGTCAGC ATAGCTGACA SUBSTITUTE SHEET - 36 - 3101 ATTCTGTCAG TAAATGCGAT TGAATAGTGA GCATTTTAGT TGTAGATAAA 3151 AGAACCGTCA GCATAGCTGA CAATTCTGTC AGTAATTGCG ATTGAATAGT 3201 GAGCATTTTA GTTGTAGATA AAAGAACCGT CAGCATAGCT GACAATTCTG 3251 TCAGTAATTG CGATTGAATA GTGAGCATTT TAGTTGTAGA TAAAAGAACT 3301 ATCAGCGTAA CTGACAATTC TGTCAGTAAA TATTACTGAC AAAAAGTACA 3351 AAATTACTGA CAGAATTTGT CAGAATAAAT TTTTAAAAAA GGAAATAAAA 3401 AAATGTCAGG TAAAACAATA TTTGATAAAC TTTGGGATCA GCATGTGATT 3451 GCTGGAAATG AGGGAGAACC TCAACTGCTT TATATTGACC TTCATGTTAT 3501 TCATGAGGTT ACGAGTCCGC AAGCATTTCA GGGCTTACGT GAAGCAGGAC 3551 GTCGTGTTCG GAGAAAAGAT TTGACATACG GAACTCTTGA CCACAATGTT 3601 CCAACACAAA ATATTTTTAA TATTCAAGAT TTGATTTCTA AAAAACAAAT 3651 TGATACTTTT ACTAAAAATG TCAAAGAATT TGATGTTCCA GCGGAGACTC 3701 ATGGTGGAAA AGGACAAGGA ATTGTTCACA TGGTAGCACC TGAATCTGGC 3751 AGAACTCAAC CGGGAAAAAC AATTGTTTGT GGCGATAGTC ATACCGCAAC 3801 AAATGGAGCA TTTGGTGCAA TTGCTTTTGG AATTGGTACA AGTGAAGTTG 3851 AACATGTTCT TGCAACTCAA ACCATTTGGC AAGTTAAACC CAAGCGTATG 3901 AAAATTGAAT TTCAAGGTCA TCCACAAAAA GGAATTTATA GCAAAGACTT 3951 TATCCTCGCA TTAATTGCTA AATATGGTGT GGATGCAGGT GTAGGTTATG 4001 CGGTTGAATA TAGTGGGGAT GCTATCAGTG ATTTAAGCAT GGAAGAACGG 4051 ATGACAATCT GTAACATGTC AATTGAATTT GGGGCAAAAA SUBSTITUTE SHEET - 37 - TTGGCCTGAT 4101 GAATCCTGAT GAAAAAACTT ATGACTATGT CAAAGGGCGT GAACATGCAC 4151 CTAAAAACTT TGATGAAGCT GTCAGTAAAT GGGAAAAACT TGTCAGTGAT 4201 TCTGATGCAC AATACGATAA GATTTTAAGT CTTGATGTCA GCCAGTTGAA 4251 ACCAATGGTG ACATGGGGAA CAAATCCCGG AATGGGCCTA GAATTTGGCG 4301 AAAAGTTTCC GGAAATTAAC AATGATTTGA ATTATGAACG TGCTTATCAG 4351 TACATGGATT TAAAGCCAGG CCAAACCGCT TCTGACATAG ATTTAGGCTA 4401 TATTTTCATT GGTTCTTGTA CGAATGCTAG ACTTGGTGAT TTAGAAGAAG 4451 CTGCAAAAAT TATTGGAGAC AGACATATTG CTGATGGACT GACAGGAATT 4501 GTCGTCCCTG GAAGCAGACC TGTGAAAGAA GCGGCTGAAG CACAAGGGCT 4551 TGATAAAATT TTTAAAGAAG CTGGTTTTGA ATGGCGGGAA CCGGGTTGCT 4601 CAGCCTGTCT TGGAATGAAT CCTGACCAAA TTCCAGAATA CGTTCATTGT 4651 GCTTCAACCT CTAATCGAAA TTTTGAAGGT CGTCAAGGAC ATAATGCAAG 4701 AACGCACCTG TGCTCTCCAG CTATGGCTGC TGCCGCCGCA ATCGCTGGTA 4751 AATTTGTAGA TGTTAGAATG CTCGTAACAG ATTAGTCTGT AGAAAGAAAA 4801 AAAGATGGAA AAATTCACGA TTTACAAAGG GACAAGTGTT CCAGTCATGA 4851 ACGATAATAT TGACACAGAC CAAATTATTC CTAAACAATT TTTGAAAGCA 4901 ATCGATAAAA AGGGCTTTGG GAAAAATTTA TTTTATGAAT GGCGTTATCT 4951 TAAAGATTAC GATGAGAATC CTGATTTTAT TTTGAATGCT CCAAAATACA 5001 AAAAAGCTTC TCTGTTAATT TCAGGAGATA ATTTTGGTTC GGGTTCTTCA SUBSTITUTE SHEET - 38 - 5051 AGAGAACATG CGGCATGGGC CTTATCAGAT TACGGCTTTC GGGCAATTAT 5101 TGCTGGCTCT TACTCAGATA TTTTTTATAA TAATGCTTTA AAAAATGGCT 5151 TGTTACCAAT TAAACAACCA AGAGAAGTTC TAAATCAACT GACAAAACTG 5201 TCAAGTCAAG AAGAAATTAC AATTGATTTA CCCCATCAGC TAATCATCAC 5251 AAGCCTTGGT GACTTTCATT TTGAGATTGA CCCCATTTGG AAAGACAAAT 5301 TAATTAATGG CTTAGATGAT ATTGGAATAA CTTTGCAATA TGAAGAAGCA 5351 ATCTCAGCTT ACGAACAAAA AAATCAATAA GAGCGAGCCT AAAATGACAA 5401 TTATTAATTT AAAGAATGTA AATCTTACTC GAAATAAAAA AGAAATTCTT 5451 AAAGATATTA CTTGGAAAGT AAATCCCGGC GAAAATTGGG TTATTCTGGG 5501 CCTCAACGGC TCTGGAAAAT CAAGTCTTTT GAAATTGATT TTAGCAGAAG 5551 AATGGAAAAC TTCTGGTGAA ATCACTGTTT TAAATACTCA ATTTAGAAAT 5601 GGAGAAATTC CTAAGTTGAG AAAAAGAATC AGCGTAGTTG GCTCATTTAT 5651 TGCTGAAAGA TTTCAACCAA ATATTAAGGC TGAAAACCTT GTTTATACTG 5701 GGAAATTTAA TTCGAGCATG CTCTATAAAC CCTACACAGA TCAGGAACTT 5751 GATGAGGCCC GTCAGCTTTT AAGACAAATG GGCGCAAAAT CACTTATTGG 5801 CCGAAATTAT GCCAGCCTTT CTCAAGGGGA AAAGCAAGTT CTTCTTATTG 5851 CTAGGAGCTT AATTTTAAAG CCTGAGCTTT TAATTTTGGA CGAAGCAACG 5901 AACGGTTTAG ATTTATTTGC TAAAGAAAAA TTATTAAAGC AACTGCAGCA 5951 GATTAATCAA TTAAAAACCG CACCAACACT AATTTATATT TCTCATCATC 6001 CCGATGAAAT CACTGATATT TTTACTCACC TTTTACTTTT SUBSTITUTESHEET - 39 - AAGAGAAGGA 6051 AAAGTGATTC AATCAGGGAA AAAAGAAAAC TTATTAAATG AAAAGATACT 6101 TACTGATTTT TATCAAGAAA AAGTAGAAGT TCACCGTTTT GAGCAGAAAT 6151 ATTTTGTAAT TCCTGCTAAC TGAGAAAGGA AAGCAAAAGT ATTTTATATA 6201 CTATATAGAA TATTCTGACA GATTATTGTA TTTTCATTTT TTTAGTGATA 6251 AAATAGCTCT ATGTAAATTT ACGGGGAGGT CAAAAAGATA ACATATGGAA 6301 TTCAAATATA ACGGAAAAGT TGAATCAGTG GAACTCAATA AATATTCTAA 6351 GACATTGACT CCAAGATCAA CACAACCAGC GACTCAAGCG ATGTACTACG 6401 GCATTGGTTT TAAAGATGAG GATTTCAAAA AAGCTCAGGT CGGAATCGTC 6451 AGCATGGATT GGGACGGAAA TCCATGTAAT ATGCACTTGG GAACACTTGG 6501 GAGTAAAATC AAAAGTTCTG TCAACCAAAC TGACGGATTG ATTGGACTTC 6551 AATTTCATAC TATTGGAGTT TCTGATGGAA TTGCTAACGG AAAGCTTGGC 6601 ATGAGATATT CTTTGGTCAG TCGTGAAGTT ATTGCTGACA GCATCGAAAC 6651 CAACGCTGGC GCAGAATATT ATGATGCCAT CGTTGCCATT CCCGGTTGTG 6701 ATAAAAATAT GCCCGGGTCA ATTATCGGAA TGGCTCGCTT AAATCGTCCG 6751 TCAATTATGG TCTATGGTGG AACGATTGAA CATGGCGAAT ATAAAGGTGA 6801 AAAATTAAAT ATTGTTTCGG CCTTTGAAGC TCTGGGGCAA AAAATCACTG 6851 GAAATATTTC TGATGAAGAT TATCATGGCG TTATTTGCAA TGCCATTCCA 6901 GGACAAGGTG CTTGCGGAGG AATGTACACT GCCAATACCC TGGCTGCTGC 6951 TATTGAAACT TTGGGAATGA GTTTACCTTA TTCCTCTTCC AATCCAGCAG SUBSTITUTE SHEET - 40 - 7001 TCAGTCAAGA AAAACAAGAA GAGTGTGATG ACATTGGTTT AGCCATCAAA 7051 AATTTATTAG AAAAAGATAT TAAACCAAGT GATATCATGA CCAAAGAAGC 7101 TTTTGAAAAT GCCATAACAA TTGTCATGGT CCTTGGAGGC TCAACCAATG 7151 CTGTGCTTCA TATCATTGCA ATGGCAAATG CCATTGGTGT AGAAATTACG 7201 CAAGATGATT TCCAACGTAT TTCAGATATT ATCCCTGTTC TTGGCGATTT 7251 CAAACCGAGC GGAAAATATA TGATGGAAGA TCTGCACAAA ATTGGTGGCC 7301 TTCCTGCTGT TTTGAAATAC CTACTTAAAG AAGGAAAACT TCACGGTGAT 7351 TGTTTGACCG TCACAGGTAA AACTTTGGCT GAAAATGTTG AAACAGCATT 7401 AGATTTGGAC TTTGACAGTC AAGATATTAT GCGACCACTA AAAAATCCAA 7451 TTAAAGCTAC TGGACATTTA CAAATTTTGT ACGGTAATCT TGCCCAAGGG 7501 GGTTCTGTTG CAAAAATTTC TGGTAAAGAA GGCGAATTTT TCAAAGGAAC 7551 AGCTCGTGTT TTTGACGGAG AACAACACTT TATCGATGGC ATTGAGTCTG 7601 GCCGATTGCA TGCCGGTGAT GTTGCGGTCA TTAGAAATAT TGGCCCAGTC 7651 GGAGGTCCGG GAATGCCAGA GATGTTAAAA CCAACCTCAG CATTAATTGG 7701 AGCAGGACTT GGAAAATCTT GTGCCCTAAT TACTGACGGA AGATTTTCTG 7751 GTGGCACACA CGGCTTTGTT GTGGGTCATA TCGTCCCTGA AGCAGTTGAA 7801 GGTGGGTTGA TTGGTTTAGT TGAAGATGAT GATATTATCG AAATTGATGC 7851 GGTGAATAAT AGTATTAGTT TAAAAGTTTC TAATGAAGAA ATTGCTAAAC 7901 GACGTGCCAA TTATCAAAAA CCAACCCCTA AAGCAACGCG TGGTGTTCTT 7951 GCAAAATTTG CCAAACTTAC GCGCCCCGCT AGTGAAGGTT SUBSTITUTE SHEET - 41 - GCGTTACAGA 8001 TTTATAGAAA GGTTTGAATG AAAAAAATAA AGTTAGAAAA ACCTACTTCC 8051 GGTTCCCAAC TTGTTCTCCA AACCTTAAAA GAACTTGGAG TAGAAATTAT 8101 TTTTGGTTAT CCTGGTGGGG CCATGCTCCC CTTGTATGAT GCGATTCATA 8151 ATTTTGAAGG AATTCAACAT ATTTTAGCCC GTCATGAGCA AGGAGCAACG 8201 CATGAAGCCG AAGGTTACGC TAAATCGTCT GGTAAAGTTG GTGTCGTCGT 8251 TGTTACGTCA GGACCAGGAG CGACTAATGC AGTAACCGGA ATTGCTGACG 8301 CTTATCTTGA TTCAGTCCCA TTGTTAGTTT TCACAGGTCA AGTTGGCCGT 8351 CAGTCAATTG GTAAAGATGC TTTTCAAGAA GCAGATACTG TTGGAATTAC 8401 AGCCCCAATT ACAAAATATA ATTATCAAAT TAGGGAAACC GCAGATATTC 8451 CAAGAATTGT TACAGAAGCC TATTATTTGG CAAGGACAGG ACGTCCTGGA 8501 CCAGTAGAAA TTGATTTACC AAAAGATGTT TCCACCCTTG AAGTCACTGA 8551 AATTAATGAC CCAAGCTTGA ATCTTCCTCA TTATCACGAA AGTGAAAAAG 8601 CGACTGATGA ACAATTGCAA GAATTACTGA CAGAACTTTC TGTCAGTAAA 8651 AAACCAGTCA TTATTGCTGG CGGAGGAATT AATTATTCTG GCTCAGTTGA 8701 TATTTTCAGA GCATTTGTCG AAAAATATC AATTCCAGTT GTTTCTACAT 8751 TGCTTGGCTT AGGAACATTA CCAATCAGCC ACGAATTGCA ACTAGGAATG 8801 GCAGGAATGC ACGGTTCATA CGCTGCAAAT ATGGCTTTAG TTGAAGCTGA 8851 CTATATTATT AATTTGGGAT CACGTTTTGA CGATAGAGTT GTATCCAATC 8901 CTGCAAAATT TGCTAAAAAT GCTGTCGTTG CTCATATTGA TATTGACGCT SUBSTITUTE SHEET - 42 - 8951 GCTGAACTTG GCAAAATTGT AAAAACCGAT ATTCCAATCC TTTCTGATTT 9001 GAAAGCGGCT TTAAGCAGAC TTTTGCAATT AAATAAGGTC AGGACTGACT 9051 TTAATGATTG GATTAAAACT GTCATTGAAA ATAAAGAGAA AGCACCATTT 9101 ACTTATGAGC CCCAAAACCA TGATATCCGT CCACAGGAAA CAATTAAATT 9151 AATTGGAGAA TACACTCAAG GAGATGCAAT CATTGTAACT GACGTTGGGC 9201 AACATCAAAT GTGGGTGGCG CAATATTATC CTTATAAAAA TGCAAGGCAA 9251 CTTATTACTT CTGGGGGAAT GGGAACGATG GGCTTTGGCA TTCCTGCAGC 9301 AATCGGTGCA AAGCTGGCAC AGCCAAATAA AAATGTCATT GTTTTTGTTG 9351 GCGATGGTGG CTTTCAAATG ACTAATCAAG AATTAGCATT ACTTAATGGC 9401 TACGGTATTG CAATCAAAGT TGTGCTGATT AATAATCATT CATTGGGAAT 9451 GGTACGTCAA TGGCAAGAAT CATTCTATGA AGAGCGACGT TCACAATCGG 9501 TTTTTGATGT TGAACCCAAT TTTCAATTGT TAGCCGAAGC TTATGGCATC 9551 AAACATGTTA AGTTAGATAA TCCAAAAACT TTGGCTGATG ATTTAAAAAT 9601 TATTACAGAA GATGAGCCAA TGCTTATTGA AGTTCTAATT TCAAAATCTG 9651 AGCATGTTTT ACCAATGATA CCAGCTGGAT TACACAATGA CGAAATGATT 9701 GGACTTCATT TTACTGATAA GAATGAGGAG ATAGATAATG CGTAGAATGA 9751 TTATCGCAAA ACTTCATAAC GTGACAGGAA TTATGAATCG ATTTACCGCC 9801 GTTCTCAATC GAAGGCAAGT GAACATTCTC TCAATTACCG CTGGAGTTAC 9851 AGAAAGTCAA GACTTAACTC ATACCACTTT TGTTATTGAA GTTGATCATC 9901 TTGATGAAGT AGAACAAATC ATTAAACAAT TAAATCGCTT SUBSTITUTE SHEET - 43 - AATAGATGTA 9951 ATTGAAGTAG CTGATATTAC TGATTTTCCT CATGTAGAAC GTGAAGTCGT 10001 CTTGATTAAA GTATCAGCTC CACCGACCAT TAGGGCAGAA ATTTTTACAA 10051 TGATTGAACC TTTTAGAGTA AATGTAGTTG ATGTCAATCT GGAAAATGTC 10101 ACCATTCAAT TAACGGGTGA TTCAGCAAAA ATCGAAGCAC TTATTGAGGT 10151 TGTTAGTCCT TATGGCATTC TAAATATGGC TCGGACAGGT AGTGCAGGTT 10201 TTGAGCGTGG CTAAATTTAA ATAAGTTAAC AAATAAATAG AAAAATAGAG 10251 GAAACAAAAA TGGCAGTTAC AATGTATTAT GAAGATGATG TAGAAGTATC 10301 AGCACTTGCT GGAAAGCAAA TTGCAGTAAT CGGTTATGGT TCACAAGGAC 10351 ATGCTCACGC ACAGAATTTG CGTGATTCTG GTCACAACGT TATCATTGGT 10401 GTGCGCCACG GAAAATCTTT TGATAAAGCA AAAGAAGATG GCTTTGAAAC 10451 ATTTGAAGTA GGAGAAGCAG TAGCTAAAGC TGATGTTATT ATGGTTTTGG 10501 CACCAGATGA ACTTCAACAA TCCATTTATG AAGAGGACAT CAAACCAAAC 10551 TTGAAAGCAG GTTCAGCACT TGGTTTTGCT CACGGATTTA ATATCCATTT 10601 TGGCTATATT AAAGTACCAG AAGACGTTGA CGTCTTTATG GTTGCGCCTA 10651 AGGCTCCAGG TCACCTTGTC CGTCGGACTT ATACTGAAGG TTTTGGTACA 10701 CCAGCTTTGT TTGTTTCACA CCAAAATGCA AGTGGTCATG CGCGTGAAAT 10751 CGCAATGGAT TGGGCCAAAG GAATTGGTTG TGCTCGAGTG GGAATTATTG 10801 AAACAACTTT TAAAGAAGAA ACAGAAGAAG ATTTGTTTGG AGAACAAGCT 10851 GTTCTATGTG GAGGTTTGAC AGCACTTGTT GAAGCCGGTT TTGAAACACT SUBSTITUTE SHEET - 44 - 10901 GACAGAAGCT GGATACGCTG GCGAATTGGC TTACTTTGAA GTTTTGCACG 10951 AAATGAAATT GATTGTTGAC CTCATGTATG AAGGTGGTTT TACTAAAATG 11001 CGTCAATCCA TCTCAAATAC TGCTGAGTTT GGCGATTATG TGACTGGTCC 11051 ACGGATTATT ACTGACGAAG TTAAAAAGAA TATGAAGCTT GTTTTGGCTG 11101 ATATTCAATC TGGAAAATTT GCTCAAGATT TCGTTGATGA CTTCAAAGCG 11151 GGGCGTCCAA AATTAATAGC CTATCGCGAA GCTGCAAAAA ATCTTGAAAT 11201 TGAAAAAATT GGGGCAGAGC ACGTCAAGCA ATGCCATTCA CACAATCTGG 11251 TGATGACGAT GCCTTTAAAA TCTATCAGTA ATTTCTCTTA TTGATTGAAC 11301 AAAAACATAA AAGCATTTTA TGGAGGAATG ACATAAATGA TAAGTGCCAA 11351 AGAGGTTGAA GATGCCTATG ATTTGTTAAA AGCAGTTGTC ACTAAAACAC 11401 CTTTACAATT AGACCCTTAC CTTTCCAATA AATATCAAGC AAATATTTAC 11451 TTAAAAGAAG TTGTCACTAA AACACCTTTA CAATTAGACC CTTACCTTTC 11501 CAATAAATAT CAAGCAAATA TTTACTTAAA AGAAGAAAAC TTACAGAAAG 11551 TTCGTTCTTT TAAATTACGA GGAGCTTATT ATTCTATCAG TAAATTATCT 11601 GATGAGCAAC GCTCTAAAGG AGTGGTTTGT GCCTCAGCAG GAAATCATGC 11651 ACAAGGGGTT GCTTTTGCTG CAAATCAATT AAATATTTCT GCGACAATTT 11701 TTATGCCCGT TACCACACCT AACCAAAAAA TTTCACAAGT TAAATTTTTT 11751 GGCGAAAGTC ACGTAACAAT TCGTTTAATT GGTGATACTT TTGATGAATC 11801 AGCCAGAGCA GCAAAAGCTT TTTCTCAAGA TAATGACAAA CCATTTATAG 11851 ACCCTTTTGA TGATGAAAAT GTAATTGCTG GTCAAGGGAC SUBSTITUTE SHEET - 45 - AGTGGCTTTA 11901 GAAATTTTTG CGCAAGCTAA AAAACAAGGA ATAAGTTTAG ATAAGATTTT 11951 TGTACAGATT GGTGGAGGTG GTTTAATTGC AGGAATTACT GCCTACAGTA 12001 AGGAGCGCTA TCCCCAAACT GAAATTATCG GAGTTGAAGC AAAAGGGGCA 12051 ACAAGTATGA AAGCTGCCTA CTCTGCTGGT CAGCCCGTCA CCTTGGAACA 12101 CATTGATAAA TTTGCTGACG GAATTGCGGT TGCGACTGTC GGTCAGAAAA 12151 CTTACCAACT TATTAATGAC AAAGTGAAAC AATTGCTTGC GGTTGATGAA 12201 GGTTTAATTT CTCAAACCAT ACTCGAATTG TATTCAAAAT TAGGAATTGT 12251 CGCCGAGCCA GCAGGTGCAA CATCTGTTGC CGCACTTGAA CTTATTAAAG 12301 ATGAAATCAA GGGTAAAAAT ATCGTCTGTA TCATCAGCGG CGGAAATAAT 12351 GATATTAGTC GAATGCAAGA AATTGAAGAA AGAGCTTTGG TTTATGAAGG 12401 TCTAAAACAT TATTTTGTCA TTAACTTTCC TCAAAGACCA GGATCCTTAC 12451 GAACTTTTGT CAGTGATATT TTAGGGCCAA ATGATGATAT CACCCGATTT 12501 GAGTACATCA AAAGGGCTGA TAAAGGTAAA GGACCTTGTC TTGTTGGGAT 12551 TTTACTTTCA GATGCTAGTG ATTATGATTC ATTGATTAAT CGGATTGAAA 12601 GATTTGATAA TCGTTATGTT AACTTACGTG GAAATGATAG TTTATACGAA 12651 CTTTTGGTCT AACTAACCAA TTGGTTTGAG CCATTTTCTA GTTTCAATTC 12701 TCTTTAAATC ACTAGAAATT ilvb Length: 1728 September 22, 1993 11:08 Type: N Check: 9885 1 ATGAAAAAAA TAAAGTTAGA AAAACCTACT TCCGGTTCCC AACTTGTTCT SUBSTITUTESHEET - 46 - 51 CCAAACCTTA AAAGAACTTG GAGTAGAAAT TATTTTTGGT TATCCTGGTG 101 GGGCCATGCT CCCCTTGTAT GATGCGATTC ATAATTTTGA AGGAATTCAA 151 CATATTTTAG CCCGTCATGA GCAAGGAGCA ACGCATGAAG CCGAAGGTTA 201 CGCTAAATCG TCTGGTAAAG TTGGTGTCGT CGTTGTTACG TCAGGACCAG 251 GAGCGACTAA TGCAGTAACC GGAATTGCTG ACGCTTATCT TGATTCAGTC 301 CCATTGTTAG TTTTCACAGG TCAAGTTGGC CGTCAGTCAA TTGGTAAAGA 351 TGCTTTTCAA GAAGCAGATA CTGTTGGAAT TACAGCCCCA ATTACAAAAT 401 ATAATTATCA AATTAGGGAA ACCGCAGATA TTCCAAGAAT TGTTACAGAA 451 GCCTATTATT TGGCAAGGAC AGGACGTCCT GGACCAGTAG AAATTGATTT 501 ACCAAAAGAT GTTTCCACCC TTGAAGTCAC TGAAATTAAT GACCCAAGCT 551 TGAATCTTCC TCATTATCAC GAAAGTGAAA AAGCGACTGA TGAACAATTG 601 CAAGAATTAC TGACAGAACT TTCTGTCAGT AAAAAACCAG TCATTATTGC 651 TGGCGGAGGA ATTAATTATT CTGGCTCAGT TGATATTTTC AGAGCATTTG 701 TCGAAAAATA TCAAATTCCA GTTGTTTCTA CATTGCTTGG CTTAGGAACA 751 TTACCAATCA GCCACGAATT GCAACTAGGA ATGGCAGGAA TGCACGGTTC 801 ATACGCTGCA AATATGGCTT TAGTTGAAGC TGACTATATT ATTAATTTGG 851 GATCACGTTT TGACGATAGA GTTGTATCCA ATCCTGCAAA ATTTGCTAAA 901 AATGCTGTCG TTGCTCATAT TGATATTGAC GCTGCTGAAC TTGGCAAAAT 951 TGTAAAAACC GATATTCCAA TCCTTTCTGA TTTGAAAGCG GCTTTAAGCA 1001 GACTTTTGCA ATTAAATAAG GTCAGGACTG ACTTTAATGA SUBSTITUTE SHEET - 47 - TTGGATTAAA 1051 ACTGTCATTG AAAATAAAGA GAAAGCACCA TTTACTTATG AGCCCCAAAA 1101 CCATGATATC CGTCCACAGG AAACAATTAA ATTAATTGGA GAATACACTC 1151 AAGGAGATGC AATCATTGTA ACTGACGTTG GGCAACATCA AATGTGGGTG 1201 GCGCAATATT ATCCTTATAA AAATGCAAGG CAACTTATTA CTTCTGGGGG 1251 AATGGGAACG ATGGGCTTTG GCATTCCTGC AGCAATCGGT GCAAAGCTGG 1301 CACAGCCAAA TAAAAATGTC ATTGTTTTTG TTGGCGATGG TGGCTTTCAA 1351 ATGACTAATC AAGAATTAGC ATTACTTAAT GGCTACGGTA TTGCAATCAA 1401 AGTTGTGCTG ATTAATAATC ATTCATTGGG AATGGTACGT CAATGGCAAG 1451 AATCATTCTA TGAAGAGCGA CGTTCACAAT CGGTTTTTGA TGTTGAACCC 1501 AATTTTCAAT TGTTAGCCGA AGCTTATGGC ATCAAACATG TTAAGTTAGA 1551 TAATCCAAAA ACTTTGGCTG ATGATTTAAA AATTATTACA GAAGATGAGC 1601 CAATGCTTAT TGAAGTTCTA ATTTCAAAAT CTGAGCATGT TTTACCAATG 1651 ATACCAGCTG GATTACACAA TGACGAAATG ATTGGACTTC ATTTTACTGA 1701 TAAGAATGAG GAGATAGATA ATGCGTAG ilvn Length: 477 September 22, 1993 11:09 Type: N Check: 6061 1 ATGCGTAGAA TGATTATCGC AAAACTTCAT AACGTGACAG GAATTATGAA 51 TCGATTTACC GCCGTTCTCA ATCGAAGGCA AGTGAACATT CTCTCAATTA 101 CCGCTGGAGT TACAGAAAGT CAAGACTTAA CTCATACCAC TTTTGTTATT 151 GAAGTTGATC ATCTTGATGA AGTAGAACAA ATCATTAAAC AATTAAATCG SUBSTITUTE SHEET - 48 - 201 CTTAATAGAT GTAATTGAAG TAGCTGATAT TACTGATTTT CCTCATGTAG 251 AACGTGAAGT CGTCTTGATT AAAGTATCAG CTCCACCGAC CATTAGGGCA 301 GAAATTTTTA CAATGATTGA ACCTTTTAGA GTAAATGTAG TTGATGTCAA 351 TCTGGAAAAT GTCACCATTC AATTAACGGG TGATTCAGCA AAAATCGAAG 401 CACTTATTGA GGTTGTTAGT CCTTATGGCA TTCTAAATAT GGCTCGGACA 451 GGTAGTGCAG GTTTTGAGCG TGGCTAA ilvbn Length: 2231 September 22, 1993 11:12 Type: N Check: 7704 1 ATTTATAGAA AGGTTTGAAT GAAAAAAATA AAGTTAGAAA AACCTACTTC 51 CGGTTCCCAA CTTGTTCTCC AAACCTTAAA AGAACTTGGA GTAGAAATTA 101 TTTTTGGTTA TCCTGGTGGG GCCATGCTCC CCTTGTATGA TGCGATTCAT 151 AATTTTGAAG GAATTCAACA TATTTTAGCC CGTCATGAGC AAGGAGCAAC 201 GCATGAAGCC GAAGGTTACG CTAAATCGTC TGGTAAAGTT GGTGTCGTCG 251 TTGTTACGTC AGGACCAGGA GCGACTAATG CAGTAACCGG AATTGCTGAC 301 GCTTATCTTG ATTCAGTCCC ATTGTTAGTT TTCACAGGTC AAGTTGGCCG 351 TCAGTCAATT GGTAAAGATG CTTTTCAAGA AGCAGATACT GTTGGAATTA 401 CAGCCCCAAT TACAAAATAT AATTATCAAA TTAGGGAAAC CGCAGATATT 451 CCAAGAATTG TTACAGAAGC CTATTATTTG GCAAGGACAG GACGTCCTGG 501 ACCAGTAGAA ATTGATTTAC CAAAAGATGT TTCCACCCTT GAAGTCACTG 551 AAATTAATGA CCCAAGCTTG AATCTTCCTC ATTATCACGA AAGTGAAAAA 601 GCGACTGATG AACAATTGCA AGAATTACTG ACAGAACTTT CTGTCAGTAA SUBSTITUTE SHEET - 49 - 651 AAAACCAGTC ATTATTGCTG GCGGAGGAAT TAATTATTCT GGCTCAGTTG 701 ATATTTTCAG AGCATTTGTC GAAAAATATC AAATTCCAGT TGTTTCTACA 751 TTGCTTGGCT TAGGAACATT ACCAATCAGC CACGAATTGC AACTAGGAAT 801 GGCAGGAATG CACGGTTCAT ACGCTGCAAA TATGGCTTTA GTTGAAGCTG 851 ACTATATTAT TAATTTGGGA TCACGTTTTG ACGATAGAGT TGTATCCAAT 901 CCTGCAAAAT TTGCTAAAAA TGCTGTCGTT GCTCATATTG ATATTGACGC 951 TGCTGAACTT GGCAAAATTG TAAAAACCGA TATTCCAATC CTTTCTGATT 1001 TGAAAGCGGC TTTAAGCAGA CTTTTGCAAT TAAATAAGGT CAGGACTGAC 1051 TTTAATGATT GGATTAAAAC TGTCATTGAA AATAAAGAGA AAGCACCATT 1101 TACTTATGAG CCCCAAAACC ATGATATCCG TCCACAGGAA ACAATTAAAT 1151 TAATTGGAGA ATACACTCAA GGAGATGCAA TCATTGTAAC TGACGTTGGG 1201 CAACATCAAA TGTGGGTGGC GCAATATTAT CCTTATAAAA ATGCAAGGCA 1251 ACTTATTACT TCTGGGGGAA TGGGAACGAT GGGCTTTGGC ATTCCTGCAG 1301 CAATCGGTGC AAAGCTGGCA CAGCCAAATA AAAATGTCAT TGTTTTTGTT 1351 GGCGATGGTG GCTTTCAAAT GACTAATCAA GAATTAGCAT TACTTAATGG 1401 CTACGGTATT GCAATCAAAG TTGTGCTGAT TAATAATCAT TCATTGGGAA 1451 TGGTACGTCA ATGGCAAGAA TCATTCTATG AAGAGCGACG TTCACAATCG 1501 GTTTTTGATG TTGAACCCAA TTTTCAATTG TTAGCCGAAG CTTATGGCAT 1551 CAAACATGTT AAGTTAGATA ATCCAAAAAC TTTGGCTGAT GATTTAAAAA 1601 TTATTACAGA AGATGAGCCA ATGCTTATTG AAGTTCTAAT SUBSTITUTE SHEET - 50 - TTCAAAATCT 1651 GAGCATGTTT TACCAATGAT ACCAGCTGGA TTACACAATG ACGAAATGAT 1701 TGGACTTCAT TTTACTGATA AGAATGAGGA GATAGATAAT GCGTAGAATG 1751 ATTATCGCAA AACTTCATAA CGTGACAGGA ATTATGAATC GATTTACCGC 1801 CGTTCTCAAT CGAAGGCAAG TGAACATTCT CTCAATTACC GCTGGAGTTA 1851 CAGAAAGTCA AGACTTAACT CATACCACTT TTGTTATTGA AGTTGATCAT 1901 CTTGATGAAG TAGAACAAAT CATTAAACAA TTAAATCGCT TAATAGATGT 1951 AATTGAAGTA GCTGATATTA CTGATTTTCC TCATGTAGAA CGTGAAGTCG 2001 TCTTGATTAA AGTATCAGCT CCACCGACCA TTAGGGCAGA AATTTTTACA 2051 ATGATTGAAC CTTTTAGAGT AAATGTAGTT GATGTCAATC TGGAAAATGT 2101 CACCATTCAA TTAACGGGTG ATTCAGCAAA AATCGAAGCA CTTATTGAGG 2151 TTGTTAGTCC TTATGGCATT CTAAATATGG CTCGGACAGG TAGTGCAGGT 2201 TTTGAGCGTG GCTAAATTTA AATAAGTTAA C SUBSTITUTE SHEET

Claims: - 51 - CLAIMS 1. Nucleic acid, characterized in that it comprises all or part or an analogue of the DNA sequence bounded by the nucleotides located at positions 8018 and 10211 of Figure 2, and in that it codes for one or both subunits of a protein capable of converting pyruvate to α-acetolactate. 2. Nucleic acid according to Claim 1, characterized in that it comprises all or part or an analogue of the DNA sequence bounded by the nucleotides located at positions 8018 and 9742, and in that it codes for a subunit of a protein capable of converting pyruvate to α-acetolactate. 3. Nucleic acid according to Claim 1, characterized in that it comprises all or part or an analogue of the DNA sequence bounded by the nucleotides located at positions 9738 and 10211 of Figure 2, and in that it codes for a subunit of a protein capable of converting pyruvate to α-acetolactate. 4. Nucleic acid according to Claim l, characterized in that it comprises the DNA sequence bounded by the nucleotides located at positions 8018 and 9742 and the DNA sequence bounded by the nucleotides located at positions 9738 and 10211, and in that it codes for both subunits of a protein capable of converting pyruvate to α-acetolactate. 5. Nucleic acid, characterized in that it comprises at least one nucleotide sequence according to one of Claims 1 to 4, combined with at least one promoter under the control of which transcription of the said sequence is capable of being implemented and, where appropriate, at least one DNA sequence coding for one or more transcrip¬ tion termination signals. 6. Recombinant vector capable of transforming a host cell, the said vector containing at least one nucleic acid according to one of Claims 1 to 5, under the control of regulatory elements permitting expression of this nucleic acid in the host cell. 7. Method for the transformation of a microorganism, in particular of the type capable of being SUBSTITUTESHEET - 52 - used as leaven, characterized in that at least one nucleic acid according to Claim 5 is introduced directly into the DNA of the said microorganism. 8. Method for the transformation of a microorganism, in particular of the type capable of being used as leaven, characterized in that at least one recombinant vector according to Claim 6 is introduced into the said microorganism. 9. Microorganism, in particular of the type capable of being used as leaven, characterized in that it is transformed with at least one nucleic acid according to one of Claims 1 to 5. 10. Microorganism according to Claim 9, characterized in that its genes coding for lactate dehydrogenase and/or α-acetolactate decarboxylase are inactivated. 11. Protein possessing α-acetolactate synthase activity, characterized in that it consists of a first polypeptide comprising all or part of the polypeptide sequence of Figure 2 encoded by the nucleic acid according to Claim 2, and a second polypeptide comprising all or part of the polypeptide sequence of Figure 2 encoded by the nucleic acid according to Claim 3. SUBSTITUTE SHEET

Inventors: EHRLICH STANISLAV, GODON JEAN-JACQUES, RENAULT PIERRE, EHRLICH, STANISLAV, GODON, JEAN-JACQUES, RENAULT, PIERRE
Applicants: AGRICULTURAL & FOOD RES, EHRLICH STANISLAV, GODON JEAN JACQUES, RENAULT PIERRE, AGRICULTURAL AND FOOD RESEARCH COUNCIL
Application year: 1993
Main classification: C12N15
IPC codes: C12N1, C12N9, C12N15, C12P7