Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Patent
1995-07-21
1997-07-01
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
435 74, 4352523, 43525233, 4352528, 4352529, 4353201, C12N 988
Patent
active
056437790
DESCRIPTION:
BRIEF SUMMARY
This application is the U.S. national stage application of PCT International Application No. PCT/GB93/02012, filed 27 Sep. 1993, which claims priority of French Patent Application 92/11470, filed 25 September 1992.
The invention relates to a nucleic acid coding for an .alpha.-acetolactate synthase, as well as to vectors containing this nucleic acid and to the use of these vectors for transforming microorganisms in which the production of .alpha.-acetolactate and diacetyl will be promoted.
Diacetyl is a flavouring sought in the production of food products such as butter, creme fraiche 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.
.alpha.-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 characterized in Escherichia coli (3), Salmonella typhimurium (3), Bacillus subtilis (5, 6, 7), Corynebacterium typhimurium (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 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. glutamicum 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-CD, 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 NCDO2118 (AFRC, Institute of Food Research Reading Laboratory, Shinfield, Reading, Berks, United Kingdom). The genes were characterized by cloning, complementation in E. coli and B. subtilis and sequence analysis. Nine structural genes are assembled on a 12-kb DNA fragment in the following order: leuABCDilvDBNCA.
Among these genes, the invention relates to nucleic acid sequence coding
REFERENCES:
patent: 5420021 (1995-05-01), Marugg et al.
Starrenburg et al., "Citrate Fermentation by Lactococcus and Leuconostoc spp.," Applied and Environmental Microbiology, 57(12):3535-3540 (Dec. 1991).
Snoep et al., "Isolation, Characterization, and Physiological Role of the Pyruvate Dehydrogenase Complex and .alpha.-Acetolactate Synthase of Lactococcus lactis subsp. lactis bv. diacetylactis," Journal of Bacteriology, 174(14):4838-4841 (Jul. 1992).
Zahler et al., ".alpha.-Acetolactate Synthesis by Bacillus subtilis," Biosynth. Branched Chain Amino Acids, Proc. Workshop 1988, pp. 25-32 (1990).
Godon et al. "Branched-Chain Amino Acid Biosynth. Gene in . . . " J. Bacteriol. 174, 6580-6589. Oct. 1992.
Hugenholtz et al. "Diacetyl production by different . . . " Appl. Microbiol. Biotechnol. 38, 17-22. 1992.
Ehrlich Stanislav
Godon Jean-Jacques
Renault Pierre
Biotechnology and Biological Sciences Research Council
Nashed Nashaat T.
Wax Robert A.
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