P EF-TU expression units
View Patent ↗The present invention relates to the use of nucleic acid sequences for regulating the transcription and expression of genes, the novel promoters and expression units themselves, methods for altering or causing the transcription rate and/or expression rate of genes, expression cassettes comprising the expression units, genetically modified microorganisms with altered or caused transcription rate and/or expression rate, and methods for preparing biosynthetic products by cultivating the genetically modified microorganisms.
1. A method for preparing lysine by cultivating a genetically modified Corynebacterium glutamicum comprising
(1) a nucleic acid molecule having promoter activity, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:1; or
(2) an expression unit comprising either
a) the nucleic acid molecule of any 1 wherein said nucleic acid molecule is functionally linked to a nucleic acid sequence which ensures the translation of ribonucleic acids, or
b) the nucleic acid molecule of SEQ ID NO:2;
wherein the nucleic acid molecule of (1) or the expression unit of (2) alters, regulates or causes the expression of at least one endogenous gene, compared with the wild type;
and wherein the at least one endogenous gene is selected from the group consisting of nucleic acids encoding an aspartate kinase, nucleic acids encoding an aspartate-semialdehyde dehydrogenase, nucleic acids encoding a diaminopimelate dehydrogenase, nucleic acids encoding a diaminopimelate decarboxylase, nucleic acids encoding a dihydrodipicolinate synthetase, nucleic acids encoding a dihydrodipicolinate reductase, nucleic acids encoding a glyceraldehyde-3-phosphate dehydrogenase, nucleic acids encoding a 3-phosphoglycerate kinase, nucleic acids encoding a pyruvate carboxylase, nucleic acids encoding a triosephosphate isomerase, nucleic acids encoding a transcriptional regulator LuxR, nucleic acids encoding a transcriptional regulator LysR1, nucleic acids encoding a transcriptional regulator LysR2, nucleic acids encoding a malate-quinone oxidoreductase, nucleic acids encoding a glucose-6-phosphate dehydrogenase, nucleic acids encoding a 6-phosphogluconate dehydrogenase, nucleic acids encoding a transketolase, nucleic acids encoding a transaldolase, nucleic acids encoding a lysine exporter, nucleic acids encoding a biotin ligase, nucleic acids encoding an arginyl-tRNA synthetase, nucleic acids encoding a phosphoenolpyruvate carboxylase, nucleic acids encoding a fructose-1,6-bisphosphatase, nucleic acids encoding a protein OpcA, nucleic acids encoding a 1-phosphofructokinase and nucleic acids encoding a 6-phosphofructokinase.
2. The method of claim 1 , wherein the genetically modified Corynebacterium glutamicum has an increased activity, as compared with the wild type, of at least one activity selected from the group consisting of aspartate kinase activity, aspartate-semialdehyde dehydrogenase activity, diaminopimelate dehydrogenase activity, diaminopimelate decarboxylase activity, dihydrodipicolinate synthetase activity, dihydrodipicolinate reductase activity, glyceraldehyde-3-phosphate dehydrogenase activity, 3-phosphoglycerate kinase activity, pyruvate carboxylase activity, triosephosphate isomerase activity, activity of the transcriptional regulator LuxR, activity of the transcriptional regulator LysR1, activity of the transcriptional regulator LysR2, malate-quinone oxidoreductase activity, glucose-6-phosphate deydrogenase activity, 6-phosphogluconate dehydrogenase activity, transketolase activity, transaldolase activity, lysine exporter activity, arginyl-tRNA synthetase activity, phosphoenolpyruvate carboxylase activity, fructose-1,6-bisphosphatase activity, protein OpcA activity, 1-phosphofructokinase activity, 6-phosphofructokinase activity and biotin ligase activity.
3. The method of claim 1 or 2 , wherein the genetically modified Corynebacterium glutamicum has a reduced activity, as compared with the wild type, of at least one activity selected from the group consisting of threonine dehydratase activity, homoserine O-acetyltransferase activity, O-acetylhomoserine sulfhydrylase activity, phosphoenolpyruvate carboxykinase activity, pyruvate oxidase activity, homoserine kinase activity, homoserine dehydrogenase activity, threonine exporter activity, threonine efflux protein activity, asparaginase activity, aspartate decarboxylase activity and threonine synthase activity.
4. The method of claim 1 or 2 , wherein the lysine is isolated from the cultivation medium.
5. The method of claim 4 , wherein the lysine is purified.
6. The method of claim 3 , wherein the lysine is isolated from the cultivation medium.
7. The method of claim 6 , wherein the lysine is purified.
8. The method of claim 1 , wherein the nucleic acid molecule of (1) or the expression unit of (2) alters the expression of the at least one gene.
9. The method of claim 1 , wherein the nucleic acid molecule of (1) or the expression unit of (2) regulates the expression of the at least one gene.
10. The method of claim 1 , wherein the nucleic acid molecule of (1) or the expression unit of (2) causes the expression of the at least one gene.
11. The method of claim 1 , wherein the nucleic acid molecule having promoter activity consists of the nucleotide sequence of SEQ ID NO:1.
12. The method of claim 1 , wherein the expression unit consists of the nucleotide sequence of SEQ ID NO:2.