Inducible high expression system
View Patent ↗The present invention provides a vector containing a DNA construct containing a first promoter that is a nitrilase gene promoter, a multicloning site and a nitrilase transcription regulatory protein gene (particularly preferably a structural gene is ligated into a multicloning site and a second promoter is placed at the upstream of the nitrilase transcription regulatory protein gene), and the like. The present invention affords a high expression system capable of expressing a large amount of useful proteins by actinomycete host represented by actinomycete belonging to the genus Streptomyces and the like beneficially used for the production of useful proteins.
1. A DNA construct comprising a first promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, operably linked to a nitrilase gene from Rhodococcus rhodochrous J 1 and a second promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, wherein the second promoter is placed upstream of a nitrilase transcription regulatory protein gene from Rhodococcus rhodochrous J 1.
2. A DNA construct comprising a first promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, operably linked to a cloning or multi-cloning site and a second promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, wherein the second promoter is placed upstream of a nitrilase transcription regulatory protein gene from Rhodococcus rhodochrous J 1.
3. The DNA construct of claim 1 , further comprising a terminator region upstream of the first promoter and/or the second promoter.
4. A vector comprising both the DNA construct of claim 1 , and a DNA involved in a regulatory function of intracellular autonomous replication of actinomycete.
5. The vector of claim 4 , further comprising a DNA involved in a regulatory function of intracellular autonomous replication of a bacterial cell other than actinomycete.
6. The vector of claim 4 , wherein the actinomycete belongs to a genus other than the genus Rhodococcus.
7. The vector of claim 4 , wherein the actinomycete belongs to the genus Streptomyces.
8. The vector of claim 5 , wherein the bacterial cell other than actinomycete is Eseherichia coli.
9. A transformant transformed with the vector of claim 4 .
10. A transformant transformed with the vector of claim 5 .
11. A transformant belonging to actinomycete, which has been transformed with a vector comprising both a DNA construct comprising a first promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, operably linked to a structural gene and a second promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, wherein the second promoter is placed upstream of the nitrilase transcription regulatory protein gene from Rhodococcus rhodochrous J 1, and a DNA involved in a regulatory function of intracellular autonomous replication of actinomycete.
12. A transformant belonging to actinomycete, into which a DNA construct comprising a first promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, operably linked to a structural gene and a second promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, wherein the second promoter is placed upstream of the nitrilase transcription regulatory protein gene from Rhodococcus rhodochrous J 1, has been introduced.
13. A structural gene expression system which comprises expression of a vector comprising both a DNA construct comprising a first promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, operably linked to the structural gene and a second promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, wherein the second promoter is placed upstream of the nitrilase transcription regulatory protein gene from Rhodococcus rhodochrous J 1 and a DNA involved in a regulatory function of intracellular autonomous replication of actinomycete, under the control of an inducer in an actinomycete host.
14. A structural gene expression system which comprises expression of the structural gene in an actinomycete host, into which a DNA construct comprising a first promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, operably linked to the structural gene and a second promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, wherein the second promoter is placed upstream of the nitrilase transcription regulatory protein gene from Rhodococcus rhodochrous J 1, has been introduced, under the control of an inducer.
15. A production method of a gene product encoded by a structural gene, which comprises expression of a vector comprising both a DNA construct comprising a first promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, operably linked to the structural gene and a second promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, wherein the second promoter is placed upstream of the nitrilase transcription regulatory protein gene from Rhodococcus rhodochrous J 1 and a DNA involved in a regulatory function of intracellular autonomous replication of actinomycete, in an actinomycete under the control of an inducer.
16. A production method of a gene product encoded by a structural gene, which comprises expression, under the control of an inducer, of the structural gene in a host belonging to actinomycete into which a DNA construct comprising a first promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, operably linked to the structural gene and a second promoter that is a nitrilase gene promoter consisting of the sequence of SEQ ID NO: 15, wherein the second promoter is placed upstream of the nitrilase transcription regulatory protein gene from Rhodococcus rhodochrous J 1, has been introduced.
17. The DNA construct of claim 2 , further comprising a terminator region upstream of the first promoter and/or the second promoter.
18. A vector comprising both the DNA construct of claim 2 , and a DNA involved in a regulatory function of intracellular autonomous replication of actinomycete.
19. The vector of claim 18 , further comprising a DNA involved in a regulatory function of intracellular autonomous replication of a bacterial cell other than actinomycete.
20. The vector of claim 18 , wherein the actinomycete belongs to a genus other than the genus Rhodococcus.
21. The vector of claim 18 , wherein the actinomycete belongs to the genus Streptomyces.
22. The vector of claim 19 , wherein the bacterial cell other than actinomycete is Escherichia coli.
23. A transformant transformed with the vector of claim 18 .
24. A transformant transformed with the vector of claim 19 .
25. The transformant of claim 11 , wherein the structural gene is a nitrilase gene.
26. The transformant of claim 12 , wherein the structural gene is a nitrilase gene.
27. The system of claim 13 , wherein the structural gene is a nitrilase gene.
28. The system of claim 14 , wherein the structural gene is a nitrilase gene.
29. The production method of claim 15 , wherein the structural gene is a nitrilase gene.
30. The system of claim 13 , wherein the actinomycete belongs to the genus Streptomyces.
31. The system of claim 14 , wherein the actinomycete belongs to the genus Streptomyces.
32. The method of claim 15 , wherein the actinomycete belongs to the genus Streptomyces.
33. The method of claim 16 , wherein the actinomycete belongs to the genus Streptomyces.