CS270751B1 - Recombinant Plaamides pSA2O and pXT2, encoding the synthesis of alpha amylase alpha starch fluid in streptomycetes - Google Patents

Abstract

The solution concerns recombined plasmids pSA2o and pXT2 encoding the synthesis of starch liquefying alpha amylase in streptomycetes, which are characterized by the presence of a 3.2 kb restriction fragment in Streptomyces limosus for plasmid pSA2o and the presence of a single intervention site for the restriction endonuclease EcoRI created on the basis of the cleavage of the 2 kb EcoRI restriction fragment from plasmid pSA2o and restriction maps. Both plasmids were prepared by the in vitro gene manipulation technique. In addition to the production of alpha amylase, the recombined plasmids pSA2o and pXT2 encode the resistance of streptomycetes cells to the antibiotic thiostrepton. Based on the restriction maps of these plasmids, it is possible to isolate the genetic information for the synthesis of alpha amylase from the recombined plasmids and transfer it to other streptomycetes replicons. Plasmid pSA2o is preserved in the Streptomyces lividans AS2o strain, which is deposited in the Czechoslovak Collection of Microorganisms of the J. E. Purkyně University in Brno under the number CCM 4oo9. Plasmid pXT2, which is preserved in the Streptomyces lividans AS2 strain, is deposited in the Czechoslovak Collection of Microorganisms of the J. E. Purkyně University in Brno under the number CCM 4ol2.

Description

The invention ee relates to recombinant plasmids pSA20 and pXT2 encoding the synthesis of starch liquefying α-anylase in a streptonycete cell.

Streptomycetes produce a number of exoenzymes in the surrounding medium. It is therefore advantageous to use them as host cells in the cloning of genetic information encoding the synthesis of α-anylase, which is an industrially important enzyme. The source of genetic information for cloning a functional α-awylase gene in streptomycetes was, in the only successful work so far, the chromosome of the Streptomyces hygroscopicus strain. Cloning was performed in plasmids pIJ7o2 (McKillop et al. FEMS Microbiol. Lett. 36: 3, 1986). The resulting plasmid encoding d-amylase synthesis is 8.6 kb in size and the size of the chromosome insert carrying the α-amylase gene alone is 2.8 kb.

The invention relates to recombinant plasmids pSA20 and pXT2 encoding the synthesis of starch liquefying alpha amylase in streptomycetes characterized by the presence of a 3.2 kb restriction fragment from Streptomyces limosus for plasmid pSA20 and the presence of a single EcoRI restriction endonuclease restriction site. 2 kb in size from plasmids pSA20.

Construction of recombinant plasmids pSA20 and pXT2 encoding α-amylase synthesis

Fragments of the α-amylase-producing strain of Streptomyces limosus and the plasmid vector pIJ622 were used to construct new genetic information encoding α-amylase synthesis in a streptomycete cell. The resulting recombinant plasmids pSA20 and pXT2 encoding the synthesis of α-amylase starch liquefying are 9 kb and 7 kb in size. In both described embodiments, the incorporation of the α-amylase gene into the plasmid vector resulted in an increase in α-amylase production due to the increased number of α-amylase gene due to the high copy number of recombinant plasmids pSA20 and pXT2 in the streptomycete host.

Own construction of recombinant plasmids pSA2o and pXT2

The 3.2 kb Sau3AI fragment encoding α-amylase synthesis was ligated into the multicopy plasmid vector pIJ622 at its single BglII site. The resulting plasmid pSA20 transformed into a streptomycete host, strain Streptomyces lividans TK24, encodes α-amylase eynthesis, which leads to overproduction of α-amylase by this strain due to the increased copy number (4-6o) of the amylase gene. pSA20 has the same restriction map with the previously constructed plasmid pSA28 between the PvuII and KpnI restriction sites located in the inserted chromosomal DNA, but differs in the terminal regions of the insert.

Because insertion into the EcoRI site located in the cloned chromosomal DNA fragment in the recombinant plasmid pSA2o was found not to affect the synthesis of plasmid-encoded α-amylase, plasmid pSA2o was truncated by 2.0 kb by digesting the EcoRI-EcoRI restriction fragment and resealing the reduced T4 DNA. ligase. The newly generated plasmid pXT2 is 7 kb in size and averages the number of copies in one streptomycete cell (measured per chromosome equivalent). Plasmid pXT2 inserted into recipient protoplasts of Streptomyces lividans TK24 encodes intensive α-amylase synthesis by a streptomycete host. The stability of the pXT2 plasmids in the host cell is identical to the stability of the starting pSA20 plasmids and reaches -99% in cultures without selection pressure.

Plasmids pSA20 and pXT2 encoded by α-amylase have a molecular weight of 48 kb and the final starch cleavage products by this enzyme are oligosaccharides.

Illustrations of the construction of the recombinant plasmids pSA20 and pXT2 are shown in Figures 1 and 2, where the chromosomal DNA insert of the S. limosus strain encoding α-amylase production and the weakly plasmid vector DNA are marked.

Following the introduction of plasmids pSA20 into the host strain Streptomyces lividans TK24, a new strain of Streptomyces lividans AS20 was created, showing as new properties the presence of DNA molecules of plasmids pSA20, the ability to synthesize alpha amylase and the resistance of cells to thiostrepton.

CS 27o751 Bl

The lividane AS2 strain was generated by transferring plasmid pXT2 to the Streptomyces lividane TK24 host strain. The cells of the S. lividane AS2 strain contain the plasmid pXT2, which has a characteristic molecular weight and encodes alpha amylase synthesis and thiostrepton resistance in the cell of the host strain.

Both plasmids pSA2o and pXT2 are stored in Streptomyces lividane strains AS2o and AS2 stored in the Czechoslovak collection of microorganisms of the J. E. Purkyně University in Brno under the numbers CCM 4oo9 and CCM 4ol2.

Claims (1)

OBJECT OF THE INVENTION Recombinant plasmids pSA20 and pXT2, encoding the synthesis of starch liquefying alpha amylase in streptomycetes, characterized by the presence of a 3.2 kt restriction fragment from Streptomyces limosua for plasmid pSA20 and the presence of a single EcoRI restriction endonuclease restriction site. 2 kb from plasmid pSA20 and restriction maps and maintained in Streptomyces lividans AS20 CCM 4oo9 and Streptomyces lividane CCM 4ol2 strains.