PURPOSE: To obtain the subject gene DNA, isolated from a coryneform bacterium such as Brevibacterium.flavum MJ-233, having a specific base sequence and capable of providing a transformant remarkably improved in productivity of L-isoleucine, L-valine, etc.
CONSTITUTION: Brevibacterium.flavum MJ-233 which is a corynebacterium is cultured in a culture medium till the latter period of the logarithmic growth phase and the microbial cell is collected and suspended in a buffer solution containing a lysozyme. Protenase K(R) and sodium dodecyl sulfate are then added to carry out the lysis. The resultant lysate is subsequently extracted with a phenol/chloroform solution. Ethanol is added to the extract solution to recover a DNA, which is then treated with a restriction enzyme, bound to a cloning vector and inserted into Escherichia coli to perform transformation. The obtained transformant is subsequently cloned to sort out a positive clone. A plasmid is recovered from the obtained strain and treated with a restriction enzyme to afford the objective gene DNA, capable of coding an acetohydroxy acid synthase derived from the coryneform bacterium and expressed by the formula, etc.
KOBAYASHI MIKI
YUGAWA HIDEAKI
Next Patent: HYBRID-PLASMID FOR EXPRESSION OF 38K DALTON ANTIGEN OF MYCOBACTERIUM TUBERCULOSIS, ESCHERICHIA COLI ...