Patent ID: 6599745
Filing Date: 2003-07-29
Classification: C07K,C12N,C12Q,C12Y

Abstract:
A method for preparing and selecting a mutant strain of a microorganism, wherein said mutant strain possesses a mutation that enhances a predetermined part of metabolism in comparison to a strain of said microorganisms not possessing said mutation, said method comprising the following steps:(a) introducing into a microorganism a nucleic acid cassette comprising: (i) a nucleic acid sequence encoding a bidirectional marker; (ii) a basic transcriptional unit operatively linked to the nucleic acid sequence encoding the bidirectional marker; and (iii) an inducible enhancer or activator sequence linked to the basic transcriptional unit in such a manner that upon induction of said inducible enhancer or activator sequence the bidirectional marker is expressed, said inducible enhancer or activator sequence being an inducible enhancer or activator sequence of a gene associated with metabolic activity, wherein said microorganism does not exhibit the phenotype associated with expression of the bidirectional marker prior to introduction of the nucleic acid cassette into said microorganism;(b) culturing the microorganism resulting from step (a) under conditions wherein the inducible enhancer or activator sequence of the nucleic acid cassette is active, and under conditions wherein the bidirectional marker is expressed; (c) selecting a transformant that exhibits the phenotype corresponding to expression of the bidirectional marker gene under the culturing conditions of step (b); (d) subjecting the transformant selected in step (c) to mutagenesis; (e) culturing the mutagenized transformant of step (d) under conditions acceptable for a strain with a phenotype corresponding to expression of the bidirectional marker and under conditions that in a non-mutated transformant of step (c) result in non-expression of the bidirectional marker, and in the presence of a metabolisable substrate for the predetermined part of metabolism; and (f) selecting a strain resulting from the culturing step (e) that exhibits a phenotype corresponding to expression of the bidirectional marker gene under selection conditions that for a non-mutated transformant of step (c) result in non-expression of the bidirectional marker.