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Two strategies have been used for targeted integration at the lys2 locus of Penicillium chrysogenum. In the first strategy the disruption of lys2 was obtained by a single crossing over between the endogenous lys2 and a fragment of the same gene located in an integrative plasmid. lys2-disrupted mutants were obtained with 1.6% efficiency when the lys2 homologous region was 4.9 kb, but no homologous integration was observed with constructions containing a shorter homologous region. Similarly, lys2-disrupted mutants were obtained by a double crossing over (gene replacement) with an efficiency of 0.14% by using two lys2 homologous regions of 4.3 and 3.0 kb flanking the pyrG marker. No homologous recombination was observed when the selectable marker was flanked by short lys2 homologous DNA fragments. The disruption of lys2 was confirmed by Southern blot analysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. The lys2-disrupted mutants lacked alpha-aminoadipate reductase activity (encoded by lys2) and showed specific penicillin yields double those of the parental nondisrupted strain, Wis 54-1255. The alpha-aminoadipic acid precursor is channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at the alpha-aminoadipate reductase level.

Gene Targeting inPenicillium chrysogenum: Disruption of thelys2Gene Leads to Penicillin Overproduction