Gene targeting in Penicillium chrysogenum: Disruption of the lys2 gene leads to penicillin overproduction

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 fragme nt of the same gene located in an integrative plasmid, lys2-disrupted mutan ts were obtained with 1.6% efficiency when the lys2 homologous region was 4 .9 kb, but no homologous integration was observed with constructions contai ning a shorter homologous region. Similarly, lys2-disrupted mutants were ob tained 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 py rG marker. No homologous recombination was observed when the selectable mar ker was flanked by short lys2 homologous DNA fragments, The disruption of l ys2 was confirmed by Southern blot analysis of three different lysine auxot rophs obtained by a single crossing over or gene replacement. The lys2-disr upted mutants lacked alpha-aminoadipate reductase activity (encoded by lys2 ) and showed specific penicillin yields double those of the parental nondis rupted strain, Wis 54-1255, The cw-aminoadipic acid precursor is channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at t he alpha-aminoadipate reductase level.