Overexpression of the lys1 gene in Penicillium chrysogenum: homocitrate synthase levels, alpha-aminoadipic acid pool and penicillin production

Homocitrate synthase activity (encoded by the lys1 gene) catalyzes the firs t step of the lysine and penicillin pathway and is highly sensitive to feed back regulation by L-lysine. The transcript levels of the lys1 gene and the homocitrate synthase activity are high during the growth phase and decreas e during the antibiotic production phase, except in the high penicillin pro ducer strain AS-P-99 which maintained high levels of homocitrate synthase a ctivity in cultures at 96 h and 120 h. The lys1 gene was overexpressed in P enicillium chrysogenum using additional copies of lys1 with its own promote r or under the control of the pcbC promoter in either autonomously replicat ing or integrative vectors. Transformants containing 3 to 32 additional cop ies of the lys1 gene were selected. Some of these transformants, particular ly TI-C4 (integrative) and TAR-L9 (with autonomously replicating plasmids) showed very high levels of lys1 transcript and, in the case of TAR-L9, high levels of homocitrate synthase activity in cultures of 120 h. However, the se transformants did not show increased alpha-aminoadipate or lysine pools. A mutant P. chrysogenum L(-)G(-) disrupted in the lys2 gene (therefore lac king the lysine branch of the pathway) showed increased a-aminoadipate leve ls and produced higher levels of penicillin than non-disrupted control stra ins. Overexpression of the lys1 gene in the L-G- mutant resulted in high ho mocitrate synthase levels but no additional increase of the a-aminoadipate pool or penicillin production levels. These results suggest that after ampl ification of the homocitrate synthase levels there are other limiting steps in the common stem of the lysine and penicillin pathways.