Quantitative analysis of Penicillium chrysogenum Wis54-1255 transformants overexpressing the penicillin biosynthetic genes

The low penicillin-producing, single gene copy strain Wis54-1255 was used t o study the effect of overexpressing the penicillin biosynthetic genes in P enicillium chrysogenum. Transformants of Wis54-1255 were obtained with the amdS expression-cassette using the four combinations: pcbAB, pcbC, pcbC-pen DE, and pcbAB-pcbC-penDE of the three penicillin biosynthetic genes. Transformants showing an increased penicillin production were investigated during steady-state continuous cultivations with glucose as the growth-limi ting substrate. The transformants were characterized with respect to specif ic penicillin productivity, the activity of the two pathway enzymes delta-( L-alpha -aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicil lin N synthetase (IPNS) and the intracellular concentration of the metaboli tes: 6-(L-ol-aminoadipyl)-L-cysteinyl-D-valin (ACV), bis delta-(L-D-aminoad ipyl)-L-cysteinyl-D-valin (bisACV), isopenicillin N (IPN), glutathione (GSH ), and glutathione disulphide (GSSG). Transformants with the whole gene clu ster amplified showed the largest increase in specific penicillin productiv ity (r(p))-124% and 176%, respectively, whereas transformation with the pcb C-penDE gene fragment resulted in a decrease in r(p) of 9% relative to Wis5 4-1255. A marked increase in r(p) is clearly correlated with a balanced amp lification of both the ACVS and IPNS activity or a large amplification of e ither enzyme activity. The increased capacity of a single enzyme occurs sur prisingly only in the transformants where all the three biosynthetic genes are overexpressed but is not found within the group of pcbAB or pcbC transf ormants. The indication of the pcbAB and pcbC genes being closely regulated in fungi might explain why high-yielding strains of P. chrysogenum have be en found to contain amplifications of a large region including the whole pe nicillin gene cluster and not single gene amplifications. Measurements of t he total ACV concentration showed a large span of variability, which reflec ted the individual status of enzyme overexpression and activity found in ea ch strain. The ratio ACV:bisACV remained constant, also at high ACV concent rations, indicating no limitation in the capacity of the thioredoxin-thiore doxin reductase (TR) system, which is assumed to keep the pathway intermedi ate LLD-ACV in its reduced state. The total GSH pool was at a constant leve l of approx. 5.7 mM in all cultivations. (C) 2001 John Wiley & Sons, Inc.