Brakhage A A, Browne P, Turner G
Lehrstuhl für Mikrobiologie, Universität München, Germany.
Mol Gen Genet. 1994 Jan;242(1):57-64. doi: 10.1007/BF00277348.
To analyse the regulation of the biosynthesis of the secondary metabolite penicillin in Aspergillus nidulans, a strain with an inactivated acvA gene produced by targeted disruption was used. acvA encodes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), which catalyses the first step in the penicillin biosynthetic pathway. To study the effect of the inactivated acvA gene on the expression of acvA and the second gene, ipnA, which encodes isopenicillin N synthase (IPNS), A. nidulans strain XEPD, with the acvA disruption, was crossed with strain AXB4A carrying acvA-uidA and ipnA-lacZ fusion genes. Ascospores with the predicted non-penicillin producing phenotype and a hybridization pattern indicating the presence of the disrupted acvA gene, and the fusion genes integrated in single copy at the chromosomal argB locus were identified. Both fusion genes were expressed at the same level as in the non-disrupted strain. Western blot analysis (immunoblotting) revealed that similar amounts of IPNS enzyme were present in both strains from 24 to 68 h of a fermentation run. In the acvA disrupted strain, IPNS and acyl-CoA: 6-aminopenicillanic acid acyltransferase (ACT) specific activities were detected, excluding a sequential induction mechanism of regulation of the penicillin biosynthesis gene ipnA and the third gene aat.
为了分析构巢曲霉中次生代谢产物青霉素生物合成的调控机制，使用了一株通过靶向破坏产生的acvA基因失活的菌株。acvA编码δ-（L-α-氨基己二酰基）-L-半胱氨酰-D-缬氨酸合成酶（ACVS），它催化青霉素生物合成途径的第一步。为了研究失活的acvA基因对acvA以及第二个基因ipnA（编码异青霉素N合成酶，IPNS）表达的影响，将携带acvA-uidA和ipnA-lacZ融合基因的AXB4A菌株与acvA基因被破坏的构巢曲霉XEPD菌株进行杂交。鉴定出具有预测的不产青霉素表型且杂交模式表明存在被破坏的acvA基因以及在染色体argB位点单拷贝整合的融合基因的子囊孢子。两个融合基因的表达水平与未被破坏的菌株相同。蛋白质免疫印迹分析（免疫印迹法）显示，在发酵运行24至68小时期间，两种菌株中存在相似量的IPNS酶。在acvA基因被破坏的菌株中，检测到了IPNS和酰基辅酶A：6-氨基青霉烷酸酰基转移酶（ACT）的比活性，排除了青霉素生物合成基因ipnA和第三个基因aat的顺序诱导调控机制。
