Liu G, Casqueiro J, Bañuelos O, Cardoza R E, Gutiérrez S, Martín J F
Area of Microbiology, Faculty of Biology, University of León, 24071 León, Spain.
J Bacteriol. 2001 Mar;183(5):1765-72. doi: 10.1128/JB.183.5.1765-1772.2001.
Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-gamma-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH(2)) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-gamma-lyase.
通过对产黄顶头孢霉应用双标记富集技术，该丝状真菌的靶向基因破坏效率提高了10倍。通过双标记技术破坏mecB基因在筛选的转化体中有5%获得成功。通过基因替换获得的突变体T6和T24经Southern印迹分析显示mecB基因失活，且无胱硫醚-γ-裂解酶活性。在添加甲硫氨酸的MDFA培养基中，这些突变体的头孢菌素产量低于对照菌株产黄顶头孢霉C10。然而，在不含甲硫氨酸的沈氏限定发酵培养基（MDFA）中，亲本菌株产黄顶头孢霉C10与突变体T6和T24的头孢菌素产量没有差异。这些结果表明，通过转硫途径供应半胱氨酸是高水平头孢菌素生物合成所必需的，但在不添加甲硫氨酸的培养基中低水平生产这种抗生素时并非必需。因此，产黄顶头孢霉中用于头孢菌素生物合成的半胱氨酸来源于自养途径（SH(2)）和逆向转硫途径。头孢菌素生物合成基因pcbC的甲硫氨酸诱导水平在亲本菌株和mecB突变体中是相同的，这表明诱导效应不是由胱硫醚-γ-裂解酶介导的。
