Tatsuno Satoshi, Arakawa Kenji, Kinashi Haruyasu
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan.
Biosci Biotechnol Biochem. 2009 Dec;73(12):2712-9. doi: 10.1271/bbb.90591. Epub 2009 Dec 7.
Extensive mutations of lankacidin synthase genes were carried out to analyze the modular-iterative mixed polyketide biosynthesis of lankacidin. Three ketoreductase domains (lkcC-KR, lkcF-KR1, and lkcF-KR2) were inactivated by in-frame deletion and site-directed mutagenesis of their active sites. The mutants ceased or diminished lankacidin production, indicating that the three KR domains are functional in lankacidin biosynthesis. However, all of the KR mutants failed to accumulate the expected unreduced metabolites. Mutational analysis of two tandemly aligned acyl carrier protein domains (lkcC-ACP1 and lkcC-ACP2) revealed that either ACP is sufficient for lankacidin production. Disruption and complementation experiments on three unique genes/domain (lkcD for acyltransferase, lkcB for dehydratase, and lkcC-MT for a C-methyltransferase domain) suggested that their gene products function iteratively during lankacidin biosynthesis.
对兰卡杀菌素合成酶基因进行了广泛的突变，以分析兰卡杀菌素的模块化迭代混合聚酮生物合成。通过框内缺失和对其活性位点进行定点诱变，使三个酮还原酶结构域（lkcC-KR、lkcF-KR1和lkcF-KR2）失活。突变体停止或减少了兰卡杀菌素的产生，表明这三个KR结构域在兰卡杀菌素生物合成中具有功能。然而，所有的KR突变体都未能积累预期的未还原代谢产物。对两个串联排列的酰基载体蛋白结构域（lkcC-ACP1和lkcC-ACP2）的突变分析表明，任何一个ACP都足以产生兰卡杀菌素。对三个独特基因/结构域（酰基转移酶的lkcD、脱水酶的lkcB和C-甲基转移酶结构域的lkcC-MT）进行的破坏和互补实验表明，它们的基因产物在兰卡杀菌素生物合成过程中进行迭代发挥作用。
