Leiser A, Birch A, Robinson J A
Institute of Organic Chemistry, University of Zurich, Switzerland.
Gene. 1996 Oct 24;177(1-2):217-22. doi: 10.1016/0378-1119(96)00305-8.
The catabolism of branched chain amino acids, especially valine, appears to play an important role in furnishing building blocks for macrolide antibiotic biosynthesis. To determine for the first time the importance of valine dehydrogenase (vdh) in polyether antibiotic biosynthesis, the vdh gene from Streptomyces cinnamonensis has been cloned and sequenced. The enzyme (M(r)37,718 Da) has been produced in large amounts in an active form in the E. coli cytoplasm using a T7 RNA-polymerase expression system. Upon inactivation of the gene in S. cinnamonensis by a double-crossover mechanism, a hyg::vdh mutant was isolated that was devoid of vdh activity. Upon growth in chemically defined media, as well as a complex medium optimised for monensin production, the mutant and wild-type grew equally well and reached the same levels of monensin production. In both strains a valine transaminase activity could be detected that provides an alternative route for converting valine into 2-oxoisovaleric acid. The results show that vdh is not essential for normal growth of S. cinnamonensis, and its inactivation does not significantly affect normal levels of monensin production in this strain.
支链氨基酸的分解代谢，尤其是缬氨酸的分解代谢，似乎在为大环内酯类抗生素生物合成提供构建模块方面发挥着重要作用。为了首次确定缬氨酸脱氢酶（vdh）在聚醚类抗生素生物合成中的重要性，来自肉桂链霉菌的vdh基因已被克隆并测序。使用T7 RNA聚合酶表达系统，该酶（分子量37,718 Da）已在大肠杆菌细胞质中大量以活性形式产生。通过双交换机制使肉桂链霉菌中的该基因失活后，分离出了一个缺失vdh活性的hyg::vdh突变体。在化学限定培养基以及为莫能菌素生产优化的复合培养基中生长时，突变体和野生型生长情况相同，且莫能菌素产量达到相同水平。在两种菌株中都能检测到缬氨酸转氨酶活性，该活性为将缬氨酸转化为2-氧代异戊酸提供了一条替代途径。结果表明，vdh对于肉桂链霉菌的正常生长不是必需的，其失活对该菌株中莫能菌素的正常产量没有显著影响。
