Baltz R H, Seno E T
Antimicrob Agents Chemother. 1981 Aug;20(2):214-25. doi: 10.1128/AAC.20.2.214.
We isolated numerous mutants of Streptomyces fradiae blocked in tylosin biosynthesis after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. These mutants were classified into nine groups, based upon the tylosin-like compounds produced and upon cofermentation analyses. More than 80% of the mutants isolated produced no tylosin-like compounds, and the majority of these were blocked only in the formation of tylactone. Four classes of mutants blocked in the biosynthesis or addition of tylosin sugars were isolated; tylA mutants were blocked in the formation of all three tylosin sugars, whereas tylB, tylC, and tylD mutants were blocked specifically in the biosynthesis or the addition of mycaminose, mycarose, and 6-deoxy-d-allose, respectively. Two classes of mutants (tylH and tylI) blocked in specific oxidations of tylactone and two classes (tylE and tylF) blocked in specific O-methylations of demethylmacrocin and macrocin were also characterized. Cofermentation and bioconversion studies with these mutants suggested the following relationships: (i) the tylosin sugars are derived from a common intermediate; (ii) tylactone is the first intermediate which can be excreted in appreciable quantities; (iii) the addition of mycaminose to the C-5 hydroxyl group of tylactone must precede oxidations at C-20 and C-23; (iv) oxidation at C-20 normally precedes the attachment of mycarose to the 4' hydroxyl position of mycaminose; and (v) 6-deoxy-d-allose is added to the C-23 hydroxyl position of the lactone and subsequently O-methylated at 2''' and 3''' positions. The O-methylations appear to be the final two steps in tylosin biosynthesis, and the 2''' O-methylation must occur before the 3''' O-methylation can take place. All of the tyl mutants except the tylG mutants produced relatively high levels of tylosin-like intermediates or shunt products. Mutants blocked in specific steps other than 3''' O-methylation, including a mutant blocked in 2''' O-methylation of demethylmacrocin, produced normal levels of macrocin O-methyltransferase. Mutants apparently containing specific tylosin structural gene mutations produced normal levels of aerial mycelia and spores, produced low levels of tylosin aldehyde reductase, and were resistant to high levels of tylosin. However, three atypical tylG mutants produced no tylosin-like compounds, could not cosynthesize tylosin with any other tyl mutant, could not bioconvert tylactone or macrocin to tylosin, and produced no macrocin O-methyltransferase. These three mutants produced elevated levels of tylosin aldehyde reductase. In addition, one was very succeptible to tylosin and did not produce aerial mycelia or spores.
我们通过N-甲基-N'-硝基-N-亚硝基胍诱变，分离出了许多在泰乐菌素生物合成过程中受阻的弗氏链霉菌突变体。根据所产生的泰乐菌素类化合物以及共发酵分析，这些突变体被分为九组。分离出的突变体中超过80%不产生泰乐菌素类化合物，其中大多数仅在泰内酯的形成过程中受阻。分离出了四类在泰乐菌素糖的生物合成或添加过程中受阻的突变体；tylA突变体在所有三种泰乐菌素糖的形成过程中受阻，而tylB、tylC和tylD突变体分别在霉胺糖、鼠李糖和6-脱氧-D-阿洛糖的生物合成或添加过程中特异性受阻。还鉴定出了两类在泰内酯的特定氧化过程中受阻的突变体（tylH和tylI）以及两类在去甲基马罗星和马罗星的特定O-甲基化过程中受阻的突变体（tylE和tylF）。对这些突变体进行的共发酵和生物转化研究表明了以下关系：（i）泰乐菌素糖源自一个共同中间体；（ii）泰内酯是第一个能够大量分泌的中间体；（iii）霉胺糖添加到泰内酯的C-5羟基上必须先于C-20和C-23的氧化；（iv）C-20的氧化通常先于鼠李糖连接到霉胺糖的4'羟基位置；（v）6-脱氧-D-阿洛糖添加到内酯的C-23羟基位置，随后在2'''和3'''位置进行O-甲基化。O-甲基化似乎是泰乐菌素生物合成的最后两步，并且2''' O-甲基化必须在3''' O-甲基化发生之前进行。除tylG突变体外，所有tyl突变体都产生了相对高水平的泰乐菌素类中间体或旁路产物。在除3''' O-甲基化以外的特定步骤中受阻的突变体，包括一个在去甲基马罗星的2''' O-甲基化过程中受阻的突变体，产生了正常水平的马罗星O-甲基转移酶。明显含有特定泰乐菌素结构基因突变的突变体产生了正常水平的气生菌丝和孢子，产生了低水平的泰乐菌素醛还原酶，并且对高水平的泰乐菌素具有抗性。然而，三个非典型tylG突变体不产生泰乐菌素类化合物，不能与任何其他tyl突变体共同合成泰乐菌素，不能将泰内酯或马罗星生物转化为泰乐菌素，并且不产生马罗星O-甲基转移酶。这三个突变体产生了升高水平的泰乐菌素醛还原酶。此外，其中一个对泰乐菌素非常敏感，并且不产生气生菌丝或孢子。
