Evidence for the unusual condensation of a diketide with a pentulose in the methylenomycin biosynthetic pathway of Streptomyces coelicolor A3(2)
Evidence for the unusual condensation of a diketide with a pentulose in the methylenomycin biosynthetic pathway of Streptomyces coelicolor A3(2)
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DOI：
10.1002/cbic.200500243
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发表时间：
2005-12-01
期刊：
影响因子：
3.2
通讯作者：
Challis, GL
中科院分区：
文献类型：
--
作者：
Corre, C;Challis, GL
21662005 Wiley-VCH Verlag GmbH&Co.KGaA，Weinheim ChemBioChem 2005，6,2166-2170或U9在完全介质(包含多种碳源的复杂介质)中，并导致低掺入水平(∼0.1%)。在这些研究中，我们无法确定1的C-2和C-7的代谢来源，因为天蓝色链霉菌U9在最低限度的培养基中不产生1和2。因此，我们尝试了另一种方法，涉及利用含有引导天蓝色链霉菌生物合成的基因簇的粘粒转化变青链霉菌1326的实验。最近，天蓝色链霉菌的整个巨型线性质粒SCP1的序列已经确定，其中包含指导1和2的生物合成的mmy簇。用整合的粘粒C73 787(包含来自SCP1基因组文库的mmy簇)转化的天蓝色链霉菌M145已被报道产生亚甲基霉素。[7]在这里，我们提供了明确的化学证据，证明了用粘粒C73 787转化的变铅青链霉菌1326在补充的最低限度的培养基中产生了1和2，并报告了进一步的掺入实验，揭示了C-2、C-3、C-4、C-7和C-8的异常代谢起源，至今仍不清楚。从最低限度培养的变铅青链霉菌1326(C73 787)酸化培养上清液的有机提取物中分离得到亚甲基霉素1和2，经1H和13C核磁共振谱鉴定。通过HMQC、HMBC和Difference-NOE实验对2的1H和13CNMR谱中的信号进行了明确的归属。在这些实验过程中，当2储存在酸性氯仿中时，观察到3的产生，但不能被鉴定为发酵的直接产物，这可能是酸促进2脱羧基的结果。
2166 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2005, 6, 2166–2170 or U9 in complete medium (a complex medium containing multiple carbon sources) and resulted in low incorporation levels (∼ 0.1%). In these studies, we were unable to identify the metabolic origin of C-2 and C-7 of 1 because S. coelicolor U9 does not produce 1 and 2 in minimal media. Thus, we tried an alternative approach involving experiments utilising Streptomyces lividans 1326 transformed with a cosmid containing the gene cluster that directs methylenomycin biosynthesis in S. coelicolor.Recently, the sequence of the entire giant linear plasmid SCP1 of S. coelicolor, which contains the mmy cluster that directs the biosynthesis of 1 and 2, has been determined.[6] On the basis of a bioassay, S. coelicolor M145 transformed with the integrative cosmid C73 787 (containing the mmy cluster—from a genomic library of SCP1) has been reported to produce methylenomycins.[7] Here we present unambiguous chemical evidence that S. lividans 1326 transformed with cosmid C73 787 produces 1 and 2 in a supplemented minimal medium and report further incorporation experiments that reveal the unusual metabolic origin of C-2, C-3, C-4, C-7 and C-8, which has remained unclear until now. Methylenomycins 1 and 2 were isolated from an organic extract of the acidified culture supernatant of S. lividans 1326 (C73 787) grown in the minimal medium, and identified by 1H and 13C NMR spectroscopy. The signals in the 1H and 13C NMR spectra of 1 have been unambiguously assigned previously.[5] The signals in the 1H and 13C NMR spectra of 2 were unambiguously assigned by using HMQC, HMBC and difference-NOE experiments. In the course of these experiments, the production of 3, which could not be identified as a direct product of the fermentation, was observed when 2 was stored in acidic chloroform, presumably as a result of acid-promoted decarboxylation of 2.