Cloning of the biosynthetic gene cluster for naphthoxanthene antibiotic FD-594 from Streptomyces sp TA-0256
Cloning of the biosynthetic gene cluster for naphthoxanthene antibiotic FD-594 from Streptomyces sp TA-0256
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DOI：
10.1038/ja.2010.145
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发表时间：
2011-01-01
影响因子：
3.3
通讯作者：
Eguchi, Tadashi
中科院分区：
文献类型：
--
作者：
Kudo, Fumitaka;Yonezawa, Takanori;Eguchi, Tadashi
FD-594是一种独特的吡喃并[4‘，3’：6，7]萘并[1，2-b]黄杂环己烯聚酮，具有2，6-二脱氧糖的三糖结构。在本研究中，我们克隆了产生菌Fd-594生物合成基因簇。TA-0256，研究其生物合成。已鉴定的PNX基因簇全长38143bp，由40个开放阅读框组成，包括一个最小的PKS基因、TDP-橄榄糖生物合成基因、两个糖基转移酶基因、两个甲基转移酶基因和许多加氧酶/还原酶基因。大多数编码在PNX簇中的酶被合理地分配到FD-594的一条合理的生物合成途径上，在该途径中，可能由依赖于黄素腺嘌呤二核苷酸(FAD)的单加氧酶催化的Baeyer-Villiger类型氧化的独特的开环过程导致了独特的黄原烯结构。为了阐明Pnx基因在FD-594生物合成中的作用，用在大肠杆菌中表达的重组蛋白对糖基转移酶PnxGT2和甲基转移酶PnxMT2进行了鉴定。结果表明，PnxGT2以TDP-橄榄糖为糖基供体，催化三重橄榄糖转移到FD-594苷元上，得到三橄榄糖苷。令人惊讶的是，在PnxGT2酶反应中，除了预期的三橄榄糖苷外，还检测到了四橄榄糖苷和五橄榄糖苷。据我们所知，PnxGT2是次生代谢中第一个邻接的寡糖形成糖基转移酶。此外，将PnxMT2和S-腺苷-L-蛋氨酸加入PnxGT2反应混合物中，得到天然的FD-594，证实PnxGT2反应产物是预期的区域特异性糖基化化合物。因此，已鉴定的PNX基因簇似乎与FD-594的生物合成有关。《抗生素杂志》，第123-132号；doi：10.1038/ja.2010.145；2010年11月24日在线出版
FD-594 is an unique pyrano[4',3':6,7]naphtho[1,2-b]xanthene polyketide with a trisaccharide of 2,6-dideoxysugars. In this study, we cloned the FD-594 biosynthetic gene cluster from the producer strain Streptomyces sp. TA-0256 to investigate its biosynthesis. The identified pnx gene cluster was 38 143 bp, consisting of 40 open reading frames, including a minimal PKS gene, TDP-olivose biosynthetic genes, two glycosyltransferase genes, two methyltransferase genes and many oxygenase/reductase genes. Most of these enzymes coded in the pnx cluster were reasonably assigned to a plausible biosynthetic pathway for FD-594, in which an unique ring opening process via Baeyer-Villiger-type oxidation catalyzed by a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase, is speculated to lead to the unique xanthene structure. To clarify the involvement of pnx genes in the FD-594 biosynthesis, a glycosyltransferase, PnxGT2, and a methyltransferase, PnxMT2, were characterized enzymatically with the recombinant proteins expressed in Escherichia coli. As a result, PnxGT2 catalyzed the triple olivose transfers to the FD-594 aglycon with TDP-olivose as the glycosyl donor to afford triolivoside. Surprisingly, in the PnxGT2 enzymatic reaction, tetraolivoside and pentaolivoside were significantly detected along with the expected triolivoside. To our knowledge, PnxGT2 is the first contiguous oligosaccharide-forming glycosyltransferase in secondary metabolism. Furthermore, addition of PnxMT2 and S-adenosyl-L-methionine into the PnxGT2 reaction mixture afforded natural FD-594 to confirm that the PnxGT2 reaction product was the expected regiospecifically glycosylated compound. Consequently, the identified pnx gene cluster appears to be involved in FD-594 biosynthesis. The Journal of Antibiotics (2011) 64, 123-132; doi:10.1038/ja.2010.145; published online 24 November 2010