背景:天然四胺是具有表现出宽范围生物活性的四胺酸(吡咯烷-2,4-二酮)部分的杂化聚酮化合物家族。微生物中四聚体的生物合成通常由杂合聚酮合成酶(PKS)和非核糖体肽合成酶(NRPS)机制指导,通过在细菌中募集反式或顺式作用的硫酯酶样Dieckmann环化酶来形成四酰胺酸环。有一组具有独特骨架的3-(2H-吡喃-2-亚基)吡咯烷-2,4-二酮的四胺酸盐,它们的生物合成逻辑还有待研究。
结果:这里,四甲酸型化合物溴碘酮(BPD)及其新类似物,具有3-(2H-吡喃-2-亚基)吡咯烷-2,4-二酮的罕见骨架,从海绵共生细菌链霉菌LHW50302中发现。基因缺失和突变互补表明BPDs的产生与PKS-NRPS生物合成基因簇(BGC)相关,其中通过定点突变鉴定了Dieckmann环化酶基因bpdE。根据生物信息学分析,BPDs的四酰胺酸部分应该在由两个离散蛋白组成的非典型NRPS模块上形成,包括BpdC的C(缩合)-A(腺苷酸化)-T(巯基化)结构域和BpdD的A-T结构域。进一步的定点诱变分析证实了BpdC中A域的自然沉默和两个T域的功能必要性,因此,这表明在两个NRPS亚基的T结构域之间应该发生异常的氨基酰基替硫作用。此外,LuxR型调节基因的表征导致BPDs产量增加了7到8倍。该研究提出了具有3-(2H-吡喃-2-亚基)吡咯烷-2,4-二酮骨架的天然分子的第一个生物合成案例。使用BpdD作为探针的基因组挖掘揭示了单独的NRPS亚基之间的氨基酰基转硫醇化应该发生在自然界中的特定NRPS群体中。
BACKGROUND: Natural tetramates are a family of hybrid polyketides bearing tetramic acid (pyrrolidine-2,4-dione) moiety exhibiting a broad range of bioactivities. Biosynthesis of tetramates in microorganisms is normally directed by hybrid polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) machineries, which form the tetramic acid ring by recruiting trans- or cis-acting thioesterase-like Dieckmann cyclase in bacteria. There are a group of tetramates with unique skeleton of 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione, which remain to be investigated for their biosynthetic logics.
RESULTS: Herein, the tetramate type compounds bripiodionen (BPD) and its new analog, featuring the rare skeleton of 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione, were discovered from the sponge symbiotic bacterial Streptomyces reniochalinae LHW50302. Gene deletion and mutant complementation revealed the production of BPDs being correlated with a PKS-NRPS biosynthetic gene cluster (BGC), in which a Dieckmann cyclase gene bpdE was identified by sit-directed mutations. According to bioinformatic analysis, the tetramic acid moiety of BPDs should be formed on an atypical NRPS module constituted by two discrete proteins, including the C (condensation)-A (adenylation)-T (thiolation) domains of BpdC and the A-T domains of BpdD. Further site-directed mutagenetic analysis confirmed the natural silence of the A domain in BpdC and the functional necessities of the two T domains, therefore suggesting that an unusual aminoacyl transthiolation should occur between the T domains of two NRPS subunits. Additionally, characterization of a LuxR type regulator gene led to seven- to eight-fold increasement of BPDs production. The study presents the first biosynthesis case of the natural molecule with 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione skeleton. Genomic mining using BpdD as probe reveals that the aminoacyl transthiolation between separate NRPS subunits should occur in a certain population of NRPSs in nature.