Research Center for Marine Drugs, Department of Pharmacy, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China.
Department of Chemistry, University of Aberdeen, Aberdeen, AB24 3UE, UK.
Microb Cell Fact. 2024 Mar 21;23(1):87. doi: 10.1186/s12934-024-02364-7.
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.
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.
天然四氢盐是一类含有四氢酸（吡咯烷-2,4-二酮）部分的杂合聚酮，具有广泛的生物活性。微生物中四氢盐的生物合成通常由杂合聚酮合酶（PKS）和非核糖体肽合成酶（NRPS）机制指导，这些机制通过在细菌中招募反式或顺式作用硫酯酶样狄克曼环化酶来形成四氢酸环。有一组四氢盐具有独特的 3-(2H-吡喃-2-亚基)吡咯烷-2,4-二酮骨架，其生物合成逻辑仍有待研究。
本文从海绵共生细菌链霉菌 reniochalinae LHW50302 中发现了四氢盐类化合物 bripiodionen（BPD）及其新类似物，其具有罕见的 3-(2H-吡喃-2-亚基)吡咯烷-2,4-二酮骨架。基因缺失和突变体互补实验表明，BPD 的产生与一个 PKS-NRPS 生物合成基因簇（BGC）相关，其中通过定点突变鉴定出一个狄克曼环化酶基因 bpdE。根据生物信息学分析，BPDs 的四氢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（腺苷酰化）-T（硫醇化）结构域和 BpdD 的 A-T 结构域。进一步的定点突变分析证实了 BpdC 的 A 结构域的自然沉默和两个 T 结构域的功能必要性，因此表明两个 NRPS 亚基的 T 结构域之间应该发生不寻常的氨酰基转硫反应。此外，对 LuxR 型调节基因的表征导致 BPDs 产量增加了七到八倍。该研究首次报道了具有 3-(2H-吡喃-2-亚基)吡咯烷-2,4-二酮骨架的天然分子的生物合成案例。使用 BpdD 作为探针的基因组挖掘表明，在自然界中，某些 NRPS 群体中应该发生独立 NRPS 亚基之间的氨酰基转硫反应。
