DataSheet_1_Discovery of a gene cluster for the biosynthesis of novel cyclic peptide compound, KK-1, in Curvularia clavata.docx

KK-1, a cyclic depsipeptide with 10 residues produced by a filamentous fungus Curvularia clavata BAUA-2787, is a promising pesticide active compound with high activity against many plant pathogens, especially Botrytis cinerea. As a first step toward the future mass production of KK-1 through synthetic biological approaches, we aimed to identify the genes responsible for the KK-1 biosynthesis. To achieve this, we conducted whole genome sequencing and transcriptome analysis of C. clavata BAUA-2787 to predict the KK-1 biosynthetic gene cluster. We then generated the overexpression and deletion mutants for each cluster gene using our originally developed transformation system for this fungus, and analyzed the KK-1 production and the cluster gene expression levels to confirm their involvement in KK-1 biosynthesis. As a result of these, a region of approximately 71 kb was found, containing 10 open reading frames, which were co-induced during KK-1 production, as a biosynthetic gene cluster. These include kk1B, which encodes nonribosomal peptide synthetase with a domain structure that is consistent with the structural features of KK-1, and kk1F, which encodes a transcription factor. The overexpression of kk1F increased the expression of the entire cluster genes and, consequently, improved KK-1 production, whereas its deletion decreased the expression of the entire cluster genes and almost eliminated KK-1 production, demonstrating that the protein encoded by kk1F regulates the expressions of the other nine cluster genes cooperatively as the pathway-specific transcription factor. Furthermore, the deletion of each cluster gene caused a reduction in KK-1 productivity, indicating that each gene is involved in KK-1 production. The genes kk1A, kk1D, kk1H, and kk1I, which showed a significant decrease in KK-1 productivity due to deletion, were presumed to be directly involved in KK-1 structure formation, including the biosynthesis of the constituent residues. kk1C, kk1E, kk1G, and kk1J, which maintained a certain level of KK-1 productivity despite deletion, were possibly involved in promoting or assisting KK-1 production, such as extracellular transportation and the removal of aberrant units incorporated into the peptide chain.

KK-1是由丝状真菌棒形弯孢霉（Curvularia clavata）BAUA-2787产生的10残基环酯肽（cyclic depsipeptide），是一种极具应用前景的农药活性化合物，对多种植物病原菌尤其是灰葡萄孢菌（Botrytis cinerea）具有优异抑菌活性。为通过合成生物学手段实现KK-1的未来大规模量产，本研究首先旨在鉴定参与KK-1生物合成的相关基因。为此，我们对棒形弯孢霉BAUA-2787开展全基因组测序与转录组分析，以预测KK-1生物合成基因簇（biosynthetic gene cluster）。随后，我们利用本研究针对该真菌开发的原创转化系统，针对每个簇内基因构建过表达突变体与敲除突变体，并通过检测KK-1产量及簇内基因表达水平，验证这些基因是否参与KK-1的生物合成过程。研究结果显示，一段长度约71 kb的区域包含10个开放阅读框（open reading frame），该区域在KK-1合成过程中被协同诱导，即为KK-1生物合成基因簇。该基因簇包含kk1B与kk1F：其中kk1B编码非核糖体肽合成酶（nonribosomal peptide synthetase），其结构域组成与KK-1的结构特征相符；kk1F则编码转录因子。对kk1F进行过表达可提升整个基因簇内所有基因的表达水平，进而提高KK-1产量；而敲除kk1F则会降低整个基因簇的基因表达，几乎完全阻断KK-1的合成。这表明kk1F编码的蛋白作为途径特异性转录因子，可协同调控其余9个簇内基因的表达。此外，对每个簇内基因进行单基因敲除均会降低KK-1的合成效率，表明所有簇内基因均参与KK-1的产生过程。其中，kk1A、kk1D、kk1H与kk1I在敲除后会导致KK-1产量显著下降，推测其直接参与KK-1的结构形成过程，包括其组成残基的生物合成。而kk1C、kk1E、kk1G与kk1J在敲除后仍可维持一定水平的KK-1产量，推测其可能参与促进或辅助KK-1的合成过程，例如胞外运输以及移除掺入肽链的异常单元。