Production of α-keto carboxylic acid dimers in yeast by overexpression of NRPS-like genes from Aspergillus terreus
Production of α-keto carboxylic acid dimers in yeast by overexpression of NRPS-like genes from Aspergillus terreus
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DOI：
10.1007/s00253-017-8719-1
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发表时间：
2018-02-01
影响因子：
5
通讯作者：
Li, Shu-Ming
中科院分区：
文献类型：
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作者：
Huehner, Elisabeth;Backhaus, Katja;Li, Shu-Ming
非核糖体肽合成酶（NRPSs）是微生物合成具有生物学和药理活性的天然产物肽骨架的关键酶。单模的类nrps酶通常包括腺苷化(A)、硫代化(T)和硫代酯酶（TE）结构域。与NRPSs相比，它们不含任何缩合结构域，通常催化α -酮羧酸二聚化，从而为进一步修饰提供了多种支架。在本研究中，我们在酿酒酵母中建立了nrps样基因的表达体系。通过对土曲霉中四个已知基因的表达，证实了它们的预测功能，并实现了每升培养35 mg的产量。此外，来自同一真菌的ATEG_03090的表达，编码最后一个未被表征的具有A-T-TE结构域的nrps样酶，导致苯醌衍生物atromentin的形成。所有积累产物都被分离出来，并通过核磁共振和质谱分析对其结构进行了鉴定。这项研究为基因功能的证明提供了一个方便的系统，并为合成生物学奠定了基础，因为可以引入其他编码修饰酶的基因。
Non-ribosomal peptide synthetases (NRPSs) are key enzymes in microorganisms for the assembly of peptide backbones of biologically and pharmacologically active natural products. The monomodular NRPS-like enzymes comprise often an adenylation (A), a thiolation (T), and a thioesterase (TE) domain. In contrast to the NRPSs, they do not contain any condensation domain and usually catalyze a dimerization of alpha-keto carboxylic acids and thereby provide diverse scaffolds for further modifications. In this study, we established an expression system for NRPS-like genes in Saccharomyces cerevisiae. By expression of four known genes from Aspergillus terreus, their predicted function was confirmed and product yields of up to 35 mg per liter culture were achieved. Furthermore, expression of ATEG_03090 from the same fungus, encoding for the last uncharacterized NRPS-like enzyme with an A-T-TE domain structure, led to the formation of the benzoquinone derivative atromentin. All the accumulated products were isolated and their structures were elucidated by NMR and MS analyses. This study provides a convenient system for proof of gene function as well as a basis for synthetic biology, since additional genes encoding modification enzymes can be introduced.