Biosynthesis of angelyl-CoA in Saccharomyces cerevisiae
Biosynthesis of angelyl-CoA in Saccharomyces cerevisiae
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DOI：
10.1186/s12934-018-0925-8
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发表时间：
2018-05-12
影响因子：
6.4
通讯作者：
Weber, Nora
中科院分区：
文献类型：
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作者：
Callari, Roberta;Fischer, David;Weber, Nora
背景：天使酸部分是许多生物活性产品中必不可少的修饰。这些产品通常被称为天使酸盐，一些研究已经证明了它们的治疗作用，包括抗炎和抗癌作用。然而，由于提取率低，它们在治疗学开发中的可用性受到限制。化学合成已经实现，但其复杂性阻碍了应用，因此微生物生产可能提供一个有前途的替代方案。在这里，我们设计了出芽酵母酿酒酵母，以产生天使酶a，这是一种酶a活化形式的天使酸。结果：在酵母生产angelyl-CoA的过程中，我们首先表达了最近在酿酒酵母中Streptomyces sp. SF2575生物合成簇ssf中发现的基因。最初采用外源饲喂丙酸和异源表达链霉菌丙酰辅酶a合成酶的方法来提高细胞内丙酰辅酶a的水平，使angelyl-CoA的产量达到5 mg/L左右。用链霉菌衍生的羧化酶替代链霉菌丙酰辅酶a羧化酶可使angelyl-CoA含量达到6.4 mg/L。体内分析鉴定了该途径中重要的中间体，包括甲基丙二酰辅酶a和3-羟基-2-甲基丁基辅酶a。此外，添加丙二酸甲酯和表达CoA连接酶可以合成甲基丙二酸甲酯，并与部分ssf途径一起支持约1.5 mg/L的angelyl-CoA滴度。最后，将天使酸喂给表达植物酰基辅酶a连接酶的酵母，可使天使酸的产量达到约40 mg/L。结论：我们的研究结果证明了外源羧酸前体在酵母中生物合成了angelyl-CoA。这是对ssf基因活性的首次报道。我们设想，我们的方法将提供一个平台，更可持续地生产药用上重要的化合物类天使。
Background: The angelic acid moiety represents an essential modification in many biologically active products. These products are commonly known as angelates and several studies have demonstrated their therapeutic benefits, including anti-inflammatory and anti-cancer effects. However, their availability for use in the development of therapeutics is limited due to poor extraction yields. Chemical synthesis has been achieved but its complexity prevents application, therefore microbial production may offer a promising alternative. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce angelyl-CoA, the CoA-activated form of angelic acid.Results: For yeast-based production of angelyl-CoA we first expressed genes recently identified in the biosynthetic cluster ssf of Streptomyces sp. SF2575 in S. cerevisiae. Exogenous feeding of propionate and heterologous expression of a propionyl-CoA synthase from Streptomyces sp. were initially employed to increase the intracellular propionyl-CoA level, resulting in production of angelyl-CoA in the order of 5 mg/L. Substituting the Streptomyces sp. propionyl-CoA carboxylase with a carboxylase derived from Streptomyces coelicolor resulted in angelyl-CoA levels up to 6.4 mg/L. In vivo analysis allowed identification of important intermediates in the pathway, including methyl-malonyl-CoA and 3-hydroxyl-2-methyl-butyryl-CoA. Furthermore, methyl-malonate supplementation and expression of matB CoA ligase from S. coelicolor allowed for methyl-malonyl-CoA synthesis and supported, together with parts of the ssf pathway, angelyl-CoA titres of approximately 1.5 mg/L. Finally, feeding of angelic acid to yeasts expressing acyl-CoA ligases from plant species led to angelyl-CoA production rates of approximately 40 mg/L.Conclusions: Our results demonstrate the biosynthesis of angelyl-CoA in yeast from exogenously supplied carboxylic acid precursors. This is the first report on the activity of the ssf genes. We envision that our approach will provide a platform for a more sustainable production of the pharmaceutically important compound class of angelates.