De novo metabolic engineering of a designed biosynthetic pathway for complex terpenoids in yeast

生产复杂天然产物的工程微生物系统将为化学合成过程或从天然资源中提取提供一种高效和环保的替代方法。对于许多天然产物来说，潜在生物合成途径的识别取得了重大进展。微生物宿主体内几种天然产物的代谢途径工程已成为可能。然而，由于不相容的底物特异性、密码子使用模式、调控、所需的底物压力等原因，已鉴定的生物合成基因在这种代谢工程方法中的应用往往受到限制。体外进化被证明是有效定制生物催化剂的有力工具。此外，体外进化的使用将大大扩展工程生物合成途径的合理设计。这里提出的项目希望以类杉二萜为例，利用定制的生物催化剂，研究合理设计生物合成途径的从头代谢工程概念。在红豆杉中，类杉生物合成途径被证明是一个相互连接的生物合成网络，具有许多代谢转移，导致许多密切相关的生物合成产物。利用已确定的生物合成基因，该项目试图建立一个线性合理设计的酵母类taxoid产品的生物合成途径。这一尝试代表了目前临床使用的紫杉烷（Taxol & taxoere）供应充足和可持续生产的长期问题的解决方案。此外，该项目将为合成具有优异生物活性的新型第三代临床紫杉烷开辟新的途径。除了解决高氧萜类化合物的代谢工程，特别是利用细胞色素P450依赖的单加氧酶，该项目还将为酵母的代谢工程提供新的见解。因此，拟议的项目将代表酵母代谢工程的重大进展，用于生产具有挑战性的复杂精细化学品的细胞工厂。

Engineered microbial systems producing complex natural products would provide an efficient and environmental friendly alternative to chemical synthetic processes or the extraction from natural sources. For many natural products the identification of the underlying biosynthetic pathways mad emajor advances. And the metabolic pathway engineering for several natural products in microbial hosts is now becoming possible. However, the use of the identified biosynthetic genes for such metabolic engineering approaches is often limited, due to incompatible substrate specificity, codon usage patterns, regulation, required substrate pressure etc. In vitro evolution proved to be a powerful tool for the efficient tailoring of biocatalysts. In addition, the use of in vitro evolution will allow to a significant extend the rational design of the engineered biosynthetic pathway. The here proposed project wants to examine the concept of de novo metabolic engineering of rational designed biosynthetic pathways, using tailor-made biocatalysts, on the example of the taxoid diterpenoids. In Taxus the taxoid biosynthetic pathway proved to be an anastamosing Biosynthetic network, with many metabolic diversions, leading to many closely related biosynthetic products. Using the identified biosynthetic genes the project attempts to establish a linear rational designed biosynthetic pathway for defined taxoid products in yeast. This attempt represents a solution for the long-standing problem of adequate supply and sustainable production of the currently used clinically taxanes (Taxol & Taxotere). In addition, the projekt will open new avenues for the synthesis of novel, third generation clinical taxanes with superior biological activity. Besides addressing the metabolic engineering of a highly oxygenated terpenoid, using cytochrome P450 dependent monooxygenases, in particular, the projekt will also deliver new insight into the metabolic engineering of yeast in general. Therefore, the proposed projekt will represent a major advancement in the metabolic engineering of yeast for the use as cell factories for the production of challenging, complex fine chemicals.