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

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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.