Metabolic engineering for the production of functionalized terpenoids in heterologous hosts

Abstract

Terpenoids are a large and diverse group of secondary metabolites which natively play essential biochemical roles, and have been adopted for a wide range of uses by humans. These molecules are of interest scientifically due to their often complex structures, as well as commercially, with market value exceeding $50 billion annually. Many of these compounds are derived from natural sources, such as plants, which are prone to long life cycles, low productivity, as well as climate and market variability. As such, production in heterologous microbial systems through metabolic engineering stands as an attractive alternative to truly natural production and allows for increased control over biosynthetic pathways enabling improved yields, titer, and productivity. The anti-cancer molecule Taxol (Paclitaxel) stands as one of the most medically and economically important terpenoids. However, despite decades of extensive study, its biosynthesis remains poorly understood. In this work we report on investigations into the reconstruction of the early Taxol bioysnthetic pathway. Early successes toward pathway reconstitution within E. coli and S. cerevisiae led to the identification of an early bottleneck, identified to arise from unpredictable behavior of the first oxygenation enzyme, taxadiene-5o-hydroxylase (CYP725A4), which produces a range of undesired products. We present chemical and biological evidence of an unreported epoxidase activity of taxadiene-5a-hydroxylase that puts into question the previously proposed radical-rebound mechanism. We demonstrate that the poor selectivity of taxadiene-5o-hydroxylase arises from the non-selective degradation of an epoxide intermediate produced via a selective oxidation step, rather than from promiscuous oxidation, as previously proposed. We support these conclusions by demonstrating variable enzyme behavior in differing hosts and conditions, similarity of products and product ratios generated from chemical epoxidation and by taxadiene-5a-hydroxylase, and differing enzymatic activity on alternative taxadiene isomers. We next systematically investigate three methodologies, terpene cyclase engineering, P450 engineering, and hydrolase-enzyme screening to overcome this early pathway selectivity bottleneck. We demonstrate that engineering of taxadiene synthase, upstream of the promiscuous oxidation step, acts as a practical methodology for selectivity improvement. Through mutagenesis we achieve a 2.4-fold improvement in yield and selectivity for an alternative cyclization product, taxa-4(20)-11(12)-diene; and for the Taxol precursor taxadien-5a-ol, when co-expressed with CYP725A4. This work lays the foundation for the elucidation, engineering, and improved production of Taxol and early Taxol precursors. In addition to bottlenecks in the downstream production of terpenoids such as Taxol, upstream flux derived from the microbial methylerythritol phosphate (MEP) pathway also imposes limitations on culture productivity and titer. Thus, efforts were also focused on developing alternative biosynthetic routes to terpenoids. This led to the investigation of a novel route, which relies on phosphorylation of the bulk chemical feedstock isopentenol, to generate isopentenyl disphosphate (IPP), the building block of all terpenoids. Following proofs-of-concept experiments demonstrating pathway functionality, we turned our attention to methodologies to improve flux through this pathway. As few high-throughput methodologies for the quantification of terpenoid production have been previously described, this led to the development of a genetically-encoded terpenoid-responsive biosensor. Furthermore, we develop theoretical framework and proof-of concepts for the application of evolution-based approaches to the optimization of the pathway.