Isoprenoid Pathway Optimization for Taxol Precursor Overproduction in Escherichia coli

Abstract

Taxol (paclitaxel) is a potent anticancer drug first isolated from the Taxus brevifolia Pacific yew tree. Currently, cost-efficient production of Taxol and its analogs remains limited. Here, we report a multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene-the first committed Taxol intermediate-approximately 1 gram per liter (~15,000-fold) in an engineered Escherichia coli strain. Our approach partitioned the taxadiene metabolic pathway into two modules: a native upstream methylerythritol-phosphate (MEP) pathway forming isopentenyl pyrophosphate and a heterologous downstream terpenoid-forming pathway. Systematic multivariate search identified conditions that optimally balance the two pathway modules so as to maximize the taxadiene production with minimal accumulation of indole, which is an inhibitory compound found here. We also engineered the next step in Taxol biosynthesis, a P450-mediated 5α-oxidation of taxadiene to taxadien-5α-ol. More broadly, the modular pathway engineering approach helped to unlock the potential of the MEP pathway for the engineered production of terpenoid natural products.Taxol (paclitaxel) and its structural analogs are among the most potent and commercially successful anticancer drugs (1). Taxol was first isolated from the bark of the Pacific yew tree (2), and early-stage production methods required sacrificing two to four fully grown trees to secure sufficient dosage for one patient (3). Taxol's structural complexity limited its chemical synthesis to elaborate routes that required 35 to 51 steps, with a highest yield of 0.4% (4-6). A semisynthetic route was later devised in which the biosynthetic intermediate baccatin III, isolated from plant sources, was chemically converted to Taxol (7). Although this approach and subsequent plant cell culture-based production efforts have decreased the need for harvesting the yew tree, production still depends on plant-based processes (8), with

Synthetic RNA devices to expedite the evolution of metabolite-producing microbes

“…S8). These results further imply that finetuning control of gene expression depending on the expression system is critical for pathway optimization 5,32 . In case of the control experiment performed with library constructed by using WLN4 harbouring RiboNULL, no particular promoter sequences are enriched after the fourth enrichment cycle ( Supplementary Fig.…”

Downstream reactions and engineering in the microbially reconstituted pathway for Taxol

“…With regards to Taxol, both S. cerevisiae and E. coli have been used to generate early stage intermediates in the downstream pathway (Ajikumar et al 2010;Dejong et al 2006;Engels et al 2008;Huang et al 2001). Both hosts are strong candidates for heterologous biosynthesis as a function of their rapid growth rates (compared to plant cells) and range of molecular biology tools to facilitate and optimize metabolic engineering efforts.…”

“…Over the last 10 years, the emergence of facilitating technical developments for hosts like E. coli and S. cerevisiae have continually fueled and allowed increasing attempts at heterologous natural product biosynthesis with great success. For example, gene synthesis and chromosomal engineering methodology helped expedite the heterologous transfer and reconstitution process for artemisinin (an antimalarial compound) and Taxol intermediate production (Ajikumar et al 2010;Martin et al 2003;Ro et al 2006). The downstream pathways for complex isoprenoid compounds typically feature several steps catalyzed by plant cytochrome P450 enzymes and a partner reductase Jennewein et al 2005;Kaspera and Croteau 2006).…”

“…The most recent efforts derive both from the focused work on Taxol biosynthesis and the more general advances in metabolic engineering and gene expression that have occurred over the last 10 years. A combination of metabolic engineering and optimized gene expression led to improved (~300 mg/L) production of taxadiene from E. coli (Ajikumar et al 2010). Here, metabolic engineering was used to balance the flow of carbon between upstream and downstream pathways as a way to optimize final titers.…”

“…Approximating this membrane localization in E. coli will clearly be challenging. However, taking the lead from numerous groups that have focused on E. coli gene expression of mammalian and plant P450 enzymes (Barnes 1996;Barnes et al 1991;Chang et al 2007;Leonard et al 2006;Pritchard et al 2006;Yun et al 2006), functional expression of the first Taxol pathway P450 was accomplished (Ajikumar et al 2010). Doing so required truncation of the native transmembrane region (with three different truncation lengths tested) and further modification of the N-terminal portion of the enzyme to include an eight amino acid membrane anchor peptide sequence (MALLLAVF) which has proven functional in E. coli (Barnes et al 1991).…”