Removal of Ribosome Stalling Motifs to Improve Oxygenation of Key Intermediate in the Taxol Pathway

Taxol is an effective natural product chemotherapeutic applicable for the treatment of multiple cancers. However, production of Taxol has proven to be a significant challenge for society, as bark extraction, chemical synthesis, and semisynthesis using plant cell cultures all have issues with either yield or scalability. To address this problem, synthetic biologists have generated a strategy to produce Taxol precursors in rapid-growing microbial hosts by the heterologous expression of Taxol pathway enzymes. Although this approach has been successful in generating initial intermediates of the pathway, there is a distinct bottleneck that occurs at the first oxygenation of taxadiene in Eschericha coli. Previous work indicated that only minor changes in expression of this P450 enzyme (CYP725A4) severely undermined overall cellular protein production, resulting in poor pathway productivity. Global protein expression effects hinted that some property of this enzyme was inhibiting protein synthesis, perhaps at the level of the ribosome. Recently, XPPX motifs, two consecutive prolines in an amino acid sequence, have been found to induce ribosome stalling. Indeed, CYP725A4 contains three such motifs. Here, we mutate away these XPPX motifs to probe their impact on protein expression and oxygenation efficiency.