The following three slides represent my drafts for my final project. Project one involves decaffeinating drinks using bacterial strains, project 2 and 3 are similiar in nature as both are small molecule drugs which I aim to synthesise using bacteria. Although this is ambitious I have also found that a mutual precursor such as diterpene could be made instead of the complete drug.
Paclitaxel is a popular chemotherapy in several cancers such as ovarian, breast, and lung. However, the current production of it remains unsustainable from an environmental and economic perspective and can be optimised using biosynthesis. The most common way it is made on an industrial scale is via semi-synthesis by extraction of 10-deacetylbaccatin III from the European Yew (Taxus baccata) or other similar trees and eventually ending up with paclitaxel. This often is not extremely efficient (paclitaxel after extraction 10%), and contributes to environmental strain as it takes long for yew trees to mature driving up costs further Current biosynthesis is not particularly effective either, some work has been done in generating the taxol precursors using E. coli (10.1126/science.1191652). The complete synthesis is difficult due to the inefficiency of enzymatic reactions. One of the current bottlenecks in production is the first oxidation step catalysed by taxadiene 5α-hydroxylase (CYP725A4). This enzyme converts taxadiene into taxadien‑5α‑ol but exhibits low catalytic efficiency and poor selectivity, resulting in the formation of multiple undesired side products. Heterologous expression of taxadiene‑5α‑hydroxylase (CYP725A4) results in a high side‑product to main‑product ratio and low taxadien‑5α‑ol titres due to the formation of multiple oxygenated taxane derivatives, thereby limiting metabolic flux toward paclitaxel precursors and hindering efficient microbial production (doi.org/10.1186/s12934-022-01922-1).
“The challenge for biosynthesis of paclitaxel lies on the insufficient precursor, such as taxadien-5α-ol” (Wu, QY et al. doi.org/10.1186/s40643-022-00569-5)
I want to optimise the CYP725A4‑catalysed oxidation step in paclitaxel biosynthesis, which currently exhibits low selectivity due to competing reaction pathways in its active site. This may be achieved through enzyme engineering approaches such as active site analysis, molecular docking, rational mutation prediction, or by exploring alternative enzyme variants. Improving this early biosynthetic step could increase taxadien‑5α‑ol production and enhance the overall efficiency and sustainability of microbial paclitaxel synthesis.
After some discussion with my node TAs, I settled on paclitaxel as my final project. My three goals are as follows;
Aim 1: Identify and design CYP725A4 variants with improved efficiency using DNA construct design, rational mutation prediction, active-site analysis, molecular docking, and AI
Aim 2: Experimentally test the best CYP725A4 variants in a heterologous expression system and compare product distributions to determine if product formation improves relative to current variants
Aim 3: Enable more efficient and sustainable microbial paclitaxel production by reducing a major bottleneck in the biosynthetic pathway, decreasing dependence on plant derived intermediates