Production of novel taxane intermediates of anticancer drug Taxol using microbial consortia

Abstract

Paclitaxel —commercially known as taxol— is one of the most widely used drugs to treat different types of cancer. Its production using microbial platforms has been challenging due to the complexity of the molecule and the difficulty of expressing functional enzymes. Expression of many heterologous enzymes in microorganisms often leads to an increased metabolic burden that usually causes growth and production decrease. Division of Labour can offer great advantages as the metabolic burden can be divided into two or more specialised organisms.

In this work, the use of microbial consortia to produce oxygenated and acetylated paclitaxel precursors was examined. The aim of Chapter 3 was specialisation by engineering various GAL80 mutant strains which could produce taxanes without the need for galactose for induction, and to further expand the Taxol metabolic pathway by incorporating the enzyme Taxane-10-Hydroxylase (T10ßOH). Using glucose showed a decreased Taxadiene production of up to 50% in comparison to galactose, probably because MIG1 is believed to cause repression in the presence of this sugar. Sucrose and fructose yielded very poor taxadiene results around 90% lower than galactose to produce taxanes. Conversely, using raffinose yielded the best production for Taxa-4, 11-dien-5a-yl acetate (T5αAc) at 72 ± 15 mg/L which represented a 7-fold increase compared to previous literature data. Despite the good production results with raffinose and galactose, the T10ßOH product was not detected.

The next section aimed to develop microbial consortia and an inoculum engineering strategy to enable the production of T10ß-ol. Different plasmids carrying the T5αOH, TAT and T10OH enzymes were assembled for expression in an engineered ethanologenic E. coli capable of growing on xylose. As result, the product of the T10ßOH enzyme was detected and quantified for the first time using the consortia. A production of 8 ± 0.30 mg/L of T10ßol was found when fermenting glucose with a yeast strain with GAL80 deletion which was only capable of producing taxadiene proving that the pathway was efficiently divided. Finally, as a second approach, two plasmids carrying TAT and T10ßOH enzymes were assembled to form a consortium integrated by two S. cerevisiae strains. The inoculation ratio of 3:1 (EJ2 10ß: EJ2 TAT) resulted in a production of 26.5 ±5 mg/L of Taxa-4, 11-dien-5α-acetoxy-10-ol (T10ßol), the highest ever reported for this compound. It is important to mention that this consortium was the most ambitious of all as it involved 2 different yeast strains plus 3 different bacteria strains, which to the best of our knowledge is one of the most successful and complex consortia including five microorganisms diving a synthetic pathway and achieving true synergy to divide the metabolic burden among the consortia.

Following the detection of T10ßol, we aimed to test a novel enzyme candidate (T1ßOH) to produce the next paclitaxel intermediate taxadiene-5a-acetoxy-1a,10b-diol (T1ß-ol) using optimized co-cultures described in chapter 4. After adjusting inoculation ratios and other parameters based on the evidence obtained from previous experiments we managed to detect and quantify the new T1ß-ol compound with an estimated production of 45 ±3 mg/L.

The strategies of inoculum engineering, microbial consortia and conversion towards constitutive expression have shown to be very suitable to expand and optimize the paclitaxel metabolic pathway. These strategies also proved effective for the detection and production of a new compound highlighting the potential for its application in other metabolic routes practically and rapidly.