The Choice of Metabolic Engineering Over Chemical Synthesis; Taxol as an example.
1. Total synthesis
Total synthesis of taxol, albeit very difficult was achieved in 1992 (Holton et al., Nicolaou et al.,), having remained a challenged for over 20 years since it was first isolated. The total synthesis developed by the two teams had yields of 91% (Holton) over 41 steps from (-) patchino, and 85% yield through 51 steps from butene diol (Nicolaou).
While this was a breakthrough in organic chemistry, and total synthesis, it was not and is still not a solution to the taxol supply. This is due to the complicated nature, the many steps required to get to taxol, and the insignificant yields from such a process (~0.4% at most), which make it commercially non-viable. This prompts the need to look for other solutions, and a more commercially viable solution when compared to total synthesis; the semi synthesis of taxol.
2. The Semi-Synthetic Approach: A Solution to The Difficulty in Supply
After discovering and successfully testing the efficacy of taxol to treat cancer, there resulted a huge ecological problem; The highest amounts of taxol could be harvested from the barks of the yew tree, and this led to the death of the plant. And for a single dose, bark needed to be harvested from between 2-4 fully grown trees. Added to this was the fact that the yew species is a slow growing species, and the extraction process lengthy and very expensive
Therefore, in 1988, a semi-synthetic approach to taxol via 10-Deacetyl baccatin 111 was described by Denis and Greene et al., The process had a lot of merit in that the 10-Deacetyl baccatin 111 was more abundantly available in the plants, and that it was found in higher amounts in the leaves. This meant that it could be harvested sustainably without leading to the death of the source plants.
This method though greatly refined is one of the main routes of commercial production of taxol today. What makes it even more attractive is the ability to extract the 10-DAB even from nursery cultivated yew trees (Liu et al). In pursuit of this, large farms were developed in China, Germany etc. where different Taxus species are planted and harvested every two months for their twigs and needles and 10DAB extracted for a semi-synthetic conversion to taxol.
An alternative route to taxol through 7-?-Xylosyl-10-deacetyltaxol, found in the bark of the yew trees, was described by Rao V. in 1997. This compound had been known to be a potential pre-cursor to taxol, but its conversion had not been achieved due to difficulty in hydrolyzing the xylose residue while maintaining the stability of the compound. Removal of xylosyl moiety from 7- ?-xylosyl-10-deacetyltaxol converts it into 10-deacetyltaxol which can be further converted to paclitaxel through acetylation. (Chattopadhyay et al 2002)
3. Production of Taxol in Plants
The commercial production of taxol currently is fully done using plant systems, with slight variations in the techniques. The biotech firm Phyton Biotech produces Taxol commercially using plant cell-suspension cultures through huge bioreactors with a capacity of up to 75000L (Huang & McDonald 2009).
Callus formation candidates are selected from young needle tissue -young needles that have been collected within two months have higher content of paclitaxel than older ones. Once the callus is established in the solid media, it is suspended in a liquid medium favorable for cell growth (Tabata H. 2004).
Production of taxol and other related taxanes is promoted strongly by silver thiosulfate and methyl jasmonates jasmonates are important in signal transduction processes to regulate genes related to defense. Endogenous levels of jasmonates increase with response to mechanical forces, wounding and pathogen attacks. Maximal induction of methyl jasmonate in baccatin III and taxol accumulation was achieved at the concentration of 100 mmol L and above, growth of the suspension cultures was inhibited by as much as 20-25% (Tabata H. 2004).
The plant-based production efforts have been developed through the years, with the improvement in different parts of the operation; callus growth induction, induction of production of secondary metabolites including taxol, different types of media, improvement of bioreactor design etc.
The table below adapted from Y. Li et al. 2015 has a brief summary of the particular Taxus species used, the different engineering strategies utilized, the type of bioreactor if any and the final titer of Taxol.
Figure 3 taxol plant cell culture engineering strategies
Production constraints and the unreliability of the plant-based systems have fueled the continued research into other systems for the production of Taxol. These challenges include; each Taxus species requiring different conditions, high production costs due to the slow growth of the explants, slow proliferation of suspended cells, low titers leading to unstable yields and sensitivity to shearing (Y. Li et al. 2015).
Other issues reviewed by Liu et al. 2016 are more or less similar to the challenges mentioned above and are; culture growth inhibition due to secondary metabolite accumulation, aggregation and heterogeneity. These and other constraints lead to costly measures to tackle the challenges such as media optimization, phytohormones, adsorbants, methyl jasmonate, methyl jasmonate & ethylene, precursor and substrate feeding, cellular dispersion.
Saccharomyces cerevisiae; Choice as A Microbial Host
Known as the bakers yeast, distillers yeast, brewers yeast, wine yeast etc.; Saccharomyces cerevisiae is a yeast of the phylum Ascomycota, and genus Saccharomyces. It has wide applications as an industrial organism in the production of beer, wine, bread, pharmaceuticals and nutraceuticals.
In addition, S. cerevisiae has been used extensively for the industrial production of heterologous proteins such as Human Interferon, Hepatitis B Surface Antigens, Human Growth Hormone, Insulin, etc. This is enabled by the ability of the eukaryote to undertake post translational modifications on the proteins not much unlike mammalian cells. In comparison to mammalian cells, the faster growth rate, abilities and tools to easily modify its genome, and highly developed fermentation processes makes it an even more attractive host in the production of some proteins.
In industrial applications that require robust organisms, whose biochemistry, physiology, and genetics are well characterized; Saccharomyces cerevisiae and Escherichia coli are unmatched. Furthermore, Saccharomyces cerevisiae being the first eukaryote to be fully sequenced, made the rapid development of molecular tools easier.
It is the preferred organism of choice in genetic engineering and more recently metabolic engineering and synthetic biology, even over Escherichia coli due to its; robustness, less risk to contamination through its general tolerance to harsh industrial conditions -low pH, and high ethanol concentrations. (Nielsen, and Jewett, 2008, Kocharin et al., 2012 Chen et al., 2013 Krivoruchko et al., 2013 and Jiazhang Lian et al., 2014).