Advanced bioprocessing strategies for optimised high-value isoprenoid production using microbial cell factories
Item statusRestricted Access
Embargo end date29/07/2023
Walls, Laura Ellen
Currently over 95% of transport fuels and most pharmaceutical products are derived from petroleum. Sustainable alternatives are critical to achieving net-zero emissions without detriment to quality of life. The blockbuster chemotherapeutic, Taxol, and biojet fuel, bisabolene, are among promising renewable alternatives. However, these compounds are naturally scarce, difficult to extract and their chemical complexity often hinders total synthesis. Industrial scale production is therefore a monumental challenge. Microbial biosynthesis is an alternative, sustainable production route, which has been widely reported at laboratory scale. However, the translation of these breakthroughs to industrial bioprocesses remains a critical bottleneck. This thesis demonstrated novel accelerated bioprocessing strategies to optimise and scale up production of these valuable natural products. In the case of Taxol, low yields and the promiscuity and multispecificity of the CYP725A4 enzyme, responsible for the conversion of taxadiene to taxadien–5𝛼�����–ol, hinder further biosynthetic pathway development despite decades of research. To tackle this, a holistic Quality by Design approach was employed to optimise production of taxadien–5𝛼�����–ol and the subsequent Taxol intermediate, taxadien–5𝛼�����–yl-acetate using engineered Saccharomyces cerevisiae cell factories. Statistical Design of Experiments approaches were coupled with an industrially relevant high–throughput microbioreactor platform to screen the effect of a wide range of factors on productivity. The optimal conditions elucidated during microscale screening were subsequently validated using 1 L and 5 L bench top bioreactors. The resulting data was employed to derive a mechanistic model of the process and predict performance at increased scale. Taxane production was improved over 15-fold compared to the previous literature maximum and comparable titres were achieved at 5L scale despite a 2500-fold scale up. In addition, the key Taxadien–5𝛼�����-yl-acetate intermediate was detected for the first time in yeast cell factories with a maximum titre of 22 mg/L. Compared to Taxol, the biosynthetic pathway of bisabolene is relatively simple. Despite this commercialisation is still a monumental challenge due to its low value and economic feasibility necessitates substantially greater titres and the use of second generation feedstocks. A novel two–step bioprocess was therefore developed for the bioconversion of cellulose into bisabolene using anaerobic rumen bacteria and non-conventional yeast. The cellulolytic bacterium, Ruminococcus flavefaciens, was employed to ferment cellulose, yielding a maximum total organic acid titre of 5.15 g/L. The feasibility of these organic acids as a carbon source for Rhodosporidium toruloides was confirmed at microscale with a maximum bisabolene titre of 1055 ± 7 mg/L. The optimal process was subsequently scaled up 125–fold using mini–bioreactors and a pH controlled organic acid feeding strategy was introduced. Here bisabolene titres were improved to 7758 mg/L, the highest reported microbial titre. Finally a proof–of–concept sequential bioreactor approach was demonstrated, with a maximum bisabolene titre of 318 ± 22 mg/L. In conclusion, although isoprenoids hold great promise to alleviate some of the greatest challenges faced by humanity today, bioprocess development is a complex undertaking with unique challenges depending on the target product. This study highlighted the value of holistic, Quality by Design approaches for expedited bioprocess optimisation and scale up.