RESUMO
The recent technological advancements in synthetic biology have demonstrated the extensive potential socio-economic benefits at laboratory scale. However, translations of such technologies to industrial scale fermentations remains a major bottleneck. The existence and lack of understanding of the major discrepancies in cultivation conditions between scales often leads to the selection of suboptimal bioprocessing conditions, crippling industrial scale productivity. In this study, strategic design of experiments approaches were coupled with state-of-the-art bioreactor tools to characterize and overcome nutritional stress for the enhanced production of precursors to the blockbuster chemotherapy drug, Taxol, in S. cerevisiae cell factories. The batch-to-batch variation in yeast extract composition was found to trigger nutritional stress at a mini-bioreactor scale, resulting in profound changes in cellular morphology and the inhibition of taxane production. The cells shifted from the typical budding morphology into striking pseudohyphal cells. Doubling initial yeast extract and peptone concentrations (2×YP) delayed filamentous growth, and taxane accumulation improved to 108 mg/L. Through coupling a statistical definitive screening design approach with the state-of-the-art high-throughput micro-bioreactors, the total taxane titers were improved a further two-fold, compared to the 2×YP culture, to 229 mg/L. Filamentous growth was absent in nutrient-limited microscale cultures, underlining the complex and multifactorial nature of yeast stress responses. Validation of the optimal microscale conditions in 1L bioreactors successfully alleviated nutritional stress and improved the titers to 387 mg/L. Production of the key Taxol precursor, T5αAc, was improved two-fold to 22 mg/L compared to previous maxima. The present study highlights the importance of following an interdisciplinary approach combining synthetic biology and bioprocessing technologies for effective process optimization and scale-up.
RESUMO
As the fastest mode of transport, the aircraft is a major driver for globalization and economic growth. The development of alternative advanced liquid fuels is critical to sustainable development within the sector. Such fuels should be compatible with existing infrastructure and derived from second generation feedstocks to avoid competition with food markets. With properties similar to petroleum based fuels, isoprenoid derived compounds such as limonene, bisabolane, farnesane, and pinene dimers are of increasing interest as "drop-in" replacement jet fuels. In this review potential isoprenoid derived jet fuels and progress toward their microbial production was discussed in detail. Although substantial advancements have been achieved, the use of first generation feedstocks remains ubiquitous. Lignocellulosic biomass is the most abundant raw material available for biofuel production, however, technological constraints associated with its pretreatment and saccharification hinder its economic feasibility for low-value commodity production. Non-conventional microbes with novel characteristics including cellulolytic bacteria and fungi capable of highly efficient lignocellulose degradation and xylose fermenting oleaginous yeast with enhanced lignin-associated inhibitor tolerance were investigated as alternatives to traditional model hosts. Finally, innovative bioprocessing methods including consolidated bioprocessing and sequential bioreactor approaches, with potential to capitalize on such unique natural capabilities were considered.
