Metabolic Engineering of Saccharomyces cerevisiae for Sesquiterpene Production

Doctoral thesis, 2012

Industrial biotechnology aims to develop robust “microbial cell factories”, to produce an array of added value chemicals presently dominated by petrochemical processes. The exploitation of an efficient microbial production as sustainable technology has an important impact for our society. Sesquiterpenes are a class of natural products with a diverse range of attractive industrial proprieties. Due to economic difficulties of their production via traditional extraction processes or chemical synthesis there is interest in developing alternative and cost efficient bio-processes. Microbial cells engineered for efficient production of plant sesquiterpenes may allow for a sustainable and scalable production of these compounds. Saccharomyces cerevisiae is one of the most robust and characterized microbial platforms suitable to be exploited for bio-production. The hydrocarbon α-santalene is a precursor of sesquiterpenes with relevant commercial application and was selected as case study. Here, for the first time a S. cerevisiae strain capable of producing high levels of α-santalene was constructed through a multidisciplinary system level metabolic engineering approach. First, a minimal engineering approach was applied to address the feasibility of α-santalene production in S. cerevisiae. Successively, a rationally designed metabolic control strategy with the aim to dynamically modulate a key metabolic step to achieve optimal sesquiterpene production was applied, combined with the engineering of the main regulatory checkpoint of targeted pathway. It was possible to divert the carbon flux toward the sesquiterpene compound, and the resulting strain shows a 88-fold improvement in α-santalene productivity. A second round of strain optimization was performed using a multistep strategy focused to increase precursors and co-factor supply to manipulate the yeast metabolic network in order to further redirect the carbon toward the desired product. This approach results in an overall increase of 1.9-fold in α-santalene productivity. Furthermore, strain improvement was integrated with the development of an efficient fermentation/ downstream recovery process, resulting in a 1.4-fold improvement in productivity and a final α-santalene titer of 193 mg l-1. Finally, the substrate utilization range of the selected platform was expanded to use xylose as alternative carbon source for biorefinery compatibility, via pathway reconstruction and an evolutionary strategy approach, resulting in a strain capable of rapid growth and fast xylose consumption. The results obtained illustrate how the synergistic application of multilevel metabolic engineering and bioprocess engineering can be used to obtain a significant amount of high value sesquiterpene in yeast. This represents a starting point toward the construction of a yeast “sesquiterpene production factory” and for the development of an economically viable bio-based process that has the potential to replace the current production methods.