Metabolic Engineering of Saccharomyces Cerevisiae for Cyclic Terpenoid Production

Authors: 
Czarnotta, E., RWTH Aachen University
Knuf, C., DTU Denmark
Forster, J., Technical University of Denmark
Jacobsen, S. A., Technical University of Denmark
Polakowski, T., Organobalance GmbH

Triterpenoids are terpenoids derived from squalene and consist of six isoprene units (C30). These compounds can be isolated from many different plant sources. They occur in countless variations and can be subclassified into several groups including lanostanes, dammaranes, lupanes, oleananes, ursanes, hopanes, cycloartanes, friedelanes, cucurbitacins, and miscellaneous compounds (Mullauer et al. 2010).

Many of them or their synthetic derivatives are currently being investigated as medicinal products for various diseases. The cyclic triterpenoid betulinic acid is of special interest for the pharmaceutical and nutritional industry as it has antiretroviral, antimalarial, and anti-inflammatory properties and has potential as an anticancer agent (Muffler et al. 2011). Despite their obvious industrial potential, the application is often hindered by their low abundance in natural sources. This poses challenges in a biosustainable production of such compounds since per gram active ingredient produced a high volume of solvent is needed in the purification process.

Here, we present a novel biotechnological process for the production of betulinic acid using tailored Saccharomyces cerevisiae strains. The multi-scale optimization of this microbial process included:

  • pathway engineering by determination of optimal gene combination and dosage,
  • compartment engineering to increase the reaction space of the betulinic acid pathway, and
  • strain engineering by implementation of different push, pull and block strategies.

In parallel we developed the fermentation process and were able to boost the performance of the engineered yeast by optimization of medium composition, cultivation conditions, carbon source and mode of fermentation operation in lab scale bioreactors. Product purification was achieved by a one-step extraction with acetone.

The final process was evaluated in terms of economic and ecological efficiency and rated to be competitive with existing plant extraction procedures with potential for further performance improvement.

The engineered S. cerevisiae strain represents an ideal platform strain for the production of a variety of industrial compounds that can be derived from the betulinic acid precursors squalene and 2-oxidosqualene.

References

Muffler K, Leipold D, Scheller MC, Haas C, Steingroewer J, Bley T, Neuhaus HE, Mirata MA, Schrader J, Ulber R. 2011. Biotransformation of triterpenes. Process Biochemistry 46(1):1-15.

Mullauer FB, Kessler JH, Medema JP. 2010. Betulinic acid, a natural compound with potent anticancer effects. Anti-Cancer Drugs 21(3):215-227.