1 Department of Systems Biology, Technical University of Denmark2 Eucaryotic Molecular Cell Biology, Department of Systems Biology, Technical University of Denmark
The use of statins as cholesterol-lowering drugs is based on their ability to inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the key enzyme in the mevalonate pathway, which is responsible for the production of ergosterol in fungi and cholesterol in human. Industrial scale production of natural statins (i.e. compactin and lovastatin) and their semi-synthetic derivatives (i.e. pravastatin and simvastatin) is based on fermentation of statin-producing filamentous fungi, such as Aspergillus terreus and Penicillium solitum, however, the unique physiology and morphology make these natural producers difficult to culture in bioreactors. The production limitations associated with the use of natural producers can be overcome by heterologous expression of the biosynthetic pathway in Saccharomyces cerevisiae (1), however, it is crucial to establish a nondestructive resistance mechanism in yeast, which would overcome the undesirable effects of statins. One possible mechanism is an active export of statins, a mechanism that does not just provide the resistance but can also significantly ease the purification of the produced compounds. In order to establish export of statins from yeast we integrated a putative efflux pump-encoding gene mlcE from the P. solitum compactin biosynthetic gene cluster into S. cerevisiae genome. The resulting strain was tested for susceptibility to statins by growing the strain on media containing statins. The constructed strain showed an increased resistance to both natural statins (compactin and lovastatin), and also to a semi-synthetic statin simvastatin, when compared to the wild type strain. Expression of a mRFP-tagged MlcE show that MlcE is localized in the yeast plasma membrane. In conclusion we provide evidence indicating that MlcE is a transmembrane efflux pump, capable of exporting natural and semi-natural statins from yeast, and overexpression of MlcE in a statinproducing yeast could therefore greatly improve the commercial production of natural and semi-natural statins. Reference: (1) Xu W. et al., (2013), “LovG: The Thioesterase Required for Dihydromonacolin”, Angew. Chem. Int. Ed. 2013, 52, 6472 –6475.