Statin Resistance and Export : - development of a yeast cell factory for compactin

Abstract

Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the key enzyme in the mevalonate pathway that leads to the synthesis of cholesterol and ergosterol in animal and fungal cells, respectively. Their extensiveuse in treatment and prevention of cardiovascular diseases placed statins among the best-selling pharmaceuticals. Industrial scale production of natural statins (i.e.compactin and lovastatin) and their semi-synthetic derivatives (i.e. pravastatin andsimvastatin) is based on fermentation of statin-producing filamentous fungi, such as Penicillium sp. and Aspergillus sp.. Production limitations associated with the unique physiology and morphology of the natural producers can be overcome by heterologous expression of the pathway in a fast-growing host, such as S. cerevisiae. Future construction of S. cerevisiae cell factory for the production of high concentrations of natural statins will require the establishment of a non-destructive self-resistance mechanism to overcome the undesirable growth inhibition effects of statins. In an effort to resolve this challenge, two putative self-resistance genes, mlcD and mlcE, originating from the P. citrinum compactin gene cluster, were tested for their ability to protect yeast from statins. Chromosomal gene integration approach was used to express the genes in S. cerevisiae.This study showed that mlcD could mediate statin-resistance when expressed heterologously in S. cerevisiae, increasing yeast resistance from 0.25 mM to at least 1.24 mM. Successful complementation of Sc-HMG1 and Sc-HMG2 in yeast, in addition, proved that MlcD functions as HMGCR. A phylogenetic analysis of fungal HMGCRs revealed that HMGCRs from the known statin gene clusters (mlcD and lvrA/mokG) are likely derived from HMGCRs involved in primary metabolism. However, the occurrence of these genes in the different statin geneclusters probably did not arise from a recent duplication of the primary HMGCR inthe producing organism. A model was proposed, in which the HMGCR-encoding genes at some time during evolution were duplicated and then recruited to the statin gene clusters, a situation that has increased the chance for becoming co-regulated with the cluster and hence statin production. Collectively these results suggest that statin self-resistance is based on the HMGRS's association with the cluster, e.g.increased concentration of HMGCR at the right time, and is not due to the HMGCR being statin-insensitive. This model will require further validation by e.g. direct measurement of enzymes sensitivity to statins. Heterologous expression of mlcE, the second putative self-resistance gene from the compactin gene cluster, significantly increased S. cerevisiae’s resistance to natural statins (8.6-fold increase in growth efficiency compared to the wild-type strain). Sequence based analysis showed that MlcE likely contains 14 transmembrane spanning domains and phylogenetically it clusters together with known toxin efflux pumps from both fungi and bacteria. RFP-tagging of MlcE showed that it was localized to the plasma and vacuolar membranes in yeast. Collectively these results indicate that mlcE encodes for a transmembrane transporter, and thus likely provides the resistance to statins by secreting the compounds outside of the cells. Further testing of MlcE’s role as a self-resistance mechanism surprisingly showed that it was not only capable of protecting yeast from the negative effects of natural statins, but also semi-synthetic statins (i.e simvastatin).This work provides possible engineering strategies for improvement of future yeast based production of natural and semi-synthetic statins in yeast. Moreover, it gives new insights into the statin self-resistance mechanisms in the natural producers.