Mitochondrial Targeting of MVA Pathway Triggers Severe Inhibition of Post-Squalene Cholesterol Biosynthesis: Transcriptomic and Proteomic Insights in Yeast

Expression of the mevalonate (MVA) pathway in yeast mitochondria is deployed at commercial scale for production of squalene, because mitochondria contain abundant acetyl-CoA, the starting molecule of the MVA pathway. However, it is still unknown whether this strategy is effective in boosting the post-squalene pathway. Here the potential of this strategy is explored for production of the post-squalene chemical cholesterol, a precursor of many valuable steroidal drugs. A cholesterol-producing yeast strain, named CEN-Cho, was constructed by expressing the biosynthetic genes leading to cholesterol, accompanied by the augmentation of the cytosolic MVA pathway. The CEN-Cho strain produced 60.17 ng/mg fresh weight (FW) of squalene and 121.75 ng/mg FW of cholesterol in shake flask cultivation. When the complete MVA pathway was introduced into the mitochondria of CEN-Cho, yielding CEN-Cho-mMVA, the squalene level was increased to 325.24 ng/mg FW. Unexpectedly, the yield of cholesterol produced by CEN-Cho-mMVA was decreased to 1.5 ng/mg FW, demonstrating significant suppression on the post-squalene pathway due to the mitochondrial engineering. Comparative transcriptomic and proteomic analyses of the engineered cells provide insights into the metabolic and regulatory bottlenecks underlying this inhibition. This work reveals that the introduction of the MVA pathway into mitochondria generally inhibits the post-squalene pathway in yeast.