Metabolic compartmentalization in yeast mitochondria: Burden and solution for squalene overproduction

Harnessing mitochondria is considered as a promising method for biosynthesis of terpenes due to the adequate supply of acetyl-CoA and redox equivalents in mitochondria. However, mitochondrial engineering often causes serious metabolic burden indicated by poor cell growth. Here, we systematically analyzed the metabolic burden caused by the compartmentalization of the MVA pathway in yeast mitochondria for squalene synthesis. The phosphorylated intermediates of the MVA pathway, especially mevalonate-5-P and mevalonate-5-PP, conferred serious toxicity within mitochondria, which significantly compromised its possible advantages for squalene synthesis and was difficult to be significantly improved by routine pathway optimization. These phosphorylated intermediates were converted into ATP analogues, which strongly inhibited ATP-related cell function, such as mitochondrial oxidative respiration. Fortunately, the introduction of a partial MVA pathway from acetyl-CoA to mevalonate in mitochondria as well as the augmentation of the synthesis of mevalonate in cytosol could significantly promote the growth of yeasts. Accordingly, a combinatorial strategy of cytoplasmic and mitochondrial engineering was proposed to alleviate the metabolic burden caused by the compartmentalized MVA pathway in mitochondria and improve cell growth. The strategy also displayed the superimposed effect of cytoplasmic engineering and mitochondrial engineering on squalene production. Through a two-stage fermentation process, the squalene titer reached 21.1 g/L with a specific squalene titer of 437.1 mg/g dcw, which was the highest at present. This provides new insight into the production of squalene and other terpenes in yeasts based on the advantages of mitochondrial engineering. • Compartmentalizing the MVA pathway in yeast mitochondria can cause serious metabolic burden. • The phosphorylated metabolites of the MVA pathway are toxic within mitochondria. • Enhanced synthesis of mevalonate in cytosol can conquer the burden of mitochondrial engineering. • Cytoplasmic engineering is needed to take the full advantage of mitochondrial engineering.