Exploiting the Geranylgeranyl‐Pyrophosphate‐Sensing N‐Terminal Domain of HMG‐CoA Reductase 2 to Regulate Farnesyl Pyrophosphate Synthase (Erg20p) for Improved Monoterpene Production in <i>Saccharomyces cerevisiae</i>

Dynamic downregulation of the endogenous farnesyl pyrophosphate (FPP) synthase (Erg20p) is crucial to engineer heterologous monoterpene production in the yeast Saccharomyces cerevisiae. FPP downstream metabolite geranylgeranyl pyrophosphate (GGPP) can induce the degradation of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase 2 (Hmg2p) through its N-terminal GGPP-sensing endoplasmic reticulum transmembrane domain (Hmg2p^N) in S. cerevisiae. Here, we investigate the use of Hmg2p^N to regulate Erg20p, aiming to restrict FPP synthesis and redirect metabolic flux to monoterpene production. While using the ERG1 promoter to regulate ERG20 transcription improved monoterpene limonene by ~10-fold, combinatory fusion of Hmg2p^N to Erg20p N-terminus further improved limonene production by 40% to 0.52 g L^-1 in synthetic minimal media. This approach yielded 0.5 g L^-1 geraniol in batch cultivation, comparable to levels achieved using the N-end-rule degron K3K15 or an auxin-inducible degron to regulate Erg20p. In rich complex media, this approach was superior, leading to 2.1 g L^-1 geraniol production in semi-fed batch cultivation. In summary, the Hmg2p^N domain is an efficient tool to constrain FPP synthesis for improved monoterpene production in S. cerevisiae.