Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae

Abstract

Monoterpene production in Saccharomyces cerevisae requires the introduction of heterologous monoterpene synthases (MTSs). The endogenous farnesyl pyrosphosphate synthase (FPPS; Erg20p) competes with MTSs for the precursor geranyl pyrophosphate (GPP), which limits the production of monoterpenes. ERG20 is an essential gene that cannot be deleted and transcriptional down-regulation of ERG20 has failed to improve monoterpene production. Here, we investigated an N-degron-dependent protein degradation strategy to down-regulate Erg20p activity. Degron tagging decreased GFP protein half-life drastically to 1 h (degron K3K15) or 15 min (degrons KN113 and KN119). Degron tagging of ERG20 was therefore paired with a sterol responsive promoter to ensure sufficient metabolic flux to essential downstream sterols despite the severe destabilisation effect of degron tagging. A dual monoterpene/sesquiterpene (linalool/nerolidol) synthase, AcNES1, was used as a reporter of intracellular GPP and FPP production. Transcription of the synthetic pathway was controlled by either constitutive or diauxie-inducible promoters. A combination of degron K3K15 and the ERG1 promoter increased linalool titre by 27-fold to 11 mg L in the strain with constitutive promoter constructs, and by 17-fold to 18 mg L in the strain with diauxie-inducible promoter constructs. The sesquiterpene nerolidol remained the major product in both strains. The same strategies were applied to construct a limonene-producing strain, which produced 76 mg L in batch cultivation. The FPPS regulation method developed here successfully redirected metabolic flux toward monoterpene production. Examination of growth defects in various strains suggested that the intracellular FPP concentration had a significant effect on growth rate. Further strategies are required to balance intracellular production of FPP and GPP so as to maximise monoterpene production without impacting on cellular growth.

An Engineered Aro1 Protein Degradation Approach for Increased cis,cis -Muconic Acid Biosynthesis in Saccharomyces cerevisiae

“…Thus, aromatic amino acid synthesis must have been partially active under those conditions. An analogous degron-tagging strategy was recently reported to limit the activity of the essential Erg20 protein, thereby boosting the output of downstream monoterpene products by up to 27-fold (26). This study and the data that we have demonstrated herein showcase the effectiveness of protein degradation tags in modulating the activity of host targets in cases in which a basal level of activity must be preserved to maintain cellular fitness.…”

Production of Terpenoids by Synthetic Biology Approaches

“…Endoplasmic reticulum-associated protein degradation decreased cellular levels of ERG9, and increased the titer of sesquiterpene nerolidol to 100 mg/L (Peng et al, 2017). Also, N-degron-mediated destabilization of ERG20 improved the production of monoterpenes of 18 mg/L linalool or 76 mg/L limonene in S. cerevisiae (Peng et al, 2018). To decrease the concentration of pivotal enzyme, a synthetic degradation has been established based on Mesoplasma florum tmRNA system (Cameron and Collins, 2014).…”

The Potential Production of the Bioactive Compound Pinene Using Whey Permeate

“…The MVA pathway is native to S. cerevisiae, which initially seems promising; however, it is highly regulated and critical for cell survival. Monoterpene synthesis is more difficult to engineer into recombinant S. cerevisiae due to the presence of the farnesyl pyrophosphate synthase (Erg20p), an enzyme which competes for GPP, the precursor to pinene and other monoterpenoids [30,39]. Farnesyl pyrophosphate is an intermediate in the ergosterol production pathway and ergosterol plays a critical role in S. cerevisiae cell membrane fluidity, permeability, and structure [40].…”

“…Several strategies have been employed to overcome the necessity of ergosterol production and the enzymatic competition of the synthases for S. cerevisiae monoterpenoid production. N-degron protein degradation was employed to regulate Erg20p/ERG20; this yielded reported titers of 76 mg/L of limonene [39]. It was also shown that fusion of a terpene synthase with Erg20p increased S. cerevisiae monoterpene titers by 69% [41].…”