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• W2792972194 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−1 in the strain with constitutive promoter constructs, and by 17-fold to 18 mg L−1 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−1 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." @default.
• W2792972194 created "2018-03-29" @default.
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• W2792972194 date "2018-05-01" @default.
• W2792972194 modified "2023-10-16" @default.
• W2792972194 title "Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae" @default.
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• W2792972194 doi "https://doi.org/10.1016/j.ymben.2018.02.005" @default.
• W2792972194 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/29471044" @default.
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