초록
<P><B>Abstract</B></P> <P>Monoterpene production in <I>Saccharomyces cerevisae</I> 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. <I>ERG20</I> is an essential gene that cannot be deleted and transcriptional down-regulation of <I>ERG20</I> 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 <I>ERG20</I> 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, <I>AcNES1</I>, 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 <I>ERG1</I> promoter increased linalool titre by 27-fold to 11 mg L<SUP>−1</SUP> in the strain with constitutive promoter constructs, and by 17-fold to 18 mg L<SUP>−1</SUP> 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<SUP>−1</SUP> 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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Farnesyl pyrophosphate synthase (Erg20p) consumes the monoterpene precursor geranyl pyrophosphate. </LI> <LI> N-degron mediated protein degradation and sterol responsive promoters were applied to regulate <I>ERG20</I>/Erg20p. </LI> <LI> The mevalonate pathway was combinatorially enhanced to facilitate precursor synthesis. </LI> <LI> Destabilization of Erg20p effectively improved the production of monoterpenes. </LI> <LI> In the final engineered strains, 18 mg L<SUP>−1</SUP> linalool or 76 mg L<SUP>−1</SUP> limonene were produced. </LI> </UL> </P>