A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

Abstract

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae, Metabolic Engineering, http://dx.doi.org/10. 1016/j.ymben.2016.12.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractSesquiterpenes are C15 isoprenoids with utility as fragrances, flavours, pharmaceuticals, and potential biofuels. Microbial fermentation is being examined as a competitive approach for bulk production of these compounds. Competition for carbon allocation between synthesis of endogenous sterols and production of the introduced sesquiterpene limits yields. Achieving balance between endogenous sterols and heterologous sesquiterpenes is therefore required to achieve economical yields. In the current study, the yeast Saccharomyces cerevisiae was used to produce the acyclic sesquiterpene alcohol, trans-nerolidol. Nerolidol production was first improved by enhancing the upstream mevalonate pathway for the synthesis of the precursor farnesyl pyrophosphate (FPP). However, excess FPP was partially directed towards squalene by squalene synthase (Erg9p), resulting in squalene accumulation to 1 % biomass; moreover, the specific growth rate declined. In order to redirect carbon away from sterol production and towards the desired heterologous sesquiterpene, a novel protein destabilisation approach was developed for Erg9p. It was shown that Erg9p is located on endoplasmic reticulum and lipid droplets through a C-terminal ER-targeted transmembrane peptide. 2A PEST (rich in Pro, Glu/Asp, Ser, and Thr) sequence-dependent endoplasmic reticulum-associated protein degradation (ERAD) mechanism was established to decrease cellular levels of Erg9p without relying on inducers, repressors or specific repressing conditions. This improved nerolidol titre by 86 % to ~100 mg L -1 . In this strain, squalene levels were similar to the wild-type control strain, and downstream ergosterol levels were slightly decreased relative to the control, indicating redirection of carbon away from sterols and towards sesquiterpene production. There was no negative effect on cell growth under these conditions. Protein degradation is an efficient mechanism to control carbon allocation at flux-competing nodes in metabolic engineering applications. This study demonstrates that an engineered ERAD mechanism can be used to balance flux competition between the endogenous sterol pathway and an introduced bio-product pathways at the FPP node. The approach of protein degradation in general might be more widely applied to improve metabolic engineering outcomes.

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

“…Transcriptional down-regulation of ERG9 by chromosomal promoter replacement (using BTS1, HXT1, or CTR3 promoters) has been applied to decrease competition and redirect flux from sterols to heterologous sesquiterpenes (Paddon et al, 2013;Scalcinati et al, 2012;Xie et al, 2014). We recently demonstrated a protein degradation approach for successful down-regulation at this node using an endoplasmic reticulum associated protein degradation signal (Peng et al, 2017b), the first demonstration of engineered protein degradation for yeast metabolic engineering. Figure 1.…”

“…Previously, enzymes from the mevalonate (MVA) pathway were over-expressed to increase precursor flux for terpene synthesis (Figure 1) (Peng et al, 2017a;Peng et al, 2017b;Vickers et al, 2017). A dedicated GPP synthase has been used to increase the GPP pool for monoterpene production (Alonso-Gutierrez et al, 2013;Amiri et al, 2016;Cao et al, Liu et al, 2016;Rico et al, 2010;Sarria et al, 2014;Willrodt et al, 2014;Zhang et al, 2014).…”

“…Squalene epoxidase (ERG1) illustrates this multi-level regulation: ERG1 is upregulated by Upc2p in response to low ergosterol (Servouse and Karst, 1986;Vik and Rine, 2001), while Erg1p is degraded through the ERAD pathway resulting in a short protein half-life (Foresti et al, 2013). We have previously exploited the ERAD mechanism to regulate Erg9p, resulting in flux redirection from squalene accumulation to sesquiterpene production (Peng et al, 2017b). In the current study, we aimed to improve monoterpene production by elevating the GPP pool.…”

The Smell of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast

“…On a transcriptional level, the native promoter of the squalene synthase ERG9 gene (that encodes the enzyme that catalyses the first reaction of converting farnesyl diphosphate to ergosterol) was replaced with repressible promoters which led to subsequent lower concentrations of ERG9, facilitating the increased levels of recombinant terpenoid production [55]. Recently, a degradation tag attached to ERG9 was shown to destabilise the protein, which also led to a dramatic improvement in recombinant terpenoid production without compromising the cell viability to any significant extent [56].…”

The Smell of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast

“…On a transcriptional level, the native promoter of the squalene synthase ERG9 gene (that encodes the enzyme that catalyses the first reaction of converting farnesyl diphosphate to ergosterol) was replaced with repressible promoters which led to subsequent lower concentrations of ERG9, facilitating the increased levels of recombinant terpenoid production [54]. Recently, a degradation tag attached to ERG9 was shown to destabilise the protein, which also led to a dramatic improvement in recombinant terpenoid production without compromising the cell viability to any significant extent [55].…”