title: A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae.
abstract: Sesquiterpenes 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 re-direct 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. A 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 ~100mgL-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.
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Bingyin
authors.initials: B
authors.lastname: Peng
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072, Australia; Metabolomics Australia (Queensland Node), The University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Manuel R
authors.initials: MR
authors.lastname: Plan
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072, Australia; Metabolomics Australia (Queensland Node), The University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Chrysanthopoulos
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072, Australia; Metabolomics Australia (Queensland Node), The University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Hodson
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Lars K
authors.initials: LK
authors.lastname: Nielsen
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072, Australia. Electronic address: c.vickers@uq.edu.au.
authors.forename: Claudia E
authors.initials: CE
authors.lastname: Vickers
mesh_terms: D053898:Biosynthetic Pathways / Q000502:physiology; D004789:Enzyme Activation; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000502:physiology; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012717:Sesquiterpenes / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011106:Polyisoprenyl Phosphates; D029701:Saccharomyces cerevisiae Proteins; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; C496127:ERG9 protein, S cerevisiae; D013186:Farnesyl-Diphosphate Farnesyltransferase; C037055:nerolidol
keywords: Endoplasmic reticulum-associated protein degradation (ERAD); Metabolic engineering; Saccharomyces cerevisiae; Sesquiterpene; Trans-nerolidol