Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae

Xie, Wenping; Ye, Lidan; Lv, Xiaomei; Xu, Haoming; Yu, Hongwei

doi:10.1016/j.ymben.2014.11.007

Cited by 175 publications

(166 citation statements)

References 47 publications

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“…The fold-increase achieved using Erg9p destabilization is comparable to the fold-increase observed for other terpene end-products when using the low-glucose-repressed HXT1 promoter to control ERG9 (Scalcinati et al, 2012a;Xie et al, 2014). The advantage for protein destablization strategy is that its regulation does not depend on having a low glucose concentration or addition of a repressoras the HXT1/MET3/CRT3 promoters require.…”

Section: Destabilisation Of Erg9p Increases Nerolidol Productionmentioning

confidence: 75%

“…Transcriptional outputs can be tuned by choosing promoters with different strength and/or expression patterns, or dynamically regulated by applying synthetic molecular circuits (Peng et al, 2015;Williams et al, 2015a;Williams et al, 2015b;Xie et al, 2014). Here, we examined a novel approach to modulate metabolic flux through destabilisation of a competing enzyme at a key node.…”

Section: Resultsmentioning

confidence: 99%

“…4). In contrast to previous transcriptionally-mediated approaches to regulate squalene synthase (Asadollahi et al, 2008;Paddon et al, 2013;Scalcinati et al, 2012a;Scalcinati et al, 2012b;Xie et al, 2014), the destabilization strategy does not require either addition of repressors which may increase the cost of the bioprocess (in the case of repressible promoters), or appropriately repressing fermentation conditions (in the case of glucose-responsive promoter). Destabilisation of squalene synthase does not inhibit the maximum specific growth rate.…”

Section: Discussionmentioning

confidence: 99%

“…For metabolic engineering applications, transcription/RNA-level engineering has been used to regulate the flux-competing nodes, e.g., selecting a weaker promoter (Scalcinati et al, 2012a) or applying RNA interference (Williams et al, 2015a). To direct more FPP toward sesquiterpene production, transcription of the native squalene synthase gene (ERG9) is commonly decreased by replacing the native promoter with a weaker promoter or a promoter that can be specifically down-regulated (Asadollahi et al, 2008;Paddon et al, 2013;Scalcinati et al, 2012a;Scalcinati et al, 2012b;Xie et al, 2014). Here we investigated a novel alternative strategy to regulate the level of Erg9p: a protein degradation mechanism to decrease protein half-life.…”

Section: Minimising Competition For Fpp By Destabilising Squalene Synmentioning

confidence: 99%

“…Conditional down-regulation of ERG9 at the transcriptional level has therefore been used to constrain squalene/sterol synthesis and increase heterogeneous sesquiterpene production. This has been achieved by replacing the native ERG9 promoter on the genome with various repressible promoters: MET3 (repressed upon addition of methionine to the medium), CRT3 (repressed on highconcentration copper) or HXT1 (induced by high glucose and reduced under low glucose; transcription decreases after the exponential phase of batch cultivation) (Asadollahi et al, 2008;Paddon et al, 2013;Scalcinati et al, 2012a;Scalcinati et al, 2012b;Xie et al, 2014). However, these strategies require strict fermentation conditions and/or addition of repressors to achieve regulation, conditions that can increase the complexity and/or cost in industrial processes.…”

Section: Introductionmentioning

confidence: 99%

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

Peng

Plan

Chrysanthopoulos

et al. 2017

Metabolic Engineering

128

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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.

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Section: Destabilisation Of Erg9p Increases Nerolidol Productionmentioning

confidence: 75%