Metabolic engineering of Saccharomyces cerevisiae for linalool production

Amiri, p; Shahpiri, Azar; Asadollahi, Mohammad Ali; Momenbeik, Fariborz; Partow, Siavash

doi:10.1007/s10529-015-2000-4

Cited by 63 publications

(39 citation statements)

References 25 publications

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“…However, this strain showed a dramatic growth deficiency and required a 14-day cultivation (20 g L -1 glucose) to obtain this titre. In other engineered microbial strains, linalool production titres remain in the μg L -1 scale (Amiri et al, 2016;Rico et al, 2010). The highest titres for other monoterpenes in batch cultivation in engineered microorganisms are currently 41 mg L -1 for sabinene in E.coli (Zhang et al, 2014), 32 mg L -1 for pinene in E.coli (Sarria et al, 2014), 66 mg L -1 for geraniol in S. cerevisiae (Zhao et al, 2016), 69 mg L -1 for geraniol in E.coli (Liu et al, 2016), and 24 mg L -1 for limonene in Yarrowia lipolytica (Cao et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

“…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). However, yields were low compared to production of sesquiterpenes in a similar system, suggesting that the native Erg20p competes effectively with the monoterpene synthase for GPP.…”

Section: Introductionmentioning

confidence: 99%

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

Peng

Nielsen

Kampranis

et al. 2018

Metabolic Engineering

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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 in the strain with constitutive promoter constructs, and by 17-fold to 18 mg L 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 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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Peng

Nielsen

Kampranis

et al. 2018

Metabolic Engineering

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“…In addition to gene knock-out strategies, re-directing metabolic fluxes by tuning the expression of essential genes encoding competing pathway steps is another strategy often used to improve biobased production. This has traditionally been accomplished by integrating inducible or repressible promoters to control the expression of flux controlling genes amenable to transcriptional regulation [7, 29, 40]. More recently, CRISPR/dCas9 has been adopted for redirecting metabolic fluxes through transcriptional regulation of single and multiple genes [21, 22, 24, 41].…”

Section: Discussionmentioning

confidence: 99%

Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

et al. 2017

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BackgroundTranscriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site.ResultsIn this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker’s yeast Saccharomyces cerevisiae. By testing 101 guide-RNA (gRNA) structures on a total of 14 different yeast promoters, we identified the best-performing combinations based on reporter assays. Though a larger number of gRNA-promoter combinations do not perturb gene expression, some gRNAs support expression perturbations up to ~threefold. The best-performing gRNAs were used for single and multiplex reprogramming strategies for redirecting flux related to isoprenoid production and optimization of TAG profiles. From these studies, we identified both constitutive and inducible multiplex reprogramming strategies enabling significant changes in isoprenoid production and increases in TAG.ConclusionTaken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also identify a large number of gRNA positions in 14 native yeast target pomoters that do not affect expression, suggesting the need for further optimization of gRNA design tools and dCas9 engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0664-2) contains supplementary material, which is available to authorized users.

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“…4). Several terpene synthases from fungal, plant, and bacterial origin have been reported to produce linalool (Crowell et al, 2002;Dudareva et al, 1996;Jia et al, 1999;Landmann et al, 2007;Pichersky et al, 1995) but only a few studies have shown microbial production of this molecule at low titers (Amiri et al, 2016;Thanasomboon et al, 2012). We cloned the truncated 3R-linalool synthase, in which the plastidial transit peptide was deleted, from M. citrate (Crowell et al, 2002) into the 1P, 2P, and 2P Ã systems, and they were transformed and tested for production in both wild type and the ispA mutant DH1 host strains.…”

Section: Production Of Other Jet Fuel Precursor Monoterpenesmentioning

confidence: 99%

Production of jet fuel precursor monoterpenoids from engineered Escherichia coli

Mendez-Perez

Alonso-Gutierrez

et al. 2017

Biotech & Bioengineering

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Monoterpenes (C 10 isoprenoids) are the main components of essential oils and are possible precursors for many commodity chemicals and high energy density fuels. Monoterpenes are synthesized from geranyl diphosphate (GPP), which is also the precursor for the biosynthesis of farnesyl diphosphate (FPP). FPP biosynthesis diverts the carbon flux from monoterpene production to C 15 products and quinone biosynthesis. In this study, we tested a chromosomal mutation of E. coli's native FPP synthase (IspA) to improve GPP availability for the production of monoterpenes using a heterologous mevalonate pathway. Monoterpene production at high levels required not only optimization of GPP production but also a basal level of FPP to maintain growth. The optimized strains produced two jet fuel precursor monoterpenoids 1,8-cineole and linalool at the titer of 653 mg/L and 505 mg/L, respectively, in batch cultures with 1% glucose. The engineered strains developed in this work provide useful resources for the production of high-value monoterpenes. This article is protected by copyright. All rights reserved Acc e p ted P r e p r i nt

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Metabolic engineering of Saccharomyces cerevisiae for linalool production

Abstract: Although overexpression of tHMG1 and downregulation of ERG9 enhanced linalool titers threefold in the engineered yeast strain, alleviating linalool toxicity is necessary for further improvement of linalool biosynthesis in yeast.

Cited by 63 publications

References 25 publications

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

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

Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Production of jet fuel precursor monoterpenoids from engineered Escherichia coli

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