Dynamic control of ERG9 expression for improved amorpha-4,11-diene production in Saccharomyces cerevisiae

Yuan, Jifeng; Ching, Chi-Bun

doi:10.1186/s12934-015-0220-x

Cited by 94 publications

(83 citation statements)

References 30 publications

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“…The stability of squalene synthase also means that the squalene synthase node is less sensitive to down-regulation using transcriptional approaches (Yuan and Ching, 2015), because a decrease in cellular squalene synthase levels after transcritpional inhibition depends on subsequent dilution of the Erg9p protein through cell division (Sekar et al, 2016). As a novel metabolic engineering strategy, protein destabilization might be able to effect a rapid reduction in absolute enzyme concentration (Jungbluth et al, 2010), thereby quickly diminishing competition at specific nodes.…”

Section: Introductionmentioning

confidence: 99%

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: Introductionmentioning

confidence: 99%

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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“…Processes of this kind have been employed to produce different target compounds like succinate, lactate, or amino acids . The recent advent of new experimental techniques for the dynamic regulation of metabolic fluxes has greatly broadened the spectrum of dynamic process strategies and includes, for example, genetic toggle switches and tunable cell density sensors for controlling metabolic fluxes . Apart from applications for increasing volumetric productivity, two‐stage fermentations (TSFs) are also useful or even essential if a knockout of a gene for redirecting flux from biomass to product involves essential genes .…”

Section: Introductionmentioning

confidence: 99%

When Do Two‐Stage Processes Outperform One‐Stage Processes?

Klamt

Mahadevan

Hädicke

2017

Biotechnology Journal

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Apart from product yield and titer, volumetric productivity is a key performance indicator for many biotechnological processes. Due to the inherent trade-off between the production of biomass as catalyst and of the actual target product, yield and volumetric productivity cannot be optimized simultaneously. Therefore, in combination with genetic techniques for dynamic regulation of metabolic fluxes, two-stage fermentations (TSFs) with separated growth and production phase have recently gained much interest because of their potential to improve the productivity of bioprocesses while still allowing high product yields. However, despite some successful case studies, so far it has not been discussed and analyzed systematically whether or under which conditions a TSF guarantees superior productivity compared to one-stage fermentation (OSF). In this study, we use mathematical models to demonstrate that the volumetric productivity of a TSF is not automatically better than of a corresponding OSF. Our analysis reveals that the sharp decrease of the specific substrate uptake rate usually observed in (non-growth) production phases severely impacts the volumetric productivity and thus raises a big challenge for designing competitive TSF processes. We discuss possible approaches such as enforced ATP wasting to improve substrate utilization rates in the production phase by which TSF processes can become superior to OSF. We also analyze additional factors influencing the relative performance of OSF and TSF and show that OSF processes can be more appropriate if a high product yield is an economic constraint. In conclusion, a careful assessment of the trade-offs between substrate uptake rates, yields, and productivity is necessary when deciding for OSF vs. TSF processes.

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“…For this purpose, balanced expression of ERG9 using ergosterol responsive promoter [73] improved the production yield. The methionine-repressible promoter was also utilized for the same purpose.…”

Section: Balancing Gene Expression Levelmentioning

confidence: 99%

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

Park

Yang

et al. 2017

Adv. Biosys.

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Natural products have been attracting much interest around the world for their diverse applications, especially in drug and food industries. Plants have been a major source of many different natural products. However, plants are affected by weather and environmental conditions and their successful extraction is rather limited. Chemical synthesis is inefficient due to the complexity of their chemical structures involving enantioselectivity and regioselectivity. For these reasons, an alternative means of overproducing valuable natural products using microorganisms has emerged. In recent years, various metabolic engineering strategies have been developed for the production of natural products by microorganisms. Here, the strategies taken to produce natural products are reviewed. For convenience, natural products are classified into four main categories: terpenoids, phenylpropanoids, polyketides, and alkaloids. For each product category, the strategies for establishing and rewiring the metabolic network for heterologous natural product biosynthesis, systems approaches undertaken to optimize production hosts, and the strategies for fermentation optimization are reviewed. Taken together, metabolic engineering has enabled microorganisms to serve as a prominent platform for natural compounds production. This article examines both the conventional and novel strategies of metabolic engineering, providing general strategies for complex natural compound production through the development of robust microbial‐cell factories.

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Dynamic control of ERG9 expression for improved amorpha-4,11-diene production in Saccharomyces cerevisiae

Cited by 94 publications

References 30 publications

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

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

When Do Two‐Stage Processes Outperform One‐Stage Processes?

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

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