A synthetic library of RNA control modules for predictable tuning of gene expression in yeast

Babiskin, Andrew; Smolke, Christina D.

doi:10.1038/msb.2011.4

Cited by 74 publications

(78 citation statements)

References 59 publications

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“…Protein concentration is dynamically regulated in the cell by modulating the rates of transcription, RNA degradation, translation and/or protein degradation (Babiskin and Smolke, 2011;Jungbluth et al, 2010;Raina and Crews, 2010;Sekar et al, 2016). In metabolic engineering, specific protein levels are typically controlled through transcription (at the promoter level); control mechanisms at other levels, especially at the protein level, have not been well exploited in yeast.…”

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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“…The amount of protein accumulated depends upon mRNA stability, which might in turn depend on secondary structures in the 3′-UTR7. Refractory elements in the 3′ untranslated region (UTR) that increase mRNA half-life might be available.…”

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confidence: 99%

Enhancement of protein production via the strong DIT1 terminator and two RNA-binding proteins in Saccharomyces cerevisiae

Ito

Kitagawa

Yamanishi

et al. 2016

Sci Rep

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Post-transcriptional upregulation is an effective way to increase the expression of transgenes and thus maximize the yields of target chemicals from metabolically engineered organisms. Refractory elements in the 3′ untranslated region (UTR) that increase mRNA half-life might be available. In Saccharomyces cerevisiae, several terminator regions have shown activity in increasing the production of proteins by upstream coding genes; among these terminators the DIT1 terminator has the highest activity. Here, we found in Saccharomyces cerevisiae that two resident trans-acting RNA-binding proteins (Nab6p and Pap1p) enhance the activity of the DIT1 terminator through the cis element GUUCG/U within the 3′-UTR. These two RNA-binding proteins could upregulate a battery of cell-wall–related genes. Mutagenesis of the DIT1 terminator improved its activity by a maximum of 500% of that of the standard PGK1 terminator. Further understanding and improvement of this system will facilitate inexpensive and stable production of complicated organism-derived drugs worldwide.

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“…Furthermore, even if a gene network can be made to perform well in a particular strain and environment, there is no guarantee that the same network will perform well if ported to a new strain or environment (7). More generally, synthetic networks often operate in substantially different parameter regimes than expected during the design process and must, therefore, be tuned (2,(8)(9)(10)(11) before they function properly, and even retuned when used in new environments or with different hosts.…”

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confidence: 99%

Fine-tuning gene networks using simple sequence repeats

Egbert

Klavins

2012

Proc. Natl. Acad. Sci. U.S.A.

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The parameters in a complex synthetic gene network must be extensively tuned before the network functions as designed. Here, we introduce a simple and general approach to rapidly tune gene networks in Escherichia coli using hypermutable simple sequence repeats embedded in the spacer region of the ribosome binding site. By varying repeat length, we generated expression libraries that incrementally and predictably sample gene expression levels over a 1,000-fold range. We demonstrate the utility of the approach by creating a bistable switch library that programmatically samples the expression space to balance the two states of the switch, and we illustrate the need for tuning by showing that the switch's behavior is sensitive to host context. Further, we show that mutation rates of the repeats are controllable in vivo for stability or for targeted mutagenesis-suggesting a new approach to optimizing gene networks via directed evolution. This tuning methodology should accelerate the process of engineering functionally complex gene networks.gene network optimization | evolvability | synthetic biology E ngineering reliable and predictable synthetic gene networks presents unique challenges because genetic parts such as promoters, ribosome binding sites, and protein coding regions often behave unexpectedly when used in novel designs. Noise (1, 2), metabolic load (3), poorly characterized interactions with the host (4), and general uncertainty about the detailed functionality of parts conspire to limit the complexity of synthetic gene networks to a small number of interacting genes (5, 6). As a result, a complex gene network that has been predicted analytically to perform well may in fact perform poorly, if it works at all, when ultimately implemented in cells. Furthermore, even if a gene network can be made to perform well in a particular strain and environment, there is no guarantee that the same network will perform well if ported to a new strain or environment (7). More generally, synthetic networks often operate in substantially different parameter regimes than expected during the design process and must, therefore, be tuned (2, 8-11) before they function properly, and even retuned when used in new environments or with different hosts.One way to improve the performance of a poorly tuned gene network is to introduce focused variability into the design, generating a library of circuits (12-15) with the same genetic components and connectivity, but with each member of the library operating in a different parameter regime. In bacteria, for example, promoters (16, 17), ribosome binding sites (RBS) (18,19), RNA stability (20, 21), protein stability (22), and other biochemical details such as transcription factor regulation (23) or enzyme catalysis (24) can be varied to sample different regions of parameter space. Furthermore, sensitivity analysis (25) can guide the designer to parameters that, when tuned, are most likely to result in improved performance. Subject to screening or selection, the effectiveness of a tuning lib...

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A synthetic library of RNA control modules for predictable tuning of gene expression in yeast

Abstract: The authors describe a library of synthetic RNA control elements that provide programmable post-transcriptional regulation of gene expression in yeast. This toolkit is then used to study endogenous regulation of the ergosterol biosynthetic pathway.

Cited by 74 publications

References 59 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

Enhancement of protein production via the strong DIT1 terminator and two RNA-binding proteins in Saccharomyces cerevisiae

Fine-tuning gene networks using simple sequence repeats

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