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Genetic control elements based on Rnt1p hairpins. (A) Consensus elements of an Rnt1p hairpin. Color scheme is as follows: cleavage efficiency box (CEB), red; binding stability box (BSB), blue; initial binding and positioning box (IBPB), green. Black triangles represent location of cleavage sites. The clamp region is a synthetic sequence that acts to insulate and maintain the structure of the control element. (B) Schematic illustrating the mechanism by which Rnt1p hairpins act as gene control elements when placed in the 3′ UTR of a gene of interest (goi). Barrels represent protein molecules. (C) Sequences and structures of Rnt1p hairpin controls. (D) The transcript and protein levels associated with Rnt1p hairpins and their corresponding mutated tetraloop (CAUC) controls support that the observed gene regulatory activity is due to Rnt1p processing. Normalized protein expression levels are determined by measuring the median GFP levels from a cell population harboring the appropriate construct through flow cytometry analysis and values are reported relative to that from an identical construct lacking a hairpin module (no insert). Reported values and their error are calculated from the mean and standard deviation, respectively, from the three identically grown samples. Transcript levels are determined by measuring transcript levels of yEGFP3 and a housekeeping gene, ACT1, through qRT–PCR and normalizing the yEGFP3 levels with their corresponding ACT1 levels. Normalized transcript levels are reported relative to that from an identical construct lacking a hairpin module. Reported values and their error are calculated from the mean and standard deviation, respectively, from three identically prepared qRT–PCR reactions. Source data is available for this figure at www.nature.com/msb.
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Advances in synthetic biology have resulted in the development of genetic tools that support the design of complex biological systems encoding desired functions. The majority of efforts have focused on the development of regulatory tools in bacteria, whereas fewer tools exist for the tuning of expression levels in eukaryotic organisms. Here, we des...
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... The yeast Saccharomyces cerevisiae, a commonly used organism in molecular biological research and biotechnology applications (Blount et al., 2012), does not naturally express a complete RNAi pathway (Feldmann, 2011). However, upon introduction of Dcr and Ago in the yeast genome, RNA interference can be re-established (Wang et al., 2006;Drinnenberg et al., 2009) and employed in synthetic gene circuits to downregulate protein expression at the mRNA level as an alternative to ribozymes/riboswitches (Babiskin and Smolke, 2011;Groher et al., 2018;Liu et al., 2023), PUF proteins (JACKSON et al., 2004), and CRISPR-(d)Cas systems (d: DNase-deficient) (Borchardt et al., 2015;Zhang et al., 2022;Yu and Marchisio, 2023). ...
Boolean gates, the fundamental components of digital circuits, have been widely investigated in synthetic biology because they permit the fabrication of biosensors and facilitate biocomputing. This study was conducted to design and construct Boolean gates in the yeast Saccharomyces cerevisiae, the main component of which was the RNA interference pathway (RNAi) that is naturally absent from the budding yeast cells. We tested different expression cassettes for the siRNA precursor (a giant hairpin sequence, a DNA fragment—flanked by one or two introns—between convergent promoters or transcribed separately in the sense and antisense directions) and placed different components under the control of the circuit inputs (i.e., the siRNA precursor or proteins such as the Dicer and the Argonaute). We found that RNAi-based logic gates are highly sensitive to promoter leakage and, for this reason, challenging to implement in vivo. Convergent-promoter architecture turned out to be the most reliable solution, even though the overall best performance was achieved with the most difficult design based on the siRNA precursor as a giant hairpin.
... A few years later, Babiskin and Smolke [92] developed a library of 16 synthetic hairpins cleaved by Rnt1p.These novel synthetic RNA structures proved to be effective modules for regulating translation, in S. cerevisiae, upon placement in the 3′UTR of a target gene. The hairpins were designed by randomizing the 12 nucleotides inside the CEB and adding a four-G-C-pair clamp below the CEB itself, to achieve structural stability. ...
... (a) The hairpin cleaved by the Rnt1 protein consisted of three main parts: The IBPB (initial binding and positioning box); the BSB (binding stability box); and the CEB (cleavage efficiency box). The "Clamp" is a spacer sequence needed to insulate and stabilize the hairpin[92]. (b) Theophylline aptamer fused to the Rnt1p hairpin substrate in the GFP 3′UTR. ...
Among noncoding RNA sequences, riboswitches and ribozymes have attracted the attention of the synthetic biology community as circuit components for translation regulation. When fused to aptamer sequences, ribozymes and riboswitches are enabled to interact with chemicals. Therefore, protein synthesis can be controlled at the mRNA level without the need for transcription factors. Potentially, the use of chemical-responsive ribozymes/riboswitches would drastically simplify the design of genetic circuits. In this review, we describe synthetic RNA structures that have been used so far in the yeast Saccharomyces cerevisiae. We present their interaction mode with different chemicals (e.g., theophylline and antibiotics) or proteins (such as the RNase III) and their recent employment into clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas) systems. Particular attention is paid, throughout the whole paper, to their usage and performance into synthetic gene circuits.
... These riboswitches have become stable in the yeast synthetic biology toolbox (reviewed in Redden, Morse, & Alper, 2015). A particularly powerful example is an aptamer-controlled RNase III switch that was found to modulate gene expression over a 44% dynamic range based on the ligand concentration allowing tunable expression of the ergosterol pathway (Babiskin & Smolke, 2011). ...
Yeasts are essential for many processes, including the production of some of our most beloved foods and beverages. Less is known to the public about the far‐reaching impacts of yeasts on other products such as biofuels, pharmaceuticals, and industrial products. By leveraging and combining newly discovered yeast genetic diversity with now affordable and efficient genetic engineering and synthetic biology tools, academic and industrial yeast labs have designed yeast cell factories for a wide range of novel applications such as the production of medicines, components of human breast milk, heme for meat substitutes, bioplastics, and other biomaterials. This review covers the newest technologies developed for yeast research including synthetic biology and their use in the engineering of yeast cell factories for emerging applications.
... Therefore, a series of researches focus on construction of promoters and their modification by sequence randomization, promoter hybridization, and mathematic prediction. Besides transcription, metabolic flux could also be systematically optimized to enhance the production of target products by regulation of the level of translation, mainly focusing on the modification of ribosome binding site (RBS), 5′-untranslated region (5′ UTR), and RNase III cleavage of mRNA transcripts (Babiskin and Smolke 2011). It is notable that none of RBS, 5′ UTR, and RNase III cleavage of mRNA transcripts is a promoter component, although they are engineered simultaneously when engineering promoters in many cases (Babiskin and Smolke 2011;Kosuri et al. 2013). ...
... Besides transcription, metabolic flux could also be systematically optimized to enhance the production of target products by regulation of the level of translation, mainly focusing on the modification of ribosome binding site (RBS), 5′-untranslated region (5′ UTR), and RNase III cleavage of mRNA transcripts (Babiskin and Smolke 2011). It is notable that none of RBS, 5′ UTR, and RNase III cleavage of mRNA transcripts is a promoter component, although they are engineered simultaneously when engineering promoters in many cases (Babiskin and Smolke 2011;Kosuri et al. 2013). ...
... The hybrid promoter strategy was adopted to synthesize 12,563 combinations of 114 common promoters and 111 RBSs under DNA, RNA, and protein levels from the entire library (Kosuri et al. 2013). Babiskin and Smolke (2011) inserted Rnt1p hairpin as hybrid elements within the 3′ UTR of a transcript to direct cleavage to that region. A library of synthetic control modules in yeast based on randomizing the cleavage efficiency box was generated. ...
Promoter engineering is an enabling technology in metabolic engineering and synthetic biology. As an indispensable part of synthetic biology, the promoter is a key factor in regulating genetic circuits and in coordinating multi-gene biosynthetic pathways. In this review, we summarized the recent progresses in promoter engineering in microbes. Specifically, the endogenous promoters are firstly discussed, followed by the statement of the influence of nucleotides exchange on the strength of promoters explored by site-selective mutagenesis. We then introduced the promoter libraries with a wide range of strength, which are constructed focusing on core promoter regions and upstream activating sequences by rational designs. Finally, the application of promoter libraries in the optimization of multi-gene metabolic pathways for high-yield production of metabolites was illustrated with a couple of recent examples.
... Primers for the ACT1 (actin) (Babiskin and Smolke, 2011), QCR9 (Cytochrome b-c1 complex subunit 9) (Vaudano et al., 2009) and TFC1 genes (Transcription factor tau 95 kDa subunit) (Teste et al., 2009) were included as reference genes. Real time-PCR (qPCR) reactions were performed as triplicate in a LightCycler 1.5 (Roche Diagnostics GmbH, Mannheim, Germany). ...
Volatile esters are formed during alcoholic fermentation, particularly performed by yeasts. These compounds confer “fruity" attributes to drinks. Fermented beverages contain namely two groups of esters: acetate esters formed by alcohol acetyltransferase enzymes (ATF1 and ATF2) and ethyl esters produced by the acyl-CoA ethanol O-acyltransferase enzymes (EHT1 and EEB1), encoded by the ATF1, ATF2, EHT1 and EEB1 genes. The purpose of this study was to evaluate the e�ect of nitrogen addition, aeration and low temperatures on ester production during fermentation on Agave tequilana juice in a continuous system. Low temperatures increased ethyl esters production (4.5-fold) and enhanced the expression of the involved genes (more than 3.5-fold). Conversely, when compared to aeration, the e�ect was negative as esters production decreased (75%) whereas the ATF1, ATF2 and EHT1 genes were repressed. On the other hand, the addition of nitrogen favored the production of ethyl acetate. The results obtained during continuous culture with Agave tequilana juice showed that an increase in gene expression favored mainly the production of ethyl esters and not so much the production of ethyl acetate. Therefore, the three genes evaluated do not seem to be the only ones involved in the production of ethyl acetate, they are more related to the production of ethyl esters. So, even though all these compounds belong to the same family, the regulation of their production, as well as their synthesis pathways, seem to be di�erent.
... Primers for the ACT1 (actin) (Babiskin and Smolke, 2011), QCR9 (Cytochrome b-c1 complex subunit 9) (Vaudano et al., 2009) and TFC1 genes (Transcription factor tau 95 kDa subunit) (Teste et al., 2009) were included as reference genes. Real time-PCR (qPCR) reactions were performed as triplicate in a LightCycler 1.5 (Roche Diagnostics GmbH, Mannheim, Germany). ...
Volatile esters are formed during alcoholic fermentation, particularly performed by yeasts. These compounds confer “fruity" attributes to drinks. Fermented beverages contain namely two groups of esters: acetate esters formed by alcohol acetyltransferase enzymes (ATF1 and ATF2) and ethyl esters produced by the acyl-CoA ethanol O acyltransferase enzymes (EHT1 and EEB1), encoded by the ATF1, ATF2, EHT1 and EEB1 genes. The purpose of this study was to evaluate the e�ect of nitrogen addition, aeration and low temperatures on ester production during fermentation on Agave tequilana juice in a continuous system. Low temperatures increased ethyl esters production (4.5-fold) and enhanced the expression of the involved genes (more than 3.5-fold).
Conversely, when compared to aeration, the e�ect was negative as esters production decreased (75%) whereas the ATF1, ATF2 and EHT1 genes were repressed. On the other hand, the addition of nitrogen favored the production of ethyl acetate. The results obtained during continuous culture with Agave tequilana juice showed that an increase in gene expression favored mainly the production of ethyl esters and not so much the production of ethyl acetate. Therefore, the three genes evaluated do not seem to be the only ones involved in the production of ethyl acetate, they are more related to the production of ethyl esters. So, even though all these compounds belong to the same family, the regulation of their production, as well as their synthesis pathways, seem to be di�erent.
... RNA based regulators have become highly versatile and easily programmable tools for synthetic biology applications [34]. For example, another recent example in yeast involved the incorporation of an mRNA hairpin cleavage site that is targeted by the yeast Rnt1p RNAse to destabilise the 3 0 UTR of ERG9 mRNA, enabling fine-tuning of MVA pathway flux at the C15 node [35]. General synthetic biology tools that can dynamically regulate gene expression to control competition between growth and production in yeast have also been developed, but not yet applied to isoprenoid synthesis. ...
Isoprenoids (terpenes/terpenoids) have many useful industrial applications, but are often not produced at industrially viable level in their natural sources. Synthetic biology approaches have been used extensively to reconstruct metabolic pathways in tractable microbial hosts such as yeast and re-engineer pathways and networks to increase yields. Here we review recent advances in this field, focusing on central carbon metabolism engineering to increase precursor supply, re-directing carbon flux for production of C10, C15, or C20 isoprenoids, and chemical decoration of high value diterpenoids (C20). We also overview other novel synthetic biology strategies that have potential utility in yeast isoprenoid pathway engineering. Finally, we address the question of what is required in the future to move the field forwards.
... In several past experiments in yeast, secondary structures have been added to mRNAs to regulate and tune their expression. Lamping et al. recently showed that GC-rich sequences encoding hairpins can be used as modules to down-regulate expression [20], while others have combined hairpins with other forms of RNA-based translational regulation or with RNA-binding proteins that bind these motifs [28,29,30,31]. No approach, however, exists where gene expression can be predictably tuned at the translation step in eukaryotes as it can in bacteria. ...
... But because hairpin interactions are more specific and predictable this is much less of an issue in our approach, allowing the designed sequences to be used as modules. Already a set of 16 hairpin modules for S. cerevisiae 3'UTR regions has been described which modifies gene expression by altering mRNA degradation rates [31]. However, this complementary approach does not allow a priori predictions of protein expression levels based on sequence as achieved here. ...
Predictable tuning of gene expression is essential for engineering genetic circuits and for optimising enzyme levels in metabolic engineering projects. In bacteria, gene expression can be tuned at the stage of transcription, by exchanging the promoter, or at stage of translation by altering the ribosome binding site sequence. In eukaryotes, however, only promoter exchange is regularly used, as the tools to modulate translation are lacking. Working in S. cerevisiae yeast, we here describe how hairpin RNA structures inserted into the 5’ untranslated region (5’UTR) of mRNAs can be used to tune expression levels by altering the efficiency of translation initiation. We demonstrate a direct link between the calculated free energy of folding in the 5’UTR and protein abundance, and show that this enables rational design of hairpin libraries that give predicted expression outputs. Our approach is modular, working with different promoters and protein coding sequences, and it outperforms promoter mutation as a way to predictably generate a library where a protein is induced to express at a range of different levels. With this tool, computational RNA sequence design can be used to predictably fine-tune protein production, providing a new way to modulate gene expression in eukaryotes.
... 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. ...
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)
... Terminator regions are located within about 200 bp downstream of protein-coding sequences and have distinct functions, namely transcriptional termination, poly(A) addition, and post-transcriptional regulation via the 3′ -UTR 5,6 . The amount of protein accumulated depends upon mRNA stability, which might in turn depend on secondary structures in the 3′ -UTR 7 . Refractory elements in the 3′ untranslated region (UTR) that increase mRNA half-life might be available. ...
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.
