Appl Microbiol Biotechnol DOI 10.1007/s00253-014-5569-y


Small RNA regulators in bacteria: powerful tools for metabolic engineering and synthetic biology Zhen Kang & Chuanzhi Zhang & Junli Zhang & Peng Jin & Juan Zhang & Guocheng Du & Jian Chen

Received: 8 December 2013 / Revised: 22 January 2014 / Accepted: 23 January 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Small RNAs, a large class of ancient posttranscriptional regulators, have recently attracted considerable attention. A plethora of small RNAs has been identified and characterized, many of which belong to the major small noncoding RNA (sRNA) or riboswitch families. It has become increasingly clear that most small RNAs play critical regulatory roles in many processes and are, therefore, considered to be powerful tools for metabolic engineering and synthetic biology. In this review, we describe recent achievements in the identification, characterization, and application of small RNAs. We give particular attention to advances in the design and synthesis of novel sRNAs and riboswitches for metabolic engineering. In addition, a novel strategy for hierarchical control of global metabolic pathways is proposed.

Z. Kang : J. Zhang : G. Du : J. Chen Synergetic Innovation Center of Food Safety and Nutrition, Wuxi 214122, China Z. Kang : C. Zhang : J. Zhang : P. Jin : J. Zhang : G. Du : J. Chen The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China G. Du The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China Z. Kang : J. Zhang : G. Du : J. Chen Synergy Innovation Center of Modern Industrial Fermentation, Wuxi 214122, China J. Chen (*) National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China e-mail: [email protected] Z. Kang (*) Yixing Union Biochemical Co., Ltd., Wuxi 214203, China e-mail: [email protected]

Keywords Small RNA . Riboswitch . Gene expression . Posttranscriptional regulation . Metabolic engineering . Synthetic biology

Introduction In recent years, it has become evident that posttranscriptional regulation mediated by RNA regulators is critical to many cellular processes in both prokaryotic and eukaryotic kingdoms (Grosshans and Filipowicz 2008). Hundreds of candidate regulatory RNA genes in many bacteria have been predicted and characterized using a variety of experimental tools, such as direct labeling and RNA sequencing, Rnomics (shotgun cloning), DNA microarray, and Genomic SELEX technologies and bioinformatic approaches (Gottesman 2005; Li et al. 2013b; Livny and Waldor 2007; Pichon and Felden 2008). RNA regulators have been exploited for use in molecular engineering and compared with other regulatory systems; posttranscriptional regulation using small RNAs is costeffective (Altuvia and Wagner 2000). Furthermore, cells are able to use these small molecules to rapidly respond to various environmental signals and stresses. According to their mechanisms of action, these RNA regulators can be divided into two main classes: small noncoding RNAs (sRNAs) and riboswitches. In this review, we summarize recent advances in the discovery, design, synthesis, and application of sRNAs and riboswitches. We also predict future directions for the application of small RNA regulators in metabolic engineering and synthetic biology.

sRNAs in bacteria and their mechanisms of action Over the past decades, sRNAs in bacteria (especially in Escherichia coli) have been predicted and experimentally

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investigated. In 1967, the first bacterial sRNA 6S RNA, encoded by the E. coli gene ssrS, was discovered and characterized (Hindley 1967; Wassarman and Storz 2000). Many studies have shown that 6S RNA is highly abundant during transition from the exponential to the stationary growth phase (Decker and Hinton 2009; Trotochaud and Wassarman 2004; Wassarman 2007). 6S RNA can directly interact with the σ70RNA polymerase and downregulate the transcription of many σ70-dependent promoters, leading to long-term cell survival during nutrient deprivation (Geissen et al. 2010; Klocko and Wassarman 2009; Steuten et al. 2013). Based on a genomewide transcriptional analysis, Neusser et al. (2010) demonstrated that the effect of 6S RNA on transcription is not strictly confined to σ70-dependent promoters but is also involved in stationary phase adaptation. Interestingly, 6S RNA can also dissociate from σ70-RNA polymerase during nutritional upshift, enabling cells to resume rapid growth after starvation. This indicates the crucial role of 6S RNA in universal transcriptional regulation (Steuten et al. 2013; Wurm et al. 2010). In 1984, the first chromosomally encoded sRNA, MicF (174 nucleotides), which inhibits translation of the OmpF mRNA, was identified in E. coli (Mizuno et al. 1984). By 2000, about 13 sRNAs had been serendipitously discovered by traditional methods (for instance separation by polyacrylamide gel electrophoresis) (Masse et al. 2003). Since 2001, many bioinformatic methods designed to analyze intergenic genomic regions (Livny and Waldor 2007; Tjaden et al. 2006; Vejnar et al. 2013; Wassarman et al. 2001) and experimental strategies, such as comparative genomics and microarray technology, Rnomics, and deep sequencing (Altuvia 2007; Huttenhofer and Vogel 2006; Sharma and Vogel 2009), have been developed and used to identify new sRNAs at the genome level in many model organisms, including E. coli (Livny and Waldor 2007), Bacillus subtilis (Irnov et al. 2010), Pseudomonas aeruginosa (Sonnleitner et al. 2012), Staphylococcus aureus (Geissmann et al. 2009), and Listeria monocytogenes (Christiansen et al. 2006). To date, more than 80 sRNAs have been experimentally identified in E. coli. With the ongoing development of genomics, more and more sRNAs will be predicted and identified. This will not only enrich the sRNA database (Li et al. 2013b) but will also provide new insights into their mechanisms of function and their roles in cellular pathways. Bacterial trans-encoded sRNAs have much in common with eukaryotic microRNAs and small interfering RNAs with respect to their regulatory mechanisms (Mahmood et al. 2008). In most cases, their regulatory function depends on base pairings between the sRNA and its target mRNA (Beisel and Storz 2010). As a consequence, genes are negatively or positively regulated (Fig. 1a–c), which enables cells to rapidly adjusting their physiology in response to environmental changes (Repoila and Darfeuille 2009). sRNA-mRNA base

pairing primarily occurs in the 5′ untranslated regions (UTRs) of mRNAs and most often involves the ribosome binding site (RBS) (Repoila and Darfeuille 2009). The protein, Hfq, an RNA chaperone, is commonly required for base pairing. Hfq is presumed to facilitate RNA–RNA interactions and to accelerate the rate of duplex formation in cases of limited complementarity between an sRNA and its target mRNA (Waters and Storz 2009). Hfq also contributes to the protection, stabilization, and recruitment of sRNAs (Beisel and Storz 2010; Storz et al. 2004). In addition to Hfq, RNaseE also participates in some reactions (Repoila and Darfeuille 2009). For example, SgrS, an sRNA induced under stress conditions during which sugar phosphates accumulate, can target a number of mRNAs for degradation in the presence of Hfq and RNaseE, resulting in reduced uptake and enhanced efflux of sugars (Vanderpool and Gottesman 2004; Wadler and Vanderpool 2009). Moreover, sgrS can encode a SgrT protein that contributes to the same stress response (Balasubramanian and Vanderpool 2013; Wadler and Vanderpool 2007). It has been assumed that sRNA regulators act solely via one mechanism involving base pairing. However, Jørgensen et al. demonstrated that McaS in E. coli is a dual-function sRNA and acts by two different mechanisms involving base pairing and protein titration (Jorgensen et al. 2013). These results suggest that the roles and mechanisms of function of some sRNAs are still obscure. The discovery of new sRNAs and the determination of novel functional mechanisms will facilitate the artificial design and engineering of sRNAs.

Applications of sRNAs Genetic circuit engineering with sRNAs Since the discovery of sRNAs, researchers have sought to exploit them to mimic bacterial gene regulation to control genes of interest at the posttranscriptional level (Table 2). Coleman et al. (1984) were the first to develop and introduce this approach for the inhibition of target genes. By inserting artificial sequences (which complement the ribosome regions of Ipp, OmpC, and OmpA mRNAs) between two stem loops at both 5′ and 3′ termini of the naturally occurring MicF sRNA, they successfully repressed the expression of Ipp, OmpC, and OmpA. In recent years, great advances have been achieved in the emerging field of synthetic biology (Chen et al. 2010; Rodrigo et al. 2013), particularly in the field of synthetic RNA biology (Benenson 2012; Chappell et al. 2013). By analyzing the common structural characteristics of natural E. coli sRNAs, Man et al. (2011) developed a semi-rational strategy to design artificial sRNAs for specific gene silencing in Gram-negative bacteria. Many synthesized sRNAs effectively suppressed the expression of an exogenous EGFP gene and the endogenous uidA gene in E. coli. However, because of

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Fig. 1 Functional mechanisms of small RNAs regulators. a Translation activation by sRNAs with assistance of Hfq. By base pairing with mRNAs, sRNAs activate translation by promoting ribosome binding to mRNA with the assistance of Hfq which might facilitate sRNA-mRNA base pairing and promote RNA unfolding or increase the local concentrations of the sRNAs and its mRNA target; b translation repression by sRNAs with assistance of Hfq. The outstretched RBS sequence is buried when sRNAs and Hfq are present, resulting in translation inhibition; c translation repression by sRNAs with assistance of Hfq and RNase E. The

sRNA forms a ribonucleoprotein complex with the Hfq–RNAse E to act on the RBS of target mRNAs through base pairing, resulting in RNAse Edependent rapid degradation of the target mRNAs and translation inhibition; d negative regulation by riboswitch. The binding of a specific metabolite (ligand) to the conserved RNA-sensor domain alters the conformation and excludes the ribosome, resulting in inhibition of translation initiation; e positive regulation by riboswitch. Direct interaction between the ligand and the sensor domain gives rise to an unchained RBS, resulting in translation activation

the high diversity of sRNA sequences and non-stringent target base pairing, and the many factors involved in this complicated process, deficiencies in the efficacy of rational design soon became apparent. Accordingly, a high-throughput screening strategy combined with rational design was shown to be more efficient. Using a fused fluorescent protein as reporter, Sharma et al. (2012) successfully isolated and characterized two artificial sRNAs that can posttranscriptionally repress the endogenous genes, ompF and fliC, respectively. Meanwhile, Rodrigo et al. (2012) reported a fully automated methodology for the design and experimental validation of synthetic sRNAs in a cellular environment. By combining Watson–Crick interactions (Ellington 2007) with physicochemical principles and structural constraints, the authors engineered sRNA sequences that can implement predefined interactions. The authors also demonstrated that energy of formation and activation are sufficient criteria to engineer RNA interaction and regulation in bacteria (Rodrigo et al. 2012).

sRNAs as powerful tools for metabolic pathway engineering Metabolic engineering using an antisense RNA strategy has been performed in Clostridium acetobutylicum (Desai and Papoutsakis 1999). By over-expressing long antisense RNAs, the activities of the enzymes butyrate kinase, phosphotransbutyrylase, and phosphotransacetylase were significantly decreased, which resulted in increased production of acetone and butanol. Similarly, several antisense RNAs have been designed and overexpressed in E. coli to silence acetate kinase and phosphotransacetylase. This reduced carbon flux to acetate and increased heterologous gene expression (Kim and Cha 2003; Nakashima and Tamura 2009; Nakashima et al. 2006). Impressively, Srivastava et al. (2000) indirectly improved the yield of active organophosphorus hydrolase by downregulating sigma 32 (RpoH) with antisense RNA, indicating that modulation of transcriptional sigma factors with novel sRNAs might be a potential strategy

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for metabolic engineering. Although this antisense strategy has been applied, such antisense RNAs have not been thoroughly investigated with respect to unknown specificities and variable efficacies (Sharma et al. 2012). Consequently, sRNAs of short length are more attractive for gene regulation and pathway engineering. To precisely investigate the functions and roles of these sRNAs, a stringent overexpression vector was designed and constructed that uses L-arabinose as an inducer (Majdalani et al. 1998). Accordingly, Kang et al. (2012) introduced and overexpressed the sRNA, RyhB, in an aerobic platform E. coli strain (Kang et al. 2009). As predicted, high levels of the target product, succinate, were accumulated, suggesting that sRNAs with specific functions are powerful tools for metabolic engineering in E. coli. More recently, Li et al. (2013a) reported that by modulating the heme biosynthesis pathway by constitutive expression of RyhB, the titer of 5-aminolevulinic acid was increased by 16 % compared with the parental strain, which contained the genes, hemAM, hemL, and rhtA (Kang et al. 2011). By comparing the levels of the sRNA, SgrS, in E. coli K-12 (JM109 and MG1655) and E. coli B (BL21), Negrete et al. (2013) reduced acetate secretion from E. coli K-12 by over-expressing SgrS. To rationally engineer an optimized pathway for the production of target chemicals, Na et al. (2013) selected the natural sRNA, MicC, to act as a scaffold and synthesized 130 sRNAs composed of two parts: a scaffold MicC sequence and a target-binding sequence. By fine-tuning the target chromosomal gene expression, the titers of tyrosine and cadaverine were substantially increased. These results demonstrated that sRNAs are versatile tools for use in metabolic engineering and synthetic biology (Yoo et al. 2013; Chen et al. 2010; Rodrigo et al. 2013), and their application will be greatly enhanced with the development of sRNA databases (Mehta et al. 2013). Improved stress resistance with sRNAs In nature, many sRNAs are involved in controlling a variety of stress responses, including envelope stress, temperature stress, and acid stress (Hoe et al. 2013). In the enterobacter, E. coli and Salmonella, at least 12 sRNAs, InvR, MicA, MicC, MicF, OmrAB, RprA, DsrA, RseX, SdsR, CyaR, VrrA, and RybB, have been proposed as regulators of the envelope stress response (Boehm and Vogel 2012; Coornaert et al. 2010; Vogel and Papenfort 2006). For instance, DsrA, an 87-nucleotide sRNA, is recognized as a multifunctional genetic regulator in E. coli and several studies have demonstrated that this sRNA plays crucial roles in response to many environmental stresses (Lease et al. 2004). In the fermentation process, it is critical to maintain a robustness and prolonged productivity under stressful conditions. Thus, improvement of a strain’s tolerance would be beneficial for bioprocessing applications. In E. coli, the global transcription regulator, RpoS, is posttranscriptionally

upregulated by the sRNA DsrA, RprA, and ArcZ (Majdalani et al. 2002; Papenfort et al. 2009; Sledjeski et al. 1996), and downregulated by OxyS (Zhang et al. 1998). More recently, Gaida et al. (2013) reported that simultaneous overexpression of DsrA, RprA, and ArcZ substantially increased acid tolerance and provided protection against carboxylic acid and oxidative stress during the exponential phase. Similarly, applying this multi-posttranscriptional regulation strategy, Kang et al. (2008) constructed a stress-induced system for the high-level production of chemicals, including polyhydroxybutyrate and 1,3-propanediol (Liang et al. 2011b). Therefore, improvement of stress resistance with sRNAs is an effective strategy. Potential applications of sRNAs: global regulation in vivo To effectively sense and respond to rapid changes in the environment and to complete natural programmed life cycles, bacterial cells have to strictly govern complex regulatory networks. Although tremendous advances in understanding transcriptional regulatory networks have been achieved (Alon 2007; Seshasayee et al. 2006), the posttranscriptional regulatory architecture is obscure. Numerous sRNAs have been identified as global regulators (Beisel and Storz 2010; Gottesman and Storz 2011; Hoe et al. 2013) and play vital roles in controlling different genes modules (Fig. 2). In addition, many algorithms for designing RNAs with specific structures have been proposed (Benenson 2012; Levin et al. 2012; Zhao et al. 2012). Therefore, it is practicable and attractive to achieve module and global regulation with novel designed sRNAs. For instance, seven sigma factors, σ70, σ38, σ54, σ24, σ32, σ28, and σ19 (encoded by rpoD, rpoS, rpoN, rpoE, rpoH, fliA, and fecI, respectively), are present in E. coli and are required for initiating transcription of global genes (Cook and Ussery 2013). To generate a global disturbance and to construct mutant libraries, Alper et al. successfully developed a global transcription machinery engineering approach for improving cellular phenotype by directly engineering the sigma factor (Alper et al. 2006; Alper and Stephanopoulos 2007). More recently, Stevens et al. (2013) constructed an effective and general heterologous expression system by over-expressing the alternative sigma factor σ54. Hence, artificial sRNAs can be applied to improve cellular background and phenotype by indirectly controlling sigma factors (Fig. 3).

Riboswitches Riboswitches are noncoding RNA sequences located in UTRs of mRNAs and can specifically control the expression of a large number of genes in response to changing cellular conditions (Barrick and Breaker 2007; Henkin 2008; Serganov 2009). Unlike the sRNAs discussed above, riboswitches are

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Fig. 2 Global natural sRNA regulators identified in E. coli. By regulating the global transcriptional dual regulators, the natural versatile RNAs can indirectly modulate cells to resist various stresses. The sRNAs and the global mediators are highlighted in blue and brown, respectively

cis-acting and can directly bind small molecules (ligands) to cause allosteric rearrangement of their structures and a quick controllable response (Vitreschak et al. 2004). Therefore, riboswitches are considered to be one of the oldest universal genetic factors and are widely distributed in archaea, bacteria, fungi, and plants (Barrick and Breaker 2007).

Riboswitches in bacteria and their mechanisms of action Since the term “riboswitch” was proposed by Ronald Breaker in 2002 (Winkler et al. 2002a), many distinct classes of riboswitches, which respond to various metabolites, have been

Fig. 3 Schematic of global pathway regulation with artificial sRNAs in E. coli. According to the regulation mechanisms of natural sRNAs, artificial specific sRNAs towards different sigma factors σ70, σ38, σ54, σ24, σ32, σ28, and σ19 which encoded by rpoD, rpoS, rpoN, rpoE, rpoH, fliA, and fecI, respectively, can be designed, synthesized, and screened. By modulating the numbered sigma factors with artificial sRNAs, the global biological pathways can be indirectly controlled

identified and characterized in numerous bacteria (Table 1). Strikingly, without any helper protein and by undergoing only minimal alterations to their molecular architecture, riboswitches are able to regulate a large variety of biological processes, such as transcription attenuation, translation initiation, mRNA splicing, and mRNA processing. This regulation is important in several biosynthetic pathways that produce vitamins, amino acids, and purines (Coppins et al. 2007; Vitreschak et al. 2004). In general, riboswitches are composed of two domains. One is the aptamer domain, which is involved in ligand binding, and the other is the expression platform, which functions in gene control (Serganov 2009). The function of a riboswitch is typically based on the interplay between alternative conformations

Appl Microbiol Biotechnol Table 1 Riboswitches identified and characterized Riboswitches






Thiamine biosynthesis or transport Riboflavin biosynthesis and transport Cobalamin biosynthesis; transport of cobalamin Methionine biosynthesis and transport; SAM metabolism


Miranda-Rios et al. (2001)


Bacteria, archaea, fungi, and plants Bacteria

Coenzyme B12


Nou and Kadner (2000)



Grundy and Henkin (1998)

SAH Purines


Wang et al. (2008) Mandal et al. (2003)



Mandal et al. (2003)

Guanine Queuosine Lysine

Bacteria Bacteria

Mandal et al. (2003) Roth et al. (2007) Mandal et al. (2003)

Glycine GlcN6P

Bacteria Bacteria

Lipfert et al. (2007) Klein and Ferre-D'Amare (2006)

Cyclic di-GMP


Sudarsan et al. (2008)

Molybdenum ATP L-glutamine

Bacteria Bacteria

Regulski et al. (2008) Barrick et al. (2004) Ames and Breaker (2011)

FMN-element B12-element S-box

SAH G-box Adenine Guanine preQ1 L-box Glycine-element GlmS-element Cyclic di-GMP-element Molybdenum YdaO-element Glutamine-element

Purine metabolism and transport Adenine metabolism and transport Queuosine biosynthesis Lysine biosynthesis, transport and catabolism Glycine synthesis Glucosamine-6-phosphate synthesis and transport Physiologic adaptation and virulence Energy-related genes Nitrogen metabolism

Winkler et al. (2002b)

TPP thiamin pyrophosphate, FMN flavin mononucleotide, Coenzyme B 12 adenosylcobalamin, SAM S-adenosylmethionine, SAH Sadenosylhomocysteine, queuosine 7-aminomethyl-7-deazaguanine, GlcN6P glucosamine-6-phosphate

of the evolutionarily conserved metabolite-sensing domain and the variable expression platform. When levels of specific ligands (metabolites) increase, the aptamer domain of the riboswitch folds into a defined structure that shows high affinity for and produces specific contacts with the atomic groups of the ligand (Breaker 2012; Serganov and Patel 2007). As a result, the ligand–aptamer complex has a direct influence on the folding of the down-stream expression platform (which usually interacts with the ribosome binding site). In most cases, natural riboswitches negatively regulate gene expression. Direct interaction with a specific metabolite causes changes to the secondary structures of the RBS and AUG sequences, which eventually results in translation repression (Fig. 1d). To date, only three riboswitch activators, pbuE, ydhL, and gcvT, which are involved in adenine (pbuE and ydhL) and glycine (gcvT) synthesis, have been identified (Breaker 2008). With specific aptamer interaction, translation of the corresponding gene was triggered and strengthened (Fig. 1e). Although it was generally accepted that the formation of a Watson–Crick base pair between the ligand and the aptamer is specific, recent studies show that riboswitches can reprogram ligand specificity by folding diverse secondary structures (Serganov and Patel 2012), indicating that this

approach to posttranscriptional regulation is much more flexible than expected.

Application of riboswitches Driven by the identification of new natural riboswitches and increased understanding of their functional mechanisms, many studies have focused on the design and engineering of novel riboswitches for wide-ranging applications. Construction of biosensors for environmental monitoring In the past few years, a variety of natural riboswitches have been used (Table 2) to detect small molecules that can be transported into cells. Using β-galactosidase as a reporter, Desai and Gallivan (2004) subcloned the theophylline-responsive aptamer, mTCT8-4, to a location 5 bp upstream of the ribosome binding site of the β-galactosidase gene. They then generated a recombinant E. coli harboring the synthetic riboswitch to exclusively detect theophylline. This study demonstrated that an aptamer generated in vitro can be transferred into an in vivo genetic assay and maintain the same molecular recognition

Appl Microbiol Biotechnol Table 2 Application examples of sRNAs and riboswitches Research properties sRNAs Expression of an antisense RNA to silence the butyrate kinase gene Downregulation of the sigma factor 32 with antisense RNAs Expression of antisense RNAs to silence the genes pta and ackA Design and expression of effective PTasRNAs to silence the ackA gene Construction of a stress-induced system based on regulation of the sigma factor 38 Overexpression of antisense RNAs to silence the genes mutS, mutD and ndk Overexpression of the sRNA RyhB Overexpression of the sRNA RyhB Synthesis of sRNAs to repress the target chromosomal genes Overexpression of the three sRNAs DsrA, ArcZ and RprA Riboswitches Engineering of riboswitches Design of antiswitches (trans-action) Synthesis of a theophylline-response riboswitch to regulate the gene cheZ Development of ribozyme switches with a sensor domain (aptamer) and an actuator domain (hammerhead ribozyme) Cloning the sensor of the coenzyme B12-response riboswitch Combination of in vitro and in vivo selection to identify novel riboswitch Development of a novel synthetic riboswitch De novo synthesis of riboswitch modulated by theophylline Construction of a riboselector with modules of riboswitch and selection



Increase of acetone and butanol

Desai and Papoutsakis (1999)

Increase of active organophosphorus hydrolase Decrease of acetate and increase of recombinant proteins Repression of the AckA activity

Srivastava et al. (2000)

Production of chemicals such as polyhydroxybutyrate Increase of mutation frequencies

Kang et al. (2008)

Kim and Cha (2003) Nakashima et al. (2006)

Nakashima and Tamura (2009)

Increase of succinate Regulation of the heme biosynthesis and increase of 5-aminolevulinic acid Increase of tyrosine and cadaverine

Na et al. (2013)

Increase of acid stress resistance

Gaida et al. (2013)

Monitor of specific small molecules Control of eukaryotic gene expression Navigation of bacteria with small molecules

Desai and Gallivan (2004) Bayer and Smolke (2005) Topp and Gallivan (2007)

Regulation of cell growth and in vivo sensing of metabolite production

Win and Smolke (2007)

Monitor of the synthesis and the import of coenzyme B12 Construction of recombinant strains to seek and destroy atrazine Positive response to 2,4-dinitrotoluene Regulation of gene expression at the transcriptional level Enrichment of lysine and tryptophan metaboliteproducing microbes

Fowler et al. (2010)

properties. Similarly, Fowler et al. (2010) cloned the coenzyme B12 riboswitch fragment from E. coli and constructed a sensor system with green fluorescent protein as a reporter to quantitatively examine coenzyme B12 (Fig. 4b). Hence, riboswitches can be widely applied for the detection of extracellular small molecules. In bacteria, the chemo-navigation system is widely distributed and is considered to have potential applications in the areas of bioremediation, drug delivery, and synthetic biology. Applying a theophylline-sensitive riboswitch to control translation of the key enzyme, CheZ, in the chemotaxis pathway, Topp and Gallivan (2007) were able to reprogram E. coli cells to precisely follow a completely new theophylline-containing path. Recently, Sinha et al. (2010) identified a synthetic riboswitch (Fig. 4a) that can activate protein translation in response to the herbicide, atrazine, by combining in vitro and in vivo selection strategies. More importantly, they successfully reprogrammed an E. coli

Kang et al. (2012) Li et al. (2013a)

Sinha et al. (2010) Davidson et al. (2013) Wachsmuth et al. (2013) Yang et al. (2013)

strain to seek and efficiently destroy atrazine. To directly investigate specific small molecules, Ogawa (2011) used a fully rational design strategy to construct a number of novel riboswitches for the detection of theophylline, FMN, tetracycline, and sulforhodamine B in a eukaryotic cell-free translation system. More recently, Davidson et al. (2013) constructed and screened a 2,4-dinitrotoluene responsive synthetic riboswitch in E. coli using the Tobacco Etch Virus protease as a reporter. These studies demonstrate that compared with traditional protein engineering strategies, reprogramming bacterial cells with synthetic riboswitches to detect new chemical signals is highly promising. In addition to riboswitches that sense chemicals or metabolites, certain riboswitches have been shown to respond to stress signals (Dambach and Winkler 2009). Overall, riboswitches are versatile regulatory elements that merit intensive research to enable their use in environmental monitoring.

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Fig. 4 Application examples of designed riboswitches in metabolic engineering and synthetic biology. Two different patterns (positive and negative) of directors for monitoring extracellular or intracellular chemicals or signals are shown in a and b, respectively. c Selectors designed for enriching bacteria cells with specific phenotype. The selector device is composed of two parts: riboswitch module and function module. Chemicals produced by the engineered cells with an optimal pathway or added artificially interact with the selector device can block expression of

the lethal factors and result in cell growth. d Regulators designed for optimizing the specific pathways. Arrow in green designates activation (riboswitch 1 and 4) and dot in red designates repression (riboswitch 2). Riboswitch 3 is designed to respond to I4 and output signals. Accumulation of the intermediate I1 activates the last step for the end product (hexagon) synthesis. In reverse, the end product negatively regulates the branch pathway to the intermediate I4. The concentration of the end product can be reflected by the strength of signal (negatively)

Generating novel biosensors for metabolic pathway engineering

model organisms (Bayer and Smolke 2005; Topp et al. 2010; Win and Smolke 2007) and applied in many areas of metabolic engineering (biosynthesis, bioremediation, and health and medicine) (reviewed in Liang et al. 2011a) (Table 2). To simplify the engineering process, Qi et al. (2012) constructed several ligand-sensing sRNA regulators by substituting the riboswitch expression platform with naturally occurring transsRNAs to fine-tune the intracellular transcriptional cascades. Compared with previous approaches, this new strategy is much more modular, flexible, and versatile. Similarly, Ceres et al. (2013) generated novel active riboswitches by the modular selection of different readout domains (expression

Construction of genetic circuits or optimization of metabolic pathways requires precise and dynamic control of gene expression (Dahl et al. 2013). Over the past decade, many novel riboswitches that act as regulatory elements have been developed, screened, and characterized using the SELEX strategy (Kiga et al. 1998), which has recently been expertly reviewed (Blouin et al. 2009; Henkin 2008; Joyce 2007; Serganov and Patel 2007; Suess and Weigand 2008). To date, many riboswitch control systems have been constructed in several

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platforms) to regulate translation both in vitro and in vivo. More importantly, this technique enables rapid engineering of novel functional RNAs (Ceres et al. 2013). To expand the toolbox for the rational design and construction of biosynthetic and regulatory networks, riboswitches have the potential to be transcriptional regulators, although several in vivo screening approaches have failed. Excitingly, by applying the wellcharacterized theophylline aptamer as sensor, Wachsmuth et al. (2013) successfully constructed functional transcriptional riboswitches incorporating in silico designed RNA sequences as expression platforms. This work demonstrated that riboswitches can also be useful tools for regulating gene expression at the transcriptional level. Although rational metabolic engineering approaches are preferred for the optimization of target pathways and to improve product yields, the desired cellular phenotypes are not always generated because regulation of intracellular metabolic fluxes is usually much more complicated than expected (Chen and Nielsen 2013; Dahl et al. 2013; Pfleger et al. 2006). As a result, many directed evolution strategies (Alper et al. 2006; Isaacs et al. 2011; Santos and Stephanopoulos 2008; Wang et al. 2009) combined with high-throughput screening methods have been employed (Dietrich et al. 2010; Scheel and Lutke-Eversloh 2013; Tannler et al. 2008). Recently, Yang et al. (2013) developed a synthetic RNA device (called a riboselector) comprising a riboswitch (Fig. 4c) (for sensing the intracellular concentration of a specific metabolite and actuating the regulation of gene expression based on metabolite signals) and a selection module (to enable high-throughput screening) (Fowler et al. 2010; Muranaka et al. 2009) to expedite the evolution of metabolite-producing microbes. As expected, by applying this novel riboselector, they successfully enriched for pathway-optimized E. coli strains producing Llysine and L-tryptophan. Moreover, this riboselector device can provide the foundation for an efficient screening method for strain improvement.

Conclusions In bacteria, approximately 10–20 % of all genes are predicted by high-throughput computational and experimental approaches to code for RNAs with critical regulatory roles in metabolic, physiological, and pathogenic processes (Romby and Charpentier 2010). Consequently, identification of novel regulatory RNA molecules and documentation of their various functions and modes of action would greatly enrich the toolbox of metabolic engineering. Traditionally, efforts to regulate and optimize genetic circuits and metabolic pathways were mainly concentrated at the transcriptional and translational levels. In comparison, the new posttranscriptional approaches show many advantages, such as rapid response, flexible and precise control, ready restoration, and no

metabolic burden. It is, therefore, reasonable to predict that with the discovery of increased numbers of diverse natural sRNAs and riboswitches, and the development of synthetic biology and systems biology (Heinemann and Sauer 2010; Keasling 2012; Kohlstedt et al. 2010; Lee 2011; Nielsen and Pronk 2012), novel sRNAs and riboswitches with specific function can be designed, synthesized, and applied for rational, hierarchical, and dynamic regulation of metabolic pathways (Figs. 3 and 4d). This would enable the rapid development of robust strains for specific purposes. In addition, research in this area will contribute to the understanding of the control mechanisms of natural biological networks. Acknowledgments This work was financially supported by the Major State Basic Research Development Program of China (973 Program, 2012CB720802, 2014CB745103, 2013CB733902, 2013CB733602), the National Natural Science Foundation of China (31200020, 31130043), the National High Technology Research and Development Program of China (863 Program, 2011AA100905), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1135), the National Science Foundation for Post-doctoral Scientists of China (2013M540414), the Jiangsu Planned Projects for Postdoctoral Research Funds (1301010B), and the 111 Project.

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Small RNA regulators in bacteria: powerful tools for metabolic engineering and synthetic biology.

Small RNAs, a large class of ancient posttranscriptional regulators, have recently attracted considerable attention. A plethora of small RNAs has been...
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