Broad-Spectrum Gene Repression Using Scaffold Engineering of

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Research Article Cite This: ACS Synth. Biol. 2019, 8, 1452−1461

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Broad-Spectrum Gene Repression Using Scaffold Engineering of Synthetic sRNAs Minho Noh,†,# Seung Min Yoo,‡,§,# Dongsoo Yang,†,∥ and Sang Yup Lee*,†,§,∥

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Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea ‡ School of Integrative Engineering, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea § BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea ∥ Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea S Supporting Information *

ABSTRACT: Gene expression regulation in broad-spectrum range is critical for constructing cell factories and genetic circuits to balance and control system-wide fluxes. Synthetic small regulatory RNAs (sRNAs) effectively regulate gene expression at the translational level by modulating an mRNAbinding chance and sRNA abundance; however, it can control target gene expression only within the limit of the intrinsic repression ability of sRNAs. Here, we systematically mutated a SgrS scaffold as a model sRNA by dividing the Hfq-binding module of the sRNA into the three regions: the A/U-rich sequence, the stem, and the hairpin loop, and examined how efficiently the mutants suppressed DsRed2 expression. By doing this, we found that a scaffold with an altered A/U-rich sequence (CUUU) and stem length and that with altered A/U-rich sequence (GCAC) showed a 3-fold stronger and a 3-fold weaker repression than the original scaffold, respectively. For practical application of altered scaffolds, proof-of-concept experiments were performed by constructing a library of 67 synthetic sRNAs with the strongest scaffold, each one targeting a different rationally selected gene, and using this library to enhance cadaverine production in Escherichia coli, yielding in 27% increase (1.67 g/L in flask cultivation, 13.7 g/L in fed-batch cultivation). Synthetic sRNAs with engineered sRNA scaffolds could be useful in modulating gene expression for strain improvement. KEYWORDS: gene repression, Hfq-binding region, knockdown, synthetic sRNA

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physiological processes, such as carbon metabolism, amino acid metabolism, stress response, and environmental adaptation.16−19 An example of bacterial RNA regulators is transacting Hfq-dependent small regulatory RNA (sRNA), which binds at or near the ribosome-binding sites (RBSs) of mRNAs with the aid of the RNA chaperone Hfq.20−22 This binding prevents ribosomes from accessing the RBS, thereby inhibiting translation.20−22 Hfq-dependent sRNAs consist of three functional modules: an mRNA-binding region, an Hfq-binding region, and a Rho-independent transcription terminator (Figure 1A). The mRNA-binding region is complementary to a target mRNA,23 which would allow sRNA-mRNA hybridization to occur. The Hfq-binding region enhances the ability of sRNAs to bind target mRNAs, and facilitates mRNA degradation by recruiting RNase E.24−28 The Rho-independent transcription terminator is a GC-rich palindromic sequence followed by a stretch of U nucleotides that terminates the

ene expression regulation is critical for constructing cell factories and genetic circuits. This is especially important in metabolic engineering, where modulating chromosomal gene expression is a key to developing high-performance microbes for the production of chemicals, fuels, and materials. This can be achieved by balancing and controlling system-wide metabolic fluxes for enhanced product biosynthesis and cell growth.1 Because of the complexity of genetic and metabolic networks, the main challenge in this field is developing a sophisticated yet easy method for regulating target gene expression in dynamic and broad-spectrum range. In this regard, many attempts have been made to develop a gene expression system in varied levels by modifying transcriptional level-associated factor,2−10 mRNA stability and translation,11−13 and protein regulators.14,15 These methods are effective for heterogeneous genes cloned in vectors, but are still labor-intensive and time-consuming for chromosomal genes. Another effort to control gene expression involves the use of bacterial RNA regulator. Bacterial RNA regulators actively control the expression of genes involved in a wide range of © 2019 American Chemical Society

Received: April 12, 2019 Published: May 27, 2019 1452

DOI: 10.1021/acssynbio.9b00165 ACS Synth. Biol. 2019, 8, 1452−1461

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Figure 1. Schematic of the synthetic sRNA structure and the engineering of its scaffold. (A) Synthetic sRNAs compete with ribosomes for ribosome-binding sites (RBSs). Three functional elements of Hfq-dependent sRNAs are annotated: an mRNA-binding region, an Hfq-binding region, and a rho-independent transcription terminator. (B) The Hfq-binding region was divided into three elements for mutational analysis: an A/ U-rich sequence, a stem, and a loop. (C) Schematic for the analysis of the activities of synthetic sRNA mutants. Plasmids expressing the sRNAs were constructed through inverse PCR, and plasmids were transformed into E. coli cells harboring a DsRed2 expression plasmid. The repression of DsRed2 was estimated by measuring fluorescence intensities.

level of repression can be modulated by modifying the mRNAbinding sequences of the sRNA in order to change the binding energy between the sRNA and the target mRNA.31,32 This approach is simple as it is easy to design and use sRNAs; however, like the strategy above, there is a limit to modulate target gene expression in broad range beyond an intrinsic repression ability of sRNA. The potential cross-reactivity caused by the change in the mRNA-binding region should also be considered. The Rho-independent transcription terminatorbased approach can also control target gene expression. Although this region-based studies have unveiled that the poly-U tail of this region is also essential for Hfq-binding and sRNA stability,29,30 the role of the rho-independent transcription terminator is obvious. As the last functional modulebased approach, the Hfq-binding region is a potential target for modification to further modulate sRNA-mediated gene repression. Although there have been several studies on the Hfq-binding region of natural sRNAs performed through mutational analysis,24,26,28−30,39,40,46 the systematic analysis is required for improved gene regulation. In addition, the improvement of the synthetic sRNA activity by this strategy was not focused yet. In this regard, for gene expression regulation in broad-spectrum range using synthetic sRNAs, here, we report the modification of the Hfq-binding region of the synthetic sRNA SgrS-S and the application of these engineered synthetic sRNAs in modulating gene repression in Escherichia coli. The three parts of the Hfq-binding region, the A/U-rich sequence, the stem, and the hairpin loop, were systemically mutated, and the mutants were tested for their ability to repress expression using DsRed2 as a model target protein. We identified a synthetic sRNA with an altered A/Urich sequence (a UAUU to CUUU mutation) and a longer hairpin stem (a 4 nts to 6 nts mutation) that had a 3-fold stronger repression ability than that of the nonmutant SgrS-S. Also, we found that a synthetic sRNA with altered A/U-rich

synthesis of sRNAs to produce discrete sRNA molecules. Much effort has been exerted on developing a method to modulate gene repression by modifying natural sRNA scaffold.24,26,28−40 Notably, the recent development of synthetic sRNAs has paved the way for more practical and wider use of sRNA in metabolic engineering and synthetic biology that allows in trans high-throughput gene expression control without any chromosomal modification.31−42 A recently developed expanded synthetic sRNA expression platforms allowed rapid, multiplexed, and genome-scale target gene knockdown in engineered Escherichia coli without concerning plasmid compatibility.43 Also, effective knockdown of essential genes is possible by employing the sRNA technology, which is not easy using the conventional knockout techniques. CRISPR interference (CRISPRi) can also knockdown target gene expression,44 but is not as efficient as the sRNA technology. For example, CRISPRi requires expression of a large gene (encoding dCas9), which often gives metabolic burden to the host cell. Also, a recent study reported that dCas9 can directly bind to E. coli endogenous genes even without a single-guide RNA, resulting in altered cell morphology and cell growth.45 Using synthetic sRNAs, there are two strategies for controlling the expression of desired target genes. One strategy is to change sRNA abundance without disturbing sRNA scaffold, which can be achieved by employing different strength promoters or replication origins in sRNA expression vector.41 This strategy is a facile approach for controlling gene expression as it does not require additional consideration of potential cross-reactivity; however, they can modulate target gene expression only within the limit of the intrinsic repression ability of sRNAs. To expand the gene repression range of original sRNAs, the modification of three functional modules of sRNAs mentioned above could be a solution, which is the other strategy. As a first functional module-based approach, the 1453

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Figure 2. Gene repression activities of sRNAs with mutations in the Hfq-binding region. (A) Nucleotide sequences of the SgrS-S variants. Red wedges indicate the position of a nucleotide deletion. Texts with asterisks indicate inserted nucleotides. Underlined and italicized texts indicate substituted nucleotides. All possible single mutants of the boxed sequences (UAUU and AAAA) and combinations of single mutants were constructed and studied. (B) Single mutations on the A/U-rich sequence (UAUU) were examined. The mutations that increased or decreased gene repression were combined to observe the additive effects of mutations on the A/U-rich sequence (CUUU and GCAC). Mutated sequences are marked in red. (C) Stem length was increased or decreased and changes in the repression activity were observed. Stem length was changed by inserting or deleting G-C base pairs. (D) Gene repression activities of sRNA scaffolds with a single mutation in the A-rich loop. The mutations that increased or decreased gene repression were combined to observe the additive effects of mutations on the loop (GUCU and CGGG). Mutated 1454

DOI: 10.1021/acssynbio.9b00165 ACS Synth. Biol. 2019, 8, 1452−1461

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sequences are marked in red. (E) Gene repression activities of sRNAs harboring combinations of the improved modules: CUUU replacing the A/ U-rich sequence, a 6-nt long stem, and a GUCU loop. To observe changes in gene repression in detail, the promoter used for sRNA expression was changed from J23105 to the weaker promoter J23114. (F) Gene repression activities of sRNAs harboring combinations of the mutant modules: GCAC replacing the A/U-rich sequence, an 8-nt long stem, and a CGGG loop. The promoter used for sRNA expression was J23105. Insets in (B)−(F) show the dynamic range of repression capabilities of the original (open circles) and mutant sRNAs (closed circles). The gray dashed lines in all graphs indicate the repression level of the original sRNA. Data were obtained from three measurements. Fold repression was calculated by dividing the fluorescent intensities of cells without sRNAs by the fluorescent intensities from cells with original or mutant sRNAs (see Figure 1). The values displayed represent average values ± s.d. (n = 3).

an A-to-U mutation at the second nucleotide (Figure 2B). Mutations at the third and fourth nucleotides also weakened gene expression. To summarize, all mutants harboring a single nucleotide substitution were capable of gene repression up to levels ranging from 2.9- to 10-fold (inset of Figure 2B). To examine whether combining these mutations additively increases gene repression, a synthetic sRNA with a CUUU sequence replacing the A/U-rich sequence was constructed and tested for repression efficiency. This engineered sRNA repressed DsRed2 expression at a higher level (>11-fold) than the mutant with the U-to-C single mutation at the first nucleotide (10-fold). These results suggest that changing the first nucleotide of the A/U-rich sequence from U to C while assigning the rest of the three nucleotides to be U’s increases the repression capability of the sRNA substantially. The increase in repression capability resulting from having three U’s for the second to the fourth nucleotides agrees with previous findings for natural sRNA-based scaffold engineering.26,29,40 Interestingly, combining the repression-weakening single mutations led to lower repression efficiencies. For example, mutating the A/U-rich sequence to GCAC had an additive effect and reduced repression efficiency to 1.5-fold (Figure 2B). Thus, the A/U-rich sequence of the Hfq-binding region can be engineered to finely control the gene repression level on a wider spectrum of expression than that of the original sRNA scaffold. Effect of the Length of the Hairpin Stem on Gene Repression Ability. The Hfq-binding region is generally flanked by one or more hairpins.25,26,28,29,40 According to a previous study, disrupting the hairpin structure impairs Hfqbinding. Thus, the presence of a hairpin structure, regardless of the sequence of the stem, is important in maintaining the function of sRNAs.29 On the basis of this finding, we hypothesized that a more stable hairpin structure will result in an enhanced repression capability. To test this hypothesis, sRNAs of varying lengths were constructed by eliminating one G-C base pair or adding 1−4 G-C base pairs (Figure 2A) while retaining the original stem structure. These engineered sRNAs were tested for their ability to repress DsRed2 expression (Figure 2C). As stem length was increased, gene repression also increased. Repression eventually reached a maximum when the stem was 6 nts long (Figure 2C). The mutant with a 6-nt long stem dramatically repressed DsRed2 expression by up to 37-fold. As the stem length was further increased to 7 and 8 nts long, repression capability was reduced to levels lower than that of the original sRNA (Figure 2C). In addition to the hairpin structure and the preceding A/U-rich sequence, which have been previously reported to be important for sRNA function,29,40 our results suggest that the optimal length of the hairpin stem is also important in enhancing the repression capability of synthetic sRNAs.

sequence (a UAUU to GCAC mutation) had a repression capability 3 times lower than that of original sRNAs. For practical application of altered scaffold as proof-of-concept experiment, the strongest one was employed in the construction of a rationally selected sRNA library, which was in turn used to develop an E. coli strain capable of efficiently producing cadaverine, a chemical precursor in plastic production.

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RESULTS Construction of Synthetic sRNAs for Scaffold Engineering. For the synthetic sRNA scaffold used to investigate the effects of mutations on the Hfq-binding region, we selected the sRNA SgrS-S derived from SgrS, which is one of three sRNAs (MicC, MicF, and SgrS) previously reported by our group.31,32 We selected SgrS-S because it has a simple structure and its Hfq-binding region is well-characterized.29,30,41,47 In addition, its simple and stable secondary structure minimizes the number of structural variations that need to be considered before construction and after mutant generation. To investigate the effects of mutations in the Hfq-binding region on the repression ability of SgrS-S, we systematically introduced mutations to the three parts of the region: the A/ U-rich sequence and the hairpin loop and stem (Figure 1B). To examine the changes in the efficiency of repression, we used DsRed2, a red fluorescent protein, as a reporter. The ptsG binding sequence in SgrS-S was replaced with a sequence complementary to the DsRed2 gene in order to construct a synthetic sRNA capable of repressing DsRed2 expression.47 The first 24 nucleotides (nts) from the start codon of DsRed2 were targeted, as previously reported.31,32 The repression efficiency of each mutant was determined by measuring the change in fluorescence intensity (Figure 1C). Effect of Mutation in the A/U-Rich Sequence on Repression Efficiency. First, mutations were introduced to the A/U-rich sequence in the Hfq-binding region (Figure 2A). The A/U-rich sequence near the internal stem-loop is a common feature of Hfq-dependent sRNAs, such as SgrS, RybB, and DsrA. It plays an important role in Hfq-sRNA interaction and overall sRNA function.24,26,29,40 We generated mutants harboring a single nucleotide substitution at each nucleotide of the identified A/U-rich sequence of SgrS-S, UAUU,29 and assessed their ability to repress DsRed2 expression (Figure 1B). The sRNAs harboring mutations at the first and second nucleotides had an increased or comparable level of repression of DsRed2 relative to the nonmutant SgrS-S. On the other hand, sRNAs harboring mutations at the third nucleotides had decreased levels of repression compared to the original synthetic sRNA (Figure 2B). A mutant with a U-to-C mutation at the first nucleotides had the strongest repression ability, followed by a mutant with 1455

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Figure 3. Affinities between the purified Hfq-His6 and synthetic sRNA variants. (A) Schematic for the analysis of the affinities between Hfq-His6 and the synthetic sRNA variants. (B) Quantitation of synthetic sRNAs bound to Hfq-His6 using FITC-conjugated antibodies. Data were obtained from three measurements. The values displayed represent average values ± s.d. (n = 3).

Effect of Mutations in the Loop Sequence on Repression Levels. We next focused on the sequence of the hairpin loop in the Hfq-binding region. There has been a report suggesting that the affinity of Hfq to the rpoS mRNA was dependent on two single-stranded A-rich sequences and that this interaction was essential for the repression of rpoS.48 The sequence of the loop in SgrS-S also consists of a singlestranded AAAA sequence. Thus, we next examined the effects of alterations in this A-rich sequence on the repression capability of SgrS-S. To do this, we constructed all possible mutants with single-nucleotide mutations in the AAAA sequence (Figure 2D). Four mutants (CAAA, AGAA, AAGA, and AAAG) had repression levels similar to or weaker than the original sRNA (2.5- to 4-fold), while other mutants had higher repression levels than the nonmutant SgrS-S, ranging from 4fold to 8-fold (Figure 2D). To assess the combinatorial effects of these mutations, the mutations at each nucleotide resulting in the strongest repression (GAAA, AUAA, AACA, and AAAU) were incorporated into a single synthetic sRNA. This engineered sRNA, which had the AAAA sequence mutated into GUCU, had the strongest repression capability (>8-fold repression, Figure 2D). Although there has been a report showing that a UAAAAU-to-GAGCAC mutation in the loop sequence of SgrS-S results in the retention of the full gene repression capacity of the sRNA,29 our systematic mutational analysis revealed that the sequence of the loop indeed plays an important role in gene repression. One possible explanation for the role of the loop sequence is that the interaction between Hfq and sRNA is dependent on the exposed bases of the loop, like the A/U-rich sequence. On the other hand, the effect of combining mutations, which is the reduction of repression (i.e., CGGG), was not observed, and the repression efficiency of sRNAs with multiple mutations (3-fold) was similar to that of the single mutant (AAGA) with the lowest repression level (2.5-fold) (Figure 2D). In summary, all mutants tested showed gene repression levels ranging from 2.5- to 8.3-fold (inset of Figure 2D). A New sRNA Scaffold with Three Mutated Modules for Expanding the sRNA Repression Range. As detailed above, we developed sRNA scaffold mutants that could efficiently knockdown the expression of DsRed2 up to 37fold (Figure 2B−D). We speculated that this corresponds to near-complete saturation of the target mRNAs by sRNA binding. Nevertheless, as we aimed at further enhancing the

repression capabilities of sRNA scaffold mutants, we tried to increase the resolution of repression capabilities by employing a weaker promoter (J23114, MIT Registry, BBa_J23114) for sRNA expression than that (J23105, MIT Registry, BBa_J23105) used in previous experiments. The relative strengths of J23105 and J23114 are 0.24 and 0.10, respectively (http://parts.igem.org/Promoters/Catalog/Anderson). We examined the additive effect of combining the best mutations introduced in the three modules of the Hfq-binding region by constructing an sRNA with CUUU as the A/U-rich sequence, a 6-nt long stem, and GUCU as the sequence of the hairpin loop. To that end, we generated constructs with any two (CUUU A/U-rich/6-nts stem, CUUU A/U-rich/GUCU loop, and 6-nt stem/GUCU loop) or all three of the mutations (CUUU A/U-rich/6-nt stem/GUCU loop) and examined their ability to repress DsRed2 expression. The resulting mutants repressed DsRed2 expression to levels ranging from 2fold to 6-fold. The mutant with a CUUU A/U-rich sequence and a 6-nt long stem had the strongest repression, and was able to repress DsRed2 expression at a 3 times more effectively than the original sRNA (>4-fold repression, Figure 2E, column 6, Figure S1). The sRNA scaffold incorporating all three engineered modules had the second highest repression capability (>4-fold repression, Figure 2E, column 8). As the summations of the best three mutants did not lead to the highest repression capability, we also tested other combinations as well. As the A/U-rich sequence, the second best mutant (CAUU; see Figure 2B) was selected. As the hairpin loop sequence, the two second best mutants (AUAA and AACA: both showing 6.7-fold repression capability; see Figure 2D) were selected. A 6-nt long stem was selected because this mutant was significantly higher than all the other stem length mutants. Thus, we generated sRNA constructs with any two or all three of the mutations to assess their repression capabilities (Figure S2). However, none of these new combinations showed higher repression capability than that obtained with the mutant with a CUUU A/U-rich sequence and a 6-nt long stem (Figure 2E, Figure S2). We further examined whether the improved repression capability of sRNAs with an altered Hfq-binding region is associated with stronger Hfq-binding affinity. To measure the Hfq-binding affinity of sRNA scaffolds, purified Hfq was incubated with biotinylated sRNAs. The Hfq-sRNA mixture was then transferred into a streptavidin-coated multiwell plate 1456

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Figure 4. Identification of repression target genes using synthetic sRNAs derived from the engineered sRNA scaffold for enhanced cadaverine production. Schematic diagram of the cadaverine biosynthetic pathway including glycolysis, the lysine biosynthesis pathway, and a single conversion step from lysine to cadaverine by lysine decarboxylase. Changes in cadaverine production relative to the starting strain (XQ56 harboring p15CadA) are represented by colored circles. The genes and the values of the relative changes in cadaverine titers are listed in Table S2. Knocked down genes that increased cadaverine production are shown in blue. Data were obtained from three measurements.

constructed scaffolds with varying combinations of the mutant modules and measured their ability to repress DsRed2 expression (Figure 2F). Among the three single module mutants and four combined module mutants, the weakest one, which had GCAC as the A/U-rich sequence, caused a 1.4-fold reduction in DsRed2 expression, which is only 0.38 times the repression level caused by the original sRNA (Figure 2F, column 2). The other sRNAs had varied repression levels between those of the original synthetic sRNA and the sRNA with GCAC as the A/U-rich sequence. Application of the Engineered sRNA Scaffold to Cadaverine-Producing Strain. Having developed a new synthetic sRNA-based tool for gene expression control, we examined its practical usability by developing an E. coli strain capable of overproducing cadaverine, an engineering plastic precursor and a model product of metabolic engineering applications.51 For this application, 67 genes-repressing sRNAs were constructed with the altered scaffold: the strongest scaffold (CUUU A/U-rich/6-nt stem) was used as an example in this study. Sixty-seven genes related to the cadaverine biosynthesis pathway were rationally selected as target genes

and incubated to allow the formation of streptavidin−biotin complexes. After washing off residual components in the plate, sRNA-bound Hfq was quantitated using FITC-conjugated antibodies (Figure 3A). Among the four sRNA scaffolds tested (original sRNA, an sRNA scaffold with CUUU as an A/U-rich sequence, an sRNA scaffold with a 6-nt long stem, and an sRNA scaffold harboring both mutations), the sRNA scaffold with the strongest gene repression capability also had the strongest Hfq-binding affinity (Figure 3B). The amount of Hfq bound to the sRNA with the strongest repression capability was about 29% higher than the amount bound to the original sRNA. Since the amount of Hfq can be a limiting factor in the function of synthetic sRNAs in a cell, where exogenous sRNAs compete with more than 100 natural sRNAs for Hfq binding,49,50 the enhanced affinity for Hfq can contribute to the improvement of the gene repression capability of synthetic sRNAs. We also examined the effect of combining the repressionweakening mutations in the three modules of the Hfq-binding region, i.e., the sRNA having GCAC as the A/U-rich sequence, an 8-nt long stem, and CGGG as the loop sequence. We 1457

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ACS Synthetic Biology for repression (Table S1). One of these target genes is pgi, which encodes phosphoglucose isomerase. Knocking down pgi increased the production of cadaverine by 27% (1.66 g/L) relative to the starting strain (1.31 g/L)51 (Figure 4, Table S2). This gene was one of the top-ranked target genes for knockdown to enhance cadaverine production. This seems to be due to the increase in available NADPH resulting from the redirection of metabolic flux from glycolysis to the pentose phosphate pathway. Consequently, this redirection facilitates the NADPH-driven conversion of oxaloacetate to lysine, a precursor of cadaverine. The other top-ranked target gene was serA, which encodes phosphoglycerate dehydrogenase, the first enzyme in the serine biosynthesis pathway that uses 3phosphoglycerate as the starting substrate. Knocking down this gene resulted in the production of 1.67 g/L of cadaverine, the similar concentration as that obtained by knocking down pgi. Phosphoglycerate dehydrogenase functions in the conversion of 3-phosphoglycerate to 3-phosphohydroxypyruvate, a process that requires NADH. The reason behind the enhanced cadaverine production resulting from serA knockdown is unclear; thus, this gene could not be rationally selected from the sixty-seven genes as an engineering target. The target genes, target mRNA-binding sequences, and the relative cadaverine titers of the sRNA-harboring strains are listed in Table S2. Since industrial chemical production using bacteria is often performed in fed-batch mode, fed-batch cultures of the strains expressing anti-pgi or anti-serA sRNA were performed. The anti-pgi and anti-serA strains produced 9.5 and 13.7 g/L of cadaverine, respectively (Figure 2, Figure S3). The cadaverine production of the latter strain is 8.7% higher concentration than that of a strain harboring anti-murE sRNA, which had the highest cadaverine production in a previous study (12.6 g/ L).31 This exemplifies the importance and effectiveness of using a synthetic sRNA library to identify gene knockdown targets that cannot be rationally selected.

synthetic biology, strict modulators of gene expression are important because gene regulation is critical in the implementation of genetic circuits coding for the desired logic and memory functions.52−55 The enhancement of sRNA function may allow sRNA to become a more versatile tool for synthetic biology. The use of synthetic sRNA with stronger functions also allows lower spending of cellular resources. The efficient use of materials and energy is one of the important aims of metabolic engineering. The wastage of resources that could otherwise be used to produce a desired chemical can be successfully reduced by employing strong sRNA. Improved sRNA scaffolds provide a stronger control over target gene expression. Therefore, this engineered sRNA scaffold library will be useful in studying gene expression control and in the enhanced production of desired bioproducts through metabolic engineering. As a practical application, we identified new target genes for enhancing cadaverine production using the engineered sRNA scaffolds. In our previous study, we identified the target genes to be repressed for enhanced cadaverine production using the MicC scaffold.31 The strongest sRNA scaffold (CUUU A/Urich/6-nt stem) in the present study had a repression capability 4.5 times higher than the most effective scaffold identified in the previous study (Figure S1). In the present study, the repression of five targets enhanced cadaverine production by more than 10% (Figure 4). The repression of two of these genes (serA and glpX) was shown to decrease cadaverine production in the previous study.31 In addition, two target genes (pta and ilvD) identified from the previous study31 were more strongly repressed using the newly developed sRNA scaffold in this study, which further enhanced cadaverine production, despite the use of identical promoters and plasmid replication origins for the expression of these sRNAs. By developing stronger synthetic sRNA scaffolds, the limits of gene repression using synthetic sRNAs could be further extended, which would consequently allow the expansion of the spectrum of gene manipulation targets for metabolic engineering.

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DISCUSSION In this study, we generated a library of synthetic sRNA scaffolds with varying gene repression capabilities by systematically introducing mutations to the Hfq-binding region. On the basis of naturally occurring sRNAs, synthetic sRNAs that repress desired target genes have been developed, and detailed processes for the rational design and construction of synthetic sRNAs have been recently proposed.31,32 Improving the activity of sRNAs by increasing the binding energy between sRNA and mRNA has also been suggested. The binding energy of the sRNA-mRNA hybrid can be modulated by shortening or elongating the target-binding region on the sRNA, but loss of target specificity is a concern.15 In order to develop another strategy to improve the gene repression capabilities of synthetic sRNA, we focused on engineering the sRNA scaffold. Among the three functional elements of Hfq-dependent sRNAs, modification of the target mRNA-binding region is limited, and the role of the rho-independent transcription terminator in terminating transcription is obvious, as briefly discussed above. Thus, we investigated the Hfq-binding region as a potential modifiable element for the improvement of gene repression. The functional improvement of synthetic sRNA provides several advantages over synthetic biology and metabolic engineering. The engineered sRNA scaffold provides a wider range of gene repression, and even represses expression to levels beyond the intrinsic ability of the original sRNA. In

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METHODS Strains and Culture Conditions. Genotypes of strains used in this study are listed in Table S3. E. coli DH5α strain was used for construction of plasmids and for measurement of gene repression activities of synthetic sRNAs. DH5α strains were cultured in Luria−Bertani (LB) medium with appropriate antibiotics at 37 °C in a shaking incubator at 200 rpm. E. coli XQ56 strain was used to produce cadaverine.51 XQ56 strains harboring p15CadA and plasmids of synthetic sRNA library were cultured in LB medium with appropriate antibiotics at 37 °C until stationary phase. This culture was inoculated into a 300 mL baffled flask containing 50 mL of R/2 medium supplemented with 10 g/L glucose and 3 g/L (NH4)2SO4. Contents of R/2 medium are described previously.51 When required, antibiotics were added with following concentrations: 50 μg/mL of ampicillin, 25 μg/mL of kanamycin, and 17.5 μg/ mL of chloramphenicol. For fed-batch culture, flask culture (4 flasks, 200 mL, early stationary phase) was inoculated into a 6.6 L jar fermentor (Bioflo 3000; New Brunswick Scientific Co., Edison, NJ) containing 1.8 L of R/2 culture medium supplemented with 10 g/L of glucose and 3 g/L of (NH4)2SO4. To maintain pH 6.8, 14% ammonia solution was automatically added. Dissolved oxygen concentration was kept at 40% of air saturation automatically by adjusting the 1458

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ACS Synthetic Biology

and FITC-conjugated anti-6xHis antibody was purchased from Abcam (ab1206).

agitation speed and supplying of pure oxygen gas. A nutrient feeding solution was added by using the pH-stat feeding strategy when the pH rose greater than set point (pH 6.8) by 0.02 due to carbon source depletion. The feeding solution contained: 522 g/L of glucose, 170 g/L of (NH4)2SO4, and 8 g/L of MgSO4·7H2O. Samples were harvested for the measurements of OD600 and the product concentrations were determined using the culture supernatant after centrifugation at 14 000 rpm for 5 min. Plasmid Construction. Plasmids and oligonucleotides used in this study are listed in Tables S2 and S3, respectively. Plasmids expressing sRNAs used in this study were constructed by using an inverse PCR method.32 The template plasmids were pKK105SDsRed2 and pKK114SDsRed2 for the construction of scaffold mutants and pKKSEDsRed2 for the construction of synthetic sRNA library for enhanced cadaverine production. The plasmid pKKSEDsred2 was constructed using pKKSDsRed2 as a template for inverse PCR. Oligonucleotides used in the construction of synthetic sRNA library are listed in Table S4. To express E. coli Hfq for purification, Hfq-His6 was cloned into pET30a. The hfq gene was amplified from E. coli gDNA using the primers, HfqFNdeI and HfqRNcoI. The amplified DNA fragments and pET30a were digested by NdeI and NcoI and ligated. Primers, M114 F and M114 R, were used to construct pKK114MDsRed2. Gene Repression Activity Measurement Using a Fluorescence Protein. Gene repression activities of synthetic sRNAs were measured by using a red fluorescence protein, DsRed2. Briefly, DH5α cells harboring pACDsRed2 and pKKKS derivatives with synthetic sRNA expression cassettes were cultured in LB medium with appropriate antibiotics at 37 °C until stationary phase. These cultures were inoculated into 24-well plate containing 1 mL of LB medium. After incubation for 24 h at 37 °C in shaking incubator, cell densities were estimated by measuring the optical density at 600 nm (OD600). Cultured cells were diluted to be OD600 = 1 and fluorescence intensities were measured using a microplate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA). The measured intensities were modified by subtracting the fluorescence emitted from E. coli DH5α cells without DsRed2 expression. These modified intensities were normalized to the fluorescence intensity obtained from cells expressing DsRed2 without synthetic sRNA. Fold repression is calculated as fluorescence intensity measured with an empty vector divided by the fluorescence intensity measured with an sRNA expression vector. Analytical Methods. Concentrations of cadaverine were measured by high performance liquid chromatography (1100 Series HPLC, Agilent Technologies, Palo Alto, CA) by using previously described methods.51 Hfq-sRNA ELISA Assay. E. coli BL21(DE3) harboring pET30a-Hfq-His6 was cultured in LB medium at 37 °C. When OD600 reached 0.2, 1 mM IPTG was added to the culture and incubated additional 2 h. Cells were harvested by centrifugation and used for Hfq purification. Hfq was purified by the previously described procedure.56 Using purified Hfq, HfqsRNA ELISA assay was performed by the previously described protein-RNA ELISA method.57 The measured intensities were modified by subtracting the fluorescence obtained from the sample without synthetic sRNA and normalized to the fluorescence obtained from wild type SgrS-S. Streptavidin coated plates were purchased from Thermo Scientific (15119)

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.9b00165. Comparison of anti-DsRed2 sRNAs derived from various scaffolds (Figure S1), gene repression activities of sRNAs harboring combinations of the improved modules other than the combinations tested in Figure 2E,F (Figure S2), and fed-batch cultures of cadaverine producing strains with expression of improved sRNAs (Figure S3) (PDF) List of oligonucleotide sequences used in the construction of synthetic sRNA library for enhanced cadaverine production (Table S1), list of target genes of synthetic sRNA library used in metabolic engineering of cadaverine production (Table S2), strains and plasmids used in this study (Table S3), and oligonucleotides used in this study (Table S4) (XLSX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (82) 42 350 3930. ORCID

Dongsoo Yang: 0000-0002-0299-3128 Sang Yup Lee: 0000-0003-0599-3091 Author Contributions #

M.N. and S.M.Y. contributed equally. S.Y.L. and M.N. designed the research. M.N. and D.Y. performed experiments. S.M.Y., N.M., and D.Y. analyzed data and generated figures for the manuscript. M.N., S.M.Y., D.Y., and S.Y.L. wrote the manuscript. All authors read and approved the final manuscript.

Notes

The authors declare the following competing financial interest(s): The authors declare that they have conflict of interest as the synthetic sRNA technology described here is patent filed including, but not limited to KR 10-1575587, US 9388417, EP 13735942.8, CN 201380012767.X, KR 101690780, KR 10-1750855, US 15317939, CN 201480081132.X for potential commercialization. Also, the authors have conflict of interest as the expanded synthetic sRNA expression platform is of commercial interest and is patent filed including, but not limited to KR 10-2018-0073970.

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ACKNOWLEDGMENTS This work was supported by grants from the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) of Ministry of Science and ICT through the National Research Foundation of Korea.

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