Broad-Spectrum Gene Repression Using Scaffold Engineering of

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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 ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00165 • Publication Date (Web): 27 May 2019 Downloaded from http://pubs.acs.org on May 28, 2019

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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.

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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 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


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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).



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). 199x70mm (300 x 300 DPI)


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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.



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Broad-Spectrum Gene Repression using Scaffold Engineering

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of Synthetic sRNAs

5 Minho Noh,†,∥ Seung Min Yoo,‡,§,∥ Dongsoo Yang,†,¶ and Sang Yup Lee†,§, ¶,*

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†

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291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea

Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST,

10

‡

11

gu, Seoul 06974, Republic of Korea

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§

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Daejeon 34141, Republic of Korea

14

¶

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Collaborative Laboratory, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141,

16

Republic of Korea

School of Integrative Engineering, Chung-Ang University, 84 Heukseok-ro, Dongjak-

BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu,

Systems

Metabolic

Engineering

and

Systems

Healthcare

17

1


Cross-Generation

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ABSTRACT: Gene expression regulation in broad-spectrum range is critical for

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constructing cell factories and genetic circuits to balance and control system-wide fluxes.

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Synthetic small regulatory RNAs (sRNAs) effectively regulate gene expression at the

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translational level by modulating an mRNA-binding chance and sRNA abundance;

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however, it can control target gene expression only within the limit of the intrinsic

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repression ability of sRNAs. Here, we systematically mutated a SgrS scaffold as a model

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sRNA by dividing the Hfq-binding module of the sRNA into the three regions: the A/U-

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rich sequence, the stem, and the hairpin loop, and examined how efficiently the mutants

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suppressed DsRed2 expression. By doing this, we found that a scaffold with an altered

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A/U-rich sequence (CUUU) and stem length and that with altered A/U-rich sequence

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(GCAC) showed a three-fold stronger and a three-fold weaker repression than the original

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scaffold, respectively. For practical application of altered scaffolds, proof-of-concept

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experiments were performed by constructing a library of 67 synthetic sRNAs with the

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strongest scaffold, each one targeting a different rationally selected gene, and using this

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library to enhance cadaverine production in Escherichia coli, yielding in 27% increase

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(1.67 g/L in flask cultivation, 13.7 g/L in fed-batch cultivation). Synthetic sRNAs with

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engineered sRNA scaffolds could be useful in modulating gene expression for strain

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improvement.

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KEYWORDS: gene repression, Hfq-binding region, knockdown, synthetic sRNA

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2



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Gene expression regulation is critical for constructing cell factories and genetic circuits.

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This is especially important in metabolic engineering, where modulating chromosomal

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gene expression is a key to developing high-performance microbes for the production of

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chemicals, fuels and materials. This can be achieved by balancing and controlling system-

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wide metabolic fluxes for enhanced product biosynthesis and cell growth.1 Because of

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the complexity of genetic and metabolic networks, the main challenge in this field is

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developing a sophisticated yet easy method for regulating target gene expression in

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dynamic and broad-spectrum range. In this regard, much attempts have been made to

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develop a gene expression system in varied levels by modifying transcriptional level-

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associated factor,2-10 mRNA stability and translation,11-13 and protein regulators.14,15

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These methods are effective for heterogeneous genes cloned in vectors, but are still labor-

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intensive and time-consuming for chromosomal genes.

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Another effort to control gene expression involves the use of bacterial RNA

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regulator. Bacterial RNA regulators actively control the expression of genes involved in

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a wide range of physiological processes, such as carbon metabolism, amino acid

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metabolism, stress response, and environmental adaptation.16-19 An example of bacterial

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RNA regulators are trans-acting Hfq-dependent small regulatory RNAs (sRNAs), which

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bind at or near the ribosome-binding sites (RBSs) of mRNAs with the aid of the RNA

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chaperone Hfq.20-22 This binding prevents ribosomes from accessing the RBS, thereby

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inhibiting translation.20-22 Hfq-dependent sRNAs consist of three functional modules: an

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mRNA-binding region, an Hfq-binding region, and a Rho-independent transcription

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terminator (Figure 1A). The mRNA-binding region is complementary to a target

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mRNA,23 which would allow sRNA-mRNA hybridization to occur. The Hfq-binding

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region enhances the ability of sRNAs to bind target mRNAs, and facilitates mRNA 3


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degradation by recruiting RNase E.24-28 The Rho-independent transcription terminator is

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a GC-rich palindromic sequence followed by a stretch of U nucleotides that terminates

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the synthesis of sRNAs to produce discrete sRNA molecules. Much effort has been

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exerted on developing a method to modulate gene repression by modifying natural sRNA

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scaffold.24,26,28-40 Notably, the recent development of synthetic sRNAs has paved the way

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for more practical and wider use of sRNA in metabolic engineering and synthetic biology

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that allows in trans high-throughput gene expression control without any chromosomal

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modification.31-42 A recently developed expanded synthetic sRNA expression platforms

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allowed rapid, multiplexed, and genome-scale target gene knockdown in engineered

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Escherichia coli without concerning plasmid compatibility.43 Also, effective knockdown

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of essential genes is possible by employing the sRNA technology, which is not easy using

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the conventional knockout techniques. CRISPR interference (CRISPRi) can also

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knockdown target gene expression,44 but is not as efficient as the sRNA technology. For

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example, CRISPRi requires expression of a large gene (encoding dCas9) which often

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gives metabolic burden to the host cell. Also, a recent study reported that dCas9 can

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directly bind to E. coli endogenous genes even without a single-guide RNA, resulting in

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altered cell morphology and cell growth.45

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Using synthetic sRNAs, there are two strategies for controlling the expression of

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desired target genes. One strategy is to change sRNA abundance without disturbing

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sRNA scaffold, which can be achieved by employing different strength promoters or

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replication origins in sRNA expression vector.41 This strategy is a facile approach for

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controlling gene expression as it does not require additional consideration of potential

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cross-reactivity; however, they can modulate target gene expression only within the limit

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of the intrinsic repression ability of sRNAs. To expand the gene repression range of 4



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original sRNAs, the modification of three functional modules of sRNAs mentioned above

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could be a solution, which is the other strategy. As a first functional module-based

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approach, the level of repression can be modulated by modifying the mRNA-binding

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sequences of the sRNA in order to change the binding energy between the sRNA and the

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target mRNA.31,32 This approach is simple as it is easy to design and use sRNAs; however,

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like the strategy above, there is a limit to modulate target gene expression in broad range

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beyond an intrinsic repression ability of sRNA. The potential cross-reactivity caused by

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the change in the mRNA-binding region should also be considered. The Rho-independent

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transcription terminator-based approach can also control target gene expression.

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Although this region-based studies have unveiled that the poly-U tail of this region is also

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essential for Hfq-binding and sRNA stability,29,30 the role of the rho-independent

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transcription terminator is obvious. As the last functional module-based approach, the

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Hfq-binding region is a potential target for modification to further modulate sRNA-

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mediated gene repression. Although there have been several studies on the Hfq-binding

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region of natural sRNAs performed through mutational analysis,24,26,28-30,39,40,46 the

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systematic analysis is required for improved gene regulation. In addition, the

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improvement of the synthetic sRNA activity by this strategy was not focused yet. In this

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regard, for gene expression regulation in broad-spectrum range using synthetic sRNAs,

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here, we report the modification of the Hfq-binding region of the synthetic sRNA SgrS-

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S and the application of these engineered synthetic sRNAs in modulating gene repression

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in Escherichia coli. The three parts of the Hfq-binding region, the A/U-rich sequence, the

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stem, and the hairpin loop, were systemically mutated, and the mutants were tested for

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their ability to repress expression using DsRed2 as a model target protein. We identified

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a synthetic sRNA with an altered A/U-rich sequence (a UAUU to CUUU mutation) and 5


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a longer hairpin stem (a 4 nts to 6 nts mutation) that had a three-fold stronger repression

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ability than that of the non-mutant SgrS-S. Also, we found that a synthetic sRNA with

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altered A/U-rich sequence (a UAUU to GCAC mutation) had a repression capability 3

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times lower than that of original sRNAs. For practical application of altered scaffold as

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proof-of-concept experiment, the strongest one was employed in the construction of a

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rationally selected sRNA library, which was in turn used to develop an E. coli strain

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capable of efficiently producing cadaverine, a chemical precursor in plastic production.

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RESULTS

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Construction of Synthetic sRNAs for Scaffold Engineering. For the synthetic sRNA

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scaffold used to investigate the effects of mutations on the Hfq-binding region, we

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selected the sRNA SgrS-S derived from SgrS, which is one of three sRNAs (MicC, MicF,

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and SgrS) previously reported by our group.31,32 We selected SgrS-S because it has a

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simple structure and its Hfq-binding region is well-characterized. 29,30,41,47 In addition, its

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simple and stable secondary structure minimizes the number of structural variations that

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need to be considered before construction and after mutant generation.

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To investigate the effects of mutations in the Hfq-binding region on the repression

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ability of SgrS-S, we systematically introduced mutations to the three parts of the region:

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the A/U-rich sequence and the hairpin loop and stem (Figure 1B). To examine the changes

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in the efficiency of repression, we used DsRed2, a red fluorescent protein, as a reporter.

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The ptsG binding sequence in SgrS-S was replaced with a sequence complementary to

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the DsRed2 gene in order to construct a synthetic sRNA capable of repressing DsRed2

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expression.47 The first 24 nucleotides (nts) from the start codon of DsRed2 were targeted,

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as previously reported.31,32 The repression efficiency of each mutant was determined by

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measuring the change in fluorescence intensity (Figure 1C).

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Effect of Mutation in the A/U-rich Sequence on Repression Efficiency. First,

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mutations were introduced to the A/U-rich sequence in the Hfq-binding region (Figure

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2A). The A/U-rich sequence near the internal stem-loop is a common feature of Hfq-

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dependent sRNAs, such as SgrS, RybB, and DsrA. It plays an important role in Hfq-

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sRNA interaction and overall sRNA function.24,26,29,40 We generated mutants harboring a

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single nucleotide substitution at each nucleotide of the identified A/U-rich sequence of

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SgrS-S, UAUU,29 and assessed their ability to repress DsRed2 expression (Figure 1B).

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The sRNAs harboring mutations at the first and second nucleotides had an increased or

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comparable level of repression of DsRed2 relative to the non-mutant SgrS-S. On the other

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hand, sRNAs harboring mutations at the third nucleotides had decreased levels of

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repression compared to the original synthetic sRNA (Figure 2B). A mutant with a U-to-

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C mutation at the first nucleotides had the strongest repression ability, followed by a

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mutant with an A-to-U mutation at the second nucleotide (Figure 2B). Mutations at the

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third and fourth nucleotides also weakened gene expression. To summarize, all mutants

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harboring a single nucleotide substitution were capable of gene repression up to levels

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ranging from 2.9- to 10-fold (inset of Figure 2B). To examine whether combining these

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mutations additively increases gene repression, a synthetic sRNA with a CUUU sequence

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replacing the A/U-rich sequence was constructed and tested for repression efficiency.

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This engineered sRNA repressed DsRed2 expression at a higher level (>11-fold) than the

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mutant with the U-to-C single mutation at the first nucleotide (10-fold). These results

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suggest that changing the first nucleotide of the A/U-rich sequence from U to C while 7


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assigning the rest of the three nucleotides to be U’s increases the repression capability of

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the sRNA substantially. The increase in repression capability resulting from having three

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U’s for the second to the fourth nucleotides agrees with previous findings for natural

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sRNA-based scaffold engineering.26,29,40 Interestingly, combining the repression-

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weakening single mutations led to lower repression efficiencies. For example, mutating

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the A/U rich sequence to GCAC had an additive effect and reduced repression efficiency

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to 1.5-fold (Figure 2B). Thus, the A/U rich sequence of the Hfq-binding region can be

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engineered to finely control the gene repression level on a wider spectrum of expression

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than that of the original sRNA scaffold.

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Effect of the Length of the Hairpin Stem on Gene Repression Ability. The Hfq-

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binding region is generally flanked by one or more hairpins.25,26,28,29,40 According to a

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previous study, disrupting the hairpin structure impairs Hfq-binding. Thus, the presence

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of a hairpin structure, regardless of the sequence of the stem, is important in maintaining

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the function of sRNAs.29 Based on this finding, we hypothesized that a more stable

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hairpin structure will result in an enhanced repression capability. To test this hypothesis,

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sRNAs of varying lengths were constructed by eliminating one G-C base pair or adding

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1–4 G-C base pairs (Figure 2A) while retaining the original stem structure. These

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engineered sRNAs were tested for their ability to repress DsRed2 expression (Figure 2C).

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As stem length was increased, gene repression also increased. Repression eventually

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reached a maximum when the stem was 6 nts long (Figure 2C). The mutant with a 6-nt

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long stem dramatically repressed DsRed2 expression by up to 37-fold. As the stem length

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was further increased to 7 and 8 nts long, repression capability was reduced to levels

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lower than that of the original sRNA (Figure 2C). In addition to the hairpin structure and 8



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the preceding U-rich sequence, which have been previously reported to be important for

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sRNA function,29,40 our results suggest that the optimal length of the hairpin stem is also

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important in enhancing the repression capability of synthetic sRNAs.

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Effect of Mutations in the Loop Sequence on Repression Levels. We next focused

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on the sequence of the hairpin loop in the Hfq-binding region. There has been a report

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suggesting that the affinity of Hfq to the rpoS mRNA was dependent on two single-

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stranded A-rich sequences and that this interaction was essential for the repression of

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rpoS.48 The sequence of the loop in SgrS-S also consists of a single-stranded AAAA

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sequence. Thus, we next examined the effects of alterations in this A-rich sequence on

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the repression capability of SgrS-S. To do this, we constructed all possible mutants with

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single-nucleotide mutations in the AAAA sequence (Figure 2D). Four mutants (CAAA,

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AGAA, AAGA, and AAAG) had repression levels similar to or weaker than the original

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sRNA (2.5- to 4-fold), while other mutants had higher repression levels than the non-

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mutant SgrS-S, ranging from 4-fold to 8-fold (Figure 2D). To assess the combinatorial

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effects of these mutations, the mutations at each nucleotide resulting in the strongest

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repression (GAAA, AUAA, AACA, and AAAU) were incorporated into a single

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synthetic sRNA. This engineered sRNA, which had the AAAA sequence mutated into

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GUCU, had the strongest repression capability (>8-fold repression, Figure 2D). Although

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there has been a report showing that a UAAAAU-to-GAGCAC mutation in the loop

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sequence of SgrS-S results in the retention of the full gene repression capacity of the

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sRNA,29 our systematic mutational analysis revealed that the sequence of the loop indeed

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plays an important role in gene repression. One possible explanation for the role of the

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loop sequence is that the interaction between Hfq and sRNA is dependent on the exposed 9


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bases of the loop, like the A/U-rich sequence. On the other hand, the effect of combining

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mutations, which is the reduction of repression (e.g., CGGG), was not observed, and the

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repression efficiency of sRNAs with multiple mutations (3-fold) was similar to that of the

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single mutant (AAGA) with the lowest repression level (2.5-fold) (Figure 2D). In

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summary, all mutants tested showed gene repression levels ranging from 2.5- to 8.3-fold

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(inset of Figure 2D).

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A New sRNA Scaffold with Three Mutated Modules for Expanding the sRNA

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Repression Range. As detailed above, we developed sRNA scaffold mutants that could

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efficiently knockdown the expression of DsRed2 up to 37-fold (Figure 2B-D). We

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speculated that this corresponds to near-complete saturation of the target mRNAs by

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sRNA binding. Nevertheless, as we aimed at further enhancing the repression capabilities

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of sRNA scaffold mutants, we tried to increase the resolution of repression capabilities

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by employing a weaker promoter (J23114, MIT Registry, BBa_J23114) for sRNA

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expression than that (J23105, MIT Registry, BBa_J23105) used in previous experiments.

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The relative strengths of J23105 and J23114 are 0.24 and 0.10, respectively

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(http://parts.igem.org/Promoters/Catalog/Anderson). We examined the additive effect of

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combining the best mutations introduced in the three modules of the Hfq-binding region

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by constructing an sRNA with CUUU as the U-rich sequence, a 6-nt long stem, and

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GUCU as the sequence of the hairpin loop. To that end, we generated constructs with any

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two (CUUU U-rich/6-nts stem, CUUU U-rich/GUCU loop, and 6-nt stem/GUCU loop)

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or all three of the mutations (CUUU U-rich/6-nt stem/GUCU loop) and examined their

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ability to repress DsRed2 expression. The resulting mutants repressed DsRed2 expression

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to levels ranging from 2-fold to 6-fold. The mutant with a CUUU A/U-rich sequence and 10



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a 6-nt long stem had the strongest repression, and was able to repress DsRed2 expression

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at a 3 times more effectively than the original sRNA (>4-fold repression, Figure 2E,

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column 6). The sRNA scaffold incorporating all three engineered modules had the second

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highest repression capability (>4-fold repression, Figure 2E, column 8). As the

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summations of the best three mutants did not lead to the highest repression capability, we

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also tested other combinations as well. As the U-rich sequence, the second best mutant

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(CAUU; see Figure 2B) was selected. As the hairpin loop sequence, the two second best

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mutants (AUAA and AACA: both showing 6.7-fold repression capability; see Figure 2D)

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were selected. A 6-nt long stem was selected because this mutant was significantly higher

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than all the other stem length mutants. Thus, we generated sRNA constructs with any two

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or all three of the mutations to assess their repression capabilities (Figure S2). However,

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none of these new combinations showed higher repression capability than that obtained

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with the mutant with a CUUU A/U-rich sequence and a 6-nt long stem (Figure 2E, Figure

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S2).

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We further examined whether the improved repression capability of sRNAs with

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an altered Hfq-binding region is associated with stronger Hfq-binding affinity. To

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measure the Hfq-binding affinity of sRNA scaffolds, purified Hfq was incubated with

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biotinylated sRNAs. The Hfq-sRNA mixture was then transferred into a streptavidin-

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coated multi-well plate and incubated to allow the formation of streptavidin-biotin

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complexes. After washing off residual components in the plate, sRNA-bound Hfq was

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quantitated using FITC-conjugated antibodies (Figure 3A). Among the four sRNA

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scaffolds tested (original sRNA, an sRNA scaffold with CUUU as a U-rich sequence, an

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sRNA scaffold with a 6-nt long stem, and an sRNA scaffold harboring both mutations),

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the sRNA scaffold with the strongest gene repression capability also had the strongest 11


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Hfq-binding affinity (Figure 3B). The amount of Hfq bound to the sRNA with the

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strongest repression capability was about 29% higher than the amount bound to the

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original sRNA. Since the amount of Hfq can be a limiting factor in the function of

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synthetic sRNAs in a cell, where exogenous sRNAs compete with more than 100 natural

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sRNAs for Hfq binding,49,50 the enhanced affinity for Hfq can contribute to the

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improvement of the gene repression capability of synthetic sRNAs.

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We also examined the effect of combining the repression-weakening mutations in

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the three modules of the Hfq-binding region, i.e. the sRNA having GCAC as the U-rich

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sequence, an 8-nt long stem, and CGGG as the loop sequence. We constructed scaffolds

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with varying combinations of the mutant modules and measured their ability to repress

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DsRed2 expression (Figure 2F). Among the three single module mutants and four

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combined module mutants, the weakest one, which had GCAC as the A/U-rich sequence,

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caused a 1.4-fold reduction in DsRed2 expression, which is only 0.38 times the repression

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level caused by the original sRNA (Figure 2F, column 2). The other sRNAs had varied

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repression levels between those of the original synthetic sRNA and the sRNA with GCAC

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as the A/U-rich sequence.

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Application of the Engineered sRNA Scaffold to Cadaverine-Producing Strain.

272

Having developed a new synthetic sRNA-based tool for gene expression control, we

273

examined its practical usability by developing an E. coli strain capable of overproducing

274

cadaverine, an engineering plastic precursor and a model product of metabolic

275

engineering applications.51 For this application, sixty-seven genes-repressing sRNAs

276

were constructed to altered scaffold: the strongest scaffold (CUUU/U-rich/6-nt stem) was

277

used as an example in this study. Sixty-seven genes related to the cadaverine biosynthesis 12



278

pathway were rationally selected as target genes for repression (Table S1). One of these

279

target genes is pgi, which encodes phosphoglucose isomerase. Knocking down pgi

280

increased the production of cadaverine by 27% (1.66 g/L) relative to the starting strain

281

(1.31 g/L)51 (Figure 4, Table S2). This gene was one of the top-ranked target genes for

282

knockdown to enhance cadaverine production. This seems to be due to the increase in

283

available NADPH resulting from the redirection of metabolic flux from glycolysis to the

284

pentose phosphate pathway. Consequently, this redirection facilitates the NADPH-driven

285

conversion of oxaloacetate to lysine, a precursor of cadaverine. The other top-ranked

286

target gene was serA, which encodes phosphoglycerate dehydrogenase, the first enzyme

287

in the serine biosynthesis pathway that uses 3-phosphoglycerate as the starting substrate.

288

Knocking down this gene resulted in the production of 1.67 g/L of cadaverine, the similar

289

concentration as that obtained by knocking down pgi. Phosphoglycerate dehydrogenase

290

functions in the conversion of 3-phosphoglycerate to 3-phosphohydroxypyruvate, a

291

process that requires NADH. The reason behind the enhanced cadaverine production

292

resulting from serA knockdown is unclear; thus, this gene could not be rationally selected

293

from the sixty-seven genes as an engineering target. The target genes, target mRNA-

294

binding sequences, and the relative cadaverine titers of the sRNA-harboring strains are

295

listed in Supplementary Table 2. Since industrial chemical production using bacteria is

296

often performed in fed-batch mode, fed-batch cultures of the strains expressing anti-pgi

297

or anti-serA sRNA were performed. The anti-pgi and anti-serA strains produced 9.5 and

298

13.7 g/L of cadaverine, respectively (Figure 2). The cadaverine production of the latter

299

strain is 8.7% higher concentration than that of a strain harboring anti-murE sRNA, which

300

had the highest cadaverine production in a previous study (12.6 g/L).31 This exemplifies

13


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301

the importance and effectiveness of using a synthetic sRNA library to identify gene

302

knockdown targets that cannot be rationally selected.

303 304

DISCUSSION

305

In this study, we generated a library of synthetic sRNA scaffolds with varying gene

306

repression capabilities by systematically introducing mutations to the Hfq-binding region.

307

Based on naturally occurring sRNAs, synthetic sRNAs that repress desired target genes

308

have been developed, and detailed processes for the rational design and construction of

309

synthetic sRNAs have been recently proposed.31,32 Improving the activity of sRNAs by

310

increasing the binding energy between sRNA and mRNA has also been suggested. The

311

binding energy of the sRNA-mRNA hybrid can be modulated by shortening or elongating

312

the target-binding region on the sRNA, but loss of target specificity is a concern.15 In

313

order to develop another strategy to improve the gene repression capabilities of synthetic

314

sRNA, we focused on engineering the sRNA scaffold. Among the three functional

315

elements of Hfq-dependent sRNAs, modification of the target mRNA-binding region is

316

limited, and the role of the rho-independent transcription terminator in terminating

317

transcription is obvious, as briefly discussed above. Thus, we investigated the Hfq-

318

binding region as a potential modifiable element for the improvement of gene repression.

319

The functional improvement of synthetic sRNA provides several advantages over

320

synthetic biology and metabolic engineering. The engineered sRNA scaffold provides a

321

wider range of gene repression, and even represses expression to levels beyond the

322

intrinsic ability of the original sRNA. In synthetic biology, strict modulators of gene

323

expression are important because gene regulation is critical in the implementation of

324

genetic circuits coding for the desired logic and memory functions.52-55 The enhancement 14



325

of sRNA function may allow sRNA to become a more versatile tool for synthetic biology.

326

The use of synthetic sRNA with stronger functions also allows lower spending of cellular

327

resources. The efficient use of materials and energy is one of the important aims of

328

metabolic engineering. The wastage of resources that could otherwise be used to produce

329

a desired chemical can be successfully reduced by employing strong sRNA. Improved

330

sRNA scaffolds provide a stronger control over target gene expression. Therefore, this

331

engineered sRNA scaffold library will be useful in studying gene expression control and

332

in the enhanced production of desired bioproducts through metabolic engineering.

333

As a practical application, we identified new target genes for enhancing cadaverine

334

production using the engineered sRNA scaffolds. In our previous study, we identified the

335

target genes to be repressed for enhanced cadaverine production using the MicC

336

scaffold.31 The strongest sRNA scaffold (CUUU U-rich/6-nt stem) in the present study

337

had a repression capability 4.5 times higher than the most effective scaffold identified in

338

the previous study (Figure S1). In the present study, the repression of six targets enhanced

339

cadaverine production by more than 10% (Figure 4). The repression of three of these

340

genes (serA, glpX, and pyrL) was shown to decrease cadaverine production in the

341

previous study.31 In addition, two target genes (pta and ilvD) identified from the previous

342

study31 were more strongly repressed using the newly developed sRNA scaffold in this

343

study, which further enhanced cadaverine production, despite the use of identical

344

promoters and plasmid replication origins for the expression of these sRNAs. By

345

developing stronger synthetic sRNA scaffolds, the limits of gene repression using

346

synthetic sRNAs could be further extended, which would consequently allow the

347

expansion of the spectrum of gene manipulation targets for metabolic engineering.

348 15


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349


METHODS

350

Strains and Culture Conditions. Genotypes of strains used in this study are listed

351

in Table S3. E. coli DH5α strain was used for construction of plasmids and for

352

measurement of gene repression activities of synthetic sRNAs. DH5α strains were

353

cultured in Luria-Bertani (LB) medium with appropriate antibiotics at 37 °C in a shaking

354

incubator at 200 rpm. E. coli XQ56 strain was used to produce cadaverine.51 XQ56 strains

355

harboring p15CadA and plasmids of synthetic sRNA library were cultured in LB medium

356

with appropriate antibiotics at 37 °C until stationary phase. This culture was inoculated

357

into a 300 ml baffled flask containing 50 mL of R/2 medium supplemented with 10 g/L

358

glucose and 3 g/L (NH4)2SO4. Contents of R/2 medium are described previously.51 When

359

required, antibiotics were added with following concentration: 50 µg/mL of ampicillin,

360

25 µg/mL of kanamycin, and 17.5 µg/mL of chloramphenicol. For fed-batch culture, flask

361

culture (4 flasks, 200 mL, early stationary phase) was inoculated into a 6.6 L jar fermentor

362

(Bioflo 3000; New Brunswick Scientific Co., Edison, NJ) containing 1.8 L of R/2 culture

363

medium supplemented with 10 g/L of glucose and 3 g/L of (NH4)2SO4. To maintain pH

364

6.8, 14% ammonia solution was automatically added. Dissolved oxygen concentration

365

was kept at 40% of air saturation automatically by adjusting the agitation speed and

366

supplying of pure oxygen gas. A nutrient feeding solution was added by using the pH-

367

stat feeding strategy when the pH rose greater than set point (pH 6.8) by 0.02 due to

368

carbon source depletion. The feeding solution contained: 522 g/L of glucose, 170 g/L of

369

(NH4)2SO4, and 8 g/L of MgSO4ꞏ7H2O. Samples were harvested for the measurements of

370

OD600 and the product concentrations were determined using the culture supernatant after

371

centrifugation at 14,000 rpm for 5 min.

372 16



373

Plasmid Construction. Plasmids and oligonucleotides used in this study are listed

374

in Tables S2 and S3, respectively. Plasmids expressing sRNAs used in this study were

375

constructed by using an inverse PCR method.32 The template plasmids were

376

pKK105SDsRed2 and pKK114SDsRed2 for the construction of scaffold mutants and

377

pKKSEDsRed2 for the construction of synthetic sRNA library for enhanced cadaverine

378

production. The plasmid pKKSEDsred2 was constructed using pKKSDsRed2 as a

379

template for inverse PCR. Oligonucleotides used in the construction of synthetic sRNA

380

library are listed in Table S4. To express E. coli Hfq for purification, Hfq-His6 was cloned

381

into pET30a. The hfq gene was amplified from E. coli gDNA using the primers,

382

HfqFNdeI and HfqRNcoI. The amplified DNA fragments and pET30a were digested by

383

NdeI and NcoI and ligated. Primers, M114 F and M114 R, were used to construct

384

pKK114MDsRed2.

385 386

Gene Repression Activity Measurement Using a Fluorescence Protein. Gene

387

repression activities of synthetic sRNAs were measured by using a red fluorescence

388

protein, DsRed2. Briefly, DH5α cells harboring pACDsRed2 and pKKKS derivatives

389

with synthetic sRNA expression cassettes were cultured in LB medium with appropriate

390

antibiotics at 37 °C until stationary phase. These cultures were inoculated into 24-well

391

plate containing 1 mL of LB medium. After incubation for 24 h at 37 °C in shaking

392

incubator, cell densities were estimated by measuring the optical density at 600 nm

393

(OD600). Cultured cells were diluted to be OD600=1 and fluorescence intensities were

394

measured using a microplate reader (SpectraMax M2, Molecular Devices, Sunnyvale,

395

CA). The measured intensities were modified by subtracting the fluorescence emitted

396

from E. coli DH5α cells without DsRed2 expression. These modified intensities were 17


Page 22 of 33

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397

normalized to the fluorescence intensity obtained from cells expressing DsRed2 without

398

synthetic sRNA. Fold repression is calculated as fluorescence intensity measured with an

399

empty vector divided by the fluorescence intensity measured with an sRNA expression

400

vector.

401 402

Analytical Methods. Concentrations of cadaverine were measured by high

403

performance liquid chromatography (1100 Series HPLC, Agilent Technologies, Palo Alto,

404

CA) by using previously described method.51

405 406

Hfq-sRNA ELISA Assay. E. coli BL21(DE3) harboring pET30a-Hfq-His6 was

407

cultured in LB medium at 37 °C. When OD600 reached 0.2, 1 mM IPTG was added to the

408

culture and incubated additional 2 h. Cells were harvested by centrifugation and used for

409

Hfq purification. Hfq was purified by the previously described procedure.56 Using

410

purified Hfq, Hfq-sRNA ELISA assay was performed by the previously described

411

protein-RNA ELISA method.57 The measured intensities were modified by subtracting

412

the fluorescence obtained from the sample without synthetic sRNA and normalized to the

413

fluorescence obtained from wild type SgrS-S. Streptavidin coated plates were purchased

414

from Thermo Scientific (15119) and FITC-conjugated anti-6xHis antibody was

415

purchased from Abcam (ab1206).

416 417

ASSOCIATED CONTENT

418 419

Supporting Information

18



420

The Supporting Information is available free of charge on the ACS Publications website

421

at DOI:XXX.

422

Comparison of anti-DsRed2 sRNAs derived from various scaffolds (Figure S1), gene

423

repression activities of sRNAs harboring combinations of the improved modules

424

other than the combinations tested in Figure 2E-F (Figure S2), fed-batch cultures of

425

cadaverine producing strains with expression of improved sRNAs (Figure S3), list of

426

oligonucleotide sequences used in the construction of synthetic sRNA library for

427

enhanced cadaverine production (Table S1), list of target genes of synthetic sRNA

428

library used in metabolic engineering of cadaverine production (Table S2), strains

429

and plasmids used in this study (Table S3), and oligonucleotides used in this study

430

(Table S4). (PDF and Excel files)

431 432

AUTHOR INFORMATION

433

Corresponding Author

434

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

435 436

Author Contributions

437

S.Y.L. and M.N. designed the research. M.N. and D.Y. performed experiments. S.M.Y.,

438

N.M. and D.Y. analyzed data and generated figures for the manuscript. M.N., S.M.Y.,

439

D.Y. and S.Y.L. wrote the manuscript. All authors read and approved the final manuscript.

440

∥ M.N.

and S.M.Y. contributed equally.

441 442

Notes

19


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443

The authors declare that they have conflict of interest as the synthetic sRNA

444

technology described here is patent filed including, but not limited to KR 10-1575587,

445

US 9388417, EP 13735942.8, CN 201380012767.X, KR 10-1690780, KR 10-1750855,

446

US 15317939, CN 201480081132.X for potential commercialization. Also, the authors

447

have conflict of interest as the expanded synthetic sRNA expression platform is of

448

commercial interest and is patent filed including, but not limited to KR 10-2018-0073970.

449 450

ACKNOWLEDGMENTS

451

This work was supported by grants from the Intelligent Synthetic Biology Center through

452

the Global Frontier Project (2011-0031963) of Ministry of Science and ICT through the

453

National Research Foundation of Korea.

454 455 456

REFERENCES (1)

457

van Ooyen, J., Noack, S., Bott, M., Reth, A., and Eggeling, L. (2012) Improved

-lysine production with Corynebacterium glutamicum and systemic insight into citrate

458

L

459

synthase flux and activity. Biotechnol. Bioeng. 109, 2070–2081.

460

(2)

Brewster, R. C., Jones, D. L., and Phillips, R. (2012) Tuning promoter strength

461

through RNA polymerase binding site design in Escherichia coli. PLoS Comp. Biol. 8,

462

E1002811.

463

(3)

Kinney, J. B., Murugan, A., Callan, C. G., and Cox, E. C. (2010) Using deep

464

sequencing to characterize the biophysical mechanism of a transcriptional regulatory

465

sequence. Proc. Natl. Acad. Sci. USA 107, 9158–9163.

466

(4)

Brewster, R. C., Weinert, F. M., Garcia,H. G., Song, D., Rydenfelt, M., and

467

Phillips, R. (2014) The transcription factor titration effect dictates level of gene

468

expression. Cell 156, 1312–1323. 20



469

(5)

Page 26 of 33

Alper, H., Fischer, C., Nevoigt, E., and Stephanopoulos, G. (2005) Tuning

470

genetic control through promoter engineering. Proc. Natl. Acad. Sci. USA 102, 12678–

471

12683.

472

(6)

Zong, Y., Zhang, H. M., Lyu, C., Ji, X., Hou, J., Guo, X., Ouyang, Q., and Lou,

473

C. (2017) Insulated transcriptional elements enable precise design of genetic circuits.

474

Nat. Commun. 8, 52.

475

(7)

Cleto,

S., Jensen,

J.

V., Wendisch, V.

F., and

Lu

T.

K.

476

(2016) Corynebacterium glutamicum metabolic engineering with CRISPR interference

477

(CRISPRi). ACS Synth. Biol. 5 375–385.

478

(8)

Ji, W., Lee, D., Wong, E., Dadlani, P., Dinh, D., Huang, V., Kearns, K., Teng,

479

S., Chen, S., Haliburton, J., Heimberg, G., Heineike, B., Ramasubramanian, A., Steve

480

ns, T., Helmke, K. J., Zepeda, V., Qi, L. S., and Lim. W. A. (2014) Specific gene

481

repression by CRISPRi system transferred through bacterial conjugation. ACS Synth.

482

Biol. 3 929–931.

483

(9)

Lv, L., Ren, Y. L., Chen, J. C., Wu, Q., and Chen, G. Q. (2015) Application of

484

CRISPRi for prokaryotic metabolic engineering involving multiple genes, a case study:

485

controllable P(3HB-co-4HB) biosynthesis. Metab. Eng. 29, 160–168.

486

(10) Bruder, M. R., Pyne, M. E., Moo-Young, M., Chung, D. A., and Chou C. P.

487

(2016) Extending CRISPR-Cas9 technology from genome editing to transcriptional

488

engineering in Clostridium. Appl. Environ. Microbiol.

489

(11) Ferreira, J. P., Overton, K. W., and Wang, C. L. (2013) Tuning gene expression

490

with synthetic upstream open reading frames. Proc. Natl. Acad. Sci. USA 110, 11284–

491

11289.

492

(12) Salis, H. M., Mirsky, E. A., and Voigt, C. A. (2009) Automated design of

493

synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27,

494

946–950.

495

(13) Ringquist, S., Shinedling, S., Barrick, D., Green, L., Binkley, J., Stormo, G.

496

D., and Gold, L. (1992) Translation initiation in Escherichia coli: sequences within the

497

ribosome-binding site. Mol. Microbiol. 6, 1219–1229.

498

(14) Cameron, D. E., and Collins, J. J. (2014) Tunable protein degradation in 21


Page 27 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

499 500 501 502 503 504 505


bacteria. Nat. Biotechnol. 32, 1276–1281. (15) Cameron, D. E., Bashor, C. J., and Collins, J. J. (2014) A brief history of synthetic biology. Nat. Rev. Microbiol, 12, 381-390. (16)

regulation at the level of transcript stability. RNA Biol. 7, 140-144. (17)

Wassarman, K. M. (2002) Small RNAs in bacteria: diverse regulators of gene

expression in response to environmental changes. Cell 109, 141–144.

506

(18)

507

615–628.

508

(19)

509

Caron, M. P., Lafontaine, D. A., and Massé, E. (2010) Small RNA-mediated

Waters, L. S. and Storz, G. (2009) Regulatory RNAs in bacteria. Cell 136,

Liu, J. M. and Camilli, A. (2010) A broadening world of bacterial small RNAs.

Curr. Opin. Microbiol. 13, 18–23.

510

(20) Kawamoto, H., Koide, Y., Morita, T., and Aiba, H. (2006) Base‐pairing

511

requirement for RNA silencing by a bacterial small RNA and acceleration of duplex

512

formation by Hfq. Mol. Microbiol. 61, 1013–1022.

513 514 515 516 517 518

(21) Vogel, J. and Luisi, B. F. (2011) Hfq and its constellation of RNA. Nat. Rev. Microbiol. 9, 578–589. (22) Aiba, H. (2007) Mechanism of RNA silencing by Hfq-binding small RNAs. Curr. Opin. Microbiol. 10, 134–139. (23) Urban, J. H. and Vogel, J. (2007) Translational control and target recognition by Escherichia coli small RNAs in vivo. Nucleic Acids Res. 35, 1018–1037.

519

(24) Balbontin, R., Fiorini, F., Figueroa-Bossi, N., Casadesus, J., and Bossi, L.

520

(2010) Recognition of heptameric seed sequence underlies multi-target regulation by

521

RybB small RNA in Salmonella enterica. Mol. Microbiol. 78, 380–394.

522

(25) Geissmann, T. A. and Touati, D. (2004) Hfq, a new chaperoning role: binding

523

to messenger RNA determines access for small RNA regulator. EMBO J. 23, 396–405.

524

(26) Brescia, C. C., Mikulecky, P. J., Feig, A. L., and Sledjeski, D. D. (2003)

525

Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA

526

secondary structure. RNA 9, 33–43. 22



527

(27) Møller, T., Franch, T., Højrup, P., Keene, D. R., Bächinger, H. P., Brennan, R.

528

G., and Valentin-Hansen, P. (2002) Hfq: a bacterial Sm-like protein that mediates RNA-

529

RNA interaction. Mol. Cell 9, 23–30.

530

(28) Zhang, A., Wassarman, K. M., Ortega, J., Steven, A. C., and Storz, G. (2002)

531

The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs. Mol.

532

Cell 9, 11–22.

533

(29) Ishikawa, H., Otaka, H., Maki, K., Morita, T., and Aiba, H. (2012) The

534

functional Hfq-binding module of bacterial sRNAs consists of a double or single hairpin

535

preceded by a U-rich sequence and followed by a 3′ poly (U) tail. RNA 18, 1062–1074.

536

(30) Otaka, H., Ishikawa, H., Morita, T., and Aiba, H. (2011) PolyU tail of rho-

537

independent terminator of bacterial small RNAs is essential for Hfq action. Proc. Natl.

538

Acad. Sci. USA 108, 13059–13064.

539

(31) Na, D., Yoo, S. M., Chung, H., Park, H., Park, J. H., and Lee, S.Y. (2013)

540

Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat.

541

biotechnol. 31, 170–174.

542

(32) Yoo, S. M., Na, D., and Lee, S. Y. (2013) Design and use of synthetic

543

regulatory small RNAs to control gene expression in Escherichia coli. Nat. Protoc. 8,

544

1694–1707.

545

(33) Chen, Y., Lou, S., Fan, L., Zhang, X., and Tan, T. (2015) Control of ATP

546

concentration in Escherichia coli using synthetic small regulatory RNAs for enhanced

547

S-adenosylmethionine production. FEMS Microbiol. Lett. 362, fnv115.

548

(34) Feng, J., Gu, Y., Quan, Y., Cao, M., Gao, W., Zhang, W., Wang, S., Yang, C.,

549

and Song, C. (2015) Improved poly-γ-glutamic acid production in Bacillus

550

amyloliquefaciens by modular pathway engineering. Metab. Eng. 32, 106–115.

551

(35) Hsia, J., Holtz, W. J., Maharbiz, M. M., Arcak, M., and Keasling, J. D. (2016)

552

Modular synthetic inverters from zinc finger proteins and small RNAs. PLoS ONE 11,

553

e0149483.

554

(36) Kim, B., Park, H., Na, D., and Lee, S. Y. (2014) Metabolic engineering of

555

Escherichia coli for the production of phenol from glucose. Biotechnol. J. 9, 621–629. 23


Page 28 of 33

Page 29 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


556

(37) Libis, V. K., Bernheim, A. G., Basier, C., Jaramillo-Riveri, S., Deyell, M.,

557

Aghoghogbe, I., Atanaskovic, I., Bencherif, A. C., Benony, M., Koutsoubelis, N.,

558

Löchner, A. C., Marinkovic, Z. S., Zahra, S., Zegman, Y., Lindner, A. B., and

559

Wintermute, E. H. (2014) Silencing of antibiotic resistance in E. coli with engineered

560

phage bearing small regulatory RNAs. ACS Synth. Biol. 3, 1003–1006.

561

(38) Liu, Y., Zhu, Y., Li, J., Shin, H. D., Chen, R. R., Du, G., Liu, L., and Chen, J.

562

(2014) Modular pathway engineering of Bacillus subtilis for improved N-

563

acetylglucosamine production. Metab. Eng. 23, 42–52.

564 565

(39) Beisel, C. L., Updegrove, T. B., Janson, B. J., and Storz, G. (2012) Multiple factors dictate target selection by Hfq-binding small RNAs. EMBO J. 31, 1961–1974.

566

(40) Sauer, E., Schmidt, S., and Weichenrieder, O. (2012) Small RNA binding to

567

the lateral surface of Hfq hexamers and structural rearrangements upon mRNA target

568

recognition. Proc. Natl. Acad. Sci. USA 109, 9396–9401.

569

(41) Noh, M., Yoo, S. M., Kim, W. J., and Lee, S. Y. (2017) Fine-tuning gene

570

expression knockdown by modulating synthetic small RNA expression in Escherichia

571

coli. Cell Syst. 5, 418–426.

572

(42) Yang, D., Kim, W. J., Yoo, S. M., Choi, J. H., Ha, S. H., Lee, M. H., and Lee,

573

S. Y. (2018) Repurposing type III polyketide synthase as a malonyl-CoA biosensor for

574

metabolic engineering in bacteria. Proc. Natl. Acad. Sci. USA 115, 9835–9844.

575

(43) Yang. D., Yoo, S. M., Gu, C., Ryu, J., Lee, J. E., and Lee, S. Y. (2019)

576

Expanded synthetic small regulatory RNA expression platforms for rapid and multiplex

577

gene expression knockdown. Metab. Eng. 54, 180–190.

578

(44) Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin,

579

A. P., and Lim, W. A. (2013) Repurposing CRISPR as an RNA-guided platform for

580

sequence-specific control of gene expression. Cell 152, 1173–1183.

581

(45) Cho, S., Choe, D., Lee, E., Kim, S. C., Palsson, B., and Cho, B. K. (2018) High-

582

level dCas9 expression induces abnormal cell morphology in Escherichia coli. ACS

583

Synth. Biol. 7, 1085–1094.

24



584 585

(46) Ghodasara, A. and Voigt A. C. (2017) Balancing gene expression without library construction via a reusable sRNA pool. Nucleic Acids Res. 45, 8116-8127.

586

(47) Maki, K., Morita, T., Otaka, H., and Aiba, H. (2010) A minimal base‐pairing

587

region of a bacterial small RNA SgrS required for translational repression of ptsG

588

mRNA. Mol. Microbiol. 76, 782–792.

589 590 591 592 593 594

(48) Soper, T. J. and Woodson, S. A. (2008) The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA. RNA 14, 1907–1917. (49) Olejniczak, M. (2011) Despite similar binding to the Hfq protein regulatory RNAs widely differ in their competition performance. Biochemistry 50, 4427-4440. (50) Hussein, R. and Lim, H. N. (2011) Disruption of small RNA signaling caused by competition for Hfq. Proc. Natl Acad. Sci. USA 108, 1110-1115.

595

(51) Qian, Z. G., Xia, X. X., and Lee, S. Y. (2011) Metabolic engineering of

596

Escherichia coli for the production of cadaverine: a five carbon diamine. Biotechnol.

597

Bioeng. 108, 93–103.

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(52) Siuti, P., Yazbek, J., and Lu, T. K. (2013) Synthetic circuits integrating logic and memory in living cells. Nat. Biotechnol. 31, 448–452. (53) Bonnet, J., Yin, P., Ortiz, M. E., Subsoontorn, P., and Endy, D. (2013) Amplifying genetic logic gates. Science 340, 599–603. (54) Slusarczyk, A. L., Lin, A., and Weiss, R. (2012) Foundations for the design and implementation of synthetic genetic circuits. Nat. Rev. Genet. 13, 406–420.

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(55) Westbrook, A. M. and Lucks, J. B. (2017) Achieving large dynamic range

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control of gene expression with a compact RNA transcription-translation regulator.

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Nucleic Acids Res. 45, 5614–5624.

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(56) Maki, K., Uno, K., Morita, T., and Aiba, H. (2008) RNA, but not protein

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partners, its directly responsible for translational silencing by a bacterial Hfq-binding

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small RNA. Proc. Natl. Acad. Sci. USA 105, 10332–10337.

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(57) Gajjar, M., Candeias, M. M., Malbert-Colas, L., Mazars, A., Fujita, J.,

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Olivares-Illana, V., and Fåhraeus, R. (2012) The p53 mRNA-Mdm2 interaction controls 25


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Mdm2 nuclear trafficking and is required for p53 activation following DNA damage.

613

Cancer Cell 21, 25–35.

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26



616

Figure Legends

617 618

Figure 1. Schematic of the synthetic sRNA structure and the engineering of its scaffold.

619

(A) Synthetic sRNAs compete with ribosomes for ribosome-binding sites (RBSs). Three

620

functional elements of Hfq-dependent sRNAs are annotated: an mRNA-binding region,

621

an Hfq-binding region, and a rho-independent transcription terminator. (B) The Hfq-

622

binding region was divided into three elements for mutational analysis: an A/U-rich

623

sequence, a stem, and a loop. (C) Schematic for the analysis of the activities of synthetic

624

sRNA mutants. Plasmids expressing the sRNAs were constructed through inverse PCR,

625

and plasmids were transformed into E. coli cells harboring a DsRed2 expression plasmid.

626

The repression of DsRed2 was estimated by measuring fluorescence intensities.

627 628

Figure 2. Gene repression activities of sRNAs with mutations in the Hfq-binding region.

629

(A) Nucleotide sequences of the SgrS-S variants. Red wedges indicate the position of a

630

nucleotide deletion. Texts with asterisks indicate inserted nucleotides. Underlined and

631

italicized texts indicate substituted nucleotides. All possible single mutants of the boxed

632

sequences (UAUU and AAAA) and combinations of single mutants were constructed and

633

studied. (B) Single mutations on the A/U-rich sequence (UAUU) were examined. The

634

mutations that increased or decreased gene repression were combined to observe the

635

additive effects of mutations on the A/U-rich sequence (CUUU and GCAC). Mutated

636

sequences are marked in red. (C) Stem length was increased or decreased and changes in

637

the repression activity were observed. Stem length was changed by inserting or deleting

638

G-C base pairs. (D) Gene repression activities of sRNA scaffolds with a single mutation

639

in the A-rich loop. The mutations that increased or decreased gene repression were

640

combined to observe the additive effects of mutations on the loop (GUCU and CGGG).

641

Mutated sequences are marked in red. (E) Gene repression activities of sRNAs harboring

642

combinations of the improved modules: CUUU replacing the A/U-rich sequence, a 6-nt

643

long stem, and a GUCU loop. To observe changes in gene repression in detail, the

644

promoter used for sRNA expression was changed from J23105 to the weaker promoter

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J23114. (F) Gene repression activities of sRNAs harboring combinations of the mutant

646

modules: GCAC replacing the A/U-rich sequence, an 8-nt long stem, and a CGGG loop. 27


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647

The promoter used for sRNA expression was J23105. Insets in (B)–(F) show the dynamic

648

range of repression capabilities of the original (open circles) and mutant sRNAs (closed

649

circles). The gray dashed lines in all graphs indicate the repression level of the original

650

sRNA. Data were obtained from three measurements. Fold repression was calculated by

651

dividing the fluorescent intensities of cells without sRNAs by the fluorescent intensities

652

from cells with original or mutant sRNAs (see Figure 1). The values displayed represent

653

average values ± s.d. (n = 3).

654 655

Figure 3. Affinities between the purified Hfq-His6 and synthetic sRNA variants. (A)

656

Schematic for the analysis of the affinities between Hfq-His6 and the synthetic sRNA

657

variants. (B) Quantitation of synthetic sRNAs bound to Hfq-His6 using FITC-conjugated

658

antibodies. Data were obtained from three measurements. The values displayed represent

659

average values ± s.d. (n = 3).

660 661

Figure 4. Identification of repression target genes using synthetic sRNAs derived from

662

the engineered sRNA scaffold for enhanced cadaverine production. Schematic diagram

663

of the cadaverine biosynthetic pathway including glycolysis, the lysine biosynthesis

664

pathway, and a single conversion step from lysine to cadaverine by lysine decarboxylase.

665

Changes in cadaverine production relative to the starting strain (XQ56 harboring

666

p15CadA) are represented by colored circles. The genes and the values of the relative

667

changes in cadaverine titers are listed in Table S2. Knocked down genes that increased

668

cadaverine production are shown in blue. Data were obtained from three measurements.

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For Table of Contents Only

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Broad-Spectrum Gene Repression Using Scaffold Engineering of

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