Metabolic Engineering 19 (2013) 98–106

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Metabolic Engineering

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Design, construction and characterisation of a synthetic promoterlibrary for fine-tuned gene expression in actinomycetes

Theresa Siegl a, Bogdan Tokovenko b, Maksym Myronovskyi b, Andriy Luzhetskyy b,n

a Albert-Ludwigs-University of Freiburg, Pharmaceutical Biology and Biotechnology, Stefan-Meier-st. 19, Freiburg 79104, Germanyb Helmholtz-Institute for Pharmaceutical Research Saarland, Campus, Building C2 3, 66123 Saarbrücken, Germany

a r t i c l e i n f o

Article history:Received 26 December 2012Received in revised form2 June 2013Accepted 11 July 2013Available online 20 July 2013

Keywords:Synthetic promoterActinomycetesGusARppATranscriptomic analysisRNA-SeqErme promoter

76/$ - see front matter & 2013 Elsevier Inc. Alx.doi.org/10.1016/j.ymben.2013.07.006

esponding author.ail address: Andriy.Luzhetskyy@helmholtz-hz

a b s t r a c t

We developed a synthetic promoter library for actinomycetes based on the �10 and �35 consensussequences of the constitutive and widely used ermEp1 promoter. The sequences located upstream, inbetween and downstream of these consensus sequences were randomised using degenerate primers andcloned into an integrative plasmid upstream of the gusA reporter gene. Using this system, we createdpromoters with strengths ranging from 2% to 319% compared with ermEp1. The strongest syntheticpromoter was used in a proof-of-principle approach to achieve the overexpression of a natural type IIIpolyketide synthase. We observed high correlation between the number of gusA reporter gene RNA-Seqreads and the GusA reporter protein activity, indicating that GusA is indeed a transcription-level reportersystem.

& 2013 Elsevier Inc. All rights reserved.

1. Introduction

Synthetic biology approaches to the design and construction ofbiological systems offer enormous potential to industrial biotech-nology (Keasling, 2012). The global value of the synthetic biologymarket is expected to reach more than $10 billion by 2016.Achievement of the synthetic biology concept of engineeredbiological systems depends on the availability of standardisedbiological regulation mechanisms (e.g., synthetic promoters, RBS,terminators, ribozymes, etc.) (Yadav et al., 2012). In addition,rationally designed and well-characterised controlling elementsare invaluable tools for various metabolic engineering approachesin which the fine-tuning of gene expression is necessary (Boyleand Silver, 2012; Lynch and Gill, 2012). Promoters are the keyregulators of gene expression (Hammer et al., 2006). Therefore, aquantitatively characterised library of promoters is required toexpress a target gene. Native promoters, both constitutive andinducible, have long been used in gene expression studies andstrain engineering. Although multiple native promoters fromvarious organisms are available, they do not provide a wide andcontinuous range of transcription levels. Moreover, naturallyoccurring promoters typically interfere with the host regulatorynetwork, complicating the prediction of their transcriptional

l rights reserved.

i.de (A. Luzhetskyy).

activities. In contrast, synthetic promoters are not restricted tothe available regulatory machinery of the host strain and canprovide the widest possible range of promoter strengths. Syntheticpromoter libraries have been constructed for Escherichia coli(Braatsch et al., 2008), Saccharomyces cerevisiae (Alper et al.,2005), Pichia pastoris (Hartner et al., 2008) and Lactobacillusplantarum (Rud et al., 2006).

Actinomycetes are Gram-positive bacteria that produce manypharmaceutically relevant secondary metabolites, including herbi-cides, antibiotics, immunosuppressants, antifungal, and anticancerdrugs. Secondary metabolite production typically occurs during theearly stationary phase and is influenced by a variety of physiologicaland environmental factors acting through a complex network ofpathway-specific and pleiotropic regulators (Kieser et al., 2000).For the successful manipulation or overexpression of secondarymetabolites, it is therefore indispensable to consider the appro-priate balance between all genes involved. Hence, transcriptionalfine-tuning is essential for optimal gene expression. However, onlya few native promoters are commonly used in actinomycetes,including the constitutive ermE promoter (Bibb et al., 1985) andthe inducible tipA (Murakami et al., 1989) and tcp (Rodríguez-García et al., 2005) promoters. Several synthetic promoters havebeen constructed, but due to a lack of reliable reporter systems,these promoters have not been quantitatively characterised.Instead, promoters are only generally described as weak, mediumor strong (Seghezzi et al., 2011). Thus, there are no libraries of well-characterised synthetic promoters for actinomycetes. The aim of

T. Siegl et al. / Metabolic Engineering 19 (2013) 98–106 99

this study was therefore, to generate a library of comprehensivelycharacterised synthetic promoters, using several reporter genes(gusA, gfp, aphII, rppA), RNA sequencing (RNA-Seq) and comparingthem to described promoters like tcp and ermE. The ermE promoterregion contains two different promoters, ermEp1 and ermEp2. Itwas reported that a TGG deletion in the �35 region of the ermEp1promoter resulted in a stronger variant called ermE* (ermEp2 andermEp1 ΔTGG) (Bibb et al., 1994).

We used a popular approach based on the randomisation ofnucleotides surrounding the �10 and �35 consensus sequences(Hammer et al., 2006; Jensen and Hammer, 1998a, 1998b; Solemand Jensen, 2002) to construct a synthetic promoter library basedon the consensus sequences of the ermEp1 promoter. Thesequences upstream, in between and downstream of the �10and �35 consensus regions were randomised using degenerateprimers. The library was cloned in a derivative of pSET152, whichincludes the popular phiC31-based phage integration system tominimise deviations reflecting plasmid copy numbers.

We also used the highly sensitive beta-D-glucuronidase (GUS)reporter protein, which facilitated a visual assessment of thepromoter strength and spectrophotometrical quantification. Abroad application of the library was assessed through the compar-ison of four synthetic promoters of different strengths in threeadditional actinomycetes strains (Streptomyces albus J1074, Sac-charothrix espanaensis DSM 44229 and Salinispora tropica CNB-440). Extensive RNA-Seq analysis was used to examine thetranscriptional levels and transcriptional start points of singlereporter gene mRNAs transcribed through different syntheticpromoters and the mRNAs of two genes in a single operon.

2. Materials and methods

2.1. Strains, plasmids and media

The medium formulations and manipulations of actinomycetesand Escherichia coli were performed as previously described(Kieser et al., 2000). Mannitol soya flour medium was used forconjugation, and tryptic soy broth was used for liquid inoculation.A1 medium (Jensen and Mafnas, 2006) was used for the liquidgrowth and conjugation of Salinispora tropica CNB-440. Themedium was supplemented when appropriate with the respectiveantibiotics to final concentrations of 50 mg/ml apramycin, 50 mg/mlcarbenicillin, 150 mg/ml fosfomycin, 50 mg/ml hygromycin, and30 mg/ml kanamycin. The plasmids used in this study are sum-marised in Table 1. E. coli XL1-Blue cells (Stratagene) were used for

Table 1Plasmids used in this work.

Name Description

pGUS pSET152 derivative with aadA flanked by T4 terminatorspGUS-SPL pGUS derivative with synthetic promoter in front of gusApGUS-ermEp1 pGUS derivative with ermEp1 promoter in front of gusApGUS-ermEp1* pGUS derivative with ermEp1* promoter in front of gusApGUS-ermE pGUS derivative with ermE promoter in front of gusApGUS-ermE* pGUS derivative with ermE* promoter in front of gusApGUS-tcp pGUS derivative with tcp promoter in front of gusApGUS-SPL-aaphII pGUS derivative with synthetic promoter (72, 82, 57 or 2

and gusA in a single operonpGUS-SPL-agfp pGUS derivative with synthetic promoter (72, 82, 57 or 2

gusA in a single operonpGUS-SPL-arppA pGUS derivative with synthetic promoter 21 or ermEp1*

single operon

a number/name of (synthetic) promoter; aadA, spectinomycin resistance gene; aphII,encoding beta-glucuronidase; rppA, gene encoding type III polyketide synthase; tcp, tet

cloning, ET12567 x pUZ8002 was used for conjugation, andET12567 x pUB307 was used for tri-parental conjugation.

2.2. Generation and cloning of the synthetic promoter library

The synthetic promoter library was constructed using degen-erate primers, in which n can be any of the four nucleotides (a, t, c,g). Using the primers Spl 1 (5′-tataggtaccnnnnnnatcctannnn-nnnnnnnnnnnnnnnagccnnnnnnccgtatttgcagtac-3′) and Spl 2 (5′-ggcgctctagagaataggaacttcg-3′), the hygromycin resistance genewas amplified by PCR, digested with KpnI and XbaI (underlinedin the primer sequences, respectively) and cloned into the KpnI/XbaI sites of the digested pGUS vector, yielding pGUS-SPL (Fig. S1and S2). pGUS-SPL was then introduced into Streptomyces lividansTK24 through tri-parental conjugation. For this type of conjuga-tion, the E. coli strain ET12567 x pUB307 was used as a helperstrain to transfer the plasmids from E. coli XL-Blue cells into therecipient strain Streptomyces lividans TK24.

To directly compare the promoter activities in our syntheticpromoter library, several sequences for native and syntheticpromoters were cloned into pGUS using the approach describedabove. Spl 2 was consistently used as a reverse primer. Theforward primer included the promoter sequence (bold) for eachof the different promoters: ermEp1 (Spl ermE 5′-tataggtacccgctg-gatcctaccaaccggcacgattgtccagcccacaacccgtatttgcagtac-3′),ermEp1*¼ermEp1ΔTGG (Spl ermestar 5′-tataggtacccgctggatcc-taccaaccggcacgattgtgcccacaacccgtatttgcagtac – 3′), tetracyclineinducible promoter (Spl-tcp 5′-tataggtaccatctctatcactgatagggat-cctaccactatcaatgatagagtagccaacagccgtatttgcagtac-3′). The sequ-ence between the promoter and the start codons of the reportergenes always included the RBS sequence ggagg (Table S2).

Due to the length of the sequences, a different strategy wasapplied to amplify the entire ermE (ermEp1 and ermEp2) and ermE*(ermEp1* and ermEp2) promoter sequences. PCR amplification of thehygromycin resistance gene was performed using the primers Spl2 and bibbe2estar2 (5′-cacgtgtggaccgcgtcggtcagatcctccccgcacctct-cgccagccgtcaagatcgaccgcggctagcccgtatttgcagtaccagcg-3′), which co-ntains the ermEp2 promoter (bold) and part of the 3′ end of thehygromycin resistance gene. The resulting PCR 1276-bp fragmentwas subsequently used as a template for a second PCR reaction toamplify the entire ermE promoter sequence using the primers Spl2and bibbermE (5′-tataggtacccgctggatcctaccaaccggcacgattgtccagcc-cacaacagcatcgcggtgccacgtgtggaccgcgtcggtcagatc-3′), with the erm-Ep1 promoter shown in bold. For complete amplification of theermE* promoter, the second PCR reaction was performed with theprimers Spl 2 and bibbe2estar3 (5′-tataggtacccgctggatcctaccaaccgg-cacgattgtgcccacaacagcatcgcggtgccacgtgtggaccgcgtcggtcagatc-3′),

Reference

upstream of promoterless gusA gene (Myronovskyi et al., 2011)This workThis workThis workThis workThis workThis work

1) or ermEp1* promoter in front of aphII This work

1) or ermEp1* promoter in front of gfp and This work

promoter in front of rppA and gusA in a This work

neomycin resistance gene; gfp, gene encoding green fluorescent protein; gusA, generacycline inducible promoter.

T. Siegl et al. / Metabolic Engineering 19 (2013) 98–106100

with ermEp1* shown in bold. The resulting PCR fragments weredigested with XbaI and KpnI and ligated into the XbaI/KpnI sites ofthe digested pGUS vector to generate pGUS-ermE and pGUS-ermE*,respectively.

2.3. Cloning of further reporter genes gfp and aphII

To verify the promoter strengths using GUS activity, additionalreporter genes were cloned between the promoter region and thegusA reporter gene. The gene encoding GFP was amplified using theprimers 43 (5′-tataactagttctagaggtaccagcaacggaggtacggacatgtggagc-cacccgcagtt-3′) and 26 (5′-taatatatatctagattacttgtacagctcgtccatgc-3′),digested with KpnI and XbaI (underlined in the primer sequences,respectively) and cloned into the KpnI/SpeI sites of pGUS-SPL-72,pGUS-SPL-82, pGUS-ermEp1*, pGUS-SPL-57 and pGUS-SPL-21 toyield pGUS-SPL-72gfp, pGUS-SPL-82gfp, pGUS-ermEp1*gfp, pGUS-SPL-57gfp and pGUS-SPL-21gfp, respectively.

The gene encoding AphII was amplified using the primersneoF2 (5′-tataggtaccgagcaacggaggtacggacatgattgaacaagatggattg-ca-3′) and neoR (5′-gcgctctagatcagaagaactcgtcaagaagg-3′), dige-sted with KpnI and XbaI (underlined in the primer sequences,respectively) and cloned into the KpnI/SpeI sites of pGUS-SPL-72,pGUS-SPL-82, pGUS-ermEp1*, pGUS-SPL-57 and pGUS-SPL-21 toyield pGUS-SPL-72aphII, pGUS-SPL-82aphII, pGUS-ermEp1*aphII,pGUS-SPL-57aphII and pGUS-SPL-21aphII, respectively.

2.4. Cloning of rppA for the overexpression with a strong syntheticpromoter

The gene encoding RppA was amplified from the genome ofSaccharopolyspora erythraea using the primers rppA-f (5′-atattcta-gagtcatcggttgcctcccggggc-3′) and rppA-r (5′-cgcgtctagacaatggagg-catcggtggcagttctatg-3′), digested with XbaI (underlined in theprimer sequences) and cloned into the SpeI-digested pGUS-ermEp1* and pGUS-SPL-21 plasmids to yield pGUS-ermEp1*rppAand pGUS-SPL-21rppA, respectively.

2.5. Assessment of promoter strength (GUS assay)

The promoter strength was indirectly assessed according to theenzymatic activity of the reporter protein GUS. 1 ml of 24-hour seedcultures of the Streptomyces lividans TK24 mutant strains wasinoculated into 50 ml of TSB medium and grown in shaking flasksfor 40 h at 28 1C, which corresponds to the stationary phase. Theculture (1 ml) was pelleted, resuspended in 900 ml of GUS buffer 2(50 mM phosphate buffer [pH 7.0], 5 mM dithiothreitol [DTT], 0.1%Triton X-100, 1 mg/ml lysozyme) and incubated at 37 1C for 20 min.The lysates were diluted with 900 ml of GUS buffer 1 (50 mMphosphate buffer [pH 7.0], 5 mM DTT, 0.1% Triton X-100) andcentrifuged at 14000 rpm at 4 1C for 10 min. A 500-ml sample ofthe supernatant was mixed with 500 ml of GUS buffer 3 (50 mMphosphate buffer [pH 7.0], 5 mM DTT, 0.1% Triton X-100 supple-mented with 2 mM p-nitrophenyl-beta-D-glucuronide), and theabsorption was spectrophotometrically measured at 415 nm for30 min (Ultrospec 2100pro UV/Visible Spectrophotometer, Amer-sham Biosciences). As a reference, 500 ml of GUS buffer 1 was mixedwith 500 ml of the sample. All samples and buffers were stored onice until measurement. The slope of the resulting absorption curvewas used to calculate the enzymatic activity in units per gram dryweight. The dry weight was determined using a vacuum pump todraw 5 ml of the culture through a filter (Solvent Filter, Supor PES,Waters, USA), washing once with 5 ml of distilled water andweighing the dried filter. The following equation was used tocalculate the enzymatic activity: U/g¼2V � A415=min=14 � DW,where V equals the total end volume of the sample (1.8 ml) and

DW equals the dry weight in grams of the original sample size(1 ml).

2.6. GUS assay for the four different actinomycetes strains

For the Streptomyces lividans TK24 and Streptomyces albus J1074mutants containing the plasmids pGUS, pGUS-SPL-72, pGUS-SPL-82, pGUS-ermEp1*, pGUS-SPL-57 and pGUS-SPL-21, 107 sporeswere inoculated into 100 ml of TSB medium and grown for 42 hat 28 1C in shaking flasks. A 40-ml sample was pelleted into aFalcon tube and dried at 60 1C for 48 h. Another 40-ml sample wasused for the GUS assay. The pellet was washed once with water,and the lysis was performed in 20 ml of GUS buffer 2. The samplewas subsequently diluted to 45 ml with GUS buffer 1, and a 500-mlsample of the supernatant was measured as described above.

For the Saccharothrix espanaensis DSM 44229 mutants, 300 mlof sucrose stock was inoculated into 100 ml of TSB medium andgrown for 42 h. For the Salinispora tropica CNB-440 mutants, a1-ml sample of the seed culture was used to inoculate 100 ml ofA1 medium and grown for 42 h. For both the Saccharothrixespanaensis DSM 44229 and Salinispora tropica CNB-440 mutants,the lysozyme treatment in GUS buffer 2 was not sufficient, and aFrench press was used to obtain complete lysis. The remainingsteps of the GUS assay were performed as described above.

Because the volumes were altered, the following equationwas usedto calculate the enzymatic activity: U/g¼90 � A415=min=14 � DW

2.7. Analysis of the gfp reporter gene

Streptomyces lividans TK24 mutants were grown and treated asdescribed in the previous section “GUS assay for the four differentactinomycetes strains”. A 150-ml sample of the supernatant wastransferred in black 96-well plates, and the GFP fluorescence wasmeasured at 520 nm after excitation at 485 nm using a platereader (FLUOstar OPTIMA, BMG LABTECH).

2.8. RppA reporter protein analysis

The mutant strains were inoculated and grown as described inthe section “Assessment of promoter strength (GUS assay)”. A 1.5-ml sample of the culture was pelleted, and a 1-ml sample wasobtained to measure the absorption of the supernatant at 488 nm(Kuščer et al., 2007).

2.9. Transcriptomic data analysis (RNA-Seq)

Seed cultures of the Streptomyces albus J1074 mutants contain-ing pGUS, pGUS-SPL-72, pGUS-SPL-82, pGUS-ermEp1*, pGUS-SPL-57, pGUS-SPL-57aphII, pGUS-SPL-57gfp and pGUS-SPL-21 weregrown for 24 h and 5 ml was used to inoculate 45 ml of mainculture. After 12 h of cultivation at 28 1C in shaking flasks, 50 ml ofculture was pelleted and shipped on dry ice to Vertis Biotechno-logie AG (Freising, Germany) for RNA isolation and whole tran-scriptome shotgun sequencing (RNA-Seq). Briefly, total RNA wasisolated, followed by rRNA removal and mRNA fragmentation. ThemRNA fragments were reverse-transcribed into cDNA and sub-jected to Illumina sequencing. The preprocessing of RNA-Seqreads was performed in several steps. The leftover sequencingadapters were screened and trimmed using scythe (a naïve Bayesianclassifier of contaminants, considering base quality information:https://github.com/vsbuffalo/scythe). Using Prinseq-lite (Schmiederand Edwards, 2011), we performed a quality trimming of reads witha Phred score threshold of 20 and removed trailing poly-A stretcheslonger than five nucleotides. Leftover ribosomal RNA reads werefiltered out prior to the read alignment.

T. Siegl et al. / Metabolic Engineering 19 (2013) 98–106 101

To avoid introducing any extra biases to the read alignment,eight derivative genome sequences were constructed with theproperly integrated plasmid. Evidence for the exact plasmidintegration site within attB was identified in the RNA-Seq reads,as at least one of the attB sites of Streptomyces albus J1074 waslocated within a “pirin domain protein CDS”, which had non-zeroexpression.

The reads were aligned to corresponding edited genomesequences using Novoalign (http://www.novocraft.com/). Multiple-mapping reads were randomly distributed between matching loca-tions. The mapped read assignment to gene annotations wasperformed using in-house software. The RPKM values (reads perkilobase [of gene length] per million mapped reads) were calculatedin accordance with a previously published method (Mortazavi et al.,2008), but instead of using the “total reads in the experiment”, wenormalised the values to the “total mapped non-rRNA reads”.

To compare the transcriptomic data with the GUS activity andpromoter strength, Streptomyces albus J1074 mutants were culti-vated as described above in triplicate and subjected to a GUS assayas described in the section “Assessment of promoter strength (GUSassay)”.

3. Results

3.1. Generation of a synthetic promoter library

We created a synthetic promoter library based on the consensus�35 and �10 sequences of the ermEp1 promoter in Streptomyceslividans TK24. The use of degenerate primers and an amplifiedhygromycin resistance gene facilitated the rapid and easy cloning ofa variety of different synthetic promoters in front of the gusA reportergene. After the digested PCR fragment was ligated into the vector andthe plasmid was transformed into XL1-Blue E. coli cells, all transfor-mants contained plasmids with a synthetic promoter in front of thegusA gene. These clones were used in parallel for plasmid isolation tosequence the synthetic promoter region and for direct conjugationinto Streptomyces lividans TK24 using tri-parental conjugation. Strepto-myces lividans TK24 synthetic promoter mutants were subsequently

0

10

20

30

40

50

60

70

GU

S A

ktiv

ität U

/g

Fig. 1. Promoter strengths of the identified synthetic (light grey) and reference (dark gweight in Streptomyces lividans TK24. pGUS is an empty vector without the hygromycin rbetween the hygromycin resistance and gusA genes. In contrast, “promoter” 72 containsshows no promoter activity. Thus, “promoter” 72 acts as a more adequate negative conindependent experiments with the same exconjugant).

grown to stationary phase and subjected to a GUS assay for theindirect assessment of promoter strengths. The GUS activity wasstandardised to the dry weight, although it was constant at4.4 mg70.5 per 1 ml wet culture. Using this approach, we generateda library with 56 synthetic promoters with promoter strengths rangingfrom 2% to 319% compared with the ermEp1 promoter (100%) (Fig. 1and Table S1).

To compare the synthetic promoters to known promoters,different derivatives of the ermE promoter (ermEp1, ermEp1*,ermE and ermE*, see Fig. S3) and the tetracycline induciblepromoter (tcp) were cloned into the pGUS vector and analysedusing the GUS assay (Fig. 1). There was no significant differencebetween the ermEp1 (21.5 U/g) and ermEp1* (21.2 U/g) promotersor the ermE (39.4 U/g) and ermE* (37.6 U/g) promoters. The ermEand ermE* promoters were approximately 1.8 times stronger thanthe ermEp1 and ermEp1* promoters. Synthetic promoter 21 was1.6 times stronger than the ermE and ermE* promoters. Theuninduced tcp promoter (24.2 U/g) was slightly stronger thanthe ermEp1 and ermEp1* promoters.

3.2. Characterisation of synthetic promoters using different reportergenes and several actinomycetes hosts

For a more comprehensive analysis of the generated syntheticpromoter library, a set of weak (promoter 82), medium (promoter57) and strong (promoter 21) synthetic promoters was chosen(using the ermEp1* promoter as a reference) and subjected tofurther analyses, including a re-evaluation of promoter strengthsusing two additional reporter genes. Synthetic “promoter” 72served as negative control. These genes were introduced into thepGUS constructs between the synthetic promoters and the gusAreporter gene, generating one operon with gusA. The widely usedgreen fluorescent protein (GFP) and neomycin-resistant aminogly-coside 3′-phosphotransferase II (AphII) were selected as reporterproteins. The GFP fluorescence of the protein lysate, which wasalso used for the GUS assay, was analysed using a plate reader,while the AphII activity was determined based on the neomycinresistance in an agar diffusion assay. Both reporter proteinsshowed the prominent activity of promoter 21, but these assays

rey) promoters, as assessed from GUS reporter protein activity relative to the dryesistance gene or a promoter. C6 is a negative control without a promoter sequencea sequence the same length as the other promoters in front of the gusA gene, buttrol. The error bars indicate the standard deviations of biological triplicate (three

Fig. 2. Synthetic promoters showing similar strengths in different actinomyceteshosts. The GUS activities were measured from synthetic promoters 72, 82, 57 and21 and the ermEp1* promoter. The promoter-like sequence 72 acts as negativecontrol. The error bars indicate the standard deviations of biological triplicate.

T. Siegl et al. / Metabolic Engineering 19 (2013) 98–106102

lacked the sensitivity to detect differences between the mediumand weak promoters, including the ermEp1* promoter (data notshown).

To assess the strengths of the different synthetic promoters indistantly-related actinomycetes hosts, the same set of promoterswas employed, and the respective plasmids (pGUS-SPL-72, pGUS-SPL-82, pGUS-SPL-57, pGUS-SPL-21, pGUS-ermEp1*) were intro-duced into Streptomyces albus J1074, Saccharothrix espanaensisDSM 44229 and Salinispora tropica CNB-440 via conjugation. Allmutants were analysed through the GUS assay and exhibitedpromoter strengths similar to those previously detected in Strep-tomyces lividans TK24 (Fig. 2).

3.3. Characterisation of synthetic promoters in the context of thewhole streptomyces transcriptome using RNA-Seq analysis

The GUS activity assay indirectly reflects the promoter strengthafter transcription and translation into the functional reporterprotein. To directly assess the promoter strength at the transcrip-tional level, we performed RNA-Seq analysis, which facilitates thequantification of mRNA transcripts of any given gene in thegenome. A group of synthetic promoter constructs (72, 82, 57, 21,also 57aphII and 57gfp—please refer to the following Section 3.4for details) as well as the pGUS control and the ermEp1* promoterwere introduced into Streptomyces albus J1074, and the mutantswere grown as described in the section “Transcriptomic dataanalysis (RNA-Seq)”. Whole transcriptome sequencing revealedthat the promoter strengths were strongly correlated with thenumber of transcripts (Pearson correlation coefficient r¼0.96,p¼0.0001173, sample size 8, 95% CI 0.81–0.99).

The RNA-Seq also facilitated the identification of the transcrip-tion start sites of sufficiently expressed mRNAs, which can be usedto determine the consensus regions of the preceding promoter.We mapped the transcriptional start points of gusA mRNA afterpromoters 21, 57 and ermEp1*. For promoters 82 and 72, the readcoverage was too ambiguous to reliably identify the start points.For promoter 21 and ermEp1*, the identified transcriptional startsites were the same as those reported for the ermEp1 promoter.However, for promoter 57, the transcriptional start point of thegusA mRNA was shifted six nucleotides downstream, suggestingthat this promoter exposed different binding sequences to the RNApolymerase enzyme (Fig. 3).

To evaluate synthetic promoter strengths in the context of theoverall gene expression in the cell, we compared the transcriptionlevels of the gusA gene expressed under ermEp1* and differentsynthetic promoters with those of genes encoding ribosomalproteins. The expression levels of the ribosomal protein transcriptsrpL1, rpS12, rpL9 and rpL12 were assessed in all Streptomyces albus

mutants analysed with RNA-Seq. The mean expression valueswere calculated from all mutants and compared with those ofthe synthetic promoters. The rpL1 expression was similar to thegusA expression with promoter 57, while rpS12 and rpL9 exhibitedexpression levels similar to those of gusA with promoter 21.However, the expression of rpL12 was approximately two timeshigher than that of gusA with promoter 21 (Fig. 4).

3.4. Promoter characterisation in the operon context

Many of the genes in biosynthetic gene clusters are organisedinto operons. To successfully manipulate these clusters, it isimportant to understand the promoter activity in the context ofoperons. Therefore, different operons with the reporter genesaphII, gfp and gusA were generated to investigate the syntheticpromoter abilities in transcribing these genes. We cloned twodifferent operons downstream from a set of synthetic promoters(72, 82, 57, 21) and the ermEp1* promoter. The first operoncontained gfp and gusA, while the second operon consisted ofaphII and gusA (both constructs with the 26 bp linker tctagtcgag-caacggaggtacggac between the two CDSs, which contained an RBSsequence shown in bold, Table S2). Thus, each reporter gene waspreceded by its own RBS. The resulting Streptomyces lividans TK24mutants were subjected to the GUS assay, which revealed inter-esting variations. While the GUS activity in the gfp operon showedvalues similar to the single gusA gene directly downstream of thepromoter, the GUS activity in the aphII operon was greatlyreduced, regardless of the promoter, although promoter 21remained the strongest (Fig. 5). This result suggests that the genepreceding the gusA gene in the operon influenced the transcriptionand/or translation of GUS. To identify influential factors at thetranscriptional level, we performed RNA-Seq analysis on Strepto-myces albus J1074 pGUS-SPL-57aphII and pGUS-SPL-57gfpmutants, each containing one operon preceded by syntheticpromoter 57. The RNA-Seq data correlated perfectly with theGUS activity data. The number of gusA transcripts generated withthe gfp operon was comparable to that obtained for the single gusAgene and therefore reflects the detected GUS activity. Similarly, thereduced GUS activity in the aphII operon mirrored the equallyreduced mRNA transcript levels of the aphII and gusA operons(Fig. 6).

3.5. Comparison of flaviolin production using the strongest syntheticpromoter 21 vs. the ermEp1* promoter

To demonstrate an application of the synthetic promoterlibrary, the strongest synthetic promoter 21 was used to expressa gene for the generation of a natural compound. As a simplemodel, we chose the rppA gene, which encodes a type III polyke-tide synthase. RppA converts five malonyl-CoA molecules to1,3,6,8-tetrahydroxynaphthalene, which spontaneously oxidisesto the red-brown compound flaviolin. We cloned the rppA genefrom Saccharopolyspora erythraea between the promoter and thegusA gene in the constructs pGUS-ermEp1* and pGUS-SPL-21 togenerate pGUS-ermEp1*rppA and pGUS-SPL-21rppA, respectively.The resulting Streptomyces lividans TK24 mutants exhibited a 3.3-fold enhanced production of flaviolin with synthetic promoter 21compared with the flaviolin production of the ermEp1* promoter(Fig. S4).

4. Discussion

Using degenerate primers containing synthetic promotersequences to amplify a hygromycin resistance gene, we havepresented a rapid, easy and successful strategy for generating a

Fig. 4. Comparison of the transcription levels (expressed in RPKM, reads per kilobase [of gene length] per million mapped reads) of gusA, obtained for different syntheticpromoters, to the expression levels of genes encoding ribosomal proteins (rpL1, rpS12, and rpL12).

Fig. 5. Analysis of the expression levels of genes in operons. The strengths of thedifferent synthetic promoters (72, 82, 57 and 21) and the ermEp1* promoter wereassessed using the GUS activity assay with Streptomyces lividans TK24 mutants. Thereporter gene gusA was either located directly downstream of the promoter (gusA)or as the second gene in the operon downstream of gfp and aphII ((gfp) gusA and(aphII) gusA, respectively). pGUS is an empty vector without the hygromycinresistance gene or a promoter. The promoter-like sequence 72 acts as negativecontrol. The error bars indicate the standard deviations of biological triplicate.

Fig. 3. RNA-Seq read coverage in the vicinity of transcription start sites (TSSs, marked with thin vertical lines) after the promoters (top to bottom) 57, 21, and ermEp1*.In terms of coverage, we define TSS as the steepest coverage increase after the end of the promoter in the direction of transcription. For the promoter 57, a 5 bp shift of theTSS downstream is clearly visible (in comparison with promoters 21 and ermEp1*).

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synthetic promoter library. We analysed 56 synthetic promotersaccording to their sequence and strength, indirectly determinedthrough the activity of the highly sensitive β-D-glucuronidase, theproduct of the gusA reporter gene (Myronovskyi et al., 2011). Thesynthetic promoters were based on the �10 and �35 consensussequences of the constitutive ermEp1 promoter and exhibited awide range of promoter strengths (2% to 319%) compared with theermEp1 promoter. The promoters were 41 bp long, and therefore,could easily be incorporated in PCR primers and inserted in frontof a gene of interest. This variety of promoter strengths could beapplied to different research approaches. Strong promoters couldbe used for the overexpression of a gene of interest, whereas theexpression of genes for the production of toxic substances in highconcentrations that require only sparse expression would benefitfrom weak promoters.

Frequently, in genetic bioengineering applications, it is alsonecessary to fine-tune and balance specific gene expression due to

the complexity of regulatory networks (Boyle and Silver, 2012;Scalcinati et al., 2012). Promoters in our synthetic promoter librarywith small increments in strength could be used for such experi-ments. The strong promoters would be especially useful to research-ers working with actinomycetes to overproduce natural products viatranscription enhancements of the corresponding genes (Dangelet al., 2010).

The ermE* promoter (a stronger variant of the ermE promoter,which consists of ermEp1 and ermEp2) is one of the strongestpromoters and is widely used to overexpress genes in actinomy-cetes. The ermE promoter was originally characterised by cloningthe entire putative promoter region upstream of the ermE geneof Saccharopolyspora erythraea (including the first part of theermE gene) in front of a kanamycin resistance gene (neo) in areplicative vector in Streptomyces lividans TK24 and by measuringkanamycin phosphotransferase (KPH)-specific activity in cell-freeextracts (Bibb et al., 1994). Bibb et al. reported that a TGGdeletion in the -35 region of the ermEp1 promoter resulted in aslight increase (1.075-fold) in promoter activity (ermEp2 andermEp1ΔTGG compared with wild-type ermEp2 and ermEp1).This effect was further characterised via S1 nuclease mapping,which showed an approximately five-fold increase in the level ofermEp1 transcripts as deduced from dilutions of the plasmids. Thisupregulated variant is referred to as the ermE* promoter, whichspans more than 100 bp and contains ermEp2 and ermEp1ΔTGG(Bibb et al., 1994).

However, in the present study, no significant difference wasdetected between the strengths of the native ermE promoter andits variant ermE* or between the ermEp1 and the ermEp1*promoter. The ermE and ermE* promoters were almost two timesstronger than the ermEp1 and ermEp1* promoters, which is notsurprising because the former contain two active promotersequences (ermEp2/ermEp1 and ermEp2/ermEp1*, respectively)instead of a single promoter sequence. A 3.6-fold difference betweenthe ermE and ermEp1 promoters has been reported based on theKPH-specific activity assay (Bibb et al., 1994). In the present study, we

Fig. 6. Correlation (C) of GUS assay activities (A) to mRNA reads (B) of gusA as a single gene (72, 82, ermEp1*, 57 and 21) and in operons with gfp (57(gfp)) and aphII (57(aphII)). (C) The adjusted coefficient of determination R

2¼0.91 improved to 0.98 after removing promoter 21, which is outside the double-interquartile range (not shown).

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generated several synthetic promoters (A9, B10, 57, A3) withstrengths comparable to that of the ermE* promoter region, andsynthetic promoter 21 exhibited 1.6-fold stronger expression. Con-sidering the smaller size compared with the ermE* promoter region,these synthetic promoters are superior candidates for the overexpres-sion of genes and/or biosynthetic gene clusters in actinomycetes.

It might occur that the use of phage integration system basedplasmids, like the pGUS derivatives used in the present study, canresult in the integration of multiple plasmids in the same mutant,which in turn could lead to increased GUS activity. However, dueto the fact of consistent GUS activities of the same promoters (e.g.72, 82, 57 and 21) in four different strains (S. lividans TK24, S. albusJ1074, Salinispora tropica CNB-440 and Saccharothrix espanaensisDSM 44229) generated independently multiple integration ofplasmids are not likely.

Synthetic promoters are advantageous over native promotersbecause they are less likely to underlie intracellular regulation.Furthermore, native promoters are often not as active in aheterologous host as in the native strain (Magdevska et al., 2010;Wilkinson et al., 2002). However, the regulation of promotersoften occurs at consensus regions, where the sigma factor of theRNA-polymerase holoenzyme binds. The synthetic promoters usedin the present study contain the consensus regions of the nativeermEp1 promoter. Therefore, we wanted to exclude the possibilitythat the synthetic promoters would exhibit variable activities indifferent actinomycetes strains and selected a set of four syntheticpromoters (namely promoter 72 as negative control, the weakpromoter 82, the medium promoter 57 as well as the strongpromoter 21) and the ermEp1* promoter to assess their activitiesin three additional actinomycetes strains belonging to threedifferent orders. Saccharothrix espanaensis DSM 44229 producesthe heptadecaglycoside antibiotic saccharomicin and belongs tothe order Pseudonocardiales (Strobel et al., 2012). Salinisporatropica CNB-440 is salt-water-dependent and belongs to the orderMicromonosporales (Maldonado et al., 2005; Udwary et al., 2007),while Streptomyces lividans TK24 and Streptomyces albus J1074both belong to the order Streptomycetales (Gao and Gupta, 2012).The fact that the activities of the chosen promoters were highlysimilar in all four actinomycetes strains provides evidence that thesynthetic promoters are not subjects to intracellular regulation,but are stably and constitutively expressed to the determinedextent in a wide variety of actinomycetes. We anticipate that oursynthetic promoters will be expressed in actinomycetes strainsthat also accept the ermE promoter. The promoter strengths wereperfectly matched, considering that the promoters were expressed

in species exclusively dependent on salt water, strains resistant tolysozyme and strains with an inability to sporulate.

The synthetic promoter strength was indirectly assessed throughthe GUS enzymatic activity assay. To validate these results, we usedanother reporter gene (gfp) to measure the activity of the ermEp1*promoter and that of a set of weak, medium and strong promoters.The expression of GFP was assessed by measuring the fluorescence.Synthetic promoter 21, the strongest promoter identified in the GUSassay, exhibited the highest GFP fluorescence. The differences inGFP expression in the other tested promoters were not as pro-nounced as that of the gusA reporter gene, potentially reflecting thefact that GFP is a less sensitive reporter protein than GUS and islimited to a single photon per molecule, as opposed to theenzymatic amplification of the signal in the GUS assay(Myronovskyi et al., 2011). Any argument regarding the influenceof the gfp gene on GFP expression can be countered by the fact thatthe GUS activity assays showed highly similar results for the gusAgene directly adjacent to the promoters and for the second genedownstream of the gfp gene in the same operon.

Analogous to gfp, we cloned the neomycin resistance gene aphII inthe same operon with gusA. In all cases, the AphII expression waslow, as indicated by low levels of resistance to neomycin. Onlysynthetic promoter 21 was able to confer some resistance toneomycin, while the other tested promoters (including the ermEp1*promoter) failed to confer any considerable resistance comparedwith the negative control (pGUSaphII). The apparent low expressionof the aphII gene was also reflected by a significantly reduced gusAexpression (located in one operon downstream of aphII), as deter-mined by the GUS assay and RNA-Seq (for pGUS-SPL-57aphII).

We performed RNA-Seq to directly observe transcription,which cannot be distinguished from translational effects whenreporter protein levels are measured. RNA-Seq facilitates thequantitative analysis of gene expression and transcription initia-tion as well as the prediction of terminator and promotersequences. The RNA-Seq analysis revealed a correlation betweenthe number of mRNA transcripts of the gusA gene and the GUSactivity assayed in the same mutants of Streptomyces albus,regardless of the location of the gusA gene (directly adjacent tothe promoter or downstream of another gene in the same operon),suggesting that the GUS activity assay closely reflects the tran-scription level of the gusA gene and is therefore a good method forsurveying promoter strengths. The results obtained from the RNA-Seq analysis also provided evidence that there were no transla-tional influences causing the reduction of gusA expression in thesame operon as aphII. Using the same promoter 57 did not result

T. Siegl et al. / Metabolic Engineering 19 (2013) 98–106 105

in similar transcription levels of the aphII, gfp and gusA genes,which likely reflects the influence of mRNA secondary structureand therefore mRNA stability.

RNA-Seq facilitates a comparison of the transcription levels ofmany genes in the genome. Because they constitute an essentialpart of the ribosome, the genes encoding ribosomal proteins arethe most frequently transcribed genes in the cell. Therefore, wecompared the transcription levels of the gusA reporter geneexpressed using different (synthetic) promoters with those ofsome ribosomal protein-encoding genes. While the expressionlevels of three ribosomal genes (rpL1, rpL9 and rpS12) range amongthe expression activity of the strongest synthetic promotersidentified in our library, only rpL12 showed 2-fold higher expres-sion than gusA when transcribed using the strongest syntheticpromoter 21. Overall, this result provides evidence that the activityof the strongest synthetic promoters is comparable to that of thestrongest native promoters in the Streptomyces albus J1074genome.

The promoters were characterised according to their -10 and-35 consensus sequences, where the sigma factor of the RNApolymerase holoenzyme binds. These regions are typically identi-fied according to their distance (10 and 35 bp, respectively) fromthe transcriptional start point of the mRNA transcript, which isdesignated as +1. Thus, we were able to draw conclusions regard-ing the promoter sequences by identifying the transcriptional startpoints of the gusA transcripts expressed with different (synthetic)promoters using RNA-Seq data analysis. For promoters 72 and 82,identification of the transcriptional start point was not possibledue to low gusA expression. Promoters 21 and ermEp1* showedthe same transcriptional start points as reported for ermEp1 (Bibbet al., 1994). Because the synthetic promoters were based on theermEp1 promoter, sharing the same length and consensussequences, this result was expected. Surprisingly, the gusA mRNAtranscript expressed with synthetic promoter 57 showed a tran-scriptional start point that was shifted six nucleotides down-stream. We propose that the -10 and -35 consensus sequences ofthe 57 promoter are 5′-taccat -3′ and 5′-gagg-3′ instead of 5′-taggat-3′ and 5′-ggct-3′, respectively. No other synthetic promotersequence exhibited sequence similarities to consensus sequencesin the randomised parts of the promoter.

Typical experiments in genetic engineering include the over-expression of a gene of interest. Particularly, when working withactinomycetes, representing one of the richest sources of naturalproducts, the overproduction of a natural compound is popular(Olano et al., 2008). We showed that the strongest promoter in ourlibrary could be successfully used for similar purposes through theoverexpression of type III polyketide synthase gene rppA. RppAconverts five malonyl-CoA molecules to 1,3,6,8-tetrahydroxy-naphthalene, which spontaneously oxidises to the dark-red com-pound flaviolin. The production of flaviolin was increased 3.3-foldwhen the 21 promoter was used to express rppA compared withthe ermEp1* promoter in the Streptomyces lividans TK24 host.

In summary, a synthetic promoter library to drive gene expres-sion in actinomycetes has been constructed and comprehensivelycharacterised using several reporter genes and RNA-seq. Weanticipate that our synthetic promoter library will be of high valueto the synthetic biology community and will facilitate the drugdiscovery process in actinomycetes.

Acknowledgments

This work was supported through funding from the ERCstarting grant EXPLOGEN no.281623 to AL. The authors wouldlike to thank Bradley Moore for providing the Salinispora tropicaCNB-440 wild-type strain.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.ymben.2013.07.006.

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