Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria

Cyanobacteria are oxygenic photosynthetic prokaryotes that play important roles in the global carbon cycle. Recently, engineered cyanobacteria capable of producing various small molecules from CO2 have been developed. However, cyanobacteria are seldom considered as factories for producing proteins, mainly because of the lack of efficient strong promoters. Here, we report the discovery and verification of a super-strong promoter Pcpc560, which contains two predicted promoters and 14 predicted transcription factor binding sites (TFBSs). Using Pcpc560, functional proteins were produced at a level of up to 15% of total soluble protein in the cyanobacterium Synechocystis sp. 6803, a level comparable to that produced in Escherichia coli. We demonstrated that the presence of multiple TFBSs in Pcpc560 is crucial for its promoter strength. Genetically transformable cyanobacteria neither have endotoxins nor form inclusion bodies; therefore, Pcpc560 opens the possibility to use cyanobacteria as alternative hosts for producing heterogeneous proteins from CO2 and inorganic nutrients.

C O 2 is one of the main greenhouse gases. Cyanobacteria can convert CO 2 into organic compounds through oxygenic photosynthesis with an efficiency higher than that of terrestrial plants 1,2 . Cyanobacteria account for 20-30% of earth's photosynthetic productivity; that is, the conversion of solar energy into chemical energy 3,4 . The use of engineered cyanobacteria capable of producing fuels and other chemicals could reduce greenhouse gas emission and help to address the shortages of energy and resources 5 . As nonpathogenic aquatic photoautotrophs with a high nutritional value, some cyanobacteria are attractive hosts for producing heterologous proteins that can be used in the health, food, fodder, fertilizer, and environment sectors 6-8 . Proteins of industrial or environmental interest that have been produced in cyanobacteria include a metallothionein protein capable of absorbing heavy metals in wastewater 7 , a larvicidal protein that can kill mosquito eggs in water 8 , and a human CuZn superoxide dismutase that can be used in health products 6 . The human CuZn superoxide dismutase was expressed at a level of 3% of total soluble protein in the cyanobacterium Anacystis nidulans 6301 6 . This expression level was achieved by using a native strong promoter, P rbc343 , and an optimized SD sequence. Because this expression cassette was delivered by a replicative plasmid, the constant addition of antibiotics was required to maintain the expression level 6 .
Several heterologous genes have been expressed in cyanobacteria in an effort to create novel biosynthetic pathways to produce chemicals. Heterologous strong promoters such as P trc and P lac and native promoters such as P cpc , P rbc , and P psbA2 have been used in integrated cyanobacterial expression systems. Lan et al. 9 used P trc to express trans-enoyl-CoA reductase (Ter) for butanol production, and Guerrero et al. 10 used P lac to express ethylene-forming enzyme (EFE) for ethylene production. Lindberg et al. 11 and Bentley et al. 12 used P psbA2 to drive expression of isoprene synthase (Isps) for isoprene production in cyanobacteria. Although there was successful production of the target chemicals, the expression levels of the proteins encoded by the heterologous genes were not reported 9,10 . Recently, Angermay et al. 13 experimented with various promoters (P rnpB , P psbA2 , and P trc ) to drive expression of L-lactate dehydrogenase in cyanobacteria, and found that even powerful native and artificial promoters from Synechocystis were not strong enough to produce the enzyme in sufficient quantities.
The aim of this study was to create novel expression elements that lead to strong expression of heterologous genes in cyanobacteria. We discovered a super-strong promoter, P cpc560 , consisting of two promoters from the cpcB gene and 14 predicted transcription factor binding sites (TFBSs). Using P cpc560 , two heterologous genes were expressed in the cyanobacterium Synechocystis sp. 6803 (hereafter, S. 6803) to a level comparable to that obtained using the Escherichia coli expression system, demonstrating the strength and efficiency of P cpc560 . We further showed that the presence of multiple TFBSs is crucial for the promoter strength of P cpc560 . The discovery of this super-strong promoter will stimulate research on using cyanobacteria as hosts to produce valuable recombinant proteins from CO 2 and inorganic nutrients.

Results
Discovery of the super-strong promoter P cpc560 . Efficient expression elements are required to increase the expression level of heterologous genes. Since c-phycocyanin and ribulose-bisphosphate carboxylase/ oxygenase (Rubisco) are the major soluble proteins in cyanobacteria, expression elements from genes encoding these two proteins have been used widely to construct cyanobacterial expression vectors 6,7,[14][15][16] . For example, a metallothionein gene was expressed in the cyanobacterium Synechococcus sp. 7942 using the promoter from cpcB, the gene encoding the c-phycocyanin beta subunit 7 , while a human CuZn superoxide dismutase gene was expressed in Synechococcus sp. 7002 using the promoter and terminator from the rbc operon, which encodes Rubisco 6 .
RBSDesigner 17 predicted that the RBS in the cpcB promoter is approximately 10-times more efficient than that in the rbc promoter in terms of the predicted translation efficiency. Therefore, we chose the cpcB promoter for further investigations. The genomic sequence 1,000 bp upstream of the initiation codon of the cpcB gene (sll1577) was subjected to promoter analysis using Virtual Footprint 18 . This analysis revealed two promoters located at 135 bp and 374 bp upstream of the initiation codon of the cpcB gene. Scanning the genomic sequence 1,000 bp upstream of the initiation codon of the cpcB gene also revealed 14 TFBSs located between 381 bp and 556 bp from the initiation codon of the cpcB gene 18 (Supplementary Table S1). Negative TFBSs are usually located between 500 bp and 1,000 bp upstream of the transcription start site 19 , and some mammalian proteins such as soluble adenylyl cyclase 20 are highly similar to these proteins in cyanobacteria. Therefore, we chose the genomic sequence of 560 bp upstream of the initiation codon of cpcB as the new promoter sequence; this sequence was designated as P cpc560 . P cpc560 contains two predicted promoters and 14 predicted TFBSs ( Fig. 1).
No terminator was found in the genomic region 300 bp downstream of the stop codon of the cpcB gene. This may be because cpcB is the first gene of the cpc operon. Since the rbc terminator was used in a previous study that achieved high-level gene expression (yielding target protein at a level of 3% of total soluble protein) in a cyanobacterium 6 , we chose the terminator of rbcL, the gene encoding the Rubisco large subunit (slr0009), as the terminator (T rbcL ).
High-level expression of crotonyl-CoA-specific trans-enoyl-CoA reductase gene in S. 6803. To validate the efficacy of the newly discovered promoter P cpc560 , we used it to drive the expressions of two genes encoding important enzymes involved in metabolism in S. 6803. One enzyme was crotonyl-CoA-specific trans-enoyl-CoA reductase (Ter), a key enzyme in increasing the driving force towards butanol biosynthesis 21,22 . ter has been overexpressed in Synechococcus sp. PCC 7942 with the aim to produce butanol from CO 2 9 . A strong promoter from E. coli, P trc , was used to express a codon-optimized ter in Synechococcus sp. PCC 7942 9 . However, the activity of Ter in the crude cell extract of Synechococcus sp. PCC 7942 was 0.057 6 0.005 mmol/min/mg crude cell extract. This was 64-fold lower than that of Ter (3.7 6 0.5 mmol/min/mg) in E. coli, in which the ter gene was expressed under the control of a medium-strength promoter, P L lacO1 22 . This suggests that P trc is not an efficient promoter in cyanobacteria in terms of gene expression strength. Therefore, we tested whether the newly discovered promoter P cpc560 could significantly increase the expression level of ter in cyanobacteria.
To express ter in S. 6803, a codon-optimized ter gene (Supplementary Fig. S1) from T. denticola flanked by P cpc560 and T rbcL expression elements was integrated into the S. 6803 chromosome at the pta (slr2132) insertion site via homologous recombination. The pta gene encodes phosphotransacetylase, the first enzyme in acetate synthetic pathway, and disruption of pta does not affect cell growth 16 . Homoplasmy and gene insertion were verified by PCR and sequencing ( Fig. 2a and 2b). SDS-PAGE analysis demonstrated that ter was strongly overexpressed in the S. 6803 mutant Dpta::P cpc560 ter. The protein expression level was approximately 15% of total soluble protein (Fig. 2c), a surprisingly strong expression for cyanobacteria. The band excised from the position at approximately 43.7 kDa was confirmed as Ter from T. denticola by MALDI-TOF MS analysis (Fig. 2d).
The specific activity of Ter was determined to be 32.22 6 1.93 mmol/min/mg crude cell extract of S. 6803 strain Dpta::P cpc560 ter (Table 1). In a previous study in which the ter gene was expressed in Synechococcus sp. PCC 7942 under the control of the P trc promoter 9 , the activity of Ter was 0.057 6 0.005 mmol/min/ mg crude cell extract. When ter was expressed in E. coli BW25113 under the control of the P L lacO1 promoter, on a medium-copy number plasmid, the Ter activity was 3.7 6 0.5 mmol/min/mg crude cell extract 22 . These activities of Ter reported in the literature were measured in crude lysates obtained from different organisms cultured in various different conditions. The activity of Ter determined in our study indicates that the ter gene was strongly expressed in S. 6803 strain Dpta::P cpc560 ter.
TFBSs are crucial for P cpc560 strength. The newly discovered superstrong promoter P cpc560 differs from other commonly used promoters in that it contains 14 predicted TFBSs located between 2556 bp and 2381 bp from the initiation codon of cpcB. To investigate whether these TFBSs contribute to cpcB promoter strength, ter was expressed under the control of P cpc374 , which contained only the two promoters, in S. 6803 Dpta::P cpc374 (Fig. 2a  and 2b). Although RT-PCR confirmed ter transcription from P cpc374 ( Supplementary Fig. S2), the level of Ter protein was too low for detection by SDS-PAGE, whereas the level of Ter protein expressed from ter driven by P cpc560 was up to 15% of total soluble protein (Fig. 2c). The specific activity of Ter in strain Dpta::P cpc374 ter was only 0.12 6 0.01 mmol/min/mg crude extract (Table 1). This was 268-fold lower than that of Ter in the strain Dpta::P cpc560 ter (32.22 6 1.93 mmol/min/mg), and only slightly higher that of Ter in Synechococcus sp. PCC 7942, in which the ter gene was expressed under the control of the P trc promoter 9 . These data show that the newly discovered super-strong promoter P cpc560 is indeed much stronger than P cpc374 and P trc , and the multiple TFBSs present in P cpc560 are crucial for its promoter strength.
High-level expression of D-lactate dehydrogenase in S.6803. To investigate whether the newly discovered super-strong promoter   Expression levels of Ter or DldhE under control of strong promoter P cpc560 were approximately 15% of total soluble protein, whereas Ter expressed under control of P cpc374 in Dpta::P cpc374 ter strain was undetectable on gel. (d) Peptide mass fingerprint of bands at approximately 43.6 and 36.5 kDa. 43.6-kDa band was identified as Ter from T. denticola, 36.5-kDa band was identified as DldhE from E. coli K12. Mascot score greater than 53 (default MASCOT threshold for such searches) was accepted as significant (p value , 0.05). All strains (WT, Dpta, Dpta::P cpc374 ter, Dpta::P cpc560 ter and Dpta::P cpc560 DldhE) were continuously passaged more than 10 times without antibiotics.
www.nature.com/scientificreports SCIENTIFIC REPORTS | 4 : 4500 | DOI: 10.1038/srep04500 P cpc560 also drives strong expressions of other genes, we used it to control expression of Dldh, which encodes D-lactate dehydrogenase. The Dldh gene from E. coli K12 (designated as DldhE) flanked by P cpc560 and T rbcL was integrated into the chromosome at the pta insertion site via homologous recombination. Homoplasmy and DldhE gene insertion were verified by PCR and sequencing ( Fig. 2a  and 2b). SDS-PAGE analysis demonstrated that DldhE was strongly overexpressed in the S. 6803 mutant Dpta::P cpc560 DldhE; the protein expression level was approximately 15% of total soluble proteins (Fig. 2c). MALDI-TOF MS analysis confirmed that the band excised from the position at approximately 36.5 kDa was Dldh from E. coli (Fig. 2d). The specific activity was determined to be 51.92 6 2.31 mmol/min/mg crude extract in strain S. 6803 Dpta::P cpc560 DldhE (Table 1). In a previous study in which the DldhE gene was expressed under the control of P LtetO-1 on a lowcopy number plasmid 23 , the activity of DldhE in E. coli K12 MG1655 was 0.96 6 0.06 mmol/min/mg crude cell extract. This result confirmed that the DldhE gene was strongly expressed in S. 6803 under the control of P cpc560 .
We also investigated the genetic stability of the mutant containing the strong integrative expression cassette. The mutants Dpta::P cpc560 ter and Dpta::P cpc560 DldhE were continuously passaged 10 times in the absence of antibiotics. The expression levels of both ter and DldhE did not change during this procedure, demonstrating the stability of this expression system.

Discussion
In this study, we discovered and verified a super-strong promoter P cpc560 for efficient and strong expression of heterologous genes in cyanobacteria. The newly discovered super-strong promoter P cpc560 consists of two predicted promoters from the cpcB gene and 14 predicted TFBSs. Using P cpc560 , two heterologous genes were expressed in the cyanobacterium S. 6803 to levels of up to 15% of total soluble protein. Despite the fact that only a single copy of the target gene was inserted into the chromosome of S. 6803, the expression level of the target gene driven by P cpc560 was comparable to that which can be achieved in an E. coli expression system using low-or medium-copy number plasmids. This demonstrates that P cpc560 has potential applications for efficient production of recombinant proteins in cyanobacteria.
It is generally accepted that the genomic sequence 200-300 bp upstream of the initiation codon of a gene that is constitutively expressed at high levels can be used as a promoter sequence. Previous studies have shown that the thymine at 259 bp upstream of the initiation codon of cpcB is crucial for cpc promoter activity 24 . Recently, a genomic map of the transcriptional start sites (TSS) of S. 6803 showed that the cpcB gene is one of the genes with a long distance between its TSS and start codon 25 . Further examination revealed that most of the S. 6803 genes with a long distance between the TSS and start codon are responsive to environmental factors 25 . Since the cpcB gene is a light-and redox-responsive gene 26,27 , it is plausible that the length of the cpcB promoter is related to its responsiveness to environmental factors. P cpc560 is unusually long in that it contains not only two predicted promoters (at 135 bp and 374 bp), but also 14 predicted TFBSs located between 381 bp and 556 bp upstream of the initiation codon of cpcB. P cpc560 differs from P cpc374 in that it has an extra 186 bp DNA fragment containing the 14 predicted TFBSs. The large-scale difference in the expression levels of ter under the control of P cpc560 and P cpc374 demonstrated that the extra 186 bp DNA fragment in P cpc560 may contain positive TFBSs and that the presence of these multiple TFBSs is crucial for its promoter strength.
Transcription factors are proteins that play roles in virtually every aspect of the transcription process 28 . In prokaryotes, RNA polymerase recognizes and binds to the promoter region and initiates transcription, and a sigma factor is required for RNA polymerase to bind to the promoter. In eukaryotes, three types of eukaryotic RNA polymerases all require transcription factors to bind to the promoter sequence before transcription can be initiated 28 . Therefore, compared with eukaryotic promoters such as the yeast promoter 29 , the promoter of E. coli is rather short (approximately 30-50 bp) and TFBSs are usually not required. For instance, when we scanned the sequences of E. coli strong promoters (including P trc , P lac , and T7) that have been used to drive gene expressions in cyanobacteria previously, we found only one or two TFBSs in each of the promoters. In this study, we found 14 predicted TFBSs located between 381 bp and 556 bp upstream of the initiation codon of cpcB, and verified that the extra 186 bp DNA fragment containing multiple predicted TFBSs is crucial for P cpc560 promoter strength in cyanobacteria. This novel discovery raises the possibility that the lack of TFBSs in E. coli strong promoters may explain why they perform poorly in driving gene expression in cyanobacteria. Thus, we propose that native positive TFBSs should be considered when designing promoters to drive gene expression in cyanobacteria.
The newly discovered super-strong promoter P cpc560 will be useful for further research on the production of useful substances using transgenic cyanobacteria and the very cheap substrate CO 2 . The principle of considering TFBS in cyanobacterial promoter design will also contribute to designing strong and controllable promoters to drive the expressions of genes involved in new biosynthetic pathways from CO 2 in cyanobacteria.

Materials.
We purchased restriction endonucleases and other nucleases for plasmid construction from New England Biolabs (Beverly, MA, USA). All chemicals for enzymatic analyses were purchased from Sigma-Aldrich (St Louis, MO, USA).
Construction of expression vectors. The plasmids used and constructed in this study are listed in Supplementary Table S2. Primers are listed in Table S3. General strategies for constructing expression vectors are shown in Supplementary Fig. S3.
The sequences of P cpc560 and the T rbcL terminator were obtained from S. 6803 genomic DNA by PCR. Codon-optimized ter from Treponema denticola was ordered from Sangon Biotech (Shanghai, China). DldhE was obtained by PCR using genomic DNA of E. coli K12 as the template. Fusion PCR 30 was used to ligate DNA fragments, including P cpc560 -ter-T rbcL and P cpc560 -DldhE-T rbcL . To construct the expression vectors pSM2-P cpc560 ter and pSM2-P cpc560 DldhE, the fusion PCR products P cpc560ter-T rbcL and P cpc560 -DldhE-T rbcL were digested with Xhol and ligated into the Xhol site between two homologous arms of vector pSM2 16 . The ter gene flanked by P cpc374 and T rbcL was obtained by PCR from the constructed plasmid pSM2-P cpc560 ter and was inserted into the Xhol site of plasmid pSM2 16 .
Strains. The bacterial strains used in these experiments are listed in Supplementary  Table S2. We used E. coli DH5a to propagate constructs, and S. 6803 wild-type as the starting strain. All mutant strains were constructed by transforming S. 6803 wild-type with the plasmids listed in Supplementary Table S2, followed by subsequent selection until homoplasmy was achieved.
Transformations were performed as previously described 16 . All constructed strains are listed in Supplementary Table S2. Briefly, strain Dpta was constructed by integration of the plasmid pSM2 16 at the pta locus of wild-type S. 6803 via double crossover homologous recombination.
Transforming the wild-type strain of S. 6803 with the plasmids pSM2-P cpc560 ter and pSM2-P cpc374 ter generated strains Dpta::P cpc560 ter and Dpta::P cpc374 ter, respectively. In these strains, a part of the pta gene was replaced with the kanamycin resistance cassette and the P cpc560 -or P cpc374 -driven expression cassette of the codon-optimized gene ter from T. denticola encoding crotonyl-CoA specific transenoyl-CoA reductase.
Transforming the wild-type strain of S. 6803 with plasmid pSM2-P cpc560 DldhE generated strain Dpta::P cpc560 DldhE. In this strain, a part of pta was replaced with the kanamycin resistance cassette and the P cpc560 -driven expression cassette of the Dlactate dehydrogenase gene DldhE from E. coli K12.
Culture conditions. Wild-type and mutant lines of S. 6803 were grown in BG11 medium buffered with 10 mM HEPES (pH 8.0) at 30uC at an illumination intensity of 100 mmol photons/s/m 2 as described elsewhere 31 . Kanamycin was added to the medium at a final concentration of 10 mg/ml when necessary. To maintain strains on agar plates, the BG11 medium was supplemented with 1.5% (w/v) agar.
RT-PCR. RT-PCR was performed as previously described 16 , with modifications. Total RNA was isolated from S. 6803 wild-type (WT), Dpta, Dpta::P cpc374 ter and Dpta::P cpc560 ter cells using Redzol reagent (Qiagen, Beijing, China). Residual DNA in RNA preparations was eliminated by digestion with RNAse-free DNAse and reverse transcription reactions were performed using a Reverse Transcription kit (Qiagen). Reverse transcription products were amplified by PCR and analyzed by electrophoresis on 1.2% (w/v) agarose gels. The ter transcript was amplified using the forward primer 59-CAAGCCTTGTACCGCAAA-39 and the reverse primer 59-CGATCAAAGCGTTCCACT-39. Transcript levels of rnpB were analyzed as a positive control 32 .
Preparation of cell extract and gene expression level analysis. Cell crude extracts were prepared as described elsewhere 33 , with some modifications. We collected 10-ml samples of cultures in mid-exponential growth phase, and harvested cells by centrifugation at 14,000 g and 4uC for 2 min. Cell pellets were washed twice and then resuspended in prechilled buffer from the in vitro assay described below. Cells were disrupted by bead beating and cell debris and beads were removed by centrifugation at 14,000 g and 4uC for 30 min. Total protein concentrations of the crude extract were determined using the Bradford method with bovine serum albumin as the standard. To measure protein expression levels, crude extract was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The target protein bands were quantified using VisionWorksLS software, using the absolute quantity of the marker proteins as references. Each marker protein band contained 2 mg protein, and samples with 20 mg protein in total were loaded into each lane. The abundance of proteins in the Ter and DldhE target bands was about 1.5-fold that of the marker band, as determined by VisionWorksLS analysis. Therefore, there was approximately 3 mg Ter or DldhE on the gel. From this value, we calculated that the expression level of Ter or DldhE was approximately 15% of total soluble protein. The target protein bands were excised and subjected to in-gel-digestion and MALDI-TOF MS analysis as described in our previous report 34,35 .
Trans-2-enoyl-CoA reductase (Ter) assay. Ter activity was assayed by monitoring the decrease in absorbance at 340 nm, which corresponded to consumption of NADH as previously described 9,36 , with slight modifications. Briefly, the reaction mixture (total volume, 200 ml) contained 100 mM potassium phosphate buffer (pH 6.2), 200 mM crotonyl-CoA (Sigma), 400 mM NADH, and 0.3 mg crude extract protein from strain Dpta::P cpc560 ter or 7 mg crude extract protein from strains S. 6803, Dpta, and Dpta::P cpc374 ter. After preincubation at 30uC for 5 min, the reaction was initiated by adding the substrate. The activity was determined by monitoring the decrease in absorbance at 340 nm using a microtiter plate reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA) at 30uC. D-lactate dehydrogenase (Dldh) assay. Dldh activity was determined by monitoring the decrease of absorbance at 340 nm as previously reported 9,37 . The 200 ml reaction mixture contained 50 mM sodium phosphate (pH 6.5), 300 mM NADH, 2.5 mM MgCl 2 , 3 mg crude extract protein, and 30 mM sodium pyruvate. After preincubation at 30uC for 5 min, the reaction was initiated by adding the substrate sodium pyruvate.