Development of SyneBrick Vectors As a Synthetic Biology Platform for Gene Expression in Synechococcus elongatus PCC 7942

Cyanobacteria are oxygenic photosynthetic prokaryotes that are able to assimilate CO2 using solar energy and water. Metabolic engineering of cyanobacteria has suggested the possibility of direct CO2 conversion to value-added chemicals. However, engineering of cyanobacteria has been limited due to the lack of various genetic tools for expression and control of multiple genes to reconstruct metabolic pathways for biochemicals from CO2. Thus, we developed SyneBrick vectors as a synthetic biology platform for gene expression in Synechococcus elongatus PCC 7942 as a model cyanobacterium. The SyneBrick chromosomal integration vectors provide three inducible expression systems to control gene expression and three neutral sites for chromosomal integrations. Using a SyneBrick vector, LacI-regulated gene expression led to 24-fold induction of the eYFP reporter gene with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer in S. elongatus PCC 7942 under 5% (v/v) CO2. TetR-regulated gene expression led to 19-fold induction of the GFP gene when 100 nM anhydrotetracycline (aTc) inducer was used. Gene expression decreased after 48 h due to degradation of aTc under light. T7 RNA polymerase-based gene expression resulted in efficient expression with a lower IPTG concentration than a previously developed pTrc promoter. A library of T7 promoters can be used for tunable gene expression. In summary, SyneBrick vectors were developed as a synthetic biology platform for gene expression in S. elongatus PCC 7942. These results will accelerate metabolic engineering of biosolar cell factories through expressing and controlling multiple genes of interest.


INTRODUCTION
Increasing concerns about limited fossil fuels and the advent of global warming have drawn attention to direct conversion of CO 2 to high-value products through engineering cyanobacteria and microalgae (Ducat et al., 2011;Angermayr et al., 2015). These methods have high productivity per acre compared to terrestrial crops (Parmar et al., 2011). Metabolic engineering of cyanobacteria has been applied to development of biosolar factories from CO 2 with the aim of enhancing production of bio-products through modification of metabolism and introduction of heterologous metabolic pathways (Atsumi et al., 2009;Liu et al., 2011;Choi et al., 2016;Chwa et al., 2016;Lee et al., 2017).
To reconstruct heterologous metabolic pathways in a cyanobacterium, multiple genes must be inserted into either a plasmid or the genome. Controllable expression of those genes is required to balance metabolic flux toward desired products. Tools for controlling gene expression in cyanobacteria have been developed by engineering TetR-regulated promoters in Synechocystis sp. PCC 6803 (Huang and Lindblad, 2013). Other tools include isopropyl β-D-1-thiogalactopyranoside (IPTG) and anhydrotetracycline (aTc)-based inducible promoters and a transacting small RNA system for Synechococcus sp. PCC 7002 (Markley et al., 2015;Zess et al., 2016), designed oxygenresponsive genetic circuits in Synechocystis sp. PCC 6803 (Immethun et al., 2016), and theophylline riboswitches in Synechococcus elongatus PCC 7942 (Nakahira et al., 2013). In addition, synthetic biology-based plasmid vectors (e.g., pPMQAK1) have been developed for Synechocystis sp. PCC 6803 (Huang et al., 2010). A synthetic platform for gene expression in S. elongatus PCC 7942, a model cyanobacterium for metabolic engineering and circadian rhythms, has not been reported.
This study aimed to develop SyneBrick TM vectors as synthetic platform for controllable gene expression in S. elongatus PCC 7942 under 5% (v/v) CO 2 . The standardization of biological parts and their assembly is a core idea behind synthetic biology. Standardized assembly approaches such as BglBrick cloning do not require PCR amplification and use sequence-based homologous recombination. Thus, these cloning methods are not limited by automation or in the number of DNA fragment copies used (Hillson, 2011). BglBrick standard vectors and the derivative CoryneBrick vectors have been successfully applied for metabolic engineering of Escherichia coli and Corynebacterium glutamicum to express multiple target genes for synthetic biology (Lee et al., 2011Kang et al., 2014). Based on these synthetic biology platforms, we developed SyneBrick vectors with regulatory and promoter sections for controlling gene expression. We expect that SyneBrick vectors will facilitate metabolic engineering applications in S. elongatus PCC 7942.

Transformation of S. elongatus PCC 7942 with Synebrick Vectors
Transformation of S. elongatus PCC 7942 was as described previously (Golden et al., 1987). Cyanobacterial strains were transformed by incubating at mid-log phase (OD 730 1 to 2) with 100 ng plasmid DNA for 24 h in the dark. Cell-DNA mixed cultures were spread on BG-11 plates supplemented with appropriate antibiotics for selecting recombinants (10 µg/mL spectinomycin, 10 µg/mL kanamycin, or 3 µg/mL chloramphenicol). Single colonies were sub-cultured to prevent chromosomal segregation. PCR was used to verify chromosomal integration of targets into either NSI, NSII, or NSIII (Figure 1). DNA sequences were verified using oligonucleotides in Table S1. Genotypes of recombinant S. elongatus strains are listed in Table 1.   Table S1. DNA fragments from wild-type and strains 1, 2, 3, and 4 are shown in gel images. DNA sequences were verified.
Se12Bb11sk, Se13Bb11sc, Se23Bb11kc; and a triple integration, Se123Bb111skc. Additional neutral sites could be identified using transcriptome sequencing analysis (Ng et al., 2015). The initial SyneBrick vectors have been successfully applied to metabolic engineering (Choi et al., 2016;Chwa et al., 2016;Lee et al., 2017). To expand a selection of the SyneBrick vectors, LacI-pTrc in the standard SyneBrick vectors pSe1Bb1s-GFP and pSe2Bb1k-eYFP were replaced with transcriptional regulator TetR-pTetA and pT7 promoter, respectively. This constructed strains Se1Bb2s-GFP and Se2Bb7k-eYFP. For interpreting the features of SyneBrick vectors, the nomenclature is Se# for S. elongatus and neutral sites (#) and Bb for BglBrick vectororiginated ( Figure 1B). Annotations of BglBrick vectors are one for the trc promoter, two for the tetA promoter, seven for the T7 promoter, s for spectinomycin, k for kanamycin, and c for the chloramphenicol resistant gene. Name of genes of interest are followed by a dash.
SyneBrick vectors share the features of BglBrick/CoryneBrick vectors. A PCR step is not necessary for gene cloning and multiple gene assembly is possible with repeated enzyme digestions and ligations without choosing unique restriction enzyme sites. The details of the cloning steps have been described (Lee et al., 2011;Kang et al., 2014). In summary: A target gene of interest digested at EcoRI/BamHI sites is ligated to EcoRI/BglII sites of SyneBrick vectors (Table 1) by replacing GFP or eYFP. Each target gene with a BglBrick compatible sequence is sequentially inserted into the SyneBrick vectors in multiple gene clonings. Alternatively, multiple genes built into other BglBrick (Lee et al., 2011) or CoryneBrick vectors (Kang et al., 2014), or BglBrick-formatted genes used in Synechocystis sp. PCC 6803 (Huang et al., 2010) or S. elongatus PCC 7002 (Markley et al., 2015) can be cloned into SyneBrick vectors by one-step digestion and ligation. Target genes must be checked for the presence of EcoRI, BglII, BamHI, and XhoI sites prior to multiple-gene assembly in the vector.

Development of Controllable Gene Expression for S. elongatus PCC 7942
To test for controllable gene expression, GFP-expressing, or eYFP-expressing S. elongatus PCC 7942 strains (Se1Bb1s-eYFP, Se1Bb1s-GFP, and Se1Bb2s-GFP) were constructed after natural transformation with SyneBrick vectors. We tested the wild-type strain with IPTG up to 10 mM and found no growth inhibition (Figure 2). Subsequently, engineered strains were cultivated in BG-11 medium with 5% (v/v) CO 2 bubbling in the presence of IPTG or aTc, depending on the strain. GFP or eYFP fluorescent levels were measured.
IPTG induction up to 10 mM and aTc up to 1 mM did not cause growth inhibition of the engineered strains although slight growth inhibition with 10 mM IPTG or 1 mM aTC was observed in the engineered strains compared to the wild type. Strain Se1Bb1s-None was constructed as a control for calibrating values for gene expression of engineered strains because the wild type shows auto-fluorescence in the range of measurements (McEwen et al., 2013). Compared with a control, strains Se1Bb1s-eYFP, Se1Bb1s-GFP, and Se1Bb2s-GFP showed low levels of leaky GFP/eYFP expression in the absence of inducers (Figure 2B). When 0.01, 0.1, 1, and 10 mM of IPTG was used, higher IPTG concentrations resulted in higher induction of the reporter gene. Increased induction levels over cultivation days were also observed. Compared with 0 mM IPTG, induction of Se1Bb1s-GFP with 1 mM resulted in a 10-fold change in gene expression and 10 mM resulted in an 11-fold change. This result was consistent with the 16-fold induction of β-glucuronidase activity in a reporter assay using an IPTG-inducible pTrc gene expression system in S. elongatus PCC 7942 (Geerts et al., 1995). Compared with Se1Bb1s-GFP, Se1Bb1s-eYFP had a 24-fold higher induction with 1 mM IPTG and 26-fold with 10 mM. This discrepancy could be due to differences in signal intensities between intracellular GFP and eYFP in cyanobacteria. At least 10-fold induction was achieved with either 1 or 10 mM IPTG in S. elongatus PCC 7942 using SyneBrick vectors. For controllable gene expression with SyneBrick vectors with the 1 promoter, 1 mM IPTG induction is recommended.
Se1Bb2s-GFP with the TetR-pTetA transcriptional system was tested with four concentrations of aTc: 10, 100, 200, and 1000 nM in 5% (v/v) CO 2 . The highest induction of 19-fold occurred when 100 nM aTc was used, compared to without aTc ( Figure 2C). However, induction was decreased dramatically when more than 100 nM aTC was used. Thus, 100-nM aTC induction was recommended with SyneBrick vectors with the 2 promoter and 24-h culture. In previous studies, an aTcdependent induction system for Synechococcus sp. PCC 7002 had induction ranges of 6-and 32-fold with 1000 ng/mL ( ∼ = 2200 nM) aTc (Zess et al., 2016) until 48 h. Dynamic ranges of inductions up to 290-fold have been reported for Synechocystis sp. PCC 6803 under light-activated heterotrophic growth with 10 µg/mL ( ∼ = 22,000 nM) aTC for 48 h (Huang and Lindblad, 2013). Consistent with these results, our TetR system showed decreased gene expression after 48 h due to aTc degradation in light. However, our TetR-pTetA systems required much less aTC to induce gene expression, compared with previous studies (Huang and Lindblad, 2013;Zess et al., 2016). Thus, time-dependent gene expression in Se1Bb2s-GFP under light conditions could be possible by adding aTc in a pulse-type control mode. Engineering of our TetR-pTetA transcriptional system in S. elongatus PCC 7942 with light wavelength (i.e., red light or white light) and intensities carefully considered for SyneBrick applications with aTC as a light-sensitive inducer. Sequence modification of the pTetA promoter with tetO operators could alter intrinsic expression and induction of target genes. This could be easily incorporated into SyneBrick platform vectors.

Expanding Synthetic Biology Platforms Using T7 Gene Expression Systems
To narrow the dynamic range of IPTG induction with tunable gene expression in S. elongatus PCC 7942, T7 RNA polymerase (RNAP) and its cognate T7 promoter were developed in SyneBrick vectors (pSe2Bb7k-eYFP and pSe3Bb1c-T7RNP; Figure 3). We tested the possibility of T7 promoter-driven transcription in the absence of T7 RNAP. No gene expression was observed in Se2Bb7k-eYFP under control of the T7 promoter, indicating that the T7 promoter was not recognized by cyanobacterial RNA polymerases (data not shown). Thus, the T7 RNAP gene 1 encoding for T7 RNAP from E. coli BL21(DE3) was FIGURE 3 | Design features of SyneBrick vectors 2.0 for gene expression using the T7 promoter in S. elongatus PCC 7942. (A) Scheme for genetic control of target gene (e.g. eYFP). With IPTG, T7 RNA polymerase (T7 TNAP) was expressed and its cognate T7 promoter was used to express target genes. Sequences of original T7 promoter and sequence variants (T7.3 and T7.4) with binding and strength regions (Temme et al., 2012). (B) Construction of cyanobacterial strains using pSe2Bb7k-eYFP, pSe2Bb7.3k-eYFP, pSe2Bb7.4k-eYFP, and pSe3Bb1c-T7RNAP vectors for expression of eYFP protein under control of T7 variant promoters. Sequencing primers are in Table S1. (C) Gel images of recombinant cyanobacterial strains. Strains were verified by PCR using oligonucleotides (arrows). Sequences are in Table S1. DNA fragments from wild-type and strains 1, 2, and 3 are shown in gel images. DNA sequences were verified. integrated into Se2Bb7k-eYFP using pSe3Bb1c-T7RNP, yielding strain Se23Bb7kc-eYFP/P.
Compared with the LacI-pTrc gene expression system of Se1Bb1s-eYFP, the T7RNAP-T7 gene expression system of Se23Bb7kc-eYFP/P showed efficient induction of target gene with IPTG. Specific fluorescence intensities of Se23Bb7kc-eYFP/P increased 6-fold with 0.01 mM IPTG and 1.5-fold with 0.1 mM compared to Se1Bb1s-eYFP (Figure 4). Thus, induction of the T7RNAP-T7 gene expression system with 0.1 mM IPTG resulted in the same levels of induction as 1 mM IPTG in the LacI-pTrc gene expression system. The efficient induction with the low IPTG concentration could be due to an inherently fast mRNA elongation rate of the T7 RNA polymerase. Decreased gene expression was detected when more than 1 mM IPTG was used. The reason that expression was reduced with high concentrations of IPTG was because the RNA polyadenylation and degradation mechanisms in cyanobacteria are different from E. coli (Rott et al., 2003). Thus, orthogonal expression of the T7 polymerase gene 1 can be alternatively applied by replacing the pTrc promoter with the pTetA promoter to prevent interference with expression from the T7 promoter. The T7RNAP-T7 gene expression system induced well with 0.01 or 0.1 mM IPTG.
In addition, variants of T7 promoters (T7.3 and T7.4) in which a 5-bp strength-determining region was modified (Temme et al., 2012) were replaced with the original T7 promoter to alter promoter strength without RNA specificity (Figure 3). This generated strain Se23Bb7.3kc-eYFP/P with 82% eYFP expression compared to Se23Bb7kc-eYFP/P and Se23Bb7.4kc-eYFP/P, with 90% expression. These reductions in gene expression in cyanobacteria are different from gene expression levels in E. coli, which are 28% for T7.3 and 17% for T7.4 from T7 (Temme et al., 2012). The order of promoter strength using a T7 library in cyanobacteria was consistent with E. coli. Using a library of T7 promoter SyneBrick vectors, the process of refactoring of a gene cluster encoding for complex proteins such as αcarboxysomes would be possible as rewriting the genomes to decipher their functions in cyanobacteria. T7 lysozyme can FIGURE 4 | Gene expression analysis of engineered S. elongatus PCC 7942 using SyneBrick 2.0 vectors. (A) Growth of engineered S. elongatus PCC (pSe23Bb7kc-eYFP/P, pSe23Bb7.3kc-eYFP/P, and pSe23Bb7.4kc-eYFP/P) under 5% (v/v) CO 2 bubbling in BG-11 supplemented with indicated concentrations of IPTG, measured at OD 730 (black square, 0 mM; red circle, 0.01 mM; blue triangle, 0.1 mM; inverted pink triangle, 1 mM; green diamond, 10 mM). (B) Specific fluorescence (intensities per OD 730 ) were calculated for strains. Experiments were performed in triplicate cultures. All data are presented as mean ± standard deviation (SD) from triplicate cultures. also be used as an inhibitor for fine-tuning gene expression of the T7 promoter by controlling T7 RNA polymerase activity.
The development of clustered regulatory interspaced short palindromic repeats (CRISPR) interference technology has allowed control of gene expression by inducing dCas9 encoding genes with aTc. This method has successfully been applied to lactate production from 1% CO 2 (Gordon et al., 2016) and to repress multiple genes in the cyanobacterial genome (Yao et al., 2016). Thus, SyneBrick vectors could be further expanded to precisely control gene expression by integrating CRISPR technology for metabolic engineering of S. elongatus PCC 7942.

CONCLUSION
A synthetic biology platform of SyneBrick vectors for gene expression in S. elongatus PCC 7942 was developed with different gene inducible systems and chromosomal integration sites. LacI-pTrc, TetR-pTetA, T7RAP-T7-based SyneBrick vectors controlled expression of target genes and showed strong gene expression in S. elongatus PCC 7942 under 5% CO 2 . A synthetic T7 promoter library was used for a broad range of gene expression in S. elongatus PCC 7942. Thus, the SyneBrick vectors could be useful for expressing target genes in synthetic biology to engineer S. elongatus PCC 7942 as a biosolar cell factory for directly converting CO 2 to value-added chemicals.

AUTHOR CONTRIBUTIONS
WK and HW designed the research; WK performed the research; WK, SL, YU, SS, and HW analyzed the data; WK and HW drafted the manuscript; WK, SL, YU, SS, and HW contributed to the final manuscript.