Pioneering precision in markerless strain development for Synechococcus sp. PCC 7002

doi:10.21203/rs.3.rs-4897254/v1

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Pioneering precision in markerless strain development for Synechococcus sp. PCC 7002

https://doi.org/10.21203/rs.3.rs-4897254/v1

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Marine cyanobacteria such as Picosynechococcus sp. (formerly called Synechococcus sp.) PCC 7002 are promising chassis for photosynthetic production of commodity chemicals with low environmental burdens. Genetic engineering of cyanobacteria conventionally employs antibiotic resistance markers. However, limited availability of antibiotics is a problem for highly multigenic strain engineering. Although several markerless transformation methods have been developed for PCC 7002, they often lack versatility due to the requirement of gene disruption in the host strain. To achieve markerless transformation in Synechococcus sp. with no requirements for the host strain, this study developed a method in which temporarily introduces a mutated phenylalanyl-tRNA synthetase gene (pheS) into the genome for counter selection. Amino acid substitutions in the PheS that cause high susceptibility of PCC 7002 to the phenylalanine analogue p-chlorophenylalanine were examined, and the combination of T261A and A303G was determined as the most suitable mutation. The mutated PheS-based selection was utilized for the markerless knockout of the nblA gene in PCC 7002. In addition, the genetic construct containing the lldD and lldP genes from Escherichia coli was introduced into the ldhA gene site using the counter selection strategy, resulting in a markerless recombinant strain. The repeatability of this method was also demonstrated, suggesting it will be a powerful tool for multigenic strain engineering of cyanobacteria.

markerless

Synechococcus

phenylalanyl-tRNA synthetase

counter selection

Cyanobacteria are promising photoautotrophic chassis for sustainable chemical production, and they can convert CO₂ directly into the desired compounds. Research on cyanobacteria for the production of alcohols, diols, fatty acids, terpenoids, and organic acids, which are utilized in biofuels and pharmaceuticals, has been actively pursued [1–3]. Understanding and engineering the cyanobacterial metabolism will promote the efficiency of the CO₂ conversion, remove bottlenecks, and introduce the synthetic metabolic pathways to produce chemicals using CO₂ as the sole carbon source. For example, the production of organic acids such as succinate and d-lactate were improved in Synechocystis sp. PCC 6803 (PCC 6803) by the metabolomics-based genetic engineering approach [4, 5]. The heterologous expression of the limonene synthase gene allows for the production of monoterpene limonene in Synechococcus sp. PCC 7002 (PCC 7002), which naturally lacks the ability to produce limonene [6]. PCC 7002 is an ideal host to develop as chassis for chemical production due to its salt, thermo, and extreme high-light tolerances [7, 8]. As a marine strain, PCC 7002 doesn’t need freshwater resources, which is advantageous for mass production of low-value commodities like biofuels. These tolerances of PCC 7002 enable to grow in open pond systems and photobioreactors even in a hot environment of about 40°C [8]. Despite these valuable properties, its potential as a chassis for engineering applications is hindered by the lack of available genetic tools. Cyanobacterial strain development is typically achieved through natural transformation, as well as conjugation and electroporation methods. Regardless of the transformation method employed, all require antibiotic resistance genes for selecting recombinants. The limited number of the available selective markers restricts the genetic manipulation and the constitutive expression of marker genes may hinder growth and production. To address these issues, markerless recombination tools have been developed. As for markerless gene recombination, the SacB-based system was reported in PCC 6803 [9], and the CRISPR-based markerless tools were also reported in PCC 6803, Synechococcus UTEX 2973 (UTEX 2973), and Anabaena sp. PCC 7120 [10–13]. In PCC 7002, Begemann MB et al. reported the markerless gene deletions and insertions by disrupting the acsA gene encoding acetyl-CoA ligase and acrylate counter selection a decade ago [14]. Additionally, Kojima K et al. reported the markerless counter selection using lauric acid by disrupting the aas gene encoding acyl-acyl carrier protein synthetase [15]. These methods require disruption of the native acsA and aas genes to select mutants and lack versatility.

To establish the chassis strain, a markerless tool is needed where gene integration sites can be freely selected without disrupting specific native genes. Because the SacB-based system, utilizing sucrose counter selection, cannot be introduced to PCC 7002 [14], and the CRISPR-based system is more complex than current markerless tools for PCC 7002, we focused on PheS as a new markerless recombination system in cyanobacteria. Our aim is to establish a straightforward and effective markerless tool. The PheS-based markerless systems have been reported in various bacteria, including Thermus thermophilus [16], Bacillus [17, 18], lactic acid bacteria [19], and Streptococcus species [20–22]. Its initial report was in Burkholderia species [23], and markerless systems using mutated PheS have been developed over the last decade. The pheS gene encodes the alpha subunit of phenylalanyl-tRNA synthetase, which plays a crucial role in catalyzing the aminoacylation of tRNA and the charging of tRNA^Phe with phenylalanine. This process is essential for peptide bond formation. In Escherichia coli, an amino acid substitution at position A294G in the PheS enzyme enables the misincorporation of p-chlorophenylalanine (PCPA), a phenylalanine analog [24]. This leads to protein misfolding and either growth arrest or reduction. Miyazaki reported that mutations at positions T251A/A294G in E. coli PheS resulted in increased lethality in the presence of PCPA [25]. In this study, our objective was to establish a universal and straightforward markerless transformation method in PCC 7002 using mutated PheS. We aimed to delete the nblA gene, which encodes nonbleaching protein A, and performed markerless gene introduction at the ldhA gene site, which encodes lactate dehydrogenase. The newly developed markerless gene knockout and knockin method will not only help uncover the functions of unknown genes and metabolic regulations but also expedite the process, facilitating high-throughput modifications and rapid advancements in both research and production strategies.

Strains and culture conditions

Cyanobacterium Synechococcus sp. PCC 7002 and its recombinants were cultured in MA2 medium (1.49 g/L NaNO₃, 50 mg/L KH₂PO₄, 18 g/L NaCl, 5 g/L MgSO₄·7H₂O, 0.37 g/L CaCl₂·2H₂O, 0.6 g/L KCl, 32 mg/L Na₂EDTA·2H₂O, 8 mg/L FeCl₃·6H₂O, 34 mg/L H₃BO₃, 4.3 mg/L MnCl₂·4H₂O, 0.32 mg/L ZnCl₂, 50 µg/L Na₂MoO₄·2H₂O, 3.0 µg/L CuSO₄·5H₂O, 12 µg/L CoCl₂·6H₂O, 4.0 µg/L cobalamin, and 1 g/L tris(hydroxymethyl)aminomethane) with or without antibiotics or p-chlorophenylalanine (PCPA) (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan). The MA2 agar plate contained 1.5% agar. Cyanobacterial cells were cultured in 70 mL of MA2 in closed double-deck flasks with 2% (v/v) CO₂ at 30°C under a white fluorescence with continuous irradiation at 100 µmol/m²/s as previously described [26]. The optical density at 750 nm (OD₇₅₀) measured with a UV mini spectrophotometer (Shimadzu, Kyoto, Japan), and the cell density in the medium was also determined as dry cell weight (DCW). The cyanobacterial recombinants used in this study were listed in Table 1.

Table 1

Recombinants of *Synechococcus* sp. PCC 7002 used in this study.
Strain	Genotype	Description
Syn001	ΔnblA::KmR-wtPheS	Replacement of the nblA gene with genes for kanamycin resistance and native PheS
Syn002	ΔnblA::KmR-PheS^T261A	Replacement of the nblA gene with genes for kanamycin resistance and mutated PheS (T261A)
Syn003	ΔnblA::KmR-PheS^A303G	Replacement of the nblA gene with genes for kanamycin resistance and mutated PheS (A303G)
Syn004	ΔnblA::KmR-PheS^T261A/A303G	Replacement of the nblA gene with genes for kanamycin resistance and mutated PheS (T261A/A303G)
Syn005	ΔnblA	Markerless replacement of the nblA gene with a small fragment containing a NdeⅠ site
Syn006	ΔldhA::lldD-lldP	Markerless replacement of the ldhA gene with the lldD and lldP genes
Syn007	ΔldhA::lldD-lldP, ΔnblA::mNG	Derivative strain of Syn006 with markerless replacement of the nblA gene with the gene for mNeonGreen

Escherichia coli strain DH5α was used for gene cloning and plasmid construction. E. coli was grown in LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) at 37°C under 200 rpm agitation.

Chemicals, enzymes, and molecular biology kits

PCPA, kanamycin monosulfate, and gentamicin sulfate (Nacalai Tesque, Kyoto, Japan) were used for mutant selection, DNA polymerase for polymerase chain reaction (PCR) and In-Fusion Snap Assembly Master Mix were purchased from TOYOBO CO., LTD. (Osaka, Japan) and TaKaRa Bio Inc. (Shiga, Japan), respectively. Restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). DNA purification was performed using Wizard^®ฎ Plus SV Miniprep DNA Purification Systems and Wizard^®ฎ SV Gel and PCR Clean-Up Systems (Promega Corporation, Madison, WI, USA).

Plasmid construction

The plasmid pUC118-KmR-PheS was constructed by assembling the linearized pUC118 by double digestion of EcoRⅠ and SmaⅠ, the kanamycin resistance gene, a J23119 promoter (http://parts.igem.org/Part:BBa_J23119), the pheS gene, and the upstream and downstream regions of the nblA gene (Gene ID: SYNPCC7002_A1821) via In-Fusion cloning. The mutated pheS genes, encoding the α-subunit of phenylalanyl-tRNA synthetase (PheST261A, PheSA303G, PheST261A/A303G), were generated by PCR from pUC118-KmR-PheS. The PCR products amplified with PheS_T261A_F (5′-agctacttccccttcGCCgaaccttccgccgaa-3′) and PheS_T261A_R (5′-ttcggcggaaggttcGGCgaaggggaagtagct-3′), and PheS_A303G_F (5′-gtttatacgggcttcGGCgctgggctaggggta-3′) and PheS_A303G_R (5′-tacccctagcccagcGCCgaagcccgtataaac-3′) were self-ligated by In-Fusion cloning, resulted in pUC118-KmR-PheS_T261A and pUC118-KmR-PheS_A303G, respectively. The plasmid, pUC118-KmR-PheS_T261A/A303G, was constructed by self-ligation of the PCR product amplified from pUC118-KmR-PheS_T261A using primers PheS_A303G_F and PheS_A303G_R via In-Fusion cloning.

To construct the markerless knockout plasmid pUC118-nblA-KO, the linearized pUC118 was combined with the upstream and downstream regions of the nblA gene. An NdeⅠ site was inserted between these upstream and downstream regions.

The plasmid pUC118-GmR-PheS was constructed by replacing the kanamycin resistant gene in pUC118-KmR-PheS_T261A/A303G with a gentamicin resistant gene. To create the knockin plasmid for deleting the ldhA gene and introducing the lldD (NCBI reference sequence: NP_418062.1) and lldP (NCBI reference sequence: NP_418060.1) genes, which encode NAD-independent l-lactate dehydrogenase and lactate permease from Escherichia coli, respectively, the following components were assembled: the linearized pUC118-GmR-PheS (digested with SacⅠ and XbaⅠ), the upstream and downstream regions of the ldhA gene, the lldD and lldP genes, and a 400 bp homologous region of the lldP gene, resulting in pUC118-ldhA-KO-lldD-lldP.

Another knockin plasmid, pUC118-nblA-KO-mNG, was constructed for the deletion of the nblA gene and the introduction of the gene for mNeonGreen. This was achieved by inserting the mNeonGreen gene, the gentamicin resistance gene, the PheS_T261A/A303G gene and a 400 bp homologous region of the mNeonGreen gene into the NdeⅠ-digested pUC118-nblA-KO.

Construction of recombinant strains

Cells were cultivated until reaching an OD₇₅₀ of approximately 1.0. Subsequently, 100 µL of the culture was mixed with plasmid DNA containing the recombination fragment. The mixed culture was incubated on a rotator for 24 hours under shading. After the shading, the culture was spread on an immobilon nitrocellulose membrane (HATF08250, Merck KGaA, Darmstadt, Germany) on the agar plate and incubated at 30°C for 2 days. The membrane was transferred to the agar plate containing 100 µg/mL kanamycin or 40 µg/mL gentamicin and incubated until colonies appeared. The colonies were cultured, and transformation and segregation were confirmed by PCR.

To obtain a markerless nblA-deficient strain, a liquid culture of the recombinant strain Syn004 was mixed with pUC118-nblA-KO. After incubation in the dark, the mixture was spread on agar plate containing 20 µg/mL PCPA and incubated. The resulting colonies were then streaked onto another agar plate with 20 µg/mL PCPA. Transformation and segregation were confirmed by PCR.

For markerless knockin mutants, complete segregation was confirmed by PCR after antibiotic selection. The recombinants were cultivated in antibiotic-free medium until reaching an OD₇₅₀ of approximately 1.0. Subsequently, 200–300 µL of the culture was spread on the agar plate containing 20 µg/mL PCPA and incubated until colonies appeared. These colonies were then streaked onto an agar plate containing 20 µg/mL PCPA. The elimination of the marker genes was confirmed by PCR.

Measurement of lactate

Measurement of lactate was performed using high performance liquid chromatography (HPLC) as previously described [26]. Briefly, cultures were centrifuged at 8,000 × g for 10 min, and supernatants were subjected to HPLC. l-Lactic acid (FUJIFILM Wako Pure Chemical Corporation) was used as the quantitative standard to determine the lactate concentration using a calibration curve. The HPLC conditions were as follows: column, Aminex®HPX-87H column (Bio-Rad, Hercules, CA); column temperature, 50°C; mobile phase, 5 mM sulfuric acid; flow rate, 0.6 mL min^–1; detector, refractive index detector.

Selection of the mutated PheS and PCPA titration

The scheme of markerless transformation method is shown in Fig. 1. At the first step, the plasmid containing an antibiotic resistance gene and mutated pheS gene is integrated by replacing the target gene for disruption in the genome via conventional double crossover method at the upstream and downstream region of the target gene. The recombinants are selected with antibiotic tolerance. Then, the recombinants are re-transformed and selected with PCPA. To achieve markerless transformation in PCC 7002, both PCPA susceptibility and a functional mutated PheS specific to this strain are essential. We sought a suitable mutation in PheS to confer high PCPA susceptibility to PCC 7002. Based on previous reports by Kast et al. [27] and Miyazaki et al. [25], amino acid substitutions T261A and A303G were identified as potential mutations for enhancing PCPA susceptibility in E. coli. Considering the high amino acid sequence similarity between PheS in E. coli and PCC 7002 (69% similarity, as shown in Supplemental Fig. S1), we anticipated that these mutations would render PCC 7002 PheS susceptible to PCPA. To examine these candidate mutations, the mutated pheS genes were introduced into PCC 7002 cells with the kanamycin resistance marker. The nblA gene site was selected for markerless gene disruption since the phenotype of its disruption recombinant was studied in other cyanobacteria and it was not lethal [28, 29]. We obtained four recombinants: Syn001 (ΔnblA::KmR-wtPheS), Syn002(ΔnblA::KmR-PheS^T261A), Syn003 (ΔnblA::KmR-PheS^A303G), and Syn004 (ΔnblA::KmR-PheS^T261A/A303G), and confirmed complete segregation (Fig. 2a). Subsequently, these recombinants were spotted on agar plates containing PCPA to assess their susceptibility. The wild-type strain and Syn001 grew in the presence of 20 µg/mL PCPA (Fig. 2b). Syn002 exhibited greater susceptibility to PCPA than Syn001 but was more tolerant than Syn003 and Syn004, as both recombinants did not grow with 20 µg/mL PCPA. The appropriate PCPA concentration was 20 µg/mL. At 15 µg/mL PCPA, the susceptibility was highest in Syn004, followed by Syn003 and Syn002. Based on their growth in the presence of 15 µg/mL PCPA, the double amino acid substitutions of T261A and A303G were identified as suitable mutations that confer high susceptibility to PCPA in PCC 7002.

Establishment of the markerless knockout system

To establish the markerless knockout system, we targeted the nblA gene, which encodes the NblA (nonbleaching protein A) protein responsible for degradating phycobilisome under nitrogen- or sulfur-deprived conditions [27]. In some cyanobacteria such as UTEX 2973, deletion of the nblA gene strain results in a non-bleaching phenotype upon sulfur depletion [29] and decreased cell growth [28, 29]. We constructed a pUC118-nblA-KO plasmid designed specifically for markerless deletion of the nblA gene. This plasmid introduced an NdeⅠ site into the nblA gene, simplifying the confirmation of transformation. With the pUC118-nblA-KO plasmid, Syn004 was transformed (Fig. 3a), and the ΔnblA recombinant which lost marker genes in the genome was obtained. The elimination of the marker genes and complete segregation were confirmed by PCR (Fig. 3b). As a result, the markerless ΔnblA recombinant, Syn005, was obtained, and the markerless knockout system was successfully established.

Establishment of the markerless knockin system

Since we could not obtain the gene knockin recombinant using our original markerless knockin scheme shown in Fig. 1b, we developed an improved markerless knockin system shown in Fig. 4. In this new system, the marker genes are flanked by 400 bp homologous regions derived from the target gene for introduction. The cells were transformed via double crossover recombination, and the recombinants were selected based on antibiotic tolerance. The culture of the recombinants was then spread on the agar plate containing 20 µg/mL PCPA. Under the selection pressure of PCPA, homologous recombination occurred at the 400 bp homologous regions, resulting in recombinants where the marker genes were eliminated from the genome. To demonstrate our markerless knockin system, the genetic construct containing the lldD and lldP genes from Escherichia coli, which confers l-lactate assimilation ability to PCC 7002 [26], was introduced at the ldhA gene site as the model (Fig. 5a).

The pUC118-ldhA-KO-lldD-lldP plasmid was constructed for the markerless transformation. After introducing the pUC118-ldhA-KO-lldD-lldP plasmid into PCC 7002 by natural transformation, the candidates of the first-step recombination were selected with gentamicin, and then the integration and complete segregation were confirmed by PCR. The recombinant was subjected to the second-step recombination with PCPA selection. The complete deletion of the marker genes, gentamicin resistant gene and the mutated pheS gene, was confirmed by PCR (Fig. 5b). The phenotype of l-lactate consumption was confirmed by culturing in the presence of l-lactate (Fig. 5c). Then, the markerless l-lactate-consuming recombinant, Syn006, was obtained, and our markerless transformation system developed in this study was also shown to be available for gene knockin.

To demonstrate the usability of our markerless system developed in this study, Syn006 was transformed using the plasmid, pUC118-nblA-KO-mNG (Fig. 6a). After PCPA selection, the complete deletion of the marker genes, gentamicin resistant gene and the mutated pheS gene, was confirmed by PCR (Fig. 6b). The fluorescence of mNeonGreen was also observed in the recombinants (Supplemental Fig. S2). Therefore, the double markerless recombinant, Syn007, was successfully obtained, and our system enabled to transform PCC 7002 and its recombinants repeatedly.

In this study, we established a markerless transformation method in PCC 7002 using mutated PheS-based counter selection. This approach doesn’t require the disruption of specific genes and allows for unrestricted gene manipulation without relying on a limited number of available antibiotic resistance genes. Previous markerless methods for PCC 7002 required disrupting native acsA and aas genes. However, deleting the acsA gene increases acetate secretion[14], wasting carbon resources, while the aas gene is vital for acclimating Synechococcus elongatus strain PCC 7942 (PCC 7942) to high-light conditions [30]. Disrupting these genes may hinder PCC 7002 from becoming a production chassis strain, but the method developed in this study circumvents this issues. Unlike previous tools using acrylate or lauric acid-counter selection, which involve two transformation steps with two plasmids for gene knockout and gene knockin at multiple gene sites, our tool for gene knockin achieves this with a single transformation step and plasmid. This simplicity is advantageous for faster and more simple recombination when establishing various recombinants in PCC 7002. We employed the pheS gene as a counterselection marker for the first time in cyanobacteria. The pheS gene of PCC 7002 was utilized to obtain mutated phenylalanyl-tRNA synthetase gene, which confers PCPA susceptibility to the cyanobacterium. There was a concern regarding the replacement of the native pheS gene, as there was a possibility that the desired recombinants might not be obtained. Indeed, Xie et al. mentioned the high rate of recombination between the mutated pheS and native pheS genes in Streptococcus mutans [20]. In various bacteria, mutated and synthetic pheS genes with coding sequences comprised of synonymous codons have been used to reduce homology [16, 20, 23, 31]. However, in this study, this was not a problem, as the recombinants in which native PheS was replaced by the mutated PheS could not survive under the present PCPA selection condition, and the desired recombinants were obtained satisfactorily.

Generally, false positives often appear in the transformations of PCC 7002. In our markerless transformation scheme, we theoretically should obtain only the desired recombinants through PCPA selection. In this study, we also obtained 3 types of false positives. One was a recombinant still harboring the mutated pheS gene, which probably acquired PCPA-resistance through unintended mutations before 2nd recombination step. The others were a wild-type revertant and a heterozygous one with the wild-type and the desired genomic regions. It probably resulted from the incomplete segregation. Since false positives are excluded by PCR, the occurrence of such undesired recombinants is not a significant issue. In cyanobacteria, the SacB-based markerless system was previously reported only in PCC 6803 [9], and its usability depended on bacterium. However, the PheS-based system shows potential for widespread use due to its applicability in bacteria that cannot utilize sucrose counterselection. Recognizing the importance of robust expression of the mutated pheS gene for PCPA susceptibility [19], we employed the endogenous PheS and strong promoter. In E. coli PheS, threonine 251 plays a role in substrate recognition [32], while the substitution of alanine 294 with glycine enlarges the cavity for the para position of the aromatic ring, thereby increasing aminoacylation of tRNA using PCPA as the substrate [24]. In this study, we examined amino acid substitutions in PheS in PCC 7002. Our findings suggest that the A303G substitution is crucial for PCPA susceptibility, with the additional amino acid substitution of T261A enhancing susceptibility.

In conclusion, this study successfully established a novel markerless transformation method in PCC 7002 using the mutated pheS gene derived from PCC 7002, which was demonstrated to be effective for both gene knockout and knockin. This method does not require the disruption of specific genes, thus allowing for markerless transformation without limitations on the host PCC 7002. Moreover, our tool can be easily applied to other cyanobacteria, such as PCC 6803 and PCC 7942, which are naturally transformable like PCC 7002. The markerless transformation method is crucial as a foundational technology for developing a chassis strain, as it enables genome engineering without being constrained by marker genes. Particularly in the situations requiring gene introduction at multiple sites, our tool will prove to be an invaluable asset.

Acknowledgements

We thank Prof. Keiji Matsumoto of Kobe University for valuable suggestions.

Funding

This research was supported by the Program for Forming Japan’s Peak Research Universities (J-PEAKS) from the Japan Society for the Promotion of Science (JSPS), Invitational Fellowships for Research in Japan from JSPS, the Advanced Low Carbon Technology Research and Development Program of the Japan Science and Technology Agency (JST-ALCA) (JPMJAL1608), Kobe University Strategic International Collaborative Research Grant (Type B Fostering Joint Research), and the Cabinet Office, Government of Japan, Moonshot Research and Development Program for Agriculture, Forestry and Fisheries (funding agency: Bio-oriented Technology Research Advancement Institution)

Authors’ contributions

A.T. designed the research, performed experiments, wrote the original draft. K.I. designed the research and performed experiments. R.H. designed the research and edited manuscript. Y.K and L.D edited the manuscript. T.H. and A.K. supervised the project. All authors reviewed and consented to the manuscript.

Availability of data and materials

The data and materials supporting the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

Not applicable

Consent for publication

All authors approved the manuscript.

Competing interests

The Authors declare no conflicts of interest.

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Pioneering precision in markerless strain development for Synechococcus sp. PCC 7002

Status:

Version 1

Abstract

Figures

Introduction

Method

Strains and culture conditions

Chemicals, enzymes, and molecular biology kits

Plasmid construction

Construction of recombinant strains

Measurement of lactate

Result

Selection of the mutated PheS and PCPA titration

Establishment of the markerless knockout system

Establishment of the markerless knockin system

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1