Abstract
Nattokinase (NK) is effective in the prevention and treatment of cardiovascular disease. Cucumber is rich in nutrients with low sugar content and is safe for consumption. The aim of this study was to construct a therapeutic cucumber that can express NK, which can prevent and alleviate cardiovascular diseases by consumption. Because the Bitter fruit (Bt) gene contributes to bitter taste but has no obvious effect on the growth and development of cucumber, so the NK-producing cucumber was constructed by replacing the Bt gene with NK by using CRISPR/Cas9. The pZHY988-Cas9-sgRNA and pX6-LHA-U6-NK-T-RHA vectors were constructed and transformed into Agrobacterium tumefaciens EHA105, which was transformed into cucumber by floral dip method. The crude extract of NK-producing cucumber had significant thrombolytic activity in vitro. In addition, treatment with the crude extract significantly delayed thrombus tail appearance, and the thrombin time of mice was much longer than that of normal mice. The degrees of coagulation and blood viscosity as well as hemorheological properties improved significantly after crude extract treatment. These findings show that NK-producing cucumber can effectively alleviate thrombosis and improve blood biochemical parameters, providing a new direction for diet therapy against cardiovascular diseases.
1 Introduction
Cardiovascular diseases (CVDs), encompassing a wide range of disorders including coronary heart disease, hypertension, cardiac hypertrophy, and heart failure, are the main cause of death and disability worldwide (Wu et al., 2020). Nattokinase (NK), a serine protease secreted by Bacillus subtilis var. natto found in the Japanese traditional food natto (Suzuki et al., 2022), possesses a variety of key beneficial effects on cardiovascular system due to its strong anti-thrombus and thrombolytic activity (Wu et al., 2019). However, the smell and viscous texture of natto are not accepted by most people, and other components in natto limit the function of NK (Nishinari et al., 2018; Liu et al., 2021).
The expression of recombinant proteins in plant systems has been promoted as a cost-effective production platform (Schillberg et al., 2019), e.g., the production of tissue plasminogen activator (Abdoli Nasab et al., 2016), LTB-VP6 (Jin et al., 2022), and HBc-based virus-like particles (Moradi Vahdat et al., 2021). Cucumber is a common economic crop plant grown around the world (Rajab et al., 2023). Because cucumber is rich in vitamins, protein, inorganic salt and low in sugar (Liu et al., 2022), it is safe for patients with CVDs. The Bt gene plays an important role in the formation of bitterness in cucumber (Shang et al., 2014). The knockout of Bt has no effect on the growth and development of cucumber and could improve fruit texture. CRISPR/Cas9-mediated gene editing has been successfully applied to bacterial, yeast, plant, and mammalian cells (Schmidt et al., 2023). This approach can be used to edit the genome at a fixed point, avoiding the destruction of endogenous genes caused by the random insertion (Wen and Zhang, 2022). Therefore, the NK gene was inserted into the cucumber genome by using CRISPR/Cas9 to stably express NK and evaluate its physiological function. This study is expected to provide a new strategy for the adjuvant prevention and treatment of CVDs.
2 Materials and methods
2.1 pZHY988-Cas9-sgRNA vector preparation
According to the sequence of the cucumber Bt gene, single-guide RNAs (sgRNAs) were designed using BioTools (http://biootools.com/). The BsaI site was added to the sgRNA and two complementary sgRNA oligos were synthesised. pZHY988-Cas9 vector was linearised by BsaI (Takara, Japan). The sgRNA was cloned into the linearised pZHY988-Cas9 vector under the action of T4 DNA ligase (Takara, Japan).
2.2 Point mutation in the NK gene
B. subtilis natto (BNCC 185324) was used to obtain the NK gene. Since the start codon of NK in the B. subtilis natto genome is GTG, it must be replaced with ATG for expression in eukaryotes. Therefore, the primers NK-M1 and NK-M2 were designed to mutate the start codon of NK.
2.3 Construction of the PX6-LHA-U6-NK-T-RHA vector
The donor gene LHA-U6-NK-T-RHA was constructed by overlap PCR and connected to the pMD-19T vector. All primers used in this study are shown in Supplementary Table 1. (Supplementary files are available on the server of the Publisher). The LHA-U6-NK-T-RHA and (Marker-free) PX6 vectors were digested by XhoI and PacI. The purified LHA-U6-NK-T-RHA and the linearised pX6 fragments were ligated by T4 DNA ligase.
2.4 Transformed pZHY988-Cas9-sgRNA and pX6-LHA-U6-NK-T-RHA vectors into Agrobacterium tumefaciens EHA105
The freezing and thawing method was used to transfer PZHY988-Cas9-sgRNA and pX6-LHA-U6-NK-T-RHA vectors into Agrobacterium tumefaciens EHA105. The transformed cells were cultured on LB solid medium containing 50 mg mL−1 rifampicin and 50 mg mL−1 kanamycin at 28 °C for 48 h. The transformants were randomly selected for PCR identification.
2.5 Genetic transformation of cucumber by the floral dip method
Cucumber seedlings were quickly obtained by soaking the South China D9302 seeds in water (1:4, v/v) at 55 °C and stirring continuously. All female flowers were selected as genetic transformation receptors for the transformation. Female flowers 5–6 days before bloom were selected, and the A. tumefaciens EHA105 infection solution was dipped on the bud and ovary of these female flowers. It was ensured that the smear amount was large enough to drop the infection solution. The procedure was repeated again after 3 days. One day before flowering, the male flowers and infected female flowers were bagged and isolated, and the artificial pollination was conducted the next day. Cucumbers were picked and seeds were collected when the cucumbers were ripe (T0). Primers were designed according to the target sequences for PCR detection. The positive control (CT) was the PCR product obtained using pZHY988-Cas9 as template, and the negative control (WT) was the PCR product obtained using the wild-type cucumber genome as the template. The positive transgenic cucumber seeds of T0 were continuously planted according to the above method and T1 fruits were obtained for functional analyses.
2.6 Extraction and analysis of the crude extract of NK-producing cucumber
The fresh cucumber was cut into small pieces and ground into powder. The powder was mixed with PBS and centrifuged at 12,000 r.p.m. at 4 °C for 30 min. NK produced by cucumber was purified by hydrophobic chromatography and gel-filtration chromatography. The crude extract was precipitated using 60% (NH4)2SO4 and collected by centrifugation. Then, the crude extract was passed in PBS through a Sephadex G-75 column (Yuanye, Shanghai, China). Finally, the crude extract was transferred into a dialysis bag (molecular weight 8,000–14,000 Da, Yuanye, China) and put into distilled water at 4 °C for 24 h. The samples after dialysis were freeze-dried and subjected to SDS-PAGE to evaluate the purity. The activity of the crude extract was detected using the Botany Nattokinase ELISA Kit (Xinyu Biology, Shanghai, China) according to the manufacturer's instructions.
2.7 Thrombolytic effects of the crude extract of NK-producing cucumber in vitro
Note: ma is the mass before dissolution/g; mb is the mass after dissolution/g.
2.8 Thrombus induction and NK-producing cucumber crude extract treatment in vivo
Male Kunming (KM) mice, 4 weeks of age, specific pathogen-free, were purchased from the Laboratory Animal Center of Xuzhou Medical University. After acclimatised for 1 week, the KM mice were randomly divided into four groups: (1) normal control (NC, n = 5), normal mice treated with saline, (2) negative group (NG, n = 5), intraperitoneal injection of carrageenan (50 mg kg−1) and treated with saline, (3) positive group (PG, n = 5), intraperitoneal injection of carrageenan (50 mg kg−1) and treated with nattokinase (8,000 FU kg−1), and (4) cucumber treatment group (CG, n = 5), intraperitoneal injection of carrageenan (50 mg kg−1) and treated with crude extract (8,000 IU kg−1). The time of appearance and length of the thrombus tail within 24 h were recorded. The blood was collected and the thrombin time was measured after 48 h of treatment. Fibrinogen was measured using ELISA kits (TIANGEN, Beijing, China) according to the manufacturer's instructions. The mouse tail was cut and embedded in paraffin. HE staining was used to assess the antithrombotic ability of the crude extract. The animal study protocol was approved by the Experimental Animal Ethics Committee of Xuzhou Medical University (approval number: L20210226457).
2.9 Statistical analysis
Values are presented as means ± standard deviation (SD). The data analysis was performed using SPSS program version 17. Student's t-test was used to determine differences between two groups. Multiple groups were compared by ANOVA with Dunnett's pair-wise comparisons. A value of P < 0.05 was considered statistically significant.
3 Results and discussion
3.1 pZHY988-Cas9-sgRNA vector construction
According to the cucumber Bt gene, 12 potential sgRNAs were designed. Considering the target site on the CDS of the Bt gene, the second sgRNA was selected for further analyses (Supplementary Table 2). The BsaI site was added to both ends of sgRNA by oligo synthesis. The pZHY988-Cas9 vector and sgRNA were simultaneously digested with BsaI and then connected with T4 DNA ligase. Colony PCR revealed that the length of amplification product was consistent with the expected fragment size (Fig. 1a). Sequencing results also supported the successful construction of the pZHY988-Cas9-sgRNA vector (Fig. 1b).
Construction of the pZHY988-Cas9-sgRNA vector. a. Colony PCR of the pZHY988-Cas9-sgRNA vector. Lane M: DL15000 DNA marker; Lane 1: plasmid of pZHY988-Cas9-sgRNA; Lane 2: Colony PCR products to verify pZHY988-Cas9-sgRNA construction. b. Diagram of the pZHY988-Cas9-sgRNA vector
Citation: Acta Alimentaria 52, 1; 10.1556/066.2022.00231
3.2 Point mutation in the NK gene
A single colony was randomly selected and genomic DNAs were extracted for PCR detection. The PCR product lengths were consistent with the length of the NK gene (1146 bp) (Supplementary Fig. 1a). The positive colonies were sequenced, and the sequences were consistent with the NK sequence obtained in a search against the NCBI database. The initiation codon GTG was successfully mutated to ATG (Supplementary Fig. 1b).
3.3 Construction of the pX6-LHA-U6-NK-T-RHA vector
Overlap PCR results showed that the sizes of LHA-U6 (557 bp), T-RHA (580 bp), LHA-U6-NK (1702 bp), and LHA-U6-NK-T-RHA (2282 bp) were consistent with the expected sizes (Supplementary Fig. 2). The purified LA-U6-NK-T-RA and linearised pX6 fragments were digested by XhoI and PacI and ligated by T4 DNA ligase. LHA-U6-NK-T-RHA and pX6 were successfully connected, as determined by XhoI and PacI digestion (Fig. 2a). The PCR products of full-length LA-U6-NK-T-RHA (2282 bp) were also verified by agarose gel electrophoresis (Fig. 2b).
Construction of the vector of pX6-LHA-U6-NK-T-RHA. a. Diagram of the pX6-LHA-U6-NK-T-RHA vector. b. Verification of the pX6-LHA-U6-NK-T-RHA vector. Lane M: DL15000 DNA marker; Lane 1: Recombinant vector of pX6-LHA-U6-NK-T-RHA; Lane 2: Enzyme digestion of the pX6-LHA-U6-NK-T-RHA vector by XhoI and PacI; Lane 3: Colony PCR products to verify pZHY988-Cas9-gRNA construction; Lane 4. pX6 vector
Citation: Acta Alimentaria 52, 1; 10.1556/066.2022.00231
3.4 Transformation of pZHY988-Cas9-sgRNA and pX6-LHA-U6-NK-T-RHA vectors into A. tumefaciens EHA105
Transformants showing double resistance were randomly selected for colony PCR detection. The lengths of the PCR products were nearly 400 bp and 2600 bp, consistent with the expected lengths (Supplementary Fig. 3). These results indicate that the pZHY988-Cas9-sgRNA and pX6-LHA-U6-NK-T-RHA vectors were successfully transformed into A. tumefaciens EHA105.
3.5 Genetic transformation of cucumber and genotype analysis
The cucumber cultivation and transformation processes are shown in Supplementary Fig. 4. As many female flowers were chosen as possible to improve the success rate of floral dip transformation. The fruits were picked and seeds were harvested when the cucumbers were ripe. A total of 13 cucumber fruits were harvested and 83 cucumber seeds were obtained. All harvested seeds were cultivated, and 68 seedlings survived (T0). Cucumber genomic DNA was extracted and primers were designed according to the Cas9 sequence for PCR verification. As shown in Fig. 3a (Lanes 2, 4, 5, and 6), NK-producing cucumbers were obtained.
PCR detection of T0 plants and purification of crude extract. a. Lane M: DL2500bp DNA Marker; Lane CT: PCR products of NK; Lane WT: Wild-type cucumber; Lanes 1-6: NK-producing cucumber. b. SDS-PAGE of purified crude extract. Lane M: protein Marker; Lane 1: (NH4)2SO4 precipitation of the crude extract; Lane 2: chromatography of the crude extract; Lane 3: crude extract, unpurified
Citation: Acta Alimentaria 52, 1; 10.1556/066.2022.00231
3.6 Purification of NK
NK produced by cucumber was purified by hydrophobic chromatography and gel-filtration chromatography. As shown in Fig. 3b, the crude extract yielded many bands after purification by (NH4)2SO4 precipitation (Lane 1). The bands representing impurities were reduced after purification by gel-filtration chromatography (Lane 2), and the molecular weight of the target protein was approximately 28 kDa, consistent with that of NK (Fig. 3b).
3.7 Thrombolytic activity of the crude extract of NK-producing cucumber in vitro
NK activity of the crude extract was 202.29 ± 30.70 IU mg−1, equivalent to 7.86 ± 1.10 IU g−1 (fresh cucumber). Different doses of NK were added to blood clots and dissolution was detected. As shown in Table 1, the thrombolytic effect of the crude extract increased as the concentration increased, and there was significant difference between NK groups and the control group (P < 0.001). In addition, with the extension of the reaction time, the dissolution rate for each concentration increased and was higher than that of the control group (P < 0.001). These results showed that the crude extract had significant thrombolytic activity in vitro.
Thrombolytic effects of the crude extract of NK-producing cucumber in vitro
| Group | Crude extract (mg) | ma (g) | mb (g) | Dissolution (%) |
| Control (5h) | 0 | 0.49 ± 0.015 | 0.36 ± 0.033 | 25.69 ± 7.79 |
| Control (10h) | 0 | 0.52 ± 0.033 | 0.32 ± 0.023 | 37.17 ± 7.0 |
| Crude extract (5h) | 10 | 0.51 ± 0.042 | 0.20 ± 0.019 | 59.51 ± 4.47*** |
| Crude extract (5h) | 20 | 0.48 ± 0.063 | 0.14 ± 0.016 | 70.59 ± 5.58*** |
| Crude extract (10h) | 10 | 0.50 ± 0.047 | 0.19 ± 0.022 | 62.35 ± 1.52*** |
| Crude extract (10h) | 20 | 0.52 ± 0.072 | 0.076 ± 0.011 | 85.05 ± 3.04*** |
ma: the mass before dissolution/g; mb: the mass after dissolution/g; ***: P < 0.001 vs. control (5 h) or control (10 h).
3.8 Thrombolytic effects of NK-producing cucumber crude extract in vivo
Thrombin activity is an important factor causing thrombosis, and the thrombin time reflects the level of thrombin activity. In this study, the crude extract could significantly delay the appearance of the thrombus tail, and the thrombus length was significantly shorter than that of the control group (2.61 ± 0.40 cm vs. 5.22 ± 0.30 cm, P < 0.001) (Fig. 4a). After treatment with the crude extract of NK for 48 h, the thrombin time of mice was much longer than that of normal mice (12.71 ± 0.50 s vs. 7.99 ± 0.31 s, P < 0.001) (Fig. 4b). However, there was no significant difference in fibrinogen levels among the groups (Fig. 4c). Histomorphology of the thrombus tail showed that the arterial intimal became thicker, the lumen diameters increased, and the vessels were filled with blood clots in NG group compared with corresponding parameters in the NC group. Consistent with the changes in the PG group, crude extract treatment significantly reduced the degrees of coagulation and blood viscosity, inhibited intimal thickening, and improved hemorheological properties (Fig. 4d).
Effects of the crude extract on the thrombus length (a), thrombin time (b), fibrinogen levels (c), and histopathological characteristics (d) of the thrombus tail. Blood was collected to determine the thrombin time and fibrinogen concentration after treatment with the crude extract of NK for 48 h. The tails of mice were collected for a histopathological analysis after carrageenan injection for 24 h. ***: P < 0.001 vs. control. Bars correspond to 50 μm
Citation: Acta Alimentaria 52, 1; 10.1556/066.2022.00231
3.9 Discussion
Nattokinase is a thrombolytic drug with advantages over urokinase, streptokinase, and tissue plasminogen activator, including a low price, high efficiency, and lack of toxicity (Zhou et al., 2021). However, its taste limits its consumption. In this study, an adjuvant treatment scheme for CVD through diet therapy was designed by replacing the Bt gene with the NK gene by using CRISPR/Cas9, a simple, efficient, and versatile system in cucumber (Figs 1–2 and Supplementary Figs 1–3). Because NK can be delivered orally, cucumber as a bioreactor to produce NK can exert an adjuvant therapeutic effect by direct consumption (Wu et al., 2019). This avoids the difficulty of protein purification in plant bioreactors and the immunogenicity of plant protein injection.
“Off-target” effects are an important limitation of CRISPR/Cas9. However, these effects can be reduced by optimising gRNAs (Aquino-Jarquin, 2021). In this study, 12 sgRNAs that targeted the Bt gene of cucumber were designed, of which 9 sgRNAs had a very low probability of “off-target” effects, and one sgRNA targeting the first exon of Bt was selected in this study (Supplementary Table 2). After target gene editing using the CRISPR/Cas9 system, the homologous recombination repair system was used to replace Bt with NK. The homologous recombinant donor genes were constructed by overlap PCR, which is a PCR known to allow assembly of multiple DNA fragments into one construct (Tran et al., 2021). The extension efficiency was better when the left homologous arm was fused to the U6 promoter (LHA-U6) and the terminator was fused to the right homologous arm (T-RHA). However, the extended fusion efficiencies of larger fragments (LHA-U6-NK and LA-U6-NK-T-RHA) were reduced, and even the electrophoretic bands of PCR products were dispersed. It is possible that the overlapping region of the fusion gene was not appropriate or the fused DNA had a complex secondary structure. The length of homologous arm in eukaryotic cells greatly affects the probability of homologous recombination (Vinette and Petitclerc, 1999). However, the effect of the homologous arm length on homologous recombination efficiency in cucumber has not been reported.
4 Conclusions
In this study, 12 sgRNAs were designed using BioTools and one sgRNA was selected to connect with pZHY988-Cas9. The donor gene LHA-U6-NK-T-RHA was constructed by overlap PCR and connected with pX6. PZHY988-Cas9-sgRNA and pX6-LHA-U6-NK-T-RHA vectors were transformed into A. tumefaciens EHA105, and with the transformants the cucumbers were infected by the floral dip method. The crude extract of NK-producing cucumber has both thrombolytic and anti-coagulation functions in vivo and in vitro (Fig. 5). Therefore, NK-producing cucumber might be an ideal functional food option for the prevention and treatment of thrombosis. However, only one sgRNA for gene editing was obviously insufficient to establish the optimalised CRISPR/Cas9 system. In future studies, we will further examine the remaining sgRNAs to establish a more efficient and specific CRISPR/Cas9 gene editing system.
NK-producing cucumber was constructed using CRISPR/Cas9 and the function of its crude extract was analysed in vitro and in vivo
Citation: Acta Alimentaria 52, 1; 10.1556/066.2022.00231
Acknowledgement
This work was supported by Xuzhou Science and Technology Program (Grant number KC21273), Jiangsu Province's industry university research cooperation project (Grant number BY2022773, BY2022777), Innovation Training Program for College Students of Xuzhou University of Technology (Grant number xcx2022159).
Supplementary materials
Supplementary data to this article can be found online at https://doi.org/10.1556/066.2022.00231.
References
Abdoli Nasab, M., Jalali Javaran, M., Cusido, R.M., and Palazon, J. (2016). Purification of recombinant tissue plasminogen activator (rtPA) protein from transplastomic tobacco plants. Plant Physiology and Biochemistry, 108: 139–144.
Aquino-Jarquin, G. (2021). Current advances in overcoming obstacles of CRISPR/Cas9 off-target genome editing. Molecular Genetics and Metabolism, 134(1–2): 77–86.
Choi, J.H., Sapkota, K., Kim, S., and Kim, S.J. (2014). Starase: a bi-functional fibrinolytic protease from hepatic caeca of Asterina pectinifera displays antithrombotic potential. Biochimie, 105: 45–57.
Jin, S., Wang, T., Zhao, Y., Liu, X., Wang, Y., Jiang, L., and Zhang, Q. (2022). The heat-labile toxin B subunit of E. coli fused with VP6 from GCRV (Grass carp reovirus) was expressed and folded into an active protein in rice calli. Protein Expression and Purification, 197: 106099.
Liu, Q., Xin, D., Xi, L., Gu, T., Jia, Z., Zhang, B., and Kou, L. (2022). Novel applications of exogenous melatonin on cold stress mitigation in postharvest cucumbers. Journal of Agriculture and Food Research, 10: 100459.
Liu, Y., Han, Y., Cao, L., Wang, X., and Dou, S. (2021). Analysis of main components and prospects of natto. Advances in Enzyme Research, 9(1): 1–9.
Moradi Vahdat, M., Hemmati, F., Ghorbani, A., Rutkowska, D., Afsharifar, A., Eskandari, M.H., Rezaei, N., and Niazi, A. (2021). Hepatitis B core-based virus-like particles: a platform for vaccine development in plants. Biotechnology Reports, 29: e00605.
Nishinari, K., Fang, Y., Nagano, T., Guo, S., and Wang, R. (2018). 6 - Soy as a food ingredient. In: Yada, R.Y. (Ed.), Proteins in food processing, 2nd ed. Woodhead Publishing, pp. 149–186.
Rajab, L., Habib, W., Gerges, E., Gazal, I., and Ahmad, M. (2023). Natural occurrence of fungal endophytes in cultivated cucumber plants in Syria, with emphasis on the entomopathogen Beauveria bassiana. Journal of Invertebrate Pathology, 196: 107868.
Schillberg, S., Raven, N., Spiegel, H., Rasche, S., and Buntru, M. (2019). Critical analysis of the commercial potential of plants for the production of recombinant proteins. Frontiers in Plant Science, 10: 720–730.
Schmidt, T.J.N., Berarducci, B., Konstantinidou, S., and Raffa, V. (2023). CRISPR/Cas9 in the era of nanomedicine and synthetic biology. Drug Discovery Today, 28(1): 103375.
Shang, Y., Ma, Y., Zhou, Y., Zhang, H., Duan, L., Chen, H., Zeng, J., Zhou, Q., Wang, S., Gu, W., Liu, M., Ren, J., Gu, X., Zhang, S., Wang, Y., Yasukawa, K., Bouwmeester, H.J., Qi, X., Zhang, Z., Lucas, W.J., and Huang, S. (2014). Plant science. Biosynthesis, regulation, and domestication of bitterness in cucumber. Science, 346(6213): 1084–1088.
Suzuki, K., Nakamura, M., Sato, N., Futamura, K., Matsunaga, K., and Yagami, A. (2022). Nattokinase, a subtilisin family serine protease, is a novel allergen contained in the traditional Japanese fermented food natto. Allergology International, https://doi.org/10.1016/j.alit.2022.11.010.
Tran, P.T., Zhang, C.F., and Citovsky, V. (2021). Rapid generation of inoculum of a plant RNA virus using overlap PCR. Virology, 553: 46–50.
Vinette, C.A. and Petitclerc, D. (1999). Study of homologous recombination in relation to the length of homology on transfected bovine mammary gland cells. Theriogenology, 51(1): 428.
Wen, W. and Zhang, X.-B. (2022). CRISPR–Cas9 gene editing induced complex on-target outcomes in human cells. Experimental Hematology, 110: 13–19.
Wu, H., Wang, H., Xu, F., Chen, J., Duan, L., and Zhang, F. (2019). Acute toxicity and genotoxicity evaluations of Nattokinase, a promising agent for cardiovascular diseases prevention. Regulatory Toxicology and Pharmacology, 103: 205–209.
Wu, S., Zhang, S., Wu, X., and Zhou, X. (2020). m6A RNA Methylation in cardiovascular diseases. Molecular Therapy, 28(10): 2111–2119.
Zhou, X., Liu, L., and Zeng, X. (2021). Research progress on the utilisation of embedding technology and suitable delivery systems for improving the bioavailability of nattokinase: a review. Food Structure, 30: 100219.
