Cloning and <i>in silico</i> characterization of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase from <i>Bacillus subtilis</i>

Quang Huy Nguyen; Thi Thanh Xuan Nguyen; Minh Thu Nguyen; Luan Luong Chu

doi:10.15625/vjbt-23418

Author affiliations

Authors

Quang Huy Nguyen \(^1\) National Key Laboratory of Enzyme and Protein Technology, Faculty of Biology, Hanoi University of Science, Vietnam National University, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam https://orcid.org/0000-0001-7394-6209
Thi Thanh Xuan Nguyen \(^2\) Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Nghia Do, Hanoi, Vietnam
Minh Thu Nguyen \(^1\) National Key Laboratory of Enzyme and Protein Technology, Faculty of Biology, Hanoi University of Science, Vietnam National University, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam
Luan Luong Chu \(^1\) National Key Laboratory of Enzyme and Protein Technology, Faculty of Biology, Hanoi University of Science, Vietnam National University, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam https://orcid.org/0000-0001-9223-6798

DOI:

https://doi.org/10.15625/vjbt-23418

Keywords:

3-hexulose-6-phosphate synthase, 6-phospho-3-hexuloseisomerase, hxlA, hxlB, Bacillus subtilis ST123

Abstract

3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) are key enzymes of the ribulose monophosphate (RuMP) pathway, which plays an essential role in formaldehyde assimilation and detoxification in methylotrophic microorganisms. Although homologous genes encoding these enzymes are annotated in the genome of Bacillus subtilis, their molecular and structural characteristics remain poorly explored in this organism. In this study, taxonomic identification using Kraken2, 16S rRNA phylogenetic analysis, and multilocus sequence typing confirmed the strain as B. subtilis ST123. Moreover, the hxlA and hxlB genes encoding HPS and PHI were cloned from a local B. subtilis ST123 strain isolated from infant fecal samples. The amplified hxlA (633 bp) and hxlB (558 bp) genes were successfully cloned, sequenced, and analyzed. Sequence alignment revealed high conservation with reference B. subtilis sequences. The three-dimensional structure of HPS was predicted using AlphaFold3 and rigorously evaluated by MolProbity, ERRAT, Verify3D, and ProSa. The HPS model exhibited high overall structural quality, with a MolProbity score of 1.13, 100% residues in the Ramachandran favored region, and an ERRAT quality factor of 99.505, although localized regions of lower sequence-structure compatibility were identified by Verify3D. The PHI structure was analyzed based on an available X-ray crystal structure (PDB ID: 1M3S). Physicochemical characterization indicated that HPS is thermostable and suitable for heterologous expression, whereas PHI showed a higher instability index, suggesting potential challenges during in vitro expression. This work provides the first comprehensive cloning and in silico characterization of HPS and PHI from a Vietnamese B. subtilis strain, offering valuable insights for metabolic engineering, synthetic biology, and C1 assimilation pathway design.

Downloads

Download data is not yet available.

References

Blochlinger, K., and Diggelmann, H. (1984). Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells. Molecular and Cellular Biology, 4 (12), 2929-2931. https://doi.org/10.1128/mcb.4.12.2929-2931.1984

Phuong Thao, B., Thi Linh, N., Van Manh, N., Khanh Linh, L., Hoang Ha, C., Tien Phat, D., & Bich Ngoc, P. (2022). Optimization of Agrobacterium-mediated transformation procedure for an indica rice variety-Khang Dan 18. Vietnam Journal of Biotechnology, 20(1), 53-62. https://doi.org/10.15625/1811-4989/17200

Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. (1994). Green Fluorescent Protein as a Marker for Gene Expression. Science, 263(5148), 802-805. https://doi.org/10.1126/science.8303295

Chen, L., Cai, Y., Liu, X., Yao, W., Wu, S., and Hou, W. (2024). The RUBY reporter for visual selection in soybean genome editing. aBIOTECH, 5(2), 209-213. https://doi.org/10.1007/s42994-024-00148-6

Chu, C.C., Chu, W.C., San, S.C., and Chen, H. (1975). Establishment of an efficient medium for another culture of rice through comparative experiments on the nitrogen sources. Scientia Sinica, 18(5), 659-668. https://doi.org/10.1360/ya1975-18-5-659

Davey, M.R., and Anthony, P. (2010). Plant Cell Culture: Essential Methods (1st ed.). Wiley, https://doi.org/10.1002/9780470686522

Doyle, J.J. and J.L. Doyle (1990). Isolation of plant DNA from fresh tissue. Focus, 12:13-15.

Fish, N., Lindsey, K., and Jones, M.G.K. (1988). Plant Protoplast Fusion. Methods in molecular biology (Clifton, N.J.), 4, 481-498. https://doi.org/10.1385/0-89603-127-6:481

Gamborg, O.L., Miller, R.A., and Ojima, K. (1968). Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research, 50(5), 151-158. https://doi.org/10.1016/0014-4827(68)90403-5

He, Y., Zhang, T., Sun, H., Zhan, H., and Zhao, Y. (2020). A reporter for noninvasively monitoring gene expression and plant transformation. Horticulture Research, 7(1), 152. https://doi.org/10.1038/s41438-020-00390-1

Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal, 6(13), 3901-3907. https://doi.org/10.1002/j.1460-2075.1987.tb02730.x

Khanh Linh, L., Van Doai, N., Thi Linh, N., Quyen, P., Hoang Ha, C., Bich Ngoc, P., and Tien Phat, D. (2023). Development of CRISPR/Cas9 systems to induce targeted mutations in the promoter region of the OsSRFP1 gene in rice. Vietnam Journal of Biotechnology, 21(3), 463-475. https://doi.org/10.15625/1811-4989/19532

Lee, K., Kang, M., Ji, Q., Grosic, S., and Wang, K. (2023). New T-DNA binary vectors with NptII selection and RUBY reporter for efficient maize transformation and targeted mutagenesis. Plant Physiology, 192 (4), 2598-2603. https://doi.org/10.1093/plphys/kiad231.

Li, L., Qu, R., De Kochko, A., Fauquet, C., and Beachy, R.N. (1993). An improved rice transformation system using the biolistic method. Plant Cell Reports, 12(5), 250-255. https://doi.org/10.1007/BF00237129

Miah, M.A.A., Pathan, M.S., and Quayum, H.A. (1996). Production of salt tolerant rice breeding line via doubled haploid. Euphytica, 91(3), 285-288. https://doi.org/10.1007/BF00033089

Murashige, T., and Skoog, F. (1962). A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum, 15(3), 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

Ohira, K., Ojima, K., and Fujiwara, A. (1973). Studies on the nutrition of rice cell culture I. A simple, defined medium for rapid growth in suspension culture. Plant Cell Physiology, 14(6), 1113-1121 https://doi.org/10.1093/oxfordjournals.pcp.a074950

Ow, D.W., Wood, K.V., DeLuca, M., De Wet, J.R., Helinski, D.R., and Howell, S.H. (1986). Transient and Stable Expression of the Firefly Luciferase Gene in Plant Cells and Transgenic Plants. Science, 234(4778), 856-859. https://doi.org/10.1126/science.234.4778.856

Sallaud, C., Meynard, D., Van Boxtel, J., Gay, C., Bès, M., Brizard, J.P., et al. (2003). Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theoretical and Applied Genetics, 106(8), 1396-1408. https://doi.org/10.1007/s00122-002-1184-x

Slamet-Loedin, I.H., Chadha-Mohanty, P., and Torrizo, L. (2014). Agrobacterium-Mediated Transformation: Rice Transformation, in: Henry, R.J., Furtado, A. (Eds.), Cereal Genomics, Methods in Molecular Biology (Clifton, N.J.), 1099, 261-271. https://doi.org/10.1007/978-1-62703-715-0_21

Sun, H., Wang, S., Zhu, C., Yang, K., Liu, Y., and Gao, Z. (2023). A new biotechnology for in-planta gene editing and its application in promoting flavonoid biosynthesis in bamboo leaves. Plant Methods 19(1), 20. https://doi.org/10.1186/s13007-023-00993-4

Verma, D.K., and Srivastav, P.P. (2020). Bioactive compounds of rice (Oryza sativa L.): Review on paradigm and its potential benefit in human health. Trends in Food Science & Technology, 97, 355-365. https://doi.org/10.1016/j.tifs.2020.01.007

Wang, D., Zhong, Y., Feng, B., Qi, X., Yan, T., Liu, J., et al. (2023). The RUBY reporter enables efficient haploid identification in maize and tomato. Plant Biotechnology Journal, 21(8), 1707-1715. https://doi.org/10.1111/pbi.14071.