Abstract
Thymidine phosphorylase (TPase) plays a vital role in the biosynthesis of nucleosides and their analogs which have tremendous potential in antiviral and anticancer therapies. In this study, genome shuffling was applied to develop new strains of Lactobacillus brevis with an enhanced production of TPase. The parent organisms were mutated using ultraviolet (UV) irradiation and were shuffled by recursive pool-wise protoplast fusion. The parent protoplasts of each cycle were inactivated by UV irritation for 50 min or by heating at 60°C for 60 min. A rapid and efficient pre-screening method for determining L. brevis fusants with increased TPase production was established by adding appropriate concentrations of substrate thymidine and potassium phosphate to the culture broth based on significant differences in the absorption spectra of substrate thymidine and its product, thymine, in alkaline solution at 290 nm. Strains F3-19 and F3-36 showed high TPase activity and favorable hereditary stability and were screened out through three rounds of recursive protoplast fusion. The increase in the TPase activity of F3-19 and F3-36 was 252.6 and 260.5%, respectively, in comparison with the wild type.
Similar content being viewed by others
References
Clercq, E. D. (2004) Antiviral drugs in current clinical use. J. Clin. Virol. 30: 115–133.
Robins, R. K. (1984) The potential of nucleotide analogs as inhibitors of retroviruses and tumors. Pharm. Res. 1: 11–18.
Kadokawa, J. and S. Kobayashi (2010) Polymer synthesis by enzymatic catalysis. Curr. Opin. Chem. Biol. 14: 145–153.
Jensen, K. F. and P. Nygaard (1975) Purine nucleoside phosphorylase from Escherichia coli and Salmonella typhimurium. Eur. J. Biochem. 51: 253–265.
Shirae, H. and K. Yokozeki (1991) Purification and properties of purine nucleoside phosphorylase from Brevibacterium acetylicum ATCC 954. Agric. Biol. Chem. 55: 493–499.
Saunders, P. P., B. A. Wilson, and G. F. Saunders (1969) Purification and comparative properties of a pyrimidine nucleoside phosphorylase from Bacillus Stearothermophilus. J. Biol. Chem. 244: 3691–3697.
Avraham, Y., N. Grossowicz, and J. Yashphe (1990) Purification and characterization of uridine and thymidine phosphorylase from Lactobacillus casei. Biochim. Biophys. Acta 1040: 287–293.
Blank, J. G. and P. A. Hoffee (1975) Purification and properties of thymidine phosphorylase from Salmonella typhimurium. Arch. Biochem. Biophys. 168: 259–265.
Zinchenko, A. I., V. N. Barai, S. B. Bokut, E. I. Kvasyuk, and I. A. Mikhaiopulo (1990) Synthesis of 9-(β-D-arabinofuranosyl) guanine using whole cells of Escherichia coli. Appl. Microbiol. Biotechnol. 32: 658–661.
Fernández-Lucas, J., A. Fresco-Taboada, C. Acebal, I. de la Mata, and M. Arroyo (2011) Enzymatic synthesis of nucleosides analogues using immobilized 2'-deoxyribosyltransferase from Lactobacillus reuteri. Appl. Microbiol. Biotechnol. 91: 317–327.
Luo, W., Y. Liu, X. Zhu, W. Zhao, L. Huang, J. Cai, Z. Xu, and P. Cen (2011) Cloning and characterization of purine nucleoside phosphorylase in Escherichia coli and subsequent ribavirin biosynthesis using immobilized recombinant cells. Enz. Microb. Technol. 48: 438–444.
Biot-Pelletier, D. and V. J. Martin (2014) Evolutionary engineering by genome shuffling. Appl. Microbiol. Biotechnol. 98: 3877–3887.
Zhang, Y., K. Perry, V. A. Vinci, K. Powell, W. P. Stemmer, and S. B. del Cardayré (2002) Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415: 644–646.
Patnik, R., S. Louie, V. Gavrilovic, K. Perry, W. P. Stemmer, C. M. Ryan, and S. B. del Cardayré (2002) Genome shuffling of Lactobacillus for improved acid tolerance. Nat. Biotechnol. 20: 707–712.
El-Gendy, M. M. and A. M. El-Bondkly (2011) Genome shuffling of marine derived bacterium Nocardia sp. ALAA 2000 for improved ayamycin production. Antonie van Leeuwenhoek. 99: 773–780.
Wang, H., J. Zhang, X. Wang, W. Qi, and Y. Dai (2012) Genome shuffling improves production of the low-temperature alkalophilic lipase by Acinetobacter johnsonii. Biotechnol. Lett. 34: 145–151.
Chen, X., P. Wei, L. Fan, D. Yang, X. Zhu, W. Shen, Z. Xu, and P. Cen (2007) Generation of high-yield rapamycin-producing strains through protoplasts-related techniques. Appl. Microbiol. Biotechnol. 83: 507–512.
Yu, G., Y. Hu, M. Hui, L. Chen, L. Wang, and N. Liu (2014) Genome shuffling of Streptomyces roseosporus for improving daptomycin production. Appl. Biochem. Biotechnol. 172: 2661–2669.
Xu, B., Z. Jin, Q. Jin, N. Li, and P. Cen (2009) Improvement of pristinamycin production by genome shuffling and medium optimization for Streptomyces Pristinaespiralis. Biotechnol. Bioproc. Eng. 14: 175–179.
Li, W., G. Chen, L. Gu, W. Zeng, and Z. Liang (2014) Genome shuffling of Aspergillus niger for improving transglycosylation activity. Appl. Biochem. Biotechnol. 172: 50–61.
Friedkin, M. and D. Roberts (1954) The enzymatic synthesis of nucleosides: I. Thymidine phosphorylase in mammalian tissue. J. Biol. Chem. 207: 245–256.
Wang, Y., Y. Li, X. Pei, L. Yu, and Y. Feng (2007) Genome-shuffling improved acid tolerance and L-lactic acid volumetric productivity in Lactobacillus rhamnosus. J. Biotechnol. 129: 510–515.
Singhvi, M., D. Joshi, S. Gaikaiwari, and D. V. Gokhale (2010) Protoplast formation and regeneration in Lactobacillus delbruekii. Ind. J. Microbiol. 50: 97–100.
Lee-Wickner, L. J. and B. M. Chassy (1984) Production and regeneration of Lactobacillus casei protoplasts. Appl. Environ. Microbiol. 48: 994–1000.
Laemmi, U. K. (1970) Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 227: 680–685.
Hotchkiss, R. D. (1948) The quantitative separation of purines, pyrimidines, and nucleosides by paper chromatography. J. Biol. Chem. 175: 315–332.
Gong, J., H. Zheng, Z. Wu, T. Chen, and X. Zhao (2009) Genome shuffling: Progress and applications for phenotype improvement. Biotchnol. Adv. 27: 996–1005.
Luo, J., J. Li, D. Liu, F. Liu, Y. Wang, X. Song, and M. Wang (2012) Genome shuffling of Streptomyces gilvosporeus for improving natamycin production. J. Agirc. Food Chem. 60: 6026–6036.
Dai, M., S. Ziesman, T. Ratcliffe, R. T. Gill, and S. D. Copley (2005) Visualization of protoplast fusion and quantitation of recombination in fused protoplasts of auxotrophic strains of Escherichia coli. Metab. Eng. 7: 45–52.
Hida, H., T. Yamada, and Y. Yamada (2007) Genome shuffling of Streptomyces sp. U121 for improved production of hydroxycitric acid. Appl. Microbiol. Biotechnol. 73: 1387–1393.
Hopwood, D. A. and H. M. Wright. (1981) Protoplast fusion in Streptomyces: Fusions involving ultraviolet irradiated protoplasts. J. Gen. Microbiol. 126: 21–27.
Hopwood, D. A. and H. M. Wright (1979) Factors affecting recombinant frequency in protoplast fusions of Streptomyces coelicolor. J. Gen. Microbiol. 111: 137–143.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Li, H., Xue, F., Wang, W. et al. Genome shuffling of Lactobacillus brevis for enhanced production of thymidine phosphorylase. Biotechnol Bioproc E 20, 333–340 (2015). https://doi.org/10.1007/s12257-014-0617-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12257-014-0617-0