Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Directed Evolution of Soluble α-1,2-Fucosyltransferase Using Kanamycin Resistance Protein as a Phenotypic Reporter for Efficient Production of 2'-Fucosyllactose

  • Jonghyeok Shin (Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University) ;
  • Seungjoo Kim (Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University) ;
  • Wonbeom Park (Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University) ;
  • Kyoung Chan Jin (Department of Food Science and Technology, Chung-Ang University) ;
  • Sun-Ki Kim (Department of Food Science and Technology, Chung-Ang University) ;
  • Dae-Hyuk Kweon (Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University)
  • Received : 2022.09.13
  • Accepted : 2022.10.12
  • Published : 2022.11.28
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Abstract

2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (KanR) should be dependent on FucT2 solubility. KanR was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.

Keywords

Acknowledgement

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2017R1A6A1A03015642 and NRF-2020R1A2C2101964). This research was also supported by the Chung-Ang University Graduate Research Scholarship (Academic Scholarship for College of Biotechnology and Natural Resources) in 2022.

References

  1. Bode L. 2012. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology 22: 1147-1162. https://doi.org/10.1093/glycob/cws074
  2. Coppa GV, Zampini L, Galeazzi T, Facinelli B, Ferrante L, Capretti R, et al. 2006. Human milk oligosaccharides inhibit the adhesion to Caco-2 cells of diarrheal pathogens: Escherichia coli, Vibrio cholerae, and Salmonella fyris. Pediat. Res. 59: 377-382. https://doi.org/10.1203/01.pdr.0000200805.45593.17
  3. Thurl S, Munzert M, Henker J, Boehm G, Mller-Werner B, Jelinek J, et al. 2010. Variation of human milk oligosaccharides in relation to milk groups and lactational periods. Br. J. Nutr. 104: 1261-1271. https://doi.org/10.1017/S0007114510002072
  4. Smith-Brown P, Morrison M, Krause L, Davies PSW. 2016. Mothers secretor status affects development of childrens microbiota composition and function: a pilot study. PLoS One 11: e0161211.
  5. Han NS, Kim TJ, Park YC, Kim J, Seo JH. 2012. Biotechnological production of human milk oligosaccharides. Biotechnol. Adv. 30: 1268-1278. https://doi.org/10.1016/j.biotechadv.2011.11.003
  6. Li C, Wu M, Gao X, Zhu ZL, Li Y, Lu FP, et al. 2020. Efficient biosynthesis of 2'-fucosyllactose using an in vitro multienzyme cascade. J. Agr. Food Chem. 68: 10763-10771. https://doi.org/10.1021/acs.jafc.0c04221
  7. Lee HJ, Shin DJ, Han K, Chin YW, Park JP, Park K, et al. 2022. Simultaneous production of 2'-fucosyllactose and difucosyllactose by engineered Escherichia coli with high secretion efficiency. Biotechnol. J. 17: e2100629.
  8. Shin J, Jin YS, Park YC, Park JB, Lee YO, Kim SK, et al. 2021. Enhancing acid tolerance of Escherichia coli via viroporin-mediated export of protons and its application for efficient whole-cell biotransformation. Metab. Eng. 67: 277-284. https://doi.org/10.1016/j.ymben.2021.07.007
  9. Yu S, Liu JJ, Yun EJ, Kwak S, Kim KH, Jin YS. 2018. Production of a human milk oligosaccharide 2'-fucosyllactose by metabolically engineered Saccharomyces cerevisiae. Microb. Cell Fact. 17: 101.
  10. Liu TW, Ito H, Chiba Y, Kubota T, Sato T, Narimatsu H. 2011. Functional expression of L-fucokinase/guanosine 5'-diphosphate-L-fucose pyrophosphorylase from Bacteroides fragilis in Saccharomyces cerevisiae for the production of nucleotide sugars from exogenous monosaccharides. Glycobiology 21: 1228-1236. https://doi.org/10.1093/glycob/cwr057
  11. Chin YW, Kim JY, Lee WH, Seo JH. 2015. Enhanced production of 2'-fucosyllactose in engineered Escherichia coli BL21star(DE3) by modulation of lactose metabolism and fucosyltransferase. J. Biotechnol. 210: 107-115. https://doi.org/10.1016/j.jbiotec.2015.06.431
  12. Park Y, Shin J, Yang J, Kim H, Jung Y, Oh H, et al. 2020. Plasmid display for stabilization of enzymes inside the cell to improve wholecell biotransformation efficiency. Front. Bioeng. Biotechnol. 7: 444.
  13. Ren J, Hwang S, Shen J, Kim H, Kim H, Kim J, et al. 2022. Enhancement of the solubility of recombinant proteins by fusion with a short-disordered peptide. J. Microbiol. 60: 960-967. https://doi.org/10.1007/s12275-022-2122-z
  14. Mamipour M, Yousefi M, Hasanzadeh M. 2017. An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding. Int. J. Biol. Macromol. 102: 367-375. https://doi.org/10.1016/j.ijbiomac.2017.04.025
  15. Wang T, Badran AH, Huang TP, Liu DR. 2018. Continuous directed evolution of proteins with improved soluble expression. Nat. Chem. Biol. 14: 972-980. https://doi.org/10.1038/s41589-018-0121-5
  16. Reyes LH, Cardona C, Pimentel L, Rodriguez-Lopez A, Almeciga-Diaz CJ. 2017. Improvement in the production of the human recombinant enzyme N-acetylgalactosamine-6-sulfatase (rhGALNS) in Escherichia coli using synthetic biology approaches. Sci. Rep. 7: 5844.
  17. Han X, Ning W, Ma X, Wang X, Zhou K. 2020. Improving protein solubility and activity by introducing small peptide tags designed with machine learning models. Metab. Eng. Commun. 11: e00138.
  18. Kim C, Park M, Yang J, Shin J, Park YC, Kim SK, et al. 2021. Inducible plasmid display system for high-throughput selection of proteins with improved solubility. J. Biotechnol. 329: 143-150. https://doi.org/10.1016/j.jbiotec.2020.12.013
  19. Wang H, Wang L, Zhong BH, Dai ZJ. 2022. Protein splicing of inteins: a powerful tool in synthetic biology. Front. Bioeng. Biotechnol. 10: 810180.
  20. ang J, Kim N, Park W, Chun J, Kim S, Shin J, et al. 2022. Optimization of protein trans-splicing in an inducible plasmid display system for high-throughput screening and selection of soluble proteins. Enzyme Microb. Technol. 153: 109914.
  21. Li CK, Wen AY, Shen BC, Lu J, Huang Y, Chang YC. 2011. FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnol. 11: 92.
  22. Lee WH, Han NS, Park YC, Seo JH. 2009. Modulation of guanosine 5'-diphosphate-D-mannose metabolism in recombinant Escherichia coli for production of guanosine 5'-diphosphate-L-fucose. Bioresour. Technol. 100: 6143-6148. https://doi.org/10.1016/j.biortech.2009.07.035
  23. Baig F, Fernando LP, Salazar MA, Powell RR, Bruce TF, Harcum SW. 2014. Dynamic transcriptional response of Escherichia coli to inclusion body formation. Biotechnol. Bioeng. 111: 980-999. https://doi.org/10.1002/bit.25169
  24. Packer MS, Liu DR. 2015. Methods for the directed evolution of proteins. Nat. Rev. Genet. 16: 379-394. https://doi.org/10.1038/nrg3927
  25. Hart DJ, Tarendeau F. 2006. Combinatorial library approaches for improving soluble protein expression in Escherichia coli. Acta Crystallogr. D 62: 19-26. https://doi.org/10.1107/S0907444905036097
  26. Ren C, Wen X, Mencius J, Quan S. 2019. Selection and screening strategies in directed evolution to improve protein stability. Bioresour. Bioprocess 6: 53.
  27. Daunert S, Barrett G, Feliciano JS, Shetty RS, Shrestha S, Smith-Spencer W. 2000. Genetically engineered whale-cell sensing systems: coupling biological recognition with reporter genes. Chem. Rev. 100: 2705-2738. https://doi.org/10.1021/cr990115p
  28. Janocha S, Bichet A, Zollner A, Bernhardt R. 2011. Substitution of lysine with glutamic acid at position 193 in bovine CYP11A1 significantly affects protein oligomerization and solubility but not enzymatic activity. Biochim. Biophys. Acta 1814: 126-131. https://doi.org/10.1016/j.bbapap.2010.06.002
  29. Seitz T, Bocola M, Claren J, Sterner R. 2007. Stabilisation of a (βα)8-barrel protein designed from identical half barrels. J. Mol. Biol. 372: 114-129. https://doi.org/10.1016/j.jmb.2007.06.036
  30. Behar G, Sole V, Defontaine A, Maillasson M, Quemener A, Jacques Y, et al. 2011. Evolution of interleukin-15 for higher E. coli expression and solubility. Protein Eng. Des. Sel. 24: 283-290. https://doi.org/10.1093/protein/gzq107
  31. Rodriguez-Banqueri A, Errasti-Murugarren E, Bartoccioni P, Kowalczyk L, Peralvarez-Marin A, Palacin M, et al. 2016. Stabilization of a prokaryotic LAT transporter by random mutagenesis. J. Gen. Physiol. 147: 353-368. https://doi.org/10.1085/jgp.201511510
  32. Mo HM, Xu Y, Yu XW. 2018. Improved soluble expression and catalytic activity of a thermostable esterase using a high-throughput screening system based on a split-GFP assembly. J. Agr. Food Chem. 66: 12756-12764. https://doi.org/10.1021/acs.jafc.8b04646
  33. Biswas T, Houghton JL, Garneau-Tsodikova S, Tsodikov OV. 2012. The structural basis for substrate versatility of chloramphenicol acetyltransferase CATI. Protein Sci. 21: 520-530. https://doi.org/10.1002/pro.2036
  34. Wright GD, Thompson PR. 1999. Aminoglycoside phosphotransferases: proteins, structure, and mechanism. Front. Biosci. 4: D9-21.
  35. Tokuriki N, Stricher F, Serrano L, Tawfik DS. 2008. How protein stability and new functions trade off. PLoS Comput. Biol. 4: e1000002.
  36. Wang XJ, Minasov G, Shoichet BK. 2002. Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs. J. Mol. Biol. 320: 85-95. https://doi.org/10.1016/S0022-2836(02)00400-X
  37. DePristo MA, Weinreich DM, Hartl DL. 2005. Missense meanderings in sequence space: a biophysical view of protein evolution. Nat. Rev. Genet. 6: 678-687. https://doi.org/10.1038/nrg1672
  38. Studer RA, Christin PA, Williams MA, Orengo CA. 2014. Stability-activity tradeoffs constrain the adaptive evolution of RubisCO. Proc. Natl. Acad. Sci. USA 111: 2223-2228. https://doi.org/10.1073/pnas.1310811111
  39. Tokuriki N, Tawfik DS. 2009. Stability effects of mutations and protein evolvability. Curr. Opin. Struc. Biol. 19: 596-604. https://doi.org/10.1016/j.sbi.2009.08.003
  40. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. 2021. Highly accurate protein structure prediction with AlphaFold. Nature 596: 583-589. https://doi.org/10.1038/s41586-021-03819-2
  41. Jung SY, Park SS. 2008. Improving the expression yield of Candida antarctica lipase B in Escherichia coli by mutagenesis. Biotechnol. Lett. 30: 717-722. https://doi.org/10.1007/s10529-007-9591-3
  42. Massey-Gendel E, Zhao A, Boulting G, Kim HY, Balamotis MA, Seligman LM, et al. 2009. Genetic selection system for improving recombinant membrane protein expression in E. coli. Protein Sci. 18: 372-383. https://doi.org/10.1002/pro.39