Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Engineering of a Microbial Cell Factory for the Extracellular Production of Catalytically Active Phospholipase A2 of Streptomyces violaceoruber

  • Lee, Hyun-Jae (Department of Food Science and Technology, Chung-Ang University) ;
  • Cho, Ara (Department of Food Science and Engineering, Ewha Womans University) ;
  • Hwang, Yeji (Department of Food Science and Engineering, Ewha Womans University) ;
  • Park, Jin-Byung (Department of Food Science and Engineering, Ewha Womans University) ;
  • Kim, Sun-Ki (Department of Food Science and Technology, Chung-Ang University)
  • Received : 2020.01.30
  • Accepted : 2020.02.28
  • Published : 2020.08.28
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Abstract

Phospholipase A2 (PLA2) from Streptomyces violaceoruber is a lipolytic enzyme used in a wide range of industrial applications including production of lysolecithins and enzymatic degumming of edible oils. We have therefore investigated expression and secretion of PLA2 in two workhorse microbes, Pichia pastoris and Escherichia coli. The PLA2 was produced to an activity of 0.517 ± 0.012 U/ml in the culture broth of the recombinant P. pastoris. On the other hand, recombinant E. coli BL21 star (DE3), overexpressing the authentic PLA2 (P-PLA2), showed activity of 17.0 ± 1.3 U/ml in the intracellular fraction and 21.7 ± 0.7 U/ml in the culture broth. The extracellular PLA2 activity obtained with the recombinant E. coli system was 3.2-fold higher than the corresponding value reached in a previous study, which employed recombinant E. coli BL21 (DE3) overexpressing codon-optimized PLA2. Finally, we observed that the extracellular PLA2 from the recombinant E. coli P-PLA2 culture was able to hydrolyze 31.1 g/l of crude soybean lecithin, an industrial substrate, to a conversion yield of approximately 95%. The newly developed E. coli-based PLA2 expression system led to extracellular production of PLA2 to a productivity of 678 U/l·h, corresponding to 157-fold higher than that obtained with the P. pastoris-based system. This study will contribute to the extracellular production of a catalytically active PLA2.

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References

  1. Cerminati S, Paoletti L, Aguirre A, Peiru S, Menzella HG, Castelli ME. 2019. Industrial uses of phospholipases: current state and future applications. Appl. Microbiol. Biotechnol. 103: 2571-2582. https://doi.org/10.1007/s00253-019-09658-6
  2. De Maria L, Vind J, Oxenboll KM, Svendsen A, Patkar S. 2007. Phospholipases and their industrial applications. Appl. Microbiol. Biotechnol. 74: 290-300. https://doi.org/10.1007/s00253-006-0775-x
  3. Dijkstra AJ. 2010. Enzymatic degumming. Eur. J. Lipid Sci. Technol. 112: 1178-1189. https://doi.org/10.1002/ejlt.201000320
  4. Noel JP, Tsai MD. 1989. Phospholipase $A_2$ engineering: design, synthesis, and expression of a gene for bovine (pro)phospholipase $A_2$. J. Cell Biochem. 40: 309-320. https://doi.org/10.1002/jcb.240400307
  5. van den Bergh CJ, Bekkers AC, De Geus P, Verheij HM, de Haas GH. 1987. Secretion of biologically active porcine prophospholipase $A_2$ by Saccharomyces cerevisia: use of the prepro sequence of the alpha-mating factor. Eur. J. Biochem. 170: 241-246. https://doi.org/10.1111/j.1432-1033.1987.tb13691.x
  6. Lefkowitz LJ, Deems RA, Dennis EA. 1999. Expression of group IA phospholipase $A_2$ in Pichia pastoris: identification of a phosphatidylcholine activator site using site-directed mutagenesis. Biochemistry 38: 14174-14184. https://doi.org/10.1021/bi991432t
  7. Liu YH, Huang L, Li MJ, Liu H, Guo W, Gui S, et al. 2016. Characterization of the recombinant porcine pancreas phospholipase $A_2$ expressed in Pichia pastoris GS115 and its application to synthesis of 2-DHA-PS. Process Biochem. 51: 1472-1478. https://doi.org/10.1016/j.procbio.2016.06.023
  8. Roberts IN, Jeenes DJ, MacKenzie DA, Wilkinson AP, Sumner IG, Archer DB. 1992. Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase $A_2$ fusion protein is secreted and processed to yield mature enzyme. Gene 122: 155-161. https://doi.org/10.1016/0378-1119(92)90043-O
  9. Markert Y, Mansfeld J, Schierhorn A, Rucknagel KP, Ulbrich-Hofmann R. 2007. Production of synthetically phospholipase A2 variants created with industrial impact. Biotechnol. Bioeng. 98: 48-59. https://doi.org/10.1002/bit.21392
  10. Lathrop BK, Burack WR, Biltonen RL, Rule GS. 1992. Expression of a group II phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus in Escherichia coli: recovery and renaturation from bacterial inclusion bodies. Protein Expr. Purif. 3: 512-517. https://doi.org/10.1016/1046-5928(92)90069-9
  11. Giuliani CD, Iemma MR, Bondioli AC, Souza DH, Ferreira LL, Amaral AC, et al. 2001. Expression of an active recombinant lysine 49 phospholipase $A_2$ myotoxin as a fusion protein in bacteria. Toxicon 39: 1595-1600. https://doi.org/10.1016/S0041-0101(01)00142-8
  12. Yang WL, Peng LS, Zhong XF, Wei JW, Jiang XY, Ye LT, et al. 2003. Functional expression and characterization of a recombinant phospholipase $A_2$ from sea snake Lapemis hardwickii as a soluble protein in E. coli. Toxicon 41: 713-721. https://doi.org/10.1016/S0041-0101(03)00047-3
  13. Jin Q, Yang LX, Jiao HM, Lu B, Wu YQ, Zhou YC. 2004. Purification, gene cloning and expression of an acidic phospholipase $A_2$ from Agkistrodon shedaoensis Zhao. Acta Biochim. Biophys. Sin. 36: 27-32. https://doi.org/10.1093/abbs/36.1.27
  14. Esposito D, Chatterjee DK. 2006. Enhancement of soluble protein expression through the use of fusion tags. Curr. Opin. Biotechnol. 17: 353-358. https://doi.org/10.1016/j.copbio.2006.06.003
  15. Dennis EA, Cao J, Hsu YH, Magrioti V, Kokotos G. 2011. Phospholipase $A_2$ enzymes: physical structure, biological function, sisease implication, chemical inhibition, and therapeutic intervention. Chem. Rev. 111: 6130-6185. https://doi.org/10.1021/cr200085w
  16. Sugiyama M, Ohtani K, Izuhara M, Koike T, Suzuki K, Imamura S, et al. 2002. A novel prokaryotic phospholipase $A_2$: characterization, gene cloning, and solution structure. J. Biol. Chem. 277: 20051-20058. https://doi.org/10.1074/jbc.M200264200
  17. Liu AX, Yu XW, Sha C, Xu Y. 2015. Streptomyces violaceoruber phospholipase $A_2$: expression in Pichia pastoris, properties, and application in oil degumming. Appl. Biochem. Biotechnol. 175: 3195-3206. https://doi.org/10.1007/s12010-015-1492-7
  18. Takemori D, Yoshino K, Eba C, Nakano H, Iwasaki Y. 2012. Extracellular production of phospholipase $A_2$ from Streptomyces violaceoruber by recombinant Escherichia coli. Protein Expres. Purif. 81: 145-150. https://doi.org/10.1016/j.pep.2011.10.002
  19. Jung H-J, Kim S-K, Min W-K, Lee S-S, Park K, Park Y-C, et al. 2011. Polycationic amino acid tags enhance soluble expression of Candida antarctica lipase B in recombinant Escherichia coli. Bioprocess Biosyst. Eng. 34: 833-839. https://doi.org/10.1007/s00449-011-0533-z
  20. Jeon E-Y, Seo J-H, Kang W-R, Kim M-J, Lee J-H, Oh D-K, et al. 2016. Simultaneous enzyme/whole-cell biotransformation of plant oils into C9 carboxylic acids. ACS Catal. 6: 7547-7553. https://doi.org/10.1021/acscatal.6b01884
  21. Seo E-J, Yeon YJ, Seo J-H, Lee J-H, Bongol JP, Oh Y, et al. 2018. Enzyme/whole-cell biotransformation of plant oils, yeast derived oils, and microalgae fatty acid methyl esters into n-nonanoic acid, 9-hydroxynonanoic acid, and 1,9-nonanedioic acid. Bioresour. Technol. 251: 288-294. https://doi.org/10.1016/j.biortech.2017.12.036
  22. Seo E-J, Kim H-J, Kim M-J, Kim J-S, Park J-B. 2019. Cofactor specificity engineering of a long-chain secondary alcohol dehydrogenase from Micrococcus luteus for redox-neutral biotransformation of fatty acids. Chem. Comm. 55: 14462-14465. https://doi.org/10.1039/C9CC06447H
  23. Tschopp JF, Brust PF, Cregg JM, Stillman CA, Gingeras TR. 1987. Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res. 15: 3859-3876. https://doi.org/10.1093/nar/15.9.3859
  24. Brake AJ, Merryweather JP, Coit DG, Heberlein UA, Masiarz FR, Mullenbach GT, et al. 1984. Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 81: 4642-4646. https://doi.org/10.1073/pnas.81.15.4642
  25. Matoba Y, Sugiyama M. 2003. Atomic resolution structure of prokaryotic phospholipase $A_2$: analysis of internal motion and implication for a catalytic mechanism. Proteins 51: 453-469. https://doi.org/10.1002/prot.10360
  26. Blank K, Morfill J, Gumpp H, Gaub HE. 2006. Functional expression of Candida antarctica lipase B in Eschericha coli. J. Biotechnol. 125: 474-483. https://doi.org/10.1016/j.jbiotec.2006.04.004
  27. Jung SM, Seo JH, Lee JH, Park JB, Seo JH. 2015. Fatty acid hydration activity of a recombinant Escherichia coli-based biocatalyst is improved through targeting the oleate hydratase into the periplasm. Biotechnol. J. 10: 1887-1893. https://doi.org/10.1002/biot.201500141
  28. Jeon EY, Song JW, Cha HJ, Lee SM, Lee J, Park JB. 2018. Intracellular transformation rates of fatty acids are influenced by expression of the fatty acid transporter FadL in Escherichia coli cell membrane. J. Biotechnol. 281: 161-167. https://doi.org/10.1016/j.jbiotec.2018.07.019
  29. Liu D, Schmid RD, Rusnak M. 2006. Functional expression of Candida antarctica lipase B in the Escherichia coli cytoplasm: a screening system for a frequently used biocatalyst. Appl. Microbiol. Biotechnol. 72: 1024-1032. https://doi.org/10.1007/s00253-006-0369-7
  30. 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
  31. Kim SK, Park YC, Lee HH, Jeon ST, Min WK, Seo JH. 2015. Simple amino acid tags improve both expression and secretion of Candida antarctica lipase B in recombinant Escherichia coli. Biotechnol. Bioeng. 112: 346-355. https://doi.org/10.1002/bit.25361
  32. Kim SK, Min WK, Park YC, Seo JH. 2015. Application of repeated aspartate tags to improving extracellular production of Escherichia coli L-asparaginase isozyme II. Enzyme Microb. Technol. 79-80: 49-54. https://doi.org/10.1016/j.enzmictec.2015.06.017
  33. 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