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Combinatorial Fine-Tuning of Phospholipase D Expression by Bacillus subtilis WB600 for the Production of Phosphatidylserine

  • Huang, Tingting (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University) ;
  • Lv, Xueqin (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University) ;
  • Li, Jianghua (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University) ;
  • Shin, Hyun-dong (School of Chemical and Biomolecular Engineering, Georgia Institute of Technology) ;
  • Du, Guocheng (Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University) ;
  • Liu, Long (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University)
  • Received : 2018.06.22
  • Accepted : 2018.08.09
  • Published : 2018.12.28

Abstract

Phospholipase D has great commercial value due to its transphosphatidylation products that can be used in the food and medicine industries. In order to construct a strain for use in the production of PLD, we employed a series of combinatorial strategies to increase PLD expression in Bacillus subtilis WB600. These strategies included screening of signal peptides, selection of different plasmids, and optimization of the sequences of the ribosome-binding site (RBS) and the spacer region. We found that using the signal peptide amyE results in the highest extracellular PLD activity (11.3 U/ml) and in a PLD expression level 5.27-fold higher than when the endogenous signal peptide is used. Furthermore, the strain harboring the recombinant expression plasmid pMA0911-PLD-amyE-his produced PLD with activity enhanced by 69.03% (19.1 U/ml). We then used the online tool \RBS Calculator v2.0 to optimize the sequences of the RBS and the spacer. Using the optimized sequences resulted in an increase in the enzyme activity by about 26.7% (24.2 U/ml). In addition, we found through a transfer experiment that the retention rate of the recombinant plasmid after 5 generations was still 100%. The final product, phosphatidylserine (PS), was successfully detected, with transphosphatidylation selectivity at 74.6%. This is similar to the values for the original producer.

Keywords

References

  1. Wang XM. 2000. Multiple forms of phospholipase D in plants: the gene family, catalytic and regulatory properties, and cellular functions. Prog. Lipid Res. 39: 109-149. https://doi.org/10.1016/S0163-7827(00)00002-3
  2. Hong Y, Yuan S, Sun L, Wang X. 2018. Cytidinediphosphatediacylglycerol synthase 5 is required for phospholipid homeostasis and is negatively involved in hyperosmotic stress tolerance. Plant J. 94: 1038-1050. https://doi.org/10.1111/tpj.13916
  3. Czolkoss S, Fritz C, Holzl G, Aktas M. 2016. Two distinct cardiolipin synthases operate in agrobacterium tumefaciens. PLoS One 11: e0160373. https://doi.org/10.1371/journal.pone.0160373
  4. Takaoka R, Kurosaki H, Nakao H, Ikeda K, Nakano M. 2018. Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation. Biochim Biophys. Acta Biomembr. 1860: 245-249. https://doi.org/10.1016/j.bbamem.2017.10.011
  5. Hussain Z, Uyama T, Tsuboi K, Ueda N. 2017. Mammalian enzymes responsible for the biosynthesis of Nacylethanolamines. Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 1862: 1546-1561. https://doi.org/10.1016/j.bbalip.2017.08.006
  6. Choojit S, Bornscheuer UT, Upaichit A, H-Kittikun A. 2016. Efficient phosphatidylserine synthesis by a phospholipase D fromStreptomycessp. SC734 isolated from soil-contaminated palm oil. Eur. J. Lipid Sci. Technol. 118: 803-813. https://doi.org/10.1002/ejlt.201500227
  7. Zhou WB, Gong JS, Hou HJ, Li H, Lu ZM, Xu HY, et al. 2018. Mining of a phospholipase D and its application in enzymatic preparation of phosphatidylserine. Bioengineered 9: 80-89. https://doi.org/10.1080/21655979.2017.1308992
  8. Plonski NM, Bissoni B, Arachchilage MH, Romstedt K, Kooijman EE, Piontkivska H. 2018. Shedding light on lipid metabolism in Kinetoplastida: A phylogenetic analysis of phospholipase D protein homologs. Gene 656: 95-105. https://doi.org/10.1016/j.gene.2018.02.063
  9. Damnjanovic J, Takahashi R, Suzuki A, Nakano H, Iwasaki Y. 2012. Improving thermostability of phosphatidylinositolsynthesizing Streptomyces phospholipase D. Protein Eng. Des Sel. 25: 415-424. https://doi.org/10.1093/protein/gzs038
  10. Dressler L, Michel F, Thondorf I, Mansfeld J, Golbik R, Ulbrich-Hofmann R. 2017. Metal ions and phosphatidylinositol 4,5-bisphosphate as interacting effectors of ${\alpha}$-type plant phospholipase D. Phytochemistry 138: 57-64. https://doi.org/10.1016/j.phytochem.2017.02.024
  11. Lopez C, Bion VB, Menard O, Rousseau F, Pradel P, Besle JM. 2008. Phospholipid, sphingolipid, and fatty acid compositions of the milk fat globule membrane are modified by diet. J. Agric. Food Chem. 56: 5226-5236. https://doi.org/10.1021/jf7036104
  12. Ramrakhiani L, Chand S. 2011. Recent progress on phospholipases: different sources, assay methods, industrial potential and pathogenicity. Appl. Biochem. Biotechnol. 164: 991-1022. https://doi.org/10.1007/s12010-011-9190-6
  13. Guo Z, Vikbjerg AF, Xu XB. 2005. Enzymatic modification of phospholipids for functional applications and human nutrition. Biotechnol. Adv. 23: 203-259. https://doi.org/10.1016/j.biotechadv.2005.02.001
  14. Vance JE, Tasseva G. 2013. Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells. Biophys. Acta 1831: 543-554.
  15. Frohman MA. 2015. The phospholipase D superfamily as therapeutic targets. Trends Pharmacol. Sci. 36: 137-144. https://doi.org/10.1016/j.tips.2015.01.001
  16. Mahankali M, Alter G, Gomez-Cambronero J. 2015. Mechanism of enzymatic reaction and protein-protein interactions of PLD from a 3D structural model. Cell Signal. 27: 69-81. https://doi.org/10.1016/j.cellsig.2014.09.008
  17. Selvy PE, Lavieri RR, Lindsley CW, Brown HA. 2011. Phospholipase D: enzymology, functionality, and chemical modulation. Chem. Rev. 111: 6064-6119. https://doi.org/10.1021/cr200296t
  18. Ogino C, Kanemasu M, Hayashi Y, Kondo A, Shimizu N, Tokuyama S, et al. 2004. Over-expression system for secretory phospholipase D by Streptomyces lividans. Appl. Microbiol. Biotechnol. 64: 823-828. https://doi.org/10.1007/s00253-003-1552-8
  19. Zhang YN, Lu FP, Chen GQ, Li Y, Wang JL. 2009. Expression, purification, and characterization of phosphatidylserine synthase from Escherichia coli K12 in Bacillus subtilis. J. Agric. Food Chem. 57: 122-126. https://doi.org/10.1021/jf802664u
  20. Pappan K, Brown SA, Chapman KD, Wang XM. 1998. Substrate selectivities and lipid modulation of plant phospholipase D${\alpha}$, -${\beta}$, and -${\gamma}$. Arch. Biochem. Biophys. 353: 131-140. https://doi.org/10.1006/abbi.1998.0640
  21. Abdelkafi S, Abousalham A. 2011. Kinetic study of sunflower phospholipase D${\alpha}$: interactions with micellar substrate, detergents and metals. Plant Physiol. Biochem. 49: 752-757. https://doi.org/10.1016/j.plaphy.2011.02.002
  22. Hatanaka T, Negishi T, Mori K. 2004. A mutant phospholipase D with enhanced thermostability from Streptomyces sp. Biochim. Biophys. Acta 1696: 75-82. https://doi.org/10.1016/j.bbapap.2003.09.013
  23. Matsumoto Y, Kashiwabara N, Oyama T, Murayama K, Matsumoto H, Sakasegawa SI, et al. 2016. Molecular cloning, heterologous expression, and enzymatic characterization of lysoplasmalogen-specific phospholipase D from Thermocrispum sp. FEBS Open Bio. 6: 1113-1130. https://doi.org/10.1002/2211-5463.12131
  24. Nakazawa Y, Sagane Y, Sakurai S, Uchino M, Sato H, Toeda K, et al. 2011. Large-scale production of phospholipase D from Streptomyces racemochromogenes and its application to soybean lecithin modification. Appl. Biochem. Biotechnol. 165: 1494-1506. https://doi.org/10.1007/s12010-011-9370-4
  25. Ogino C, Daido H, Ohmura Y, Takada N, Itou Y, Kondo A, et al. 2007. Remarkable enhancement in PLD activity from Streptoverticillium cinnamoneum by substituting serine residue into the GG/GS motif. Biochim. Biophys. Acta 1774: 671-678. https://doi.org/10.1016/j.bbapap.2007.04.004
  26. Salis HM. 2011. The ribosome binding site calculator. Methods Enzymol. 498: 19-42.
  27. Stiller LM, Galinski EA, Witt EMHJ. 2018. Engineering the salt-inducible ectoine promoter region of halomonas elongata for protein expression in a unique stabilizing environment. Genes (Basel) 9: 184-200. https://doi.org/10.3390/genes9040184
  28. Ogino C, Kanemasu M, Fukumoto M, Kubo T, Yoshino T, Kondo A, et al. 2007. Continuous production of phospholipase D using immobilized recombinant Streptomyces lividans. Enzyme Microb. Technol. 41: 156-161. https://doi.org/10.1016/j.enzmictec.2006.12.011
  29. Li B, Wang J, Zhang X, Zhao B, Niu L. 2016. Aqueous-solid system for highly efficient and environmentally friendly transphosphatidylation catalyzed by phospholipase D to produce phosphatidylserine. J. Agric. Food Chem. 64: 7555-7560. https://doi.org/10.1021/acs.jafc.6b03448
  30. Duan ZQ, Hu F. 2013. Efficient synthesis of phosphatidylserine in 2 methyltetrahydrofuran. J. Biotechnol. 163: 45-49. https://doi.org/10.1016/j.jbiotec.2012.10.022
  31. Yang Sl, Duan ZQ. 2016. Insight into enzymatic synthesis of phosphatidylserine in deep eutectic solvents. Catal. Commun. 82: 16-19. https://doi.org/10.1016/j.catcom.2016.04.010
  32. Chen S, Xu L, Li Y, Hao N, Yan M. 2013. Bioconver sion of phosphatidylserine by phospholipase D from Streptomyces racemochromogenes in a microaqueous water-immiscible organic solvent. Biosci. Biotechnol. Biochem. 77: 1939-1941. https://doi.org/10.1271/bbb.130388
  33. Hama S, Ogino C, Kondo A. 2015. Enzymatic synthesis and modification of structured phospholipids: recent advances in enzyme preparation and biocatalytic processes. Appl. Microbiol. Biotechnol. 99: 7879-7891. https://doi.org/10.1007/s00253-015-6845-1
  34. Bi YH, Duan ZQ, Du WY, Wang ZY. 2015. Improved synthesis of phosphatidylserine using bio-based solvents, limonene and p-cymene. Biotechnol. Lett. 37: 115-119. https://doi.org/10.1007/s10529-014-1646-7
  35. Duan ZQ, Hu F. 2012. Highly efficient synthesis of phosphatidylserine in the eco-friendly solvent ${\gamma}$-valerolactone. Green Chem. 14: 1581-1583. https://doi.org/10.1039/c2gc35092k
  36. Brogan AP, Hallett JP. 2016. Solubilizing and stabilizing proteins in anhydrous ionic liquids through formation of protein-polymer surfactant nanoconstructs. J. Am. Chem. Soc. 138: 4494-4501. https://doi.org/10.1021/jacs.5b13425
  37. Sivapragasam M, Moniruzzaman M, Goto M. 2016. Recent advances in exploiting ionic liquids for biomolecules: Solubility, stability and applications. Biotechnol. J. 11: 1000-1013. https://doi.org/10.1002/biot.201500603
  38. Itoh T. 2017. Ionic liquids as tool to improve enzymatic organic synthesis. Chem. Rev. 117: 10567-10607. https://doi.org/10.1021/acs.chemrev.7b00158
  39. Chen W, Guo W, Gao F, Chen L, Chen S, Li D. 2017. Phospholipase A1-catalysed synthesis of docosahexaenoic acid-enriched phosphatidylcholine in reverse micelles system. Appl. Biochem. Biotechnol. 182: 1037-1052. https://doi.org/10.1007/s12010-016-2379-y
  40. Feng Y, Liu S, Jiao Y, Gao H, Wang M, Du G, et al. 2017. Enhanced extracellular production of L-asparaginase from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy. Appl. Microbiol. Biotechnol. 101: 1509-1520. https://doi.org/10.1007/s00253-016-7816-x
  41. Westers L, Westers H, Quax WJ. 2004. Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim. Biophys. Acta 1694: 299-310. https://doi.org/10.1016/j.bbamcr.2004.02.011
  42. Zalucki YM, Jennings MP. 2017. Signal peptidase I processed secretory signal sequences: Selection for and against specific amino acids at the second position of mature protein. Biochem. Biophys. Res. Commun. 483: 972-977. https://doi.org/10.1016/j.bbrc.2017.01.044
  43. Ismail NF, Hamdan S, Mahadi NM, Murad AM, Rabu A, Bakar FD, et al. 2011. A mutant L-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of Escherichia coli. Biotechnol. Lett. 33: 999-1005. https://doi.org/10.1007/s10529-011-0517-8
  44. Gennity J, Goldstein J, Inouye M. 1990. Signal peptide mutants of Escherichia coli. J. Bioenerg. Biomembr. 3: 233-269.
  45. Chou MM, Kendall DA. 1990. Polymeric sequences reveal a functional interrelationship hydrophobicity and length of signal peptides. J. Biol. Chem. 265: 2873-2880.
  46. Chen HF, Kim J, Kendall DA. 1996. Competition between functional signal peptides demonstratesvariation in affinity for the secretion pathway. J. Bacteriol. 178: 6658-6664. https://doi.org/10.1128/jb.178.23.6658-6664.1996
  47. Moser F, Broers NJ, Hartmans S, Tamsir A, Kerkman R, Roubos JA, et al. 2012. Genetic circuit performance under conditions relevant for industrial bioreactors. ACS Synth. Biol. 1: 555-564. https://doi.org/10.1021/sb3000832

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