Advertisement

Applied Microbiology and Biotechnology

, Volume 103, Issue 2, pp 793–806 | Cite as

Improving extracellular protein production in Escherichia coli by overexpressing D,D-carboxypeptidase to perturb peptidoglycan network synthesis and structure

  • Haiquan YangEmail author
  • Jinyuan Hu
  • Xiao Lu
  • Fuxiang Wang
  • Wei Shen
  • Wei Hu
  • Lingling Wang
  • Xianzhong Chen
  • Long Liu
Biotechnologically relevant enzymes and proteins
First Online:

Abstract

Most recombinant proteins in Escherichia coli are not efficiently secreted to the extracellular space. Structural stabilisation of the cell wall is essential for extracellular protein production in E. coli, for which D,D-carboxypeptidases are essential. Herein, we perturbed the peptidoglycan structure of the E. coli cell wall by overexpressing D,D-carboxypeptidase genes dacA or dacB, and investigated the effect on extracellular protein production. Overexpression of dacA or dacB promoted the accumulation of intracellular soluble peptidoglycan, altered cell morphology (shape and size) and led to the formation of transparent globular structures in E. coli cells. Compared with controls (CK), extracellular production of recombinant green fluorescent protein (GFP) was increased by 1.7- and 2.3-fold upon overexpression of dacA and dacB, respectively. Similarly, extracellular production of recombinant amylase and α-galactosidase was increased by 4.5- and 2.8-fold, respectively, upon overexpression of dacA, and by 11.9- and 2.5-fold, respectively, upon overexpression of dacB. Overexpression of dacA or dacB enhanced both the outer and inner membrane permeability of E. coli. This cell wall engineering strategy opens up a new direction for enhancing extracellular protein and chemical production in E. coli.

Keywords

Extracellular protein production Overexpression D,D-carboxypeptidase Peptidoglycan structure Membrane permeability Escherichia coli 
This is a preview of subscription content, log in to check access.

Notes

Author contributions

H.Y. designed the research; H.Y., J.H., X.L., L.W., and F.W. performed the research; X.L., W.S., F.W., and X.C. analysed the data; L.L., W.H., and H.Y. wrote the paper.

Funding information

This work was funded by National Natural Science Foundation of China (21406089), Natural Science Foundation of Jiangsu Province (BK20140152), the Open Project Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (KLIB-KF201509), the Open Project Program of the Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, China (KLCCB-KF201607, KLCCB-KF201802), 111 Project (111-2-06), and Postgraduate Education Research and Practice Project of Jiangnan University (YJSJG2017004).

Compliance with ethical standards

This article is in compliance with ethical standards, and does not contain any studies with animals performed or human participants.

Conflict of interest

The authors declare that they have no competing interest.

Supplementary material

253_2018_9510_MOESM1_ESM.pdf (625 kb)
ESM 1 (PDF 625 kb)

References

  1. Baquero MR, Bouzon M, Quintela JC, Ayala JA, Moreno F (1996) dacD, an Escherichia coli gene encoding a novel penicillin-binding protein (PBP6b) with DD-carboxypeptidase activity. J Bacteriol 178(24):7106–7111Google Scholar
  2. Barreteau H, Kovac A, Boniface A, Sova M, Gobec S, Blanot D (2008) Cytoplasmic steps of peptidoglycan biosynthesis. FEMS Microbiol Rev 32(2):168–207Google Scholar
  3. Beveridge TJ (1999) Structures of gram-negative cell walls and their derived membrane vesicles. J Bacteriol 181(16):4725–4733Google Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254Google Scholar
  5. Burstein C, Kepes A (1971) The α-galactosidase from Escherichia coli K12. Biochim Biophys Acta 230(1):52–63Google Scholar
  6. Carrio MM, Villaverde A (2002) Construction and deconstruction of bacterial inclusion bodies. J Biotechnol 96(1):3–12Google Scholar
  7. Cayley DS, Guttman HJ, Record MT Jr (2000) Biophysical characterization of changes in amounts and activity of Escherichia coli cell and compartment water and turgor pressure in response to osmotic stress. Biophys J 78(4):1748–1764Google Scholar
  8. Choi J, Lee S (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 64(5):625–635Google Scholar
  9. Chowdhury C, Nayak TR, Young KD, Ghosh AS (2010) A weak dd-carboxypeptidase activity explains the inability of PBP 6 to substitute for PBP 5 in maintaining normal cell shape in Escherichia coli. FEMS Microbiol Lett 303(1):76–83Google Scholar
  10. Demchick P, Koch AL (1996) The permeability of the wall fabric of Escherichia coli and Bacillus subtilis. J Bacteriol 178(3):768–773Google Scholar
  11. Dmitriev B, Toukach F, Ehlers S (2005) Towards a comprehensive view of the bacterial cell wall. Trends Microbiol 13(12):569–574Google Scholar
  12. Doyle RJ, Marquis RE (1994) Elastic, flexible peptidoglycan and bacterial cell wall properties. Trends Microbiol 2(2):57–60Google Scholar
  13. Egan AJF, Biboy J, Veer IV, Breukink E, Vollmer W (2015) Activities and regulation of peptidoglycan synthases. Philos Trans R Soc B 370(1679):20150031Google Scholar
  14. Frére JM, Leyh-Bouille M, Ghuysen JM, Nieto M, Perkins H (1976) Exocellular dd-carboxypeptidases-transpeptidases from Streptomyces. Methods Enzymol 45(45):610–636Google Scholar
  15. Fuwa H (1954) A new method for microdetermination of amylase activity by the use of amylose as the substrate. J Biochem 41(5):583–603Google Scholar
  16. Ghosh AS, Chowdhury C, Nelson DE (2008) Physiological functions of D-alanine carboxypeptidases in Escherichia coli. Trends Microbiol 16(7):309–317Google Scholar
  17. Höltje JV (1998) Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol Mol Biol Rev 62(1):181–203Google Scholar
  18. Horne D, Hakenbeck R, Tomasz A (1977) Secretion of lipids induced by inhibition of peptidoglycan synthesis in streptococci. J Bacteriol 132(2):704–717Google Scholar
  19. Huang KC, Mukhopadhyay R, Wen BN, Gitai Z, Wingreen NS (2008) Cell shape and cell-wall organization in Gram-negative bacteria. PNAS 105(49):19282–19287Google Scholar
  20. Kishida H, Unzai S, Roper DI, Lloyd A, Park S-Y, Tame JRH (2006) Crystal structure of penicillin binding protein 4 (dacB) from Escherichia coli, both in the native form and covalently linked to various antibiotics. Biochem 45(3):783–792Google Scholar
  21. Koch AL (1984) Shrinkage of growing Escherichia coli cells by osmotic challenge. J Bacteriol 159(3):919–924Google Scholar
  22. Koch AL, Woeste S (1992) Elasticity of the sacculus of Escherichia coli. J Bacteriol 174(14):4811–4819Google Scholar
  23. Kraft AR, Prabhu J, Ursinus A, Holtje JV (1999) Interference with murein turnover has no effect on growth but reduces β-lactamase induction in Escherichia coli. J Bacteriol 181(23):7192–7198Google Scholar
  24. Kurakake M, Okumura T, Morimoto Y (2015) Synthesis of galactosyl glycerol from guar gum by transglycosylation of α-galactosidase from Aspergillus sp. MK14. Food Chem 172:150–154Google Scholar
  25. Lee M, Hesek D, Llarrull LI, Lastochkin E, Pi H, Boggess B, Mobashery S (2013) Reactions of all Escherichia coli lytic transglycosylases with bacterial cell wall. J Am Chem Soc 135(9):3311–3314Google Scholar
  26. Lehrer RI, Barton A, Ganz T (1988) Concurrent assessment of inner and outer membrane permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry. J Immunol Methods 108(1–2):153–158Google Scholar
  27. Li B, Wang L, Su LQ, Chen S, Li ZF, Chen J, Wu J (2012) Glycine and Triton X-100 enhanced secretion of recombinant alpha-CGTase mediated by OmpA signal peptide in Escherichia coli. Biotechnol Bioprocess Eng 17(6):1128–1134Google Scholar
  28. Liu L, Yang HQ, Shin H-D, Chen RR, Li JH, Du GC, Chen J (2013) How to achieve high-level expression of microbial enzymes: strategies and perspectives. Bioeng 4(4):212–223Google Scholar
  29. Loh B, Grant C, Hancock R (1984) Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa. Antimicrob Agents Chemother 26(4):546–551Google Scholar
  30. Ma YF, Shen W, Chen XZ, Liu L, Zhou ZM, Xu F, Yang HQ (2016) Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization. J Biol Eng 10(1):13Google Scholar
  31. Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60(3):512–538Google Scholar
  32. Martin HH, Maskos C, Burger R (1975) d-alanyl-d-alanine carboxypeptidase in the cacterial form and L-form of Proteus mirabilis. FEBS J 55:465–473Google Scholar
  33. Meberg BM, Paulson AL, Priyadarshini R, Young KD (2004) Endopeptidase penicillin-binding proteins 4 and 7 play auxiliary roles in determining uniform morphology of Escherichia coli. J Bacteriol 186(24):8326–8336Google Scholar
  34. Mergulhao FJM, Monteiro GA, Cabral JMS, Taipa MA (2004) Design of bacterial vector systems for the production of recombinant proteins in Escherichia coli. J Microb Biotechnol 14(1):1–14Google Scholar
  35. Mergulhao FJM, Summers DK, Monteiro GA (2005) Recombinant protein secretion in Escherichia coli. Biotechnol Adv 23(3):177–202Google Scholar
  36. Nelson DE, Young KD (2000) Penicillin binding protein 5 affects cell diameter, contour, and morphology of Escherichia coli. J Bacteriol 182(6):1714–1721Google Scholar
  37. Nelson DE, Young KD (2001) Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteins to maintenance of cell shape in Escherichia coli. J Bacteriol 183(10):3055–3064Google Scholar
  38. Nelson DE, Ghosh AS, Paulson AL, Young KD (2002) Contribution of membrane-binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform cellular morphology of Escherichia coli. J Bacteriol 184(13):3630–3639Google Scholar
  39. Park JT, Uehara T (2008) How bacteria consume their own exoskeletons (turnover and recycling of cell wall peptidoglycan). Microbiol Mol Biol Rev 72(2):211–227Google Scholar
  40. Pollock JJ, Nguyen-Disteche M, Ghuysen JM, Coyette J, Linder R, Salton MR, Kim KS, Perkins HR, Reynolds P (1974) Fractionation of the DD-carboxypeptidase-transpeptidase activities solubilized from membranes of Escherichia coli K12, strain 44. Eur J Biochem 41(3):439–446Google Scholar
  41. Potluri LP, de Pedro MA, Young KD (2012) Escherichia coli low-molecular-weight penicillin-binding proteins help orient septal FtsZ, and their absence leads to asymmetric cell division and branching. Mol Microbiol 84(2):203–224Google Scholar
  42. Rippmann JF, Klein M, Hoischen C, Brocks B, Rettig WJ, Gumpert J, Pfizenmaier K, Mattes R, Moosmayer D (1998) Procaryotic expression of single-chain variable-fragment (scFv) antibodies: secretion in L-form cells of Proteus mirabilis leads to active product and overcomes the limitations of periplasmic expression in Escherichia coli. Appl Environ Microbiol 64(12):4862–4869Google Scholar
  43. Shimada T, Yamazaki K, Ishihama A (2013) Novel regulator PgrR for switch control of peptidoglycan recycling in Escherichia coli. Genes Cells 18(2):123–134Google Scholar
  44. Tang JB, Yang HM, Song SH, Zhu P, Ji AG (2008) Effect of glycine and Triton X-100 on secretion and expression of ZZ-EGFP fusion protein. Food Chem 108(2):657–662Google Scholar
  45. Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72(2):211–222Google Scholar
  46. Typas A, Banzhaf M, Saparoea BVDBV, Verheul J, Biboy J, Nichols RJ, Zietek M, Beilharz K, Kannenberg K, von Benchenberg M, Breukink E, den Blaauwen T, Gross CA, Vollmer W (2010) Regulation of peptidoglycan synthesis by outer-membrane proteins. Cell 143(7):1097–1109Google Scholar
  47. Typas A, Banzhaf M, Gross CA, Vollmer W (2012) From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat Rev Microbiol 10(2):123–136Google Scholar
  48. van Heijenoort J (2001) Recent advances in the formation of the bacterial peptidoglycan monomer unit. Nat Prod Rep 18(5):503–519Google Scholar
  49. Vollmer W, Seligman SJ (2010) Architecture of peptidoglycan: more data and more models. Trends Microbiol 18(2):59–66Google Scholar
  50. Vollmer W, Joris B, Charlier P, Foster S (2008) Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiol Rev 32(2):259–286Google Scholar
  51. Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as a cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta 1694(1):299–310Google Scholar
  52. Yang JB, Moyana T, Mackezie S, Xia Q, Xiang J (1998) One hundred seventy-fold increase in excretion of an FV fragment tumor necrosis factor alpha fusion protein (SFV/TNF-α) from Escherichia coli caused by the synergistic effects of glycine and triton X-100. Appl Environ Microbiol 64(8):2669–2874Google Scholar
  53. Yao X, Jericho M, Pink D, Beveridge T (1999) Thickess and elasticity of gram-negative murein sacculi measured by atomic force microscopy. J Bacteriol 181(22):6865–6875Google Scholar
  54. Young KD (2003) Bacterial shape. Mol Microbiol 49(3):571–580Google Scholar
  55. Zhang WL, Shi QC, Meroueh SC, Vakulenko SB, Mobashery S (2007) Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli. Biochem 46(35):10113–10121Google Scholar
  56. Zhang CY, Liu L, Teng LP, Chen JH, Liu J, Li JH, Du GC, Chen J (2012) Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor. Metab Eng 14(5):521–527Google Scholar
  57. Zheng HC, Yu ZX, Fu XP, Li SF, Xu JY, Song H, Ma YH (2016) High level extracellular production of a truncated alkaline β‑mannanase from alkaliphilic Bacillus sp. N16‑5 in Escherichia coli by the optimization of induction condition and fed‑batch fermentation. J Ind Microbiol Biotechnol 43(7):977–987.Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of EducationJiangnan UniversityWuxiChina
  2. 2.Institute of Modern PhysicsChinese Academy of SciencesLanzhouChina

Personalised recommendations

Cite article

Buy options