Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Permeability issues in whole-cell bioprocesses and cellular membrane engineering


Nutrient uptake and waste excretion are among the many important functions of the cellular membrane. While permitting nutrients into the cell, the cellular membrane system evolves to guide against noxious agents present in the environment from entering the intracellular milieu. The semipermeable nature of the membrane is at odds with biomolecular engineers in their endeavor of using microbes as cell factory. The cellular membrane often retards the entry of substrate into the cellular systems and prevents the product from being released from the cellular system for an easy recovery. Consequently, productivities of whole-cell bioprocesses such as biocatalysis, fermentation, and bioremediations are severely compromised. For example, the rate of whole-cell biocatalysis is usually 1–2 orders of magnitude slower than that of the isolated enzymes. When product export cannot keep pace with the production rate, intracellular product accumulation quickly leads to a halt of production due to product inhibition. While permeabilization via chemical or physical treatment of cell membrane is effective in small-scale process, large-scale implementation is problematic. Molecular engineering approach recently emerged as a much better alternative. Armed with increasingly sophisticated tools, biomolecular engineers are following nature’s ingenuity to derive satisfactory solutions to the permeability problem. This review highlights these exciting molecular engineering achievements.

This is a preview of subscription content, log in to check access.

Fig. 1


  1. Arneborg N, Salskov-Iversen AS, Mathiasen TE (1993) The effect of growth rate and other growth conditions on the lipid composition of Escherichia coli. Appl Microbiol Biotechnol 39:353–357

  2. Aso Y, Miyamoto Y, Harada KM, Momma K, Kawai S, Hashimoto W, Mikami B, Murata K (2006) Engineered membrane superchannel improves bioremediation potential of dioxin-degrading bacteria. Nat Biotechnol 24:188–189

  3. Bansal-Mutalik R, Gaikar VG (2003) Cell permeabilization for extraction of penicillin acylase from Escherichia coli by reverse micellar solution. Enzyme Microb Technol 32:14–26

  4. Breedveld MW, Zevenhuizen LP, Zehnder AJB (1992) Synthesis of cyclic β-1,2-glucans by Rhizobium leguminosarum Biovar trifolii TA-1: factors influencing excretion. J Bacteriol 174:6336–6342

  5. Burman LG, Nurudstrom K, Bloom GD (1972) Murein and the outer penetration barrier of Escherichia coli K-12, Proteus mirubilis and Pseudomonus aeruginosa. J Bacteriol 11:1364–1374

  6. Cavonas M, Torroglosa T, Kleber H, Iborra JL (2003) Effect of salt stress on crotonobetaine and D-carnitine biotransformation of L-carnitine by resting cells of Escherichia coli. J Basic Microbiol 43:259–268

  7. Chow C, Palecek S (2004) Enzyme encapsulation in permeabilized Saccharomyces cerevisiae cells. Biotechnol Prog 20:449–456

  8. Denyer SP, Maillard J-Y (2002) Cellular impermeability and uptake of biocides and antibiotics in Gram-negative bacteria. J Appl Microbiol 92(1):35S–45S

  9. Duetz WA, van Beilen JB, Witholt B (2001) Using proteins in their natural environment: potential and limitations of microbial whole-cell hydrosylations in applied biocatalysis. Curr Opin Biotechnol 12:419–425

  10. Dupont C, Clarke AJ (1991) In-vitro synthesis and O acetylation of peptidoglycan by permeabilized cells of Proteus mirabilis. J Bacteriol 3:4618–4624

  11. Faber K (1995) Biotransformations in organic chemistry—a textbook, 2nd edn. Springer, Berlin Heidelberg New York

  12. Ferrer P, Diers I, Asenjo JA, Andrews BA (1998) Yeast cell permeabilizing β-1,3 glucanases: a toll for the integration of downstream processes and metabolic engineering applications to yeast. Biotechnol Bioeng 58:321–324

  13. Fontanille P, Larroche C (2003) Optimization of isonovalal production from α-pinene oxide using permeabilized cells of Pseudomonas rhodesiae CIP 107491. Appl Microbiol Biotechnol 60:534–540

  14. French C, Keshavarz-Moore E, Ward JM (1996) Development of a simple method for the recovery of recombinant proteins from the Escherichia coli periplasm. Enzyme Microb Technol 19:332–338

  15. Gaspar P, Neves AR, Ramos A, Gasson MJ, Shearman CA, Santos H (2004) Engineering Lactococcus lactis for production of mannitol: high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system. Appl Environ Microbiol 70:1466–1474

  16. Georgiou G, Stathopoulos C, Daugherty PS, Nayak AR, Iverson BL, Curtis R (1997) Display of heterologous proteins on the surface of microorganisms: From the screening of combinatorial libraries to live recombinant vaccines. Nat Biotechnol 15:29–34

  17. Glieder A, Farinas ET, Arnold FH (2002) Laboratory evolution of a soluble, self-sufficient, highly active alknae hydroxylase. Nat Biotechnol 20:1135–1139, (Nov)

  18. Jung H, Buchholz M, Clausen J, Nietschke M, Revermann A, Schmid R, Jung K (2002) CaiT of Escherichia coli, a new transporter catalyzing L-carnitine/γ-butyrobetaine exchange. J Biol Chem 277:39251–39258

  19. Hori K, Kanek M, Tanji Y, Xing X, Unno H (2002) Construction of self-disruptive Bacillus magaterium in response to substrate exhaustion for polyhyroxybutyrate production. Appl Microbiol Biotechnol 59:211–216

  20. Isoai A, Kimura H, Reichert A, Schorgendorfer K, Nikaido K, Tohda H, Giga-Hama Y, Mutoh N, Kumagai H (2002) Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysis prepared with recombinant cells. Biotechnol Bioeng 80:22–32

  21. Kubal BS, D’Souza SF (2004) Immobilization of catalase by entrapment of permeabilized yeast cells in hen egg white using glutaraldehyde. J Biochem Biophys Methods 59:61–64

  22. Lee YJ, Kim CS, Oh DK (2004) Lactulose production by β−galactosidase in permeabilized cells of Kluyveromyces lactis. Appl Microbiol Biotechnol 64:787–793

  23. Liu Y, Hama H, Fujita Y, Kondo A, Inoue Y, Kimura A, Fukuda H (1999) Production of S-lactoylglutathione by high activity whole cell biocatalysts prepared by permeabilization of recombinant Saccharomyces cerevisiae with alcohols. Biotechnol Bioeng 64:54–60

  24. Matsumoto T, Takahash S, Kaieda M, Ueda M, Tanaka A, Fukuda H, Kondo A (2001) Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production. Appl Microbiol Biotechnol 57:515–520

  25. McGarrity JT, Armstrong (1975) The effect of salt on phosphlipid fatty acid composition in Escherichia coli K-12. Biochim Biophys Acta 398:258–264

  26. McGarrity JT, Armstrong (1981) The effect of temperature and other growth conditions on the fatty acid composition in Escherichia coli. Can J Microbiol 27:835–840

  27. Muraji M, Taniguchi H, Tatebe W, Berg H (1999) Examination of the relationship between parameters to determine electropermeability of Saccharomyces cerecisiae. Bioelectrochem Bioenerg 48:485–488

  28. Nampoothiri KM, Hoischen C, bathe B, Mockel B, Pfefferle W, Krumbach K, Sahm H, Eggeling L (2002) Expression of genes of lipid synthesis and altered lipid composition modulates l-glutamate efflux of Corynebacterium glutamicum. Appl Microbiol Biotechnol 58:89–96

  29. Neidhardt FC, Ingraham JL, Schaechter M (1990) Physiology of the bacterial cell. Sinauer Associates, Sunderland, MA

  30. Ni Y, Chen R (2004) Accelerating whole-cell biocatalysis by reducing outer membrane permeability barrier. Biotechnol Bioeng 87:804–811

  31. Ni Y, Chen R (2005) Lipoprotein mutation accelerates substrate-permeability limited toluene dioxygenase-catalyzed reaction. Biotechnol Prog 21:799–805

  32. Ni Y, Mao Z, Chen RR (2006) Outer membrane mutation effects on UDP-glucose permeability and whole-cell catalysis rate. Appl Microbiol Biotechnol 73:384–393

  33. Nikaido H (1994) Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264:382–389

  34. Nikaido H (2001) Preventing drug access to targets cell surface permeability barriers and active efflux in bacteria. Semin Cell Dev Biol 12:215–223

  35. Patnaik R, Louie S, Gavrilovic V, Perry K, Stemmer WPC, Ryan C, del Cardayré S (2002) Genome shuffling of Lactobacillus for improved acid tolerance. Nat Biotechnol 20:707–712

  36. Prescott LM, Harley JP, Klein DA (2002) Microbiology, 5th edn. McGraw–Hill, New York

  37. Reitschel ET, Kirikae T, Scjade FU, Mamat U, Schmidt G, Loppnow H, Ulmer AJ, Zahringer U, Seude U, Pydova F Di, Schreier M, Brade H (1994) Bacterial endotoxin: molecular relatioships of structure to activity and function. FASEB J 8:217–225

  38. Shepard SR, Stone C, Cook S, Bouvier A, Boyd G, Whatherly G, Lydiard D, Schrimsher J (2002) Recovery of intracellular recombinant proteins from the yeast Pichia pastoris by cell permeabilization. J Biotechnol 99:149–160

  39. Shimazu M, Mulchandani A, Chen W (2001) Cell surface display of organophosphorus hydrolase using ice nucleation protein. Biotechnol Prog 17:76–80

  40. Shokri A, Sanden AM, Larsson G (2002) Growth rate-dependent changes in Escherichia coli membrane structure and protein leakage. Appl Microbiol Biotechnol 58:386–392

  41. Silveira MM, Jonas R (2002) The biotechnological production of sorbitol. Appl Microbiol Biotechnol 59:400–409

  42. Sololovska I, Rozenberg R, Riez C, Rouxhet PG, Agathos SN, Wattiau P (2003) Carbon source-induced modifications in the mycolic acid content and cell wall permeability of Rhodococcus erythropolis E1. Appl Environ Microbiol 69:7019–7027

  43. Sroga GE, Dordick JS (2002) Strategy for in vivo screening of subtilisin E reaction specificity in E. coli periplasm. Biotechnol Bioeng 78:761–769

  44. Upadhya R, Nagajyothi, Bhat SG (2000) Stabilization of D-amino caid oxidase and catalase in permeabilized Rhodotorula gracilis cells and its application for the preparation of α-ketoacids. Biotechnol Bioeng 68:430–436

  45. Vaara M (1993) Antibiotic-supersusceptible mutants of Escherichia coli and Salmonella typhimurium. Antimicrob Agents Chemother 37:2255–2260

  46. Vaara M, Nurminen M (1999) Outer membrane permeability barrier in Escherichia coli mutants that are defective in the late acyltransferases of lipid A biosynthesis. Antimicrob Agents Chemother 43:1459–1462

  47. van der Werf MJ, Hartmans S, van den Tweel WJJ (1995) Permeabilization and lysis of Pseudomonas pseudoalcaligenes cells by triton X-100 for efficient production of D-matate. Appl Microbiol Biotechnol 43:590–594

  48. Vidal-Aroca F, Giannattasio M, Brunelli E, Vezzoli A, Plevani P, Muzi-Falsoni M, Bertoni G (2006) One-step high-throughput assay for quantitative detection of beta-galactosidase activity in intact gram-negative bacteria, yeast, and mammalian cells. BioTechniques 40:433–439

  49. Walker SA, Klaenhammer TR (2001) Leaky Lactococcus culture that externalize enzymes and antigens independently of culture lysis and secretion and export pathways. Appl Environ Microbiol 67:251–259

  50. Wouters PC, Bos AP, Ueckert J (2001) Membrane permeabilization in relation to inactivation kinetics of Lactobacillus species due to pulsed electric fields. Appl Environ Microbiol 67:3092–3101

  51. Yang RYK, Bayraktar O, Pu HT (2003) Plant-cell bioreactors with simultaneous electropermeabilization and electrophoresis. J Biotechnol 100:13–22

  52. Yamada S, Awano N, Inubushi K, Maeda E, Nakamori, Nishino K, Yamaguchi A, Takagi H (2006) Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli. Appl Environ Microbiol 72:4735–4742

Download references


This area of research in my laboratory is supported by NSF and EPA’s joint program: the Technology for a Sustainable Environment, BES 0455194.

Author information

Correspondence to Rachel Ruizhen Chen.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chen, R.R. Permeability issues in whole-cell bioprocesses and cellular membrane engineering. Appl Microbiol Biotechnol 74, 730–738 (2007).

Download citation


  • Permeability
  • Cellular membrane engineering
  • Whole-cell
  • Biocatalysis
  • Bioremediation
  • Fermentation