Applied Microbiology and Biotechnology

, Volume 103, Issue 5, pp 2053–2066 | Cite as

Engineering of lactic acid bacteria for delivery of therapeutic proteins and peptides

  • Tina Vida Plavec
  • Aleš BerlecEmail author


Lactic acid bacteria (LAB) have a long-term history of use in food industry and are becoming attractive for use in therapy on account of their safety, intrinsic beneficial health effects, and considerable biotechnological potential. The established systems for engineering are combined with novel approaches, such as CRISPR-Cas, to enable the use of LAB as vectors for delivery of various therapeutic molecules. The latter are either secreted or surface displayed and can be used for the treatment or prevention of numerous conditions: inflammatory bowel diseases, infections, autoimmune diseases, and even cancer. This review presents some recent data on engineering of LAB, with the emphasis on the most commonly used genera Lactococcus and Lactobacillus. Their use for the delivery of therapeutic proteins is discussed, while a special focus is given to the delivery of therapeutic peptides. Therapeutically relevant improvements of engineered LAB, such as containment systems, ability to visualize bacteria, or target specific host cells are also addressed. Future engineering of LAB for therapy will adopt the capabilities of synthetic biology, with first examples already emerging.


Lactic acid bacteria Lactococcus lactis Lactobacillus plantarum Engineering Protein delivery Peptide delivery 


Funding information

This work was supported by the Slovenian Research Agency (grant numbers P4-0127 and J4-9327).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain studies with human participants or animals performed by any of authors.


  1. ActoBio Therapeutics I (2018) ActoBio Therapeutics™ doses first patient in phase Ib/IIa clinical study of AG019 for the treatment of type 1 diabetes. PW Newswire. Accessed 29 Oct 2018
  2. Arora T, Wegmann U, Bobhate A, Lee YS, Greiner TU, Drucker DJ, Narbad A, Backhed F (2016) Microbially produced glucagon-like peptide 1 improves glucose tolerance in mice. Mol Metab 5(8):725–730CrossRefPubMedPubMedCentralGoogle Scholar
  3. de Azevedo M, Karczewski J, Lefevre F, Azevedo V, Miyoshi A, Wells JM, Langella P, Chatel JM (2012) In vitro and in vivo characterization of DNA delivery using recombinant Lactococcus lactis expressing a mutated form of L. monocytogenes Internalin a. BMC Microbiol 12:299CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bahey-El-Din M, Gahan CG (2011) Lactococcus lactis-based vaccines: current status and future perspectives. Hum Vaccin 7(1):106–109CrossRefPubMedGoogle Scholar
  5. Bahey-El-Din M, Casey PG, Griffin BT, Gahan CG (2010) Efficacy of a Lactococcus lactis DeltapyrG vaccine delivery platform expressing chromosomally integrated hly from Listeria monocytogenes. Bioeng Bugs 1(1):66–74. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Baradaran A, Yusoff K, Shafee N, Rahim RA (2016) Newcastle disease virus hemagglutinin neuraminidase as a potential cancer targeting agent. J Cancer 7(4):462–466CrossRefPubMedPubMedCentralGoogle Scholar
  7. Benbouziane B, Ribelles P, Aubry C, Martin R, Kharrat P, Riazi A, Langella P, Bermudez-Humaran LG (2013) Development of a stress-inducible controlled expression (SICE) system in Lactococcus lactis for the production and delivery of therapeutic molecules at mucosal surfaces. J Biotechnol 168(2):120–129. CrossRefPubMedGoogle Scholar
  8. Berlec A, Malovrh T, Zadravec P, Steyer A, Ravnikar M, Sabotic J, Poljsak-Prijatelj M, Strukelj B (2013) Expression of a hepatitis a virus antigen in Lactococcus lactis and Escherichia coli and evaluation of its immunogenicity. Appl Microbiol Biotechnol 97(10):4333–4342CrossRefPubMedGoogle Scholar
  9. Berlec A, Zavrsnik J, Butinar M, Turk B, Strukelj B (2015) In vivo imaging of Lactococcus lactis, Lactobacillus plantarum and Escherichia coli expressing infrared fluorescent protein in mice. Microb Cell Factories 14:181. CrossRefGoogle Scholar
  10. Berlec A, Skrlec K, Kocjan J, Olenic M, Strukelj B (2018) Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis. Sci Rep 8(1):1009. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Bermudez-Humaran LG, Motta JP, Aubry C, Kharrat P, Rous-Martin L, Sallenave JM, Deraison C, Vergnolle N, Langella P (2015) Serine protease inhibitors protect better than IL-10 and TGF-beta anti-inflammatory cytokines against mouse colitis when delivered by recombinant lactococci. Microb Cell Factories 14:26. CrossRefGoogle Scholar
  12. Bober JR, Beisel CL, Nair NU (2018) Synthetic biology approaches to engineer probiotics and members of the human microbiota for biomedical applications. Annu Rev Biomed Eng 20:277–300. CrossRefPubMedGoogle Scholar
  13. Bonisch E, Oh YJ, Anzengruber J, Hager FF, Lopez-Guzman A, Zayni S, Hinterdorfer P, Kosma P, Messner P, Duda KA, Schaffer C (2018) Lipoteichoic acid mediates binding of a Lactobacillus S-layer protein. Glycobiology 28(3):148–158. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Borrero J, Chen Y, Dunny GM, Kaznessis YN (2015) Modified lactic acid bacteria detect and inhibit multiresistant enterococci. ACS Synth Biol 4(3):299–306CrossRefPubMedGoogle Scholar
  15. Braat H, Rottiers P, Hommes DW, Huyghebaert N, Remaut E, Remon JP, van Deventer SJ, Neirynck S, Peppelenbosch MP, Steidler L (2006) A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn’s disease. Clin Gastroenterol Hepatol 4(6):754–759. CrossRefPubMedGoogle Scholar
  16. Bron PA, Kleerebezem M (2018) Lactic acid bacteria for delivery of endogenous or engineered therapeutic molecules. Front Microbiol 9:1821. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Brophy JA, Voigt CA (2014) Principles of genetic circuit design. Nat Methods 11(5):508–520. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Call EK, Klaenhammer TR (2013) Relevance and application of sortase and sortase-dependent proteins in lactic acid bacteria. Front Microbiol 4:73. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Caluwaerts S, Vandenbroucke K, Steidler L, Neirynck S, Vanhoenacker P, Corveleyn S, Watkins B, Sonis S, Coulie B, Rottiers P (2010) AG013, a mouth rinse formulation of Lactococcus lactis secreting human trefoil factor 1, provides a safe and efficacious therapeutic tool for treating oral mucositis. Oral Oncol 46(7):564–570CrossRefPubMedGoogle Scholar
  20. Cameron DE, Collins JJ (2014) Tunable protein degradation in bacteria. Nat Biotechnol 32(12):1276–1281CrossRefPubMedPubMedCentralGoogle Scholar
  21. Cano-Garrido O, Seras-Franzoso J, Garcia-Fruitos E (2015) Lactic acid bacteria: reviewing the potential of a promising delivery live vector for biomedical purposes. Microb Cell Factories 14:137. CrossRefGoogle Scholar
  22. Cao HP, Wang HN, Yang X, Zhang AY, Li X, Ding MD, Liu ST, Zhang ZK, Yang F (2013) Lactococcus lactis anchoring avian infectious bronchitis virus multi-epitope peptide EpiC induced specific immune responses in chickens. Biosci Biotechnol Biochem 77(7):1499–1504CrossRefPubMedGoogle Scholar
  23. Carroll IM, Andrus JM, Bruno-Barcena JM, Klaenhammer TR, Hassan HM, Threadgill DS (2007) Anti-inflammatory properties of Lactobacillus gasseri expressing manganese superoxide dismutase using the interleukin 10-deficient mouse model of colitis. Am J Physiol Gastrointest Liver Physiol 293(4):G729–G738. CrossRefPubMedGoogle Scholar
  24. Chamcha V, Jones A, Quigley BR, Scott JR, Amara RR (2015) Oral immunization with a recombinant Lactococcus lactis-expressing HIV-1 antigen on group a Streptococcus pilus induces strong mucosal immunity in the gut. J Immunol 195(10):5025–5034CrossRefPubMedPubMedCentralGoogle Scholar
  25. Chancey CJ, Khanna KV, Seegers JF, Zhang GW, Hildreth J, Langan A, Markham RB (2006) Lactobacilli-expressed single-chain variable fragment (scFv) specific for intercellular adhesion molecule 1 (ICAM-1) blocks cell-associated HIV-1 transmission across a cervical epithelial monolayer. J Immunol 176(9):5627–5636CrossRefPubMedGoogle Scholar
  26. Chowdhury MY, Li R, Kim JH, Park ME, Kim TH, Pathinayake P, Weeratunga P, Song MK, Son HY, Hong SP, Sung MH, Lee JS, Kim CJ (2014) Mucosal vaccination with recombinant Lactobacillus casei-displayed CTA1-conjugated consensus matrix protein-2 (sM2) induces broad protection against divergent influenza subtypes in BALB/c mice. PLoS One 9(4):e94051. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Daniel C, Repa A, Wild C, Pollak A, Pot B, Breiteneder H, Wiedermann U, Mercenier A (2006) Modulation of allergic immune responses by mucosal application of recombinant lactic acid bacteria producing the major birch pollen allergen bet v 1. Allergy 61(7):812–819. CrossRefPubMedGoogle Scholar
  28. Daniel C, Repa A, Mercenier A, Wiedermann U, Wells J (2007) The European LABDEL project and its relevance to the prevention and treatment of allergies. Allergy 62(11):1237–1242. CrossRefPubMedGoogle Scholar
  29. Daniel C, Poiret S, Dennin V, Boutillier D, Pot B (2013) Bioluminescence imaging study of spatial and temporal persistence of Lactobacillus plantarum and Lactococcus lactis in living mice. Appl Environ Microbiol 79(4):1086–1094. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Daniel C, Titecat M, Poiret S, Cayet D, Boutillier D, Simonet M, Sirard JC, Lemaitre N, Sebbane F (2016) Characterization of the protective immune response to Yersinia pseudotuberculosis infection in mice vaccinated with an LcrV-secreting strain of Lactococcus lactis. Vaccine 34(47):5762–5767CrossRefPubMedGoogle Scholar
  31. Danino T, Prindle A, Kwong GA, Skalak M, Li H, Allen K, Hasty J, Bhatia SN (2015) Programmable probiotics for detection of cancer in urine. Sci Transl Med 7(289):289ra84. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Del Carmen S, de Moreno de LeBlanc A, Levit R, Azevedo V, Langella P, Bermudez-Humaran LG, LeBlanc JG (2017) Anti-cancer effect of lactic acid bacteria expressing antioxidant enzymes or IL-10 in a colorectal cancer mouse model. Int Immunopharmacol 42:122–129. CrossRefPubMedGoogle Scholar
  33. Dieye Y, Usai S, Clier F, Gruss A, Piard JC (2001) Design of a protein-targeting system for lactic acid bacteria. J Bacteriol 183(14):4157–4166CrossRefPubMedPubMedCentralGoogle Scholar
  34. Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096. CrossRefPubMedGoogle Scholar
  35. van der Els S, James JK, Kleerebezem M, Bron PA (2018) Development of a versatile Cas9-driven subpopulation-selection toolbox in Lactococcus lactis. Appl Environ Microbiol.
  36. Fernandez de Palencia P, Nieto C, Acebo P, Espinosa M, Lopez P (2000) Expression of green fluorescent protein in Lactococcus lactis. FEMS Microbiol Immunol 183(2):229–234CrossRefGoogle Scholar
  37. Garcia-Cayuela T, de Cadinanos LP, Mohedano ML, de Palencia PF, Boden D, Wells J, Pelaez C, Lopez P, Requena T (2012) Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli. Appl Microbiol Biotechnol 96(1):171–181CrossRefPubMedGoogle Scholar
  38. Habimana O, Le Goff C, Juillard V, Bellon-Fontaine MN, Buist G, Kulakauskas S, Briandet R (2007) Positive role of cell wall anchored proteinase PrtP in adhesion of lactococci. BMC Microbiol 7:36CrossRefPubMedPubMedCentralGoogle Scholar
  39. Hanson ML, Hixon JA, Li W, Felber BK, Anver MR, Stewart CA, Janelsins BM, Datta SK, Shen W, McLean MH, Durum SK (2014) Oral delivery of IL-27 recombinant bacteria attenuates immune colitis in mice. Gastroenterology 146(1):210–221 e13. CrossRefPubMedGoogle Scholar
  40. Heine SJ, Franco-Mahecha OL, Chen X, Choudhari S, Blackwelder WC, van Roosmalen ML, Leenhouts K, Picking WL, Pasetti MF (2015) Shigella IpaB and IpaD displayed on L. lactis bacterium-like particles induce protective immunity in adult and infant mice. Immunol Cell Biol 93(7):641–652Google Scholar
  41. Huibregtse IL, Snoeck V, de Creus A, Braat H, De Jong EC, Van Deventer SJ, Rottiers P (2007) Induction of ovalbumin-specific tolerance by oral administration of Lactococcus lactis secreting ovalbumin. Gastroenterology 133(2):517–528. CrossRefPubMedGoogle Scholar
  42. Huynh E, Li J (2015) Generation of Lactococcus lactis capable of coexpressing epidermal growth factor and trefoil factor to enhance in vitro wound healing. Appl Microbiol Biotechnol 99(11):4667–4677CrossRefPubMedGoogle Scholar
  43. Innocentin S, Guimaraes V, Miyoshi A, Azevedo V, Langella P, Chatel JM, Lefevre F (2009) Lactococcus lactis expressing either Staphylococcus aureus fibronectin-binding protein a or Listeria monocytogenes internalin a can efficiently internalize and deliver DNA in human epithelial cells. Appl Environ Microbiol 75(14):4870–4878CrossRefPubMedPubMedCentralGoogle Scholar
  44. Kajikawa A, Zhang L, Long J, Nordone S, Stoeker L, LaVoy A, Bumgardner S, Klaenhammer T, Dean G (2012) Construction and immunological evaluation of dual cell surface display of HIV-1 gag and Salmonella enterica serovar Typhimurium FliC in Lactobacillus acidophilus for vaccine delivery. Clin Vaccine Immunol 19(9):1374–1381. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Karimi S, Ahl D, Vagesjo E, Holm L, Phillipson M, Jonsson H, Roos S (2016) In vivo and in vitro detection of luminescent and fluorescent Lactobacillus reuteri and application of red fluorescent mCherry for assessing plasmid persistence. PLoS One 11(3):e0151969. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Karlskas IL, Maudal K, Axelsson L, Rud I, Eijsink VG, Mathiesen G (2014) Heterologous protein secretion in lactobacilli with modified pSIP vectors. PLoS One 9(3):e91125. CrossRefPubMedPubMedCentralGoogle Scholar
  47. Kasarello K, Szczepankowska A, Kwiatkowska-Patzer B, Lipkowski AW, Gadamski R, Sulejczak D, Lachwa M, Bialy M, Bardowski J (2016) Effect of recombinant Lactococcus lactis producing myelin peptides on neuroimmunological changes in rats with experimental allergic encephalomyelitis. Folia Neuropathol 54(3):249–258CrossRefPubMedGoogle Scholar
  48. Kawana K, Adachi K, Kojima S, Taguchi A, Tomio K, Yamashita A, Nishida H, Nagasaka K, Arimoto T, Yokoyama T, Wada-Hiraike O, Oda K, Sewaki T, Osuga Y, Fujii T (2014) Oral vaccination against HPV E7 for treatment of cervical intraepithelial neoplasia grade 3 (CIN3) elicits E7-specific mucosal immunity in the cervix of CIN3 patients. Vaccine 32(47):6233–6239. CrossRefPubMedGoogle Scholar
  49. Kobierecka PA, Olech B, Ksiazek M, Derlatka K, Adamska I, Majewski PM, Jagusztyn-Krynicka EK, Wyszynska AK (2016) Cell wall anchoring of the Campylobacter antigens to Lactococcus lactis. Front Microbiol 7:165. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Kong W, Blanchard AE, Liao C, Lu T (2017) Engineering robust and tunable spatial structures with synthetic gene circuits. Nucleic Acids Res 45(2):1005–1014CrossRefPubMedGoogle Scholar
  51. Kosler S, Strukelj B, Berlec A (2017) Lactic acid bacteria with concomitant IL-17, IL-23 and TNFalpha-binding ability for the treatment of inflammatory bowel disease. Curr Pharm Biotechnol 18(4):318–326. CrossRefPubMedGoogle Scholar
  52. Kuipers A, Rink R, Moll GN (2009) Translocation of a thioether-bridged azurin peptide fragment via the sec pathway in Lactococcus lactis. Appl Environ Microbiol 75(11):3800–3802CrossRefPubMedPubMedCentralGoogle Scholar
  53. Kyla-Nikkila K, Alakuijala U, Saris PE (2010) Immobilization of Lactococcus lactis to cellulosic material by cellulose-binding domain of Cellvibrio japonicus. J Appl Microbiol 109(4):1274–1283. CrossRefPubMedGoogle Scholar
  54. Le Loir Y, Nouaille S, Commissaire J, Bretigny L, Gruss A, Langella P (2001) Signal peptide and propeptide optimization for heterologous protein secretion in Lactococcus lactis. Appl Environ Microbiol 67(9):4119–4127CrossRefPubMedPubMedCentralGoogle Scholar
  55. Leenay RT, Vento JM, Shah M, Martino ME, Leulier F, Beisel CL (2018) Genome editing with CRISPR-Cas9 in Lactobacillus plantarum revealed that editing outcomes can vary across strains and between methods. Biotechnol J e1700583.
  56. Lei H, Peng X, Ouyang J, Zhao D, Jiao H, Shu H, Ge X (2015) Protective immunity against influenza H5N1 virus challenge in chickens by oral administration of recombinant Lactococcus lactis expressing neuraminidase. BMC Vet Res 11:85. CrossRefPubMedPubMedCentralGoogle Scholar
  57. Li Y, Li X, Liu H, Zhuang S, Yang J, Zhang F (2014) Intranasal immunization with recombinant Lactococci carrying human papillomavirus E7 protein and mouse interleukin-12 DNA induces E7-specific antitumor effects in C57BL/6 mice. Oncol Lett 7(2):576–582. CrossRefPubMedGoogle Scholar
  58. Li HS, Piao DC, Jiang T, Bok JD, Cho CS, Lee YS, Kang SK, Choi YJ (2015) Recombinant interleukin 6 with M cell-targeting moiety produced in Lactococcus lactis IL1403 as a potent mucosal adjuvant for peroral immunization. Vaccine 33(16):1959–1967CrossRefPubMedGoogle Scholar
  59. Liang J, Aihua Z, Yu W, Yong L, Jingjing L (2010) HSP65 serves as an immunogenic carrier for a diabetogenic peptide P277 inducing anti-inflammatory immune response in NOD mice by nasal administration. Vaccine 28(19):3312–3317CrossRefPubMedGoogle Scholar
  60. Limaye SA, Haddad RI, Cilli F, Sonis ST, Colevas AD, Brennan MT, Hu KS, Murphy BA (2013) Phase 1b, multicenter, single blinded, placebo-controlled, sequential dose escalation study to assess the safety and tolerability of topically applied AG013 in subjects with locally advanced head and neck cancer receiving induction chemotherapy. Cancer 119(24):4268–4276. CrossRefPubMedGoogle Scholar
  61. Lin Y, Krogh-Andersen K, Pelletier J, Marcotte H, Ostenson CG, Hammarstrom L (2016) Oral delivery of pentameric glucagon-like peptide-1 by recombinant Lactobacillus in diabetic rats. PLoS One 11(9):e0162733. CrossRefPubMedPubMedCentralGoogle Scholar
  62. Linares DM, Alvarez-Sieiro P, del Rio B, Ladero V, Redruello B, Martin MC, Fernandez M, Alvarez MA (2015) Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis. Microb Cell Factories 14:208CrossRefGoogle Scholar
  63. Lindholm A, Smeds A, Palva A (2004) Receptor binding domain of Escherichia coli F18 fimbrial adhesin FedF can be both efficiently secreted and surface displayed in a functional form in Lactococcus lactis. Appl Environ Microbiol 70(4):2061–2071CrossRefPubMedPubMedCentralGoogle Scholar
  64. Liu S, Li Y, Deng B, Xu Z (2016) Recombinant Lactococcus lactis expressing porcine insulin-like growth factor I ameliorates DSS-induced colitis in mice. BMC Biotechnol 16:25CrossRefPubMedPubMedCentralGoogle Scholar
  65. Liu L, Zhang W, Song Y, Wang W, Zhang Y, Wang T, Li K, Pan Q, Qi X, Gao Y, Gao L, Liu C, Wang Y, He G, Wang X, Cui H (2018) Recombinant Lactococcus lactis co-expressing OmpH of an M cell-targeting ligand and IBDV-VP2 protein provide immunological protection in chickens. Vaccine 36(5):729–735CrossRefPubMedGoogle Scholar
  66. Llull D, Poquet I (2004) New expression system tightly controlled by zinc availability in Lactococcus lactis. Appl Environ Microbiol 70(9):5398–5406. CrossRefPubMedPubMedCentralGoogle Scholar
  67. Lu WW, Wang T, Wang Y, Xin M, Kong J (2016) A food-grade fimbrial adhesin FaeG expression system in Lactococcus lactis and Lactobacillus casei. Can J Microbiol 62(3):241–248CrossRefPubMedGoogle Scholar
  68. Ma Y, Liu J, Hou J, Dong Y, Lu Y, Jin L, Cao R, Li T, Wu J (2014) Oral administration of recombinant Lactococcus lactis expressing HSP65 and tandemly repeated P277 reduces the incidence of type I diabetes in non-obese diabetic mice. PLoS One 9(8):e105701. CrossRefPubMedPubMedCentralGoogle Scholar
  69. Madsen SM, Arnau J, Vrang A, Givskov M, Israelsen H (1999) Molecular characterization of the pH-inducible and growth phase-dependent promoter P170 of Lactococcus lactis. Mol Microbiol 32(1):75–87CrossRefPubMedGoogle Scholar
  70. Maguin E, Duwat P, Hege T, Ehrlich D, Gruss A (1992) New thermosensitive plasmid for gram-positive bacteria. J Bacteriol 174(17):5633–5638CrossRefPubMedPubMedCentralGoogle Scholar
  71. Mao R, Wu D, Wang Y (2016) Surface display on lactic acid bacteria without genetic modification: strategies and applications. Appl Microbiol Biotechnol 100(22):9407–9421. CrossRefPubMedGoogle Scholar
  72. Mao N, Cubillos-Ruiz A, Cameron DE, Collins JJ (2018) Probiotic strains detect and suppress cholera in mice. Sci Transl Med 10(445):eaao2586. CrossRefPubMedGoogle Scholar
  73. Marelli B, Perez AR, Banchio C, de Mendoza D, Magni C (2011) Oral immunization with live Lactococcus lactis expressing rotavirus VP8 subunit induces specific immune response in mice. J Virol Methods 175(1):28–37. CrossRefPubMedGoogle Scholar
  74. Martinez-Jaramillo E, Garza-Morales R, Loera-Arias MJ, Saucedo-Cardenas O, Montes-de-Oca-Luna R, McNally LR, Gomez-Gutierrez JG (2017) Development of Lactococcus lactis encoding fluorescent proteins, GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system. Biotech Histochem 92(3):167–174. CrossRefPubMedPubMedCentralGoogle Scholar
  75. Maxmen A (2017) Living therapeutics: scientists genetically modify bacteria to deliver drugs. Nat Med 23(1):5–7. CrossRefPubMedGoogle Scholar
  76. Michon C, Langella P, Eijsink VG, Mathiesen G, Chatel JM (2016) Display of recombinant proteins at the surface of lactic acid bacteria: strategies and applications. Microb Cell Factories 15:70. CrossRefGoogle Scholar
  77. Mierau I, Kleerebezem M (2005) 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol 68(6):705–717. CrossRefPubMedGoogle Scholar
  78. Miyoshi A, Jamet E, Commissaire J, Renault P, Langella P, Azevedo V (2004) A xylose-inducible expression system for Lactococcus lactis. FEMS Microbiol Lett 239(2):205–212. CrossRefPubMedGoogle Scholar
  79. Mohamadzadeh M, Duong T, Sandwick SJ, Hoover T, Klaenhammer TR (2009) Dendritic cell targeting of Bacillus anthracis protective antigen expressed by Lactobacillus acidophilus protects mice from lethal challenge. Proc Natl Acad Sci U S A 106(11):4331–4336. CrossRefPubMedPubMedCentralGoogle Scholar
  80. Mohamadzadeh M, Durmaz E, Zadeh M, Pakanati KC, Gramarossa M, Cohran V, Klaenhammer TR (2010) Targeted expression of anthrax protective antigen by Lactobacillus gasseri as an anthrax vaccine. Future Microbiol 5(8):1289–1296. CrossRefPubMedGoogle Scholar
  81. de Moreno de LeBlanc A, LeBlanc JG, Perdigon G, Miyoshi A, Langella P, Azevedo V, Sesma F (2008) Oral administration of a catalase-producing Lactococcus lactis can prevent a chemically induced colon cancer in mice. J Med Microbiol 57(Pt 1):100–105. CrossRefPubMedGoogle Scholar
  82. Motta JP, Bermudez-Humaran LG, Deraison C, Martin L, Rolland C, Rousset P, Boue J, Dietrich G, Chapman K, Kharrat P, Vinel JP, Alric L, Mas E, Sallenave JM, Langella P, Vergnolle N (2012) Food-grade bacteria expressing elafin protect against inflammation and restore colon homeostasis. Sci Transl Med 4(158):158ra144. CrossRefPubMedGoogle Scholar
  83. Mu D, Montalban-Lopez M, Masuda Y, Kuipers OP (2013) Zirex: a novel zinc-regulated expression system for Lactococcus lactis. Appl Environ Microbiol 79(14):4503–4508CrossRefPubMedPubMedCentralGoogle Scholar
  84. Neu T, Henrich B (2003) New thermosensitive delivery vector and its use to enable nisin-controlled gene expression in Lactobacillus gasseri. Appl Environ Microbiol 69(3):1377–1382CrossRefPubMedPubMedCentralGoogle Scholar
  85. Ng DT, Sarkar CA (2013) Engineering signal peptides for enhanced protein secretion from Lactococcus lactis. Appl Environ Microbiol 79(1):347–356. CrossRefPubMedPubMedCentralGoogle Scholar
  86. Oh JH, van Pijkeren JP (2014) CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res 42(17):e131CrossRefPubMedPubMedCentralGoogle Scholar
  87. Ohkouchi K, Kawamoto S, Tatsugawa K, Yoshikawa N, Takaoka Y, Miyauchi S, Aki T, Yamashita M, Murooka Y, Ono K (2012) Prophylactic effect of Lactobacillus oral vaccine expressing a Japanese cedar pollen allergen. J Biosci Bioeng 113(4):536–541. CrossRefPubMedGoogle Scholar
  88. Ortiz-Velez L, Britton R (2017) Genetic tools for the enhancement of probiotic properties. Microbiol Spectr 5(5):BAD-0018-2016.
  89. van Pijkeren JP, Britton RA (2012) High efficiency recombineering in lactic acid bacteria. Nucleic Acids Res 40(10):e76CrossRefPubMedPubMedCentralGoogle Scholar
  90. Pinto JP, Zeyniyev A, Karsens H, Trip H, Lolkema JS, Kuipers OP, Kok J (2011) pSEUDO, a genetic integration standard for Lactococcus lactis. Appl Environ Microbiol 77(18):6687–6690CrossRefPubMedPubMedCentralGoogle Scholar
  91. Ravn P, Arnau J, Madsen SM, Vrang A, Israelsen H (2003) Optimization of signal peptide SP310 for heterologous protein production in Lactococcus lactis. Microbiology 149(Pt 8):2193–2201. CrossRefPubMedGoogle Scholar
  92. Reardon S (2018) Genetically modified bacteria enlisted in fight against disease. Nature 558(7711):497–498CrossRefPubMedGoogle Scholar
  93. Reese KA, Lupfer C, Johnson RC, Mitev GM, Mullen VM, Geller BL, Pastey M (2013) A novel lactococcal vaccine expressing a peptide from the M2 antigen of H5N2 highly pathogenic avian influenza a virus prolongs survival of vaccinated chickens. Vet Med Int 2013:316926CrossRefPubMedPubMedCentralGoogle Scholar
  94. Robert S, Gysemans C, Takiishi T, Korf H, Spagnuolo I, Sebastiani G, Van Huynegem K, Steidler L, Caluwaerts S, Demetter P, Wasserfall CH, Atkinson MA, Dotta F, Rottiers P, Van Belle TL, Mathieu C (2014) Oral delivery of glutamic acid decarboxylase (GAD)-65 and IL10 by Lactococcus lactis reverses diabetes in recent-onset NOD mice. Diabetes 63(8):2876–2887. CrossRefPubMedGoogle Scholar
  95. Robert S, Van Huynegem K, Gysemans C, Mathieu C, Rottiers P, Steidler L (2015) Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis. Benef Microbes 6(4):591–601. CrossRefPubMedGoogle Scholar
  96. Robinson K, Chamberlain LM, Schofield KM, Wells JM, Le Page RW (1997) Oral vaccination of mice against tetanus with recombinant Lactococcus lactis. Nat Biotechnol 15(7):653–657. CrossRefPubMedGoogle Scholar
  97. van Roosmalen ML, Kanninga R, El Khattabi M, Neef J, Audouy S, Bosma T, Kuipers A, Post E, Steen A, Kok J, Buist G, Kuipers OP, Robillard G, Leenhouts K (2006) Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria. Methods 38(2):144–149. CrossRefPubMedGoogle Scholar
  98. Rottiers P, Caluwaerts S, Steidler L, Vandenbroucke K, Watkins B, Sonis S, Coulie B (2009) Effect of a mouth rinse formulation with human trefoil factor 1-secreting Lactococcus lactis in experimental oral mucositis in hamsters. J Clin Oncol 27(15S):e14570–e14570. CrossRefGoogle Scholar
  99. Shigemori S, Shimosato T (2017) Applications of genetically modified immunobiotics with high immunoregulatory capacity for treatment of inflammatory bowel diseases. Front Microbiol 8:22. CrossRefGoogle Scholar
  100. Shigemori S, Watanabe T, Kudoh K, Ihara M, Nigar S, Yamamoto Y, Suda Y, Sato T, Kitazawa H, Shimosato T (2015) Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice. Microb Cell Factories 14:189. CrossRefGoogle Scholar
  101. Shigemori S, Ihara M, Sato T, Yamamoto Y, Nigar S, Ogita T, Shimosato T (2017) Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis. Appl Microbiol Biotechnol 101(1):341–349. CrossRefPubMedGoogle Scholar
  102. Skrlec K, Pucer Janez A, Rogelj B, Strukelj B, Berlec A (2017) Evasin-displaying lactic acid bacteria bind different chemokines and neutralize CXCL8 production in Caco-2 cells. Microb Biotechnol 10(6):1732–1743. CrossRefPubMedPubMedCentralGoogle Scholar
  103. Skrlec K, Zadravec P, Hlavnickova M, Kuchar M, Vankova L, Petrokova H, Krizova L, Cerny J, Berlec A, Maly P (2018) p19-targeting ILP protein blockers of IL-23/Th-17 pro-inflammatory axis displayed on engineered bacteria of food origin. Int J Mol Sci 19(7):e1933.
  104. Škrlec K, Ručman R, Jarc E, Sikirić P, Švajger U, Petan T, Perišić Nanut M, Štrukelj B, Berlec A (2018) Engineering recombinant Lactococcus lactis as a delivery vehicle for BPC-157 peptide with antioxidant activities. Appl Microbiol Biotechnol 102:10103–10117. CrossRefPubMedGoogle Scholar
  105. Song AA, In LLA, Lim SHE, Rahim RA (2017a) A review on Lactococcus lactis: from food to factory. Microb Cell Factories 16(1):55. CrossRefGoogle Scholar
  106. Song X, Huang H, Xiong Z, Ai L, Yang S (2017b) CRISPR-Cas9(D10A) nickase-assisted genome editing in Lactobacillus casei. Appl Environ Microbiol:83(22):e01259-17.
  107. Sorvig E, Mathiesen G, Naterstad K, Eijsink VG, Axelsson L (2005) High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors. Microbiology 151(Pt 7):2439–2449. CrossRefPubMedGoogle Scholar
  108. Steidler L, Neirynck S, Huyghebaert N, Snoeck V, Vermeire A, Goddeeris B, Cox E, Remon JP, Remaut E (2003) Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat Biotechnol 21(7):785–789. CrossRefPubMedGoogle Scholar
  109. Stentz R, Jury K, Eaton T, Parker M, Narbad A, Gasson M, Shearman C (2004) Controlled expression of CluA in Lactococcus lactis and its role in conjugation. Microbiology 150(Pt 8):2503–2512CrossRefPubMedGoogle Scholar
  110. Szatraj K, Szczepankowska AK, Chmielewska-Jeznach M (2017) Lactic acid bacteria—promising vaccine vectors: possibilities, limitations, doubts. J Appl Microbiol 123(2):325–339. CrossRefPubMedGoogle Scholar
  111. Takiishi T, Cook DP, Korf H, Sebastiani G, Mancarella F, Cunha JP, Wasserfall C, Casares N, Lasarte JJ, Steidler L, Rottiers P, Dotta F, Gysemans C, Mathieu C (2017) Reversal of diabetes in NOD mice by clinical-grade proinsulin and IL-10-secreting Lactococcus lactis in combination with low-dose anti-CD3 depends on the induction of Foxp3-positive T cells. Diabetes 66(2):448–459CrossRefPubMedGoogle Scholar
  112. Toscano M, De Grandi R, Miniello VL, Mattina R, Drago L (2017) Ability of Lactobacillus kefiri LKF01 (DSM32079) to colonize the intestinal environment and modify the gut microbiota composition of healthy individuals. Dig Lives Dis 49(3):261–267. CrossRefGoogle Scholar
  113. Van Braeckel-Budimir N, Haijema BJ, Leenhouts K (2013) Bacterium-like particles for efficient immune stimulation of existing vaccines and new subunit vaccines in mucosal applications. Front Immunol 4:282PubMedPubMedCentralGoogle Scholar
  114. Vandenbroucke K, de Haard H, Beirnaert E, Dreier T, Lauwereys M, Huyck L, Van Huysse J, Demetter P, Steidler L, Remaut E, Cuvelier C, Rottiers P (2010) Orally administered L. lactis secreting an anti-TNF Nanobody demonstrate efficacy in chronic colitis. Mucosal Immunol 3(1):49–56. CrossRefPubMedGoogle Scholar
  115. Visweswaran GR, Leenhouts K, van Roosmalen M, Kok J, Buist G (2014) Exploiting the peptidoglycan-binding motif, LysM, for medical and industrial applications. Appl Microbiol Biotechnol 98(10):4331–4345. CrossRefPubMedPubMedCentralGoogle Scholar
  116. Volzing K, Borrero J, Sadowsky MJ, Kaznessis YN (2013) Antimicrobial peptides targeting gram-negative pathogens, produced and delivered by lactic acid bacteria. ACS Synth Biol 2(11):643–650CrossRefPubMedPubMedCentralGoogle Scholar
  117. de Vos WM (1999) Gene expression systems for lactic acid bacteria. Curr Opin Microbiol 2(3):289–295. CrossRefPubMedGoogle Scholar
  118. Walker DC, Klaenhammer TR (1994) Isolation of a novel IS3 group insertion element and construction of an integration vector for Lactobacillus spp. J Bacteriol 176(17):5330–5340CrossRefPubMedPubMedCentralGoogle Scholar
  119. Wang Z, Yu Q, Gao J, Yang Q (2012) Mucosal and systemic immune responses induced by recombinant lactobacillus spp. expressing the hemagglutinin of the avian influenza virus H5N1. Clin Vaccine Immunol 19(2):174–179. CrossRefPubMedPubMedCentralGoogle Scholar
  120. Wang M, Gao Z, Zhang Y, Pan L (2016) Lactic acid bacteria as mucosal delivery vehicles: a realistic therapeutic option. Appl Microbiol Biotechnol 100(13):5691–5701. CrossRefPubMedGoogle Scholar
  121. Watterlot L, Rochat T, Sokol H, Cherbuy C, Bouloufa I, Lefevre F, Gratadoux JJ, Honvo-Hueto E, Chilmonczyk S, Blugeon S, Corthier G, Langella P, Bermudez-Humaran LG (2010) Intragastric administration of a superoxide dismutase-producing recombinant Lactobacillus casei BL23 strain attenuates DSS colitis in mice. Int J Food Microbiol 144(1):35–41. CrossRefPubMedGoogle Scholar
  122. Wells JM, Wilson PW, Norton PM, Gasson MJ, Le Page RW (1993) Lactococcus lactis: high-level expression of tetanus toxin fragment C and protection against lethal challenge. Mol Microbiol 8(6):1155–1162CrossRefPubMedGoogle Scholar
  123. Wong CC, Zhang L, Li ZJ, Wu WK, Ren SX, Chen YC, Ng TB, Cho CH (2012) Protective effects of cathelicidin-encoding Lactococcus lactis in murine ulcerative colitis. J Gastroenterol Hepatol 27(7):1205–1212. CrossRefPubMedGoogle Scholar
  124. Wong CC, Zhang L, Wu WK, Shen J, Chan RL, Lu L, Hu W, Li MX, Li LF, Ren SX, Li YF, Li J, Cho CH (2017) Cathelicidin-encoding Lactococcus lactis promotes mucosal repair in murine experimental colitis. J Gastroenterol Hepatol 32(3):609–619CrossRefPubMedGoogle Scholar
  125. Wu CM, Lin CF, Chang YC, Chung TC (2006) Construction and characterization of nisin-controlled expression vectors for use in Lactobacillus reuteri. Biosci Biotechnol Biochem 70(4):757–767. CrossRefPubMedGoogle Scholar
  126. Yagnik B, Padh H, Desai P (2016) Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis. Microbes Infect 18(4):237–244CrossRefPubMedGoogle Scholar
  127. Yagnik B, Sharma D, Padh H, Desai P (2017) Immunization with r-Lactococcus lactis expressing outer membrane protein a of Shigella dysenteriae type-1: evaluation of oral and intranasal route of administration. J Appl Microbiol 122(2):493–505CrossRefPubMedGoogle Scholar
  128. Yoon SW, Lee CH, Kim JY, Kim JY, Sung MH, Poo H (2008) Lactobacillus casei secreting alpha-MSH induces the therapeutic effect on DSS-induced acute colitis in BALB/c mice. J Microbiol Biotechnol 18(12):1975–1983PubMedGoogle Scholar
  129. Yu D, Sawitzke JA, Ellis H, Court DL (2003) Recombineering with overlapping single-stranded DNA oligonucleotides: testing a recombination intermediate. Proc Natl Acad Sci U S A 100(12):7207–7212. CrossRefPubMedPubMedCentralGoogle Scholar
  130. Zadravec P, Mavrič A, Bogovič Matijašić B, Štrukelj B, Berlec A (2014) Engineering BmpA as a carrier for surface display of IgG-binding domain on Lactococcus lactis. Protein Eng Des Sel 27(1):21–27CrossRefPubMedGoogle Scholar
  131. Zadravec P, Strukelj B, Berlec A (2015a) Heterologous surface display on lactic acid bacteria: non-GMO alternative? Bioengineered 6(3):179–183. CrossRefPubMedPubMedCentralGoogle Scholar
  132. Zadravec P, Strukelj B, Berlec A (2015b) Improvement of LysM-mediated surface display of designed ankyrin repeat proteins (DARPins) in recombinant and nonrecombinant strains of Lactococcus lactis and Lactobacillus species. Appl Environ Microbiol 81(6):2098–2106. CrossRefPubMedPubMedCentralGoogle Scholar
  133. Zadravec P, Mareckova L, Petrokova H, Hodnik V, Perisic Nanut M, Anderluh G, Strukelj B, Maly P, Berlec A (2016) Development of recombinant Lactococcus lactis displaying albumin-binding domain variants against Shiga toxin 1 B subunit. PLoS One 11(9):e0162625. CrossRefPubMedPubMedCentralGoogle Scholar
  134. Zahirovic A, Lunder M (2018) Microbial delivery vehicles for allergens and allergen-derived peptides in immunotherapy of allergic diseases. Front Microbiol 9:1449. CrossRefPubMedPubMedCentralGoogle Scholar
  135. Zeng Z, Yu R, Zuo F, Zhang B, Peng D, Ma H, Chen S (2016) Heterologous expression and delivery of biologically active exendin-4 by Lactobacillus paracasei L14. PLoS One 11(10):e0165130. CrossRefPubMedPubMedCentralGoogle Scholar
  136. Zhang L, Zhang Y, Zhong W, Di C, Lin X, Xia Z (2014) Heme oxygenase-1 ameliorates dextran sulfate sodium-induced acute murine colitis by regulating Th17/Treg cell balance. J Biol Chem 289(39):26847–26858. CrossRefPubMedPubMedCentralGoogle Scholar
  137. Zhang B, Li A, Zuo F, Yu R, Zeng Z, Ma H, Chen S (2016a) Recombinant Lactococcus lactis NZ9000 secretes a bioactive kisspeptin that inhibits proliferation and migration of human colon carcinoma HT-29 cells. Microb Cell Factories 15(1):102CrossRefGoogle Scholar
  138. Zhang L, Wu WK, Gallo RL, Fang EF, Hu W, Ling TK, Shen J, Chan RL, Lu L, Luo XM, Li MX, Chan KM, Yu J, Wong VW, Ng SC, Wong SH, Chan FK, Sung JJ, Chan MT, Cho CH (2016b) Critical role of antimicrobial peptide cathelicidin for controlling Helicobacter pylori survival and infection. J Immunol 196(4):1799–1809CrossRefPubMedGoogle Scholar
  139. Zhu D, Liu F, Xu H, Bai Y, Zhang X, Saris PE, Qiao M (2015) Isolation of strong constitutive promoters from Lactococcus lactis subsp. lactis N8. FEMS Microbiol Lett 362(16):fnv107.
  140. Zhu D, Fu Y, Liu F, Xu H, Saris PE, Qiao M (2017) Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000. Microb Cell Factories 16(1):1CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiotechnologyJožef Stefan InstituteLjubljanaSlovenia
  2. 2.Faculty of PharmacyUniversity of LjubljanaLjubljanaSlovenia

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