department of biotechnology
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Biotechnology of lactic acid bacteria:

Lactic acid bacteria (LAB) have accompanied human race for centuries and constitute a part of our diet. They are among the industrially most important microorganisms. There are many reports about their beneficial health effects. They are a component of gastrointestinal flora and participate in formation of essential nutrients. They are therefore often added as probiotic food supplements. Their "generally recognized as safe" (GRAS) status has increased their importance as host organisms in biotechnology and as potential vectors for the delivery of biologicals to gastrointestinal tract.

Surface display on lactic acid bacteria

The first approach that we use includes fusion proteins, composed of three functional parts: secretion signal peptide (Usp45), displayed passenger protein and peptidoglycan binding domain (C-terminal part of the protein AcmA, containing three lysM repeats).

The second approach for surface display on L. lactis includes the search for candidate lactococcal proteins, which could serve as carriers for surface display. We determined the surface proteome by surface shaving and compared it to the bioinformatic prediction. 7 candidate proteins were cloned, expressed in L. lactis and detected on the bacterial surface. BmpA was present to the largest extent and was successfully used as a carrier for covalent surface display of a model protein. BmpA molecule was further engineered by truncating to improve surface display. Smaller variant Bmp1 has indeed improved the display, while additional shortening worsened the surface display ability.

The third approach includes heterologous surface display on non-recombinant bacteria. Secreted, surface anchor-containing fusion proteins are produced in L. lactis and non-covalently attached to the surface of Lactobacilli. Lactobacillus salivarius ATCC11741 is particularly appropriate for this type of surface display, which was shown to be improved by sub-lethal concentrations of antibiotics.

SDnonGMOBmpABmp1

Selected publications:

  • Berlec A, Zadravec P, Jevnikar Z, Štrukelj B. Identification of candidate carrier proteins for surface display on Lactococcus lactis by theoretical and experimental analyses of the surface proteome. Appl Environ Microbiol. 2011 Feb;77(4):1292-300.
  • Zadravec P, Mavrič A, Bogovič Matijasic B, Štrukelj B, Berlec A. Engineering BmpA as a carrier for surface display of IgG-binding domain on Lactococcus lactis. Protein Eng Des Sel. 2014 Jan;27(1):21-7.
  • Zadravec P, Štrukelj B, Berlec A. 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. 2015 Mar;81(6):2098-106.

Therapeutic applications of engineered lactic acid bacteria

Lactococcus lactis can be used as a vector for the delivery of therapeutic proteins to the human mucosa. Binding of delivered proteins to the bacterial surface is useful for that purpose. By replacing the B domain with TNF alpha binding affibody we obtained lactococcal cells with TNFalpha binding ability which could be used in the treatment of inflammatory bowel disease.

Another goal is the development of oral vaccines against infectious diseases. We have used L. lactis to deliver Hepatitis A antigen to the mouse intestinal tract. Recombinant bacteria elicited mucosal and systemic humoral response, albeit the response was lower than that achieved with parenterally administered purified antigen.  

We have also authored reviews on therapeutic applications of recombinant LAB and on non-immunoglobulin scaffold binders that could be applicable for LAB-mediated delivery to the intestine.

Affibody binding scheme

HAV

Selected publications:

  • Ravnikar M, Strukelj B, Obermajer N, Lunder M, Berlec A. Engineered lactic acid bacterium Lactococcus lactis capable of binding antibodies and tumor necrosis factor alpha. Appl Environ Microbiol. 2010 Oct;76(20):6928-32.
  • Berlec A, Malovrh T, Zadravec P, Steyer A, Ravnikar M, Sabotič J, Poljšak-Prijatelj M, Štrukelj B. Expression of a hepatitis A virus antigen in Lactococcus lactis and Escherichia coli and evaluation of its immunogenicity. Appl Microbiol Biotechnol. 2013 May;97(10):4333-42.
  • Berlec A, Ravnikar M, Strukelj B. Lactic acid bacteria as oral delivery systems for biomolecules. Pharmazie. 2012 Nov;67(11):891-8.
  • Škrlec K, Štrukelj B, Berlec A. Non-immunoglobulin scaffolds: a focus on their targets. Trends Biotechnol. 2015 Jul;33(7):408-18.


Imaging of lactic acid bacteria

In vivo imaging of orally administered lactic acid bacteria can provide information on the spatial and temporal distribution of bacteria in the gastrointestinal tract, and is of particular importance in monitoring of therapeutic recombinant bacteria. The bacteria were detected by expressing the infrared fluorescent protein IRFP713.The use of fluorescence tomography for spatial localization of fluorescent bacteria has been established. The expression of an additional infrared fluorescent protein IRFP682 enabled concomitant detection of two bacterial populations in live mice.
IRFP_3miceFLIT

Selected publications:

  •  Berlec A, Završnik J, Butinar M, Turk B, Štrukelj B. In vivo imaging of Lactococcus lactis, Lactobacillus plantarum and Escherichia coli expressing infrared fluorescent protein in mice. Microb Cell Fact. 2015 Nov 14;14(1):181. 

New research tools

Several applications demand high-throughput cloning in L. lactis. We described the preparation of a TA-cloning system on the basis of a nisin-controlled expression system (NICE) plasmid pNZ8148. TA-cloning enables direct ligation of Taq polymerase-amplified PCR products, which contain 3’-adenosine residues. Plasmid pNZ-T was obtained by whole plasmid PCR amplification of pNZ8148 and XcmI digestion. PCR products of various sizes were successfully cloned to pNZ-T and more than 95 % of tested colonies contained a plasmid with insert. 

TA_cloning

Selected publications:

  • Berlec A, Strukelj B.  Generating a custom TA-cloning expression plasmid for Lactococcus lactis. Biotechniques. 2012 Jan;52(1):51-3.
Expression of sweet-tasting protein brazzein in model lactic acid bacterium Lactococcus lactis

Brazzein was discovered in the west-african plant Pentadiplandra brazzeana Baillon and is the smallest sweet tasting protein. Brazzein's molecule contains 4 disulfide bridges that are responsible for the high thermal- and pH-stability of the molecule. Brazzein is 2000-fold sweeter than sucrose on a mass level. Unlike other sweet-tasting proteins, its sweetness profile resembles that of sucrose. Brazzein could be used as an alternative sweetener of natural origin due to its intensive sweet taste. The expression of brazzein in lactic acid bacteria could avoid the need to add sugar to dairy products.

brazzein3dbrazzeinNICE

Selected publications:
  • Berlec A, Jevnikar Z, Majhenic AC, Rogelj I, Strukelj B. Expression of the sweet-tasting plant protein brazein in Escherichia coli and Lactococcus lactis: a path toward sweet lactic acid bacteria. Appl Microbiol Biotechnol. 2006 Nov;73(1):158-65.
  • Berlec A, Tompa G, Slapar N, Fonović UP, Rogelj I, Strukelj B. Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis. Lett Appl Microbiol. 2008 Feb;46(2):227-31.
  • Berlec A, Strukelj B. Large increase in brazzein expression achieved by changing the plasmid /strain combination of the NICE system in Lactococcus lactis. Lett Appl Microbiol. 2009 Jun;48(6):750-5.

Contact:

Aleš Berlec
Borut Štrukelj

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