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
display on lactic acid bacteria
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
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.
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.
- 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
Therapeutic applications of engineered lactic acid bacteria
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.
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.
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.
- 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
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.
- 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
New research tools
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.
sweet-tasting protein brazzein in model lactic acid bacterium
- Berlec A, Strukelj B. Generating a custom TA-cloning expression plasmid for Lactococcus lactis. Biotechniques. 2012 Jan;52(1):51-3.
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.
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.
- 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
- 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.