150 ◾ Fundamental Food Microbiology
The multienzyme biosynthetic pathways of tetrahydrofolate from glutanyltriphosphate (GTP) in
Lac. lactis have been identified. With this information, a strain of Lac. lactis that overexpresses the
genes involved in the process has been developed. Initially, the engineered strain produces three
times more folate than the wild strain does and excretes most of it into the environment. In the
same manner, a Lac. lactis strain that produces a higher amount of riboflavin has been engineered.
This way, the nutritional value of the fermented foods can be increased.
Enhancing Proteolysis by Cell Lysis
The desirable flavor of cheese is the result of proteolysis of milk proteins by extracellular and intracellular proteolytic enzymes of a starter culture during ripening. As the intracellular enzymes are
released slowly (after death and lysis of starter-culture cells during ripening), the process is relatively slow. Methods, including metabolic engineering, are being studied to enhance the ripening
of cheese. In a metabolic engineering method, the lytic genes of bacteriophages are used to lyse
the starter cells. This has been achieved by cloning the phage genes that encode for lysin and
holin (cause cell lysis) under the nisin-induced promoter in Lac. lactis. In the presence of nisin,
the cells lyse, releasing the proteinases and peptidases, which then help accelerate the ripening
of cheese.
Protein Targeting
There is evidence now that many heterologous proteins, both from prokaryotes and eukaryotes,
can be expressed and produced in high levels in lactic acid bacteria by cloning the genes in suitable
expression vectors and using existing or new secretory signals. The products can be excreted into
the environment, attached onto the cell wall and membrane, or even remain inside the cells. The
possibilities are many and include many enzymes, antimicrobials, flavor compounds, and important bioactive molecules for use as pharmaceuticals (immunity protein vaccine) and in agriculture
(insecticides). Genes of many heterologous proteins have been cloned in Lactococcus lactis strains,
such as chicken egg lysozyme, bovine prochymosine, α-amylase, pediocin (a bacteriocin), luciferase, interleukin 2 (IL-2), IL-10, cholera toxin B, tetanus toxin C (produced by Clostridium tetani),
Internalin A protein from Listeria monocytogenes, low-calcium V antigen (LcrV) from Yersinia, and
Cry 1A insect toxin (produced by Bacillus thuringiensis). Many of the genes express at low to high
levels, but a few do not express to the level of detection. 8–10
Expression of Interleukins
A murine interleukin 2 (mIL2) expression vector, pL2MIL2, was constructed from pLET2N
(originally developed from pLET2, the lactococcal T7 polymerase-based system). The plasmid,
pL2MIL2, encodes the part of the mIL2 gene that codes for the mature protein, fused with the
lactococcal usp 45 secretory leader signal. Lac. lactis MG 1820 was transformed with this plasmid.
When grown in an appropriate growth medium, the cells synthesized the USP 45-mIL2 fusion
protein and secreted the mature mIL2 into the growth medium. The purified mIL2 was found
to be biologically as active as the natural mIL2. By similar procedures, other murine interleukins
(such as mIL-6 and mIL-10), murine interferon, and human interleukin have been produced by
Lac. lactis. 11 Recent studies have shown that daily ingestion of Lac. lactis, which can produce biologically active mIL-10 interleukin, has cured and prevented enterocolitis and alleviated allergic
diseases in mice.
The multienzyme biosynthetic pathways of tetrahydrofolate from glutanyltriphosphate (GTP) in
Lac. lactis have been identified. With this information, a strain of Lac. lactis that overexpresses the
genes involved in the process has been developed. Initially, the engineered strain produces three
times more folate than the wild strain does and excretes most of it into the environment. In the
same manner, a Lac. lactis strain that produces a higher amount of riboflavin has been engineered.
This way, the nutritional value of the fermented foods can be increased.
Enhancing Proteolysis by Cell Lysis
The desirable flavor of cheese is the result of proteolysis of milk proteins by extracellular and intracellular proteolytic enzymes of a starter culture during ripening. As the intracellular enzymes are
released slowly (after death and lysis of starter-culture cells during ripening), the process is relatively slow. Methods, including metabolic engineering, are being studied to enhance the ripening
of cheese. In a metabolic engineering method, the lytic genes of bacteriophages are used to lyse
the starter cells. This has been achieved by cloning the phage genes that encode for lysin and
holin (cause cell lysis) under the nisin-induced promoter in Lac. lactis. In the presence of nisin,
the cells lyse, releasing the proteinases and peptidases, which then help accelerate the ripening
of cheese.
Protein Targeting
There is evidence now that many heterologous proteins, both from prokaryotes and eukaryotes,
can be expressed and produced in high levels in lactic acid bacteria by cloning the genes in suitable
expression vectors and using existing or new secretory signals. The products can be excreted into
the environment, attached onto the cell wall and membrane, or even remain inside the cells. The
possibilities are many and include many enzymes, antimicrobials, flavor compounds, and important bioactive molecules for use as pharmaceuticals (immunity protein vaccine) and in agriculture
(insecticides). Genes of many heterologous proteins have been cloned in Lactococcus lactis strains,
such as chicken egg lysozyme, bovine prochymosine, α-amylase, pediocin (a bacteriocin), luciferase, interleukin 2 (IL-2), IL-10, cholera toxin B, tetanus toxin C (produced by Clostridium tetani),
Internalin A protein from Listeria monocytogenes, low-calcium V antigen (LcrV) from Yersinia, and
Cry 1A insect toxin (produced by Bacillus thuringiensis). Many of the genes express at low to high
levels, but a few do not express to the level of detection. 8–10
Expression of Interleukins
A murine interleukin 2 (mIL2) expression vector, pL2MIL2, was constructed from pLET2N
(originally developed from pLET2, the lactococcal T7 polymerase-based system). The plasmid,
pL2MIL2, encodes the part of the mIL2 gene that codes for the mature protein, fused with the
lactococcal usp 45 secretory leader signal. Lac. lactis MG 1820 was transformed with this plasmid.
When grown in an appropriate growth medium, the cells synthesized the USP 45-mIL2 fusion
protein and secreted the mature mIL2 into the growth medium. The purified mIL2 was found
to be biologically as active as the natural mIL2. By similar procedures, other murine interleukins
(such as mIL-6 and mIL-10), murine interferon, and human interleukin have been produced by
Lac. lactis. 11 Recent studies have shown that daily ingestion of Lac. lactis, which can produce biologically active mIL-10 interleukin, has cured and prevented enterocolitis and alleviated allergic
diseases in mice.