Fundamental Food Microbiology, Third Edition - Fuad Fathir

164 FUNDAMENTAL FOOD MICROBIOLOGY
of fructose-6-phosphate (in the EMP pathway) to mannitol-1-phosphate, which then
is dephosphorylated by mannitol phosphate dehydrogenase (MPDH) to mannitol.
However, 90% of it remains inside the cells. In contrast, Leuconostoc mesenteroides
secretes most of the mannitol in the environment because it has an efficient mannitol
transport system (it also produces high amounts of mannitol). Overproduction of
mannitol by an LDH-deficient Lac. lactis strain has been achieved by metabolic
engineering for overproduction of MPDH and low production of phosphofructokinase.
By introducing the mannitol-transfer system from Leu. mesenteroides, the
engineered Lac. lactis strain is able to excrete most of the mannitol in the environment.
By similar techniques, Lac. lactis strains have been developed that are able
to produce large amounts of sorbitol and tagatose. Mannitol, sorbitol, and tagatose
(as well as L-alanine, discussed previously) can be used as low-calorie sweeteners
in food.
6. Production of Folic Acid and Riboflavin
Many lactic acid bacteria, such as Lac. lactis and Str. thermophilus, while growing in
milk and other fermented foods synthesize low levels of folate, but most of it is retained
inside the cells. The multienzyme biosynthetic pathways of tetrahydrofolate from
glutanyltriphosphate (GTP) in Lac. lactis have been identified. With this information,
a strain of Lac. lactis has been developed that overexpresses the genes involved in the
process. Initially, the engineered strain produces three times more folate than the wild
strain does and excretes most of it in the environment. In the same manner, a Lac.
lactis strain has been engineered that produces higher amount of riboflavin. This way
the nutritional value of the fermented foods can be increased.
7. 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 cells
of starter cultures 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 encoding 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 peptidase, which
then help accelerate the ripening of cheese.
C. Protein Targeting
There is evidence now that many heterologous proteins, both from procaryotes and
eucaryotes, 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 in the environment, attached on 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