148 ◾ Fundamental Food Microbiology
are very efficient for cloning, transforming, and expressing genes in different lactic acid bacteria.
Several shuttle vectors have also been constructed, which can be used to transfer and express genes
between heterologous donors and recipients (e.g., pHPS9 in Figure 13.2). Because there are restrictions on the use of vectors carrying antibiotic markers to develop lactic acid bacterial strains for
use in food fermentation, several food-grade vectors have been constructed. They carry food-grade
selective markers, such as resistance to nisin, sucrose hydrolysis, and ochre suppressor, and help in
selecting out the transformed cells.
For the efficient expression of the cloned genes, a vector should have proper transcription and
translation signals upstream of the coding region. Both constitutive and inducible promoters have
been integrated in vectors, which greatly enhance the expression of the cloned genes (e.g., the
inducible lacA promoter and hisA promoter). In addition, vectors that allow the isolation of secretion signals have been constructed. Their use has enabled the secretion of heterologous proteins as
well as their anchorage to the cell wall of host cells. 3,4
Metabolic Engineering
Many lactic acid bacteria are used to produce fermented foods and desirable byproducts because
they can metabolize several carbohydrates, proteins, and lipids present in the raw materials used
in the fermentation process. In most strains, the ability to produce a desirable characteristic in a
fermented food or a valuable byproduct is quite limited. Also, the possibility of producing a novel
food or a byproduct by using these natural strains is quite low. However, an understanding of the
functions of many genes and their regulatory systems has helped conduct metabolic engineering in
lactic acid bacteria to change the metabolic pathways, and, in some instances, to produce a higher
level of byproducts and better products. This approach has a great potential to produce unique
bacterial strains, and many studies are being conducted in this field. Several examples are listed
here. 5–7
Mixed Acid Fermentation by Lactococcus lactis
Lac. lactis strains metabolize fermentable carbohydrates by the EMP pathway to produce mainly
lactic acid. In the terminal step, pyruvate is reduced to lactate by lactate dehydrogenase (LDH),
which is encoded by the ldh gene. When the ldh gene is inactivated by plasmid insertion, the
LDH-negative strain reroutes pyruvate to produce acetate, formate, ethanol, and acetoin. Ethanol
production is greatly increased by cloning the necessary genes from an ethanol-producing bacterium to the LDH-negative lactic acid bacteria. Similar results are also obtained by inactivating
both ldh genes in a Lab. plantarum strain (which has two LDH enzymes).
l(+)-Lactic Acid Production
Lactic acid bacteria, such as Lab. acidophilus, Lab. plantarum, Lab. fermentum, Lab rhamnosus,
Ped. acidilactici, and Ped. pentosaceus, that produce proportionately high quantities of lactic acid in
the EMP pathway produce a mixture of both l(+)- and d(–)-lactic acid from pyruvate because they
contain both l- and d-lactate dehydrogenases. Because l(+)-lactic acid is produced in the body, it
is preferred to d(–)-lactic acid as a food additive, and the strains mentioned are not preferred for
commercial production of lactic acid for use in food. Because many of the species mentioned are
also used to produce fermented foods, studies are being conducted to produce strains of these species that produce only l(+)-lactic acid. These species have the two genes, ldh L and ldh D, encoding
are very efficient for cloning, transforming, and expressing genes in different lactic acid bacteria.
Several shuttle vectors have also been constructed, which can be used to transfer and express genes
between heterologous donors and recipients (e.g., pHPS9 in Figure 13.2). Because there are restrictions on the use of vectors carrying antibiotic markers to develop lactic acid bacterial strains for
use in food fermentation, several food-grade vectors have been constructed. They carry food-grade
selective markers, such as resistance to nisin, sucrose hydrolysis, and ochre suppressor, and help in
selecting out the transformed cells.
For the efficient expression of the cloned genes, a vector should have proper transcription and
translation signals upstream of the coding region. Both constitutive and inducible promoters have
been integrated in vectors, which greatly enhance the expression of the cloned genes (e.g., the
inducible lacA promoter and hisA promoter). In addition, vectors that allow the isolation of secretion signals have been constructed. Their use has enabled the secretion of heterologous proteins as
well as their anchorage to the cell wall of host cells. 3,4
Metabolic Engineering
Many lactic acid bacteria are used to produce fermented foods and desirable byproducts because
they can metabolize several carbohydrates, proteins, and lipids present in the raw materials used
in the fermentation process. In most strains, the ability to produce a desirable characteristic in a
fermented food or a valuable byproduct is quite limited. Also, the possibility of producing a novel
food or a byproduct by using these natural strains is quite low. However, an understanding of the
functions of many genes and their regulatory systems has helped conduct metabolic engineering in
lactic acid bacteria to change the metabolic pathways, and, in some instances, to produce a higher
level of byproducts and better products. This approach has a great potential to produce unique
bacterial strains, and many studies are being conducted in this field. Several examples are listed
here. 5–7
Mixed Acid Fermentation by Lactococcus lactis
Lac. lactis strains metabolize fermentable carbohydrates by the EMP pathway to produce mainly
lactic acid. In the terminal step, pyruvate is reduced to lactate by lactate dehydrogenase (LDH),
which is encoded by the ldh gene. When the ldh gene is inactivated by plasmid insertion, the
LDH-negative strain reroutes pyruvate to produce acetate, formate, ethanol, and acetoin. Ethanol
production is greatly increased by cloning the necessary genes from an ethanol-producing bacterium to the LDH-negative lactic acid bacteria. Similar results are also obtained by inactivating
both ldh genes in a Lab. plantarum strain (which has two LDH enzymes).
l(+)-Lactic Acid Production
Lactic acid bacteria, such as Lab. acidophilus, Lab. plantarum, Lab. fermentum, Lab rhamnosus,
Ped. acidilactici, and Ped. pentosaceus, that produce proportionately high quantities of lactic acid in
the EMP pathway produce a mixture of both l(+)- and d(–)-lactic acid from pyruvate because they
contain both l- and d-lactate dehydrogenases. Because l(+)-lactic acid is produced in the body, it
is preferred to d(–)-lactic acid as a food additive, and the strains mentioned are not preferred for
commercial production of lactic acid for use in food. Because many of the species mentioned are
also used to produce fermented foods, studies are being conducted to produce strains of these species that produce only l(+)-lactic acid. These species have the two genes, ldh L and ldh D, encoding