Genetics of Some Beneficial Traits ◾ 149
the two LDHs. Strains of some of the species mentioned have been developed in which ldh D
expressing d-LDH has been inactivated, enabling them to produce only l(+)-lactic acid.
Diacetyl Production by Lactococcus lactis
Diacetyl is associated with a pleasant butter aroma and is used in many nondairy products to
give desirable characteristics. It is normally produced by some lactic acid bacteria, including the
biovar diacetylactis of Lac. lactis species. Other Lac. lactis species and strains produce, if at all,
very little diacetyl. Metabolic engineering has, however, enabled development of overproducing
diacetyl strains of Lac. lactis. To develop this strain, first the nox gene (NADH oxidase) from a
suitable source is cloned under the control of the nisin-inducible nisA promoter (NICE) in the
Lac. lactis strain. In the presence of a small amount of nisin, the nox gene in the strain is overexpressed. In the next step, the gene encoding for α-acetolactate decarboxylase (ALDB, which
converts α-acetolactate to acetoin, see Figure 12.5) is inactivated. This results in the accumulation
of α-acetolactate that is produced from pyruvate. Under the overexpression of the nox gene and
with inactivated ALDB, α-acetolactate is converted to diacetyl in high levels.
Alanine Production from Carbohydrates
Amino acids from proteins are generally used for growth and synthesis in lactic acid bacteria and do
not accumulate in the cells or in the environment. However, by metabolic engineering, a strain of Lac.
lactis that uses carbohydrate and ammonium sources to produce higher concentrations of alanine has
been developed. The alanine dehydrogenase gene from a suitable source was cloned under the control
of the NICE system. When the cloned vector was introduced in a LDH-negative strain and the strain
was grown in the presence of a small amount of nisin and ammonium supplements, a large amount of
carbohydrates was converted to l-alanine. Because l-alanine has a sweet taste, this strain can be used
as a starter culture to produce fermented products with different, but agreeable, tastes.
Production of Mannitol and Other Polyols
Mannitol, a sugar alcohol, is produced by some strains of lactic acid bacteria, such as Lac. lactis
and Lab. plantarum, in small amounts. It is produced by the reduction 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 that are able
to produce large amounts of sorbitol and tagatose have been developed. Mannitol, sorbitol, and
tagatose (as well as l-alanine, discussed previously) can be used as low-calorie sweeteners in food.
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 two LDHs. Strains of some of the species mentioned have been developed in which ldh D
expressing d-LDH has been inactivated, enabling them to produce only l(+)-lactic acid.
Diacetyl Production by Lactococcus lactis
Diacetyl is associated with a pleasant butter aroma and is used in many nondairy products to
give desirable characteristics. It is normally produced by some lactic acid bacteria, including the
biovar diacetylactis of Lac. lactis species. Other Lac. lactis species and strains produce, if at all,
very little diacetyl. Metabolic engineering has, however, enabled development of overproducing
diacetyl strains of Lac. lactis. To develop this strain, first the nox gene (NADH oxidase) from a
suitable source is cloned under the control of the nisin-inducible nisA promoter (NICE) in the
Lac. lactis strain. In the presence of a small amount of nisin, the nox gene in the strain is overexpressed. In the next step, the gene encoding for α-acetolactate decarboxylase (ALDB, which
converts α-acetolactate to acetoin, see Figure 12.5) is inactivated. This results in the accumulation
of α-acetolactate that is produced from pyruvate. Under the overexpression of the nox gene and
with inactivated ALDB, α-acetolactate is converted to diacetyl in high levels.
Alanine Production from Carbohydrates
Amino acids from proteins are generally used for growth and synthesis in lactic acid bacteria and do
not accumulate in the cells or in the environment. However, by metabolic engineering, a strain of Lac.
lactis that uses carbohydrate and ammonium sources to produce higher concentrations of alanine has
been developed. The alanine dehydrogenase gene from a suitable source was cloned under the control
of the NICE system. When the cloned vector was introduced in a LDH-negative strain and the strain
was grown in the presence of a small amount of nisin and ammonium supplements, a large amount of
carbohydrates was converted to l-alanine. Because l-alanine has a sweet taste, this strain can be used
as a starter culture to produce fermented products with different, but agreeable, tastes.
Production of Mannitol and Other Polyols
Mannitol, a sugar alcohol, is produced by some strains of lactic acid bacteria, such as Lac. lactis
and Lab. plantarum, in small amounts. It is produced by the reduction 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 that are able
to produce large amounts of sorbitol and tagatose have been developed. Mannitol, sorbitol, and
tagatose (as well as l-alanine, discussed previously) can be used as low-calorie sweeteners in food.
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.