Fundamental Food Microbiology, Third Edition - Fuad Fathir

GENETICS OF SOME BENEFICIAL TRAITS 161
suitable DNA restriction endonucleases and purifying it from the mixture. A suitable
plasmid (cloning vector) is selected that has one or several gene markers (such as
resistance to an antibiotic or metabolizing a carbohydrate) and a site that can be
hydrolyzed, preferably with the same restriction enzymes. The plasmid is digested
with this enzyme, mixed with the donor DNA fragment, and incubated for the
fragment to align in the opening of the plasmid DNA (Figure 12.2). The open ends
are then sealed by using suitable enzymes and nucleotide bases. This plasmid carrying
the genes from the donor can then be introduced in a bacterial cell in several
ways, most effectively by electroporation in lactic acid bacteria, as described previously.
This method is now being used to transfer genes from different sources into
lactic acid bacteria.
A. Cloning Vectors
Many vectors have been constructed by combining nucleotide segments encoding
desirable characteristics from different sources, such as the origin of replication,
highly efficient promoters, multiple cloning sites, selective markers, and inducible
systems. A vector should be stable and able to replicate in host cells. Generally,
vectors that replicate by the theta mechanism are more stable than those that replicate
by the sigma mechanism. Many small vectors have been constructed from small
cryptic plasmids of lactic acid bacteria, such as pGK12 from pWV01. They 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 recipient (e.g., pHPS9
in Figure 12.2). 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 lac A promoter and his A 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. 3,4,8,9
B. 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
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