Chemistry Reference
In-Depth Information
one-twentieth and one-quarter, respectively, of that of the original strain. Zambonelli
et al.
15
reported that when the
pld
gene from
Streptomyces
PMF was cloned and expressed in
E. coli
,
dramatic decrease in the recombinant
E. coli
cell growth accompanied by plasmid instability
was observed, suggesting possible toxic effect caused by the PLD enzyme on cell membrane.
These experiences demonstrated the importance of selecting a proper vector-host system in
order to successfully express a target gene. The genus
Streptomyces
has a very high G
+
C
content in its genomic DNA (
70%). So far, the best expression system in
Streptomyces
is
that established by Hopwood
et al.
,
8
generally involving the use of
Streptomyces
originated
vector (pIJ702) with
S. lividans
as the host. Ogino
et al.
's
18
new expression system with
the strong promoter of native
pld
stimulated the development of other industrially important
enzymes.
>
2.2.4.2
Example 2: expression of a phospholipase A
2
from
Streptomyces violaceoruber
Phospholipase A
2
(EC3.1.1.4) (PLA
2
) is an enzyme that specifically acts on the fatty acid
in sn-2 position of phospholipids. When used as a processing aid in treatment of egg yolk or
lecithin, it is kept at a constant temperature for a certain period of time. After the treatment,
the temperature of the yolk is raised in order to inactivate the enzyme, or in the case of lecithin,
further purification procedures are followed. The treated egg yolk and lecithin have improved
emulsifying properties and are widely used in food processing. Phospholipases, including
PLA
2
, are found in most cells and tissues including animal tissues that are historically
consumed by humans. A commercial PLA
2
enzyme is extracted and purified from porcine
pancreas. However, in recent years, enzymes from animal origins are less favoured because of
very high production costs and their relatively high risk of disease potency in manufacturing
and handling. Besides that, supply problems have caused some turmoil in the food industry
in several regions of the world. These problems can be prevented by producing the enzyme
using microorganisms which can be grown in a fermenter, thus preventing regional supply
limitations. There are two approaches in this respect: one is by cloning and expressing the
gene of pancreas origin into eukaryotic microorganisms such as the fungus
Aspergillus
, and
the other is to produce a real microbial enzyme.
APLA
2
enzyme from
S. violaceoruber
was purified and cloned by Sugiyama
et al.
19
The
expression efficiency of the system of Ogino as described in Section 2.4.1 was evaluated for
expression of the gene. The cloning strategy is shown in Fig. 2.3. The vector containing the
pld
expression cassette (promoter, signal peptides,
pld
gene and terminator) was spliced and
rejointed to produce pUC702-EX with the
pld
gene and the signal peptide removed (details
not shown). The process involves using restriction enzymes to digest and ligate the DNA and
PCR techniques to combine different DNA molecules. The
pla2
gene and its 5
upstream
sequences including a site for peptidase recognition (encoding a signal peptide) were isolated
from the chromosome DNA of
S. violaceoruber
by using the PCR technique, and an
SphI
restriction site was introduced at both ends of the gene in order to ligate it to the expression
plasmid pUC702-EX to construct pUC702-EX-PLA
2
. After transformation into host cells of
S. violaceoruber
, the enzyme activity of the cultivated cells was found more than 30 times
higher than that of the original host. Compared with the result reported on the expression
of a second PLA
2
gene from
S. violaceoruber
into
E. coli
host, in which case the enzyme
has been produced in
E. coli
as inclusion bodies within the cell,
20
the vector-host system of
pld
promoter of
S. lividans
(reclassified as
S. violaceoruber
) proves powerful in expressing
genes of the
Streptomyces
origin.
