Genetic Manipulation - Environmental Biotechnology: Theory and Application - page 223

the existent metabolic capability or to introduce new pathways. This has various
applications, from the improved degradation of contaminants, to the production of
enzymes for industry, thus making a process less damaging to the environment.
One such experimental example taken from 'clean technology' with potential
for the manufacturing industry, is a strain of Eschericia coli into which was
engineered some 15 genes originating from Pseudomonas . These were introduced
to construct a pathway able to produce indigo for the dyeing of denim, commented
on by Bialy (1997). The traditional method requires the use of toxic chemicals
with the associated safety measures and inherent pollution problems. Similar
technologies were investigated in the early 1980s, by Amgen in the USA and
Zeneca in the UK, but were not pursued due to questionable profitability. Whether
or not this route will now be taken up by industry remains to be seen (BMB 1995).
Recombinant Yeast
Yeast, being unicellular eukaryotes, has become popular for cloning and express-
ing eukaryotic genes. These are fairly simple to propagate, some species being
amenable to culture in much the same way as bacteria. Yeast cells are surrounded
by a thick cell wall which must be removed to permit entry of DNA into the
cell. There are several types of plasmid vector available for genetic engineer-
ing, some of which have been constructed to allow replication in both bacteria
and yeast (Beggs 1981). All have a region which permits integration into the
host yeast genome by recombination. This occurs by alignment of the sequences
complementary between the host genome and the incoming plasmid DNA. Two
crossover events then take place which effectively swap over a piece of host
DNA with the plasmid DNA. A similar process occurs in the construction of
recombinant Baculoviruses.
Recombinant Viruses
The insect virus, Baculovirus, has been shown to be the method of choice for
the overexpression of genes in many applications of molecular biology. The viral
genome is large relative to bacterial plasmids and so DNA manipulations are nor-
mally carried out on a plasmid maintained in Eschericia coli . Introduction of the
reconstructed gene, or group of genes, to the Baculovirus DNA occurs by recom-
bination in much the same way as described for the formation of recombinant
yeast. One example of interest to environmental biotechnology is the replacement
of p10, one of the two major Baculovirus proteins, polyhedrin being the other,
by the gene for a scorpion neurotoxin, with a view to improving the insecticidal
qualities of the virus, sketched in Figure 9.3 (Stewart et al . 1991). The 'promot-
ers' at the start of the gene, referred to in the figure, are the regions of RNA
which regulate protein synthesis, from none at all, to maximum expression.