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394 Metabolic Engineering

many organisms there are also strong regulated promoters is therefore laborious to disrupt genes on all the chromo-
available, e.g., the GAL7 promoter of S. cerevisiae and the some pairs. In principle the loxP-kanMX-loxP disruption
TAKA-amylase promoter of Aspergillus oryzae, but the cassette can be used to disrupt the same gene on several
application of these promoters generally requires more chromosomes by repetitive use. Alternatively, disruption
advanced fermentation strategies. For the production of of the same gene present on several chromosomes can be
heterologous proteins in E. coli the lac promoter has often done by isolation of haploid spores, disruption of the genes
been used, since with this promoter it is possible to induce in these haploid strains (or isolation of haploid strains with
the expression by addition of isopropyl-β-D-thiogalactose the proper gene disruption), and subsequent reisolation of
(IPTG). a polyploid strain through sexual crossing of the haploid
Very often metabolic engineering is carried out with strains. With this procedure it is, however, almost impossi-
laboratory strains since they are much easier to work ble to introduce specific genetic changes without altering
with, especially when it comes to genetic engineering. the overall genetic makeup of the cell, and the effect of a
Here it is possible to introduce specific genetic changes, gene disruption can be difficult to evaluate. An alternative
and this enable comparison of strains that are otherwise strategy for silencing of gene expression is to apply RNA-
isogenic. For this purpose very specific disruption cas- antisense techniques (Fig. 3B). If the antisense gene frag-
settes have been developed in certain organisms, e.g., the ment is expressed from a strong promoter, there may be
loxP-kanMX-loxP disruption cassette for gene disruption produced sufficient antisense mRNA to silence expression
in S. cerevisiae (see Fig. 3A). The advantage of this cas- from several gene copies, and this technique is therefore
sette is that the dominant marker can be looped out, and attractive in polyploid industrial strains. Normally, strate-
thereby the cassette can be used for sequential disrup- gies for metabolic engineering developed in laboratory
tion of several genes. In S. cerevisiae there is a very high strains can easily be transferred to industrial strains, but
frequency of homologous recombination, and only short in some cases a successful strategy in a laboratory strain
fragments (down to 50 base pairs) of the genes are re- will not work in an industrial strain due to a very different
quired for flanking the resistance marker. For many other genetic background. It is therefore often necessary to eval-
microorganisms the frequency of homologous recombi- uate strategies in both laboratory and industrial strains.
nation is much lower, and larger fragments are required With the rapid development in techniques for gene
[for filamentous fungi fragments up to 5 kilobase (kb) may cloning, it has become possible to recruit genes from
be needed]. Some industrial strains are polyploid, and it many different organisms. The increasing access to new

FIGURE 3 Methods for silencing gene expression. (A) Gene disruption by loop-in-loop-out method. Within a part of
the gene is cloned a resistance marker (e.g., the kanMX gene that ensures resistance towards the antibiotic G418
in S. cerevisiae). The resistance marker is flanked by two directed repeats (or loxP sequences) and fragments of the
gene to be disrupted. Upon transformation there may occur homologous recombination, and the resistance marker
is inserted within the gene, which hereby becomes disrupted. The insert can be looped out again through crossover
between the two directed repeats. The end result is a disrupted gene, and since the resistance marker has been
looped out it can be used again for another disruption. (B) Silencing of gene expression by expression of an antisense
mRNA fragment. A part of the gene is inserted in the opposite direction (often behind a strong promoter), and upon
expression an antisense mRNA is formed. This may hybridize with the sense mRNA and hereby the translation
process is prevented.