Biology Reference
In-Depth Information
By comparing transcriptome profiles between different strains of
interest or between cells cultured under different conditions, one can
elucidate possible regulatory circuits and identify potential target genes
to be manipulated for strain improvement. There have been numerous
papers on the use of DNA microarrays to obtain global transcriptome
profiles and consequently to understand cellular gene expression reg-
ulations and physiological changes in microbial strains of industrial
relevance. Among them, Gonzalez et al. [26] carried out transcriptome
profiling of two ethanologenic
Escherichia coli
strains that show differ-
ent levels of ethanol tolerance. From the comparison of transcriptome
profiles, several genes and potential mechanisms responsible for ethanol
tolerance, such as loss of FNR function and enhanced metabolism of
glycine, serine, and pyruvate, were identified. This example demon-
strates the usefulness of transcriptome profiling in understanding the
mechanisms behind the observed expression and physiological
changes. Several excellent review papers have been published [27,28].
However, there are not yet many examples of strain improvement
based on transcriptome profiling. Nevertheless, several examples have
recently appeared, and more examples will likely follow. In one such
example, transcriptome profiles of recombinant
E. coli
producing
human insulin-like growth factor I fusion protein (IGF-I
f
) were analyzed.
Recombinant
E. coli
was cultured in an industrially relevant fed-batch
mode during which samples were taken before and after induction for
transcriptome profiling. The results of comparative transcriptome
analyses were used to identify target genes that may improve IGH-I
f
production [29]. Among the
200 genes that were downregulated after
induction, those involved in amino acid/nucleotide biosynthetic path-
ways were selected as the first targets to be manipulated. As will be
descried later, the expression of these genes was found to be down-
regulated during the high cell density culture (HCDC) of
E. coli
[30].
Amplification of one of these genes, the
prsA
gene encoding the phospho-
ribosyl pyrophosphate synthetase, allowed the enhanced production of
IGH-I
f
. However, it was found that cell growth was negatively affected
by the
prsA
overexpression. The
glpF
gene encoding glycerol trans-
porter was then selected and amplified together with the
prsA
gene,
resulting in an increase of IFG-I
f
production from 1.8 to 4.3 g/l [29].
As shown in this example, the target genes to be manipulated
to improve a strain can be selected from the genes prescreened by the
analysis of transcriptome profiles. However, one should be aware that
this approach of selecting genes from transcriptome profiles is not an
easy and straightforward task. In the worst case of the above example,
one should try amplification of many more genes out of 200 downreg-
ulated genes in different combinations to achieve a desired goal. Often,
however, this is not the case. One can reduce the number of potential
target genes to 10-20 based on previous knowledge and experience
∼
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