DARPA BioSpice Project
Progress Report
October 1, 2001-July 12, 2002
Jay Keasling
Chemical Engineering
University of California, Berkeley
The goal of our work is to develop high through-put genomic, proteomic, and
metabolomic profiling of native, mutant, and engineered model microorganisms,
namely Escherichia coli and Bacillus subtilis. The data from
these complex profiles can then be analyzed and compared to computer
simulations to predict complex microbial phenotypes under a number of
different environmental conditions.
To that end, we have developed DNA arrays for all 4,290 open reading frames
in the E. coli genome and are currently developing similar arrays for
B. subtilis. We are using the E. coli arrays to examine the
response of the host to the introduction of heterologous metabolic pathways
and to mutations in key regulatory genes.
Finally, we are developing in vivo, fluorescent reporter systems to
monitor the expression of multiple genes simultaneously.
This system will allow us to deduce a cellular response cascade, such as
that controlling sporulation in B. subtilis.
To test our arrays, we have engineered E. coli with a heterologous
mevalonate pathway from Saccharomyces cerevisiae and tested its affect
on expression of E. coli's native genes.
In addition, we have monitored flux through the pathway by measuring endpoint
of the heterologous metabolic pathway. We find that the heterologous metabolic
pathways are highly expressed when induced (as evidenced by the green spots
corresponding to the mevalonate pathway genes (pmk, idi, mk, mpd))
(Figures 1 and 2).
While the expression of most of E. coli's native genes are expressed at
the same level whether the mevalonate pathway genes are induced or not (as
evidenced by the large number of yellow spots on the array), a select few of
E. coli's native genes are down-regulated during induction of the
mevalonate pathway (as evidenced by the red spots (yaaF, dxs)).
These genes encode the enzymes for E. coli's non-mevalonate pathway.
Figure 1.
Example DNA expression arrays of E. coli carrying the heterologous
mevalonate pathway in the presence of inducer for the pathway and in the
absence of inducer.
Figure 2.
A plot of gene expression levels from Figure 1 during induction of the
heterologous mevalonate pathway (Alexa 546 (green)) relative to the non-induced
state (Alexa 647 (red)).
Those data that fall on the straight line correspond to genes whose expression
is relatively unchanged during induction.
Those date that are significantly above the line correspond to genes that are
highly expressed during induction, whereas those data below the line correspond
to genes that are expressed more highly in the uninduced state than in the
induced state.
To examine the effect of the heterologous pathway on metabolic flux and the
flexibility of the metabolic network to perturbation, we measured the
end-point of that metabolic pathway, namely production of an isoprenoid
(Figure 3).
With the engineered mevalonate pathway from S. cerevisiae, the engineered
E. coli produced approximately an order of magnitude more lycopene
than the strain with the native (DXP) pathway only.
Incidentally, there is little difference in the growth rates of the two strains.
This analysis indicates that there is a significant amount of flexibility in
native metabolism.
Finally, all analyses were performed in triplicate so that we have a good
understanding of error in the metabolite profiles for modeling purposes.
Figure 3.
Heterologous mevalonate pathway (red) and native pathway (green) and amounts of
lycopene produced from the two pathways.
The amount of lycopene produced from the heterologous pathway is nearly an
order-of-magnitude higher than from the native pathway.
Conclusions.
We have constructed DNA arrays for E. coli and tested them by assessing
global cellular gene expression in a strain engineered with a heterologous
metabolic pathway.
In addition, we have shown that a metabolomics approach can be used to assess
the flexibility of the bacterial metabolic network. Both types of analyses
will be extremely important in assessing the response of E. coli and
B. subtilis to a number of environmental conditions.
Future work.
We are beginning to construct DNA arrays for all B. subtilis ORF's and
will use these to study gene expression during the onset of sporulation.
We also plan to assess metabolism in this bacterium.
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