Glycolytic Flux in Escherichia coli:
A Gene Array Perspective Comparing Glucose & Xylose
L.O. Ingram
University of Florida
The simplicity of the fermentation process (anaerobic with
pH, temperature, and agitation control) in ethanologenic
Escherichia coli KO11 and LY01 makes this an attractive system
to investigate the utility of gene arrays for biotechnology
applications. Using this system, gene expression, glycolytic
flux and growth rate have been compared in glucose-grown and
xylose-grown cells. Although the initial metabolic steps
differ, ethanol yields from both sugars were essentially
identical on a weight basis and little carbon was diverted to
biosynthesis. A total of 27 genes changed by more than 2-fold
in both strains. These included induction of xylose-specific
operons (xylE, xylFGHR, and xylAB) regulated by XylR and the
cyclic AMP-CRP system, and repression of Mlc-regulated genes
encoding glucose uptake (ptsHIcrr, ptsG) and mannose uptake (manXYZ)
during growth on xylose. However, expression of genes encoding
central carbon metabolism and biosynthesis differed by less
than 2-fold. Simple statistical methods were used to
investigate these more subtle changes. The reproducibility
(coefficient of variation of 12%) of expression measurements (mRNA
as cDNA) was found to be similar to that typically observed
for in vitro measurements of enzyme activities. Using a
student t-test, many smaller but significant sugar-dependent
changes were identified (p<0.05 in both strains). A total
of 276 genes were more highly expressed during growth on
xylose; 307 genes were more highly expressed with glucose.
Slower growth (lower ATP yield) on xylose was accompanied by
decreased expression of 62 genes encoding the biosynthesis of
small molecules (amino acids, nucleotides, cofactors, and
lipids), transcription, and translation; 5 genes were
expressed at a higher level. In xylose-grown cells, 90
genes associated with the transport, catabolism and regulation
of pathways for alternative carbon sources were expressed at
higher levels than in glucose-grown cells, consistent with a
relaxation of control by the cyclic AMP-CRP regulatory system.
Changes in expression ratios for genes encoding the
Embden-Meyerhof-Parnas (EMP) pathway were in excellent
agreement with calculated changes in flux for individual
metabolites. Flux through all but one step was predicted to be
higher during glucose fermentation, pyruvate kinase.
Expression levels (glucose/xylose) were higher in
glucose-grown cells for all EMP genes except the isoenzymes
encoding pyruvate kinase (pykA and pykF). Expression of both
isoenzymes was generally higher during xylose fermentation but
statistically higher in both strains only for pykF encoding
the fructose-6-phosphate activated isoenzyme, a key metabolite
connecting pentose metabolism to the EMP pathway. The
coordinated changes in expression of genes encoding the EMP
pathway suggest the presence a common regulatory system, and
that flux control within the EMP pathway may be broadly
distributed. In contrast, expression levels for genes encoding
the Pentose-Phosphate pathway were statistically similar
regardless of sugar.
Return to Table
of Contents
