葡萄糖代谢调控网络
孙 群
导师：杨志荣
小组其他成员： 李力 冯燕丽
most multicellular microorganisms
metabolize glucose by respiration
rather than fermentation?
Metabolism of glucose
• Glucose:
– The primary and preferred fuel for
eukaryotic microorganisms.
– Metabolized by a highly conserved series
of connected enzymatic reactions,
• Subjected to selection pressure
during evolution
Aerobic vs. anaerobic pathway
• Both are used by microorganisms to obtain
energy from glucose, in the form of ATP
• But at different rates and efficiencies
– Aerobic (respiration) proceeds at a lower rate
and with a high yield – multicellular
microorganisms
– Anaerobic (fermentation) operates at higher
rates but with lower yield – Unicellular
microorganisms
• Research on metabolic regulation in
eukaryotic microorganisms
Considering the revolution
• Cells with a higher rate but lower
yield of ATP production may gain a
selective advantage when competing
for shared energy resources
• Selection pressure imposed by energy
limitation and the high ATP yield of
respiration may have favored the
evolutionary transition from
unicellular to undifferentiated
multicellular organisms
J. Biol. Chem., Vol. 277(16): 13983-13988, April 19, 2002
Elucidation of the Metabolic Fate of Glucose in the
Filamentous Fungus Trichoderma reesei Using
Expressed Sequence Tag (EST) Analysis and cDNA
Microarrays
Felipe S. Chambergo, Eric D. Bonaccorsi, Ari J. S. Ferreira, Augusto S. P. Ramos, José
Ribamar Ferreira Júnior , José Abrahão-Neto, João P. Simon Farah, and Hamza El-Dorry
Departments of Biochemistry and Chemistry, Institute of Chemistry, University of São Paulo, Brazil
Experiment Model – Saccharomyces
cerevisiae
• Unicellular yeast
• Preferentially ferments glucose, even
in the presence of oxygen, although
uses both pathways depending on the
metabolic state of the cell – diauxic
shift
Experiment Model –Trichoderma
reesei
• Filamentous and cellulolytic fungus
• Different from S. cerevisiae at
natural habitats and nutritional
requirements.
• Economic importance:
– Enzymes produced are used in the
textile, food, and paper industries
– Chitinolytic enzymes – biocontrol agents
against plant-pathogenic fungi
Expressed Sequence Tag (EST)
• For Trichoderma reesei, EST data base
was established by using the
complementary DNA microarray
technology to analyze the gene
expression profile during glucose
exhaustion
• Compared it to the temporal program of
gene expression accompanying the
metabolic shift from fermentation to
respiration in S. cerevisiae
Experimental procedure
• Media, growth conditions, and metabolite
analysis – supplemented with glucose
• cDNA library – a unidirectional cDNA
library
• DNA sequencing
• Computational analysis
• Microarray analysis
Results
cDNA Library Analysis (I)
• The partial sequences were obtained.
– The clusters ranged in size from
2 (177 clusters) to 90 (1 cluster) sequences
• The data represent an increase of more
than 10-fold of T. reesei expressed
genes amount in the data base.
cDNA Library Analysis (II)
• Using BLASTX and a stringency score 80,
the total number of ESTs that could be
classified as:
– Functional: show similarity to proteins with
known function – encode putative protein
sequences
– Unclassified: show similarity to a sequence with
known function but do not fall into any of the
classification schemes utilized
– Unknown: show similarity to sequences of
unknown function
– No matches: have no significant similarity to
any protein sequences in the data bases
Fig. 1 Classification of the 1151 unique transcripts of T. reesei.
Fig. 2. Glucose concentration and cell density profiles during
growth of T. reesei in glucose-rich medium.
Gene Expression Analysis during
Glucose Exhaustion
• Sufficient coverage had been
achieved
• Compared transcript populations from
cells harvested when glucose reached
83 mM to those expressed at various
times as the glucose level declined
Genes coding for TCA enzymes
• It is expected that, in glucose-rich
medium, glycolysis increase but TCA
decrease.
• while
– Many genes coding for TCA enzymes were not
or only partially repressed (Citrate synthase
and ketoglutarate dehydrogenase) in glucoserich medium
No Change
Slight ↑
Fig. 4. Comparison of the expression profiles of genes for enzymes that
participate in key metabolic processes involved in the utilization of metabolites
during glucose exhaustion in T. reesei and S. cerevisiae.
Genes coding for glycolytic enzymes
• Low glucose concentration does not
repress or slightly decrease the
abundances of glycolytic enzymes
• But for enolase (→ PEP): shows the
same trend as the change of [glucose]
– Highly expressed in high [glucose]
– Markedly repressed on depletion of sugar
The same trend
as glucose
Fig. 4. Comparison of the expression profiles of genes for enzymes that
participate in key metabolic processes involved in the utilization of metabolites
during glucose exhaustion in T. reesei and S. cerevisiae.
Glycolytic transcripts
• As in S. cerevisiae, up-regulation of the
glycolytic transcripts in the presence of
glucose will increase the flow of
metabolites through the glycolytic pathway
to yield pyruvate
• Fact: two enzymes involved in the first
steps of the pentose phosphate pathwayare
expressed only at relatively low levels in
the presence of glucose
– glucose-6-phosphate dehydrogenase
– 6-phosphogluconate dehydrogenase
磷酸葡糖
酸脱水酶
glyconeogenesis
Fig. 3. Expression profile of genes repressed by glucose.
The fate of pyruvate
• In S.
– [glucose] ↑ → TCA enzyme↓
– Pyruvate → acetyaldehyde
• In T.
– [glucose] ↑ → TCA enzyme ↑
– Pyruvate ↓ due to oxidation
In glucose-rich
medium→ethanol
One gene
repressed, and
one keeps the
same activity
Acetate produced
when glucose
depleted
Fig. 4. Comparison of the expression profiles of genes for enzymes that
participate in key metabolic processes involved in the utilization of metabolites
during glucose exhaustion in T. reesei and S. cerevisiae.
In T., acetate is produced
by ALD (one gene
repressed, and one keeps
the same activity)
In S., ethanol is
produced by ADH
which is not affected by
[glucose]
Fig. 5. Production of ethanol and acetate in T. reesei after the addition of glucose.
The fate of acetaldehyde
• In S. cerevisiae, not converted to
acetate due to strong repression by
glucose
alcohol dehydrogenase
acetaldehyde + NADH → ethanol + NAD+
• Essential for anaerobic metabolism:
generates NAD+ required for glycolysis
• In T., acetaldehyde → ethanol + acetate
(ALD1 keeps the same activity; NAD+ is not
regenarated for anaerobic)
Coding peptide of
cytochrome C
oxidase
Fig. 7. Map of T. reesei mtDNA and effect of glucose on the expression of
mitochondrial and nuclear transcripts coding for mitochondrial proteins.
Respiration or fermentation?
• In S.
– when [glucose] ↑ → PDC↑ → acetyaldehye↑ →
ethanol ↑(NAD+ essential to anaerobic is
satisfied)
– [glucose] ↑→ mitochondrial genes repressed →
respiration shut off
• In T., both mitochondrial and nuclear- genes
are aboundant or decreased slightly when
[glucose] ↓ → respiration keeps on in
glucose-rich medium
Application
?
Cellulose → glucose → ethanol
Thank you!
Media, Growth Conditions, and
Metabolite Analysis
• T. reesei, strain QM 9414, was obtained from the
American Type Culture Collection (ATCC 26921). A
0.5-liter inoculum (containing 107 spores/ml) was
added to 10 liters of culture medium supplemented
with glucose at a final concentration of 100 mM. The
culture was maintained at 28°C with constant
agitation and aeration. Aliquots of the culture were
withdrawn and mycelium was collected by filtration
and frozen in liquid nitrogen.
• In the culture supernatants, Glucose concentration
was measured using a SERA-PAK kit (Bayer); ethanol
and acetate were measured enzymatically using the
TC acetic acid and TC ethanol kits obtained from
Roche Molecular Biochemicals.
cDNA Library
Total cellular RNA was extracted from glycerolgrown T. reesei cultures by the guanidium
isothiocyanate procedure, and poly(A)+ RNA was
purified using oligo(dT) chromatography. A
unidirectional cDNA library was constructed in
the Uni-ZAP XR vector. In vivo excision of
pBluescript plasmids was performed in Escherichia
coli SOLR (Stratagene). To assess the quality of
the library, the ratio of recombinants to nonrecombinants and the average size of the cDNA
inserts were determined by PCR analysis of the
DNA from 96 individual clones
DNA Sequencing
Mitochondrial DNA was isolated by cesium
chloride/bisbenzimide density gradient
centrifugation. Shotgun libraries were
constructed from sheared mitochondrial DNA
cloned into pUC18. Plasmid DNA from individual
colonies was prepared with the Concert rapid
plasmid miniprep system, and DNA sequencing
reactions performed using the BigDye terminator
cycle sequencing kit and the M13 reverse and
M13 primers. For ESTs,1 single-pass sequences of
the 5' ends of cDNAs were performed. Samples
were loaded on an ABI 377 DNA sequencer for
automated sequence analysis.
Computational Analysis
Sequences were edited for each EST using
the program phred+phrap+consed. Only ESTs
with a minimum length of 150 bases and a
phred quality value of at least 20 were
considered for further analysis. Edited
sequences were translated and used as query
sequences to search the GenBankTM nonredundant protein data base by using the
program BLASTX at the National Center for
Biotechnology Information (NCBI). Scores
80 were considered to be significant, and the
top-scoring genes were used to group the
transcripts by their putative function.
Microarray Analysis
Inserts were amplified by PCR in a 96-well
format using M13 reverse and M13 ( 20)
primers (Stratagene). PCR products were then
purified in a 96-well filtration plate using the
Millipore MultiScreen Assay System. Each PCR
product was verified by agarose gel
electrophoresis and was considered correct if
the amplified product resulted in a single band.
These DNAs were spotted on glass slides and
hybridized with fluorescently labeled cDNA
prepared by reverse transcription in the
presence of Cy3 or Cy5-labeled deoxyuridine
triphosphate.