Intermediates and gene expression associated with central carbon
metabolism
Cells are able to assess the magnitude of metabolic flux by tuning the
rate of metabolic reactions, consequently regulating cellular physiology
under various stressful conditions (Litsios, Ortega, Wit, & Heinemann,
2018). During CVEF with glucose as the sole carbon source and major
products such as ethanol, CO2, biomass, and glycerol,
the central carbon metabolism pathways of S. cerevisiae cells
mainly constitute the Embden-Meyerhof-Parnas pathway (EMP), the pentose
phosphate pathway (PPP) and the tricarboxylic acid cycle (TCA) (Figure
S3). Thus, metabolomics and transcriptomic datasets of yeast were
integrated and interpreted via a pathway-based method, referring to 26
metabolites and 90 genes related to central carbon metabolism, whose
responses in an oscillation period were visualized in Figure 3.
Glucose firstly fluxes into EMP to provide ATP and precursors for cell
growth. EMP can be divided into an energy-consuming phase (–2 mol
ATP/mol glucose from GLCin to GAP) and an
energy-producing phase (4 mol ATP/mol glucose from GAP to PYR). Most of
the pyruvate produced is converted into ethanol in the condition of low
dissolved oxygen. The PPP as the source of NADPH and pentose contains an
oxidative pathway (from G6P to Ri5P) and a non-oxidative pathway (from
Ri5P to F6P and GAP). The TCA is a series of enzyme-catalyzed chemical
reactions that form a key part of aerobic respiration in cells. As to
the changing tendency of carbon metabolism during the oscillation
(Figure 3), it was weakened during the P2 to P3 phase (especially the
EMP and TCA processes) while being enhanced during the P3 to P5 with the
decreasing residual glucose and increasing ethanol concentration.
The significant decline of most metabolites with associated
down-regulated gene expression was observed in P3/P2, but the opposite
phenomenon happened in P5/P4. The reason may be found in Figure 1 where
biomass increased 3.3 g/L from P2 to P3, but decreased 3.32 g/L from P4
to P5 was observed. In addition, more changed metabolites and gene
expression appeared in P3/P2 and P5/P4 than that observed for P2/P1 and
P4/P3 and this may be due to the biomass difference between the adjacent
samples. From Figure 1, the biomass changes between P2 and P3, P4 and P5
were about two-fold of that between P1 and P2, P3 and P4. The pool of
cofactors and ATP was enhanced in P2/P1 and P5/P4, but decreased from P2
to P4, which reflected the activity of the energy metabolic reactions.
In spite of an expected sinusoidal oscillation found in Figure 3, there
are many additional details that can be elucidated by analyzing the
metabolites and genes one by one (Figure 4).
Figure 4A showed the specific concentration and changing trend of each
metabolite, most of which are involved in Figure 3, except adenylate
energy charge (EC). Throughout the oscillation cycle, ATP was the
dominant intracellular adenine nucleotide and the adenylate energy
charge (EC = ([ATP] + 0.5*[ADP])/([ATP] + [ADP] +
[AMP])) remained high (>0.9), which indicates that the
cell has a strong potential for phosphoryl transfer and works as the
‘normally metabolizing’ cell during the CVEF. In addition, we also found
that the changing tendency of EC is identical with biomass (Figure 1),
which seems to indicate that a higher EC value is benifitial for cell
proliferation. As described in Figure S1, most profiles of genes and
metabolites are able to be divided into four patterns of sin function:
0π, π/2, π, 3π/2 phases. As shown in Figure 4B, the changing tendencies
of 16 out of 22 genes related to EMP pathway presented a π/2 phase,
while the rest five genes (HXK1 , HXT1,2,4 and PFK1 )
showed a 3π/2 phase, 0π phase and π phase, respectively. This inferred
that the processes of glucose transport, glucose phosphorylation, and
the catalysis of PFK could be the nodes for the oscillation regulation
of intracellular metabolites. Genes PFK1 and PFK2 encoded
the 4α- and 4β-subunits which compose the heterooctameric
enzyme(PFK)in yeast and both types of subunits contributing equally to
catalysis instead of that one of the subunits functioning preferentially
in allosteric regulation (Heinisch, Boles, & Timpel, 1996). It’s
interesting that PFK2 was not induced more drastically between P2
and P3/4 as PFK1 was (Figure 4B), which seems to indicate thatPFK1 is upregulated more rapidly and variably than PFK2 in
response to the increased substrate concentration. In addition, PFK
activity is subject to allosteric control mechanism, with inhibition by
ATP and activation by AMP (Heinisch et al., 1996). Here, the change
trend of ATP (0π phase) is contrary to PFK1 (π phase), which
reflects its inhibitory effect on PFK allosteric control. But the change
trend of activator AMP is not consistent with PFKs , probably
because its absolute oscillation change values are much lower than that
of ATP, making its effect was not shown intuitively in the overall trend
of PFKs . Two distinct changing tendencies for the upper and down
neighboring metabolites of PFK, G6P and F6P are in π/2 phase while FBP
and GAP are in 0π phase. A margin of π/2 phase between their profiles
was assumed to be induced by PFK activity to catalyze F6P to produce
FBP, indicating that PFK is the key rate-limiting enzyme in the
energy-consuming phase of the glycolytic pathway. This description is
similar to the mechanism proposed for the metabolic oscillation in
continuous culture or yeast extracts (Boiteux, Goldbeter, & Hess, 1975;
Papagiannakis, Niebel, Wit, & Heinemann, 2017; Thoke et al., 2018).
In ethanol production after the EMP pathway, the RPKM value ofADH4 was always over 2000, peaking at P3 with a value of 4300,
while PDC1 and ADH1 presented a π/2 phase, consistent with
most genes of the EMP pathway, contrary with PDC5,6 andADH4 (Figure 4B). The expression profiles of ADH4 and PDC5
are π/2 phase earlier than ethanol, and the overall expression level ofADH4 is much higher than that of PDC5 , suggesting that
Adh4p might be responsible for ethanol formation during CVEF. The geneADH4 encodes the zinc-dependent alcohol dehydrogenase isoenzyme,
and its transcription is induced in response to zinc deficiency, which
was consistent with the changing tendency of the expression level of
zinc deficiency related gene ZPS1 (Figure S2). The expressions ofADH1 and PDC1 were regulated by the same regulatory
factors, while that of ADH4 and PDC5 and PDC6 by a
different kind. It could be assumed that the regulation of the
expression of gene ADH1 and PDC1 were closely related to
the flux changes of catabolism, while the regulation of the expression
of gene ADH4 and PDC5 and PDC6 were closely related
to the flux changes of anabolism and zinc level.
In addition, yeast cells produced 10.2-13.7 g/L glycerol during CVEF,
and the changing tendency of the extracellular glycerol level showed a π
phase pattern, which was similar to PFK1 but contrary to theGPD1, HOR2 and the ORP value (Figure S4), while those ofGPD2 and RHR2 showed π/2 phase pattern (Figure 4B). The
expression of GPD1 and GPD2 are regulated by osmotic
stress and redox homeostasis, and studies have shown that the HOG
pathway where the GPD1 and GPD2 are located can respond to
ethanol stress (Klein, Swinnen, Thevelein, & Nevoigt, 2017; Udom,
Chansongkrow, Charoensawan, & Auesukaree, 2019), suggesting that the
glycerol production during VHG oscillation was induced by osmotic stress
and the redox environment.
The expression level of genes related to PPP was far below that in the
EMP (Figure 4B), suggesting that cellular anabolism might be repressed
during CVEF. The oxidation stage of the PPP pathway is from G6P to 6PG,
and finally Ri5P. Both steps generate NADPH, which provides reducing
power and precursor metabolites for the cell’s biosynthesis pathways.
The changing tendency of 6PG showed π phase pattern, similar to biomass
(Figure 1), indicating that yeast cell growth is closely related to
NADPH synthesis. Furthermore, the expression level of ALD4, ALD6 ,MAE1 (Figure S5), and ADH4 exhibited a 3π/2 phase pattern,
ahead of NADH and NADPH. The products of ALD4, ALD6 andMAE1 are involved in catalyzing cofactor NAD(P)H generation
steps, suggesting that cofactor NAD(P)H generation might be the driving
force of PPP flux and biomass accumulation.
As to the TCA cycle, in this study, CVEF was conducted with aeration of
only 0.05 vvm, therefore, the metabolites fluxed into the TCA cycle
might be at low concentration, which was confirmed by transcriptional
analysis result that showed the expression levels of genes related to
TCA cycle were much lower than these related to EMP pathway.