Results and Discussion
Combined over-expression
of ZWF1 and SOL3 improves the NADPH supply and thus increasing
α-farnesene production inP. pastorisX33-30*
The oxiPPP is the main inherent route for NADPH generation in P.
pastoris , which catalyzed by glucose-6-phosphate dehydrogenase (ZWF1),
6-gluconolactonase (SOL3), 6-phosphogluconate dehydrogenase (GND2) and
D-ribulose-5-phosphate 3-epimerase (RPE1).11, 18 In
order to increase the NADPH availability for α-farnesene biosynthesis,
the key enzymes in oxiPPP were optimized to overexpress in a α-farnesene
high-producing strain P. pastoris X33-30*. Firstly, we
respectively overexpressed the single gene zwf1 (encoding ZWF1),sol3 (encoding SOL3), gnd2 (encoding GND2) and rpe1(encoding RPE1) in strain X33-30*, resulted strains X33-30*Z, X33-30*S,
X33-30*G and X33-30*R. Compared with the strain X33-30*, strains
X33-30*Z and X33-30*S showed the increased NADPH concentration whereas
the strains X33-30*G and X33-30*R showed no visible difference in NADPH
concentration both at 24 h and at 72 h (Fig. 2a). Correspondingly,
strains X33-30*Z and X33-30*S also showed the increased α-farnesene
production, increased by about 6.5% and 12.0% as compared with strain
X33-30* at 72 h, respectively (Fig. 2b). The similar results were also
found in previous researches, in which overexpression of ZWF1 or SOL3
increased the foreign protein production in P. pastoris25-27 and the terpenoid production in S.
cerevisiae 28-29 because of the increased
intracellular NADPH level. ZWF1 and SOL3 catalyzed the first and the
second steps of the oxiPPP and were inhibited by NADPH and
ATP,11, 25 which catalyzed the rate-limiting steps in
oxiPPP.26 In addition, the native expression level ofsol3 in P. pastoris was low.30Therefore, these may be why strain X33-30*Z with overexpression ZWF1 and
strain X33-30*S with overexpression of SOL3 increased the NADPH
availability and thus increasing α-farnesene production. It should be
noted that overexpression of GND2 had no positive effects on increasing
the NADPH availability and α-farnesene production (Fig. 2), which was
similar with the results reported by Kim et al. 31 and
Nocon et al. 26 but different to the results reported
by Prabhu and Veeranki 25. Rebnegger et al.30 pointed out that gnd2 shows the high
expression level while sol3 shows the low expression level inP. pastoris . Based on these, we speculated that GND2 was not a
rate-limiting enzyme and only overexpression of GND2 did not enhance the
carbon flux in oxiPPP.
To further analyze the synergetic effects of these key genes in oxiPPP
on NADPH and α-farnesene production, we tried out different expression
combination ways of these genes looking for the highest performing
combos. As can be seen from Fig. 3a, the resulting strain X33-30*ZSGR
(i.e., combined overexpression of ZWF1, SOL3, GND2 and RPE1) showed the
highest intracellular NADPH level, followed by strain X33-30*ZSG (i.e.,
combined overexpression of ZWF1, SOL3 and GND2). Interestingly, combined
overexpression of GND2 and RPE1 (i.e., strain X33-30*GR) had no obvious
effect on increasing NADPH level (Fig. 3a), which was similar to single
overexpression of GND2 or RPE1 (Fig. 2a). This may be down to the
bottlenecks of the rate-limiting step, which catalyzed by ZWF1 and
SOL3.26 However, it should be noted that the
α-farnesene production was not increased with the increase of the
intracellular NADPH level in the corresponding strain (Fig. 3b). Among
the test strains, strain X33-30*ZS (i.e., strain X33-31) produced the
highest α-farnesene (i.e., 2.54±0.21 g/L) in spite of the third highest
NADPH level (Fig. 3). In contrast, although the strains X33-30*ZSGR and
X33-30*ZSG showed the top two NADPH level (Fig. 3a), they showed the
worst α-farnesene production, even lower than the original strain
X33-30* (Fig 2b and Fig. 3b). Nocon et al. 26 and
Prabhu and Veeranki 25 also found the similar results,
in which combined overexpression of ZWF1, SOL3, RPE1 or/and GND2 would
be detrimental to foreign protein production. It may be that excess
overexpression of the key enzymes in oxiPPP may imbalance the PPP flux26 or disturb acetyl-CoA biosynthetic pathway31 and thus negatively impacting on product formation.
Thus, the strain X33-31 was selected to further modify to increase
α-farnesene production.
Inactivation of
glucose-6-phosphate isomerase disturbs the cell growth and thus
negatively affecting α-farnesene biosynthesis in P. pastorisX33-31
Previous research indicated that
overexpression of the transcription factor STB5 increased cytosolic
NADPH concentrations because STB5 upregulated the expression of most
genes in the PPP and repressed the expression of glucose-6-phosphate
isomerase-coding gene in glycolysis.31-32 Maybe since
STB5 is a basal regulator of the PPP,33 overexpression
of STB5 did not increase the protopanaxadiol
production.31 PGI (encoded by pgi ) competes
with ZWF1 for glucose-6-phosphate, which catalyzes glucose-6-phosphate
to form frucose-6-phosphate (Fig. 1). In order to investigate the effect
of PGI on α-farnesene production, the PGI was inactivated in strain
X33-31, resulting in strain X33-31ΔP. As a control, strain X33-30*ΔP
(i.e., deletion of pgi in strain X33-30*) was also done.
Unfortunately, inactivation of PGI in strain X33-31 had negative effect
on α-farnesene production, in which the resultant strain X33-31ΔP only
produced about 5% of α-farnesene compared with the strain X33-31
(0.13±0.06 g/L vs. 2.54±0.21 g/L) (Fig. 4a). In addition, inactivation
of PGI increased the intracellular NADPH level but repressed the cell
growth and (Fig. 4b, c). In the past, Qin et al. 34also found that expression of pgi controlled by the ultra-low
intensity promoter NAT2p in Saccharomyces cerevisiae decreased
the cell growth and 3-hydroxypropionic acid production. The possible
reasons could be that PGI plays an important role in the central carbon
metabolism in yeast.35 In addition, Aguilera &
Zimmermann 36 pointed out that inactivation of PGI inS. cerevisiae prevents growth on glucose. However, it should be
noted that inactivation of PGI in strain X33-30* had little effect on
increasing α-farnesene production in spite of the decrease of the cell
growth (Fig. 4). There results indicated that inactivation of PGI
enforced the carbon flux into PPP and thus increasing the NADPH
availability for α-farnesene biosynthesis. Since the surplus NADPH
cannot be re-oxidized, the PGI-deficient strain did not grow on
glucose.37 Thus, we speculated the reason the strain
X33-30*ΔP did not obviously decrease the cell growth is that more NADPH
was used to biosynthesize α-farnesene. The previous results have
reinforced this speculation. For example, Fiaux et al.38 restored the cell growth on glucose of
PGI-deficient S. cerevisiae mutant by heterologous expression of
transhydrogenase UdhA from E. coli . However, although the strain
X33-30*ΔP showed the increased α-farnesene production as compared with
the strain X33-30*, its final titer of α-farnesene was still lower than
that of strain X33-31 (2.23±0.18 g/L vs. 2.54±0.21 g/L) (Fig. 4a),
indicating that inactivation of glucose-6-phosphate isomerase is not the
best strategy to increase α-farnesene production.
Low intensity expression of POS5 from S. cerevisiaebalances the NADPH/NADH ratio and thus promoting α-farnesene
biosynthesis in P. pastoris
It is well known that the intracellular NADH level is higher than the
intracellular NADPH level.39-41 Previous research
indicated that heterologous expression of NADH kinase POS5 provides
another source of NADPH except for the oxiPPP in
yeast.11, 28 To further promote the α-farnesene
production by optimizing the NADPH supply, we introduced the cPOS5
targeting in the cytosol from S. cerevisiae in strain X33-31.
Interestingly, the resultant strain X33-32 with gene cPOS5controlled by promoter PGAP showed the bad cell growth
and α-farnesene production, but it showed the increased productivity of
NADPH and α-farnesene (Fig. 5). POS5 catalyzed the NADH to form NADPH,
thereby reducing cell energy resources.11 In addition,
excess NADPH in cell would repress the cell growth, glucose consumption
and products production.40, 42 These comments may be
an underlying cause of the decreased cell growth and α-farnesene
production.
To try to dissolve this problem, we then decreased the expression level
of POS5 by replacing PGAP with a series of weak
promoters. Based on the previous reports, the relative intensity of the
promoters PPISI, PGPM1,
PMET3, and
PPGK1 were 40%, 15~40%, 13%, and
0~10% as compared with that of the
PGAP, respectively43-45. An increased
α-farnesene production (i.e., 2.77±0.18 g/L) was obtained in strain
X33-35 with gene cPOS5 controlled by PMET3, which
increased by about 9.1% as compared with strain X33-31 (i.e., 2.54±0.21
g/L)(Fig. 5c). Correspondingly, strain X33-35 also exhibited the high
intracellular NADPH level (Fig. 5b). In addition, decreasing the
expression level of POS5 restored the cell growth as compared
with the strain X33-32 (Fig. 5a), indicating that excess NADPH in cells
would be detrimental to cell growth. These results indicated that low
intensity expression of cPOS5 in strain X33-31 benefits to maintain the
right amount of NADPH for cell growth and α-farnesene production.
Although the strain X33-34 exhibited the high NADPH concentration and
cell growth (Fig. 5a, b), it should be noted that strain X33-34 did not
produce more α-farnesene than that of strain X33-31 (i.e., 2.56±0.26 g/L
vs.2.54±0.21 g/L)(Fig. 5c), indicating that another limiting factor
hampered the α-farnesene biosynthesis in strain X33-34. As can be seen
from Fig. 1, 9 molecules of ATP are need to produce 1 molecule of
α-farnesene. ATP is mainly produced by NADH oxidation via ETP under
aerobic conditions.22 Therefore, we speculated that
ATP availability is another limiting factor for further increasing
α-farnesene production.
Overexpression of adenine
phosphoribosyltransferase enhances the precursor AMP supply and thus
increasing the ATP availability and α-farnesene production in P.
pastoris
ATP can be synthesized either by substrate level phosphorylation (SLP)
or by ETP in aerobic respiring bacteria, and the ETP is the main route
for ATP generation using NADH as electron donor.22, 46In the process of ETP, AMP or/and ADP was used as the substrate for ATP
biosynthesis.22 Therefore, increasing the AMP or ADP
supply could increase the ATP availability in theory. To test this
theory, the endogenous adenine phosphoribosyltransferase (APRT) was
overexpressed in strain X33-35, resulting in strain X33-37. APRT
catalyzes the formation of AMP from adenine and
5-phospho-α-Dribose-1-diphosphate (PRPP),47 and we
found that the intracellular ATP level of strain X33-37 was 9.4% higher
than that of strain X33-35 while the intracellular NADH level was
slightly lower than that of strain X33-35 (Table 3). Similar results
were also found in previous reports, in which the mutatedCorynebacterium glutamicum with inactivation of AMP nucleosidase
showed the increased intracellular ATP level and the decreased
intracellular NADH level.20 The most likely is that
more NADH was used for ATP biosynthesis through ETC because of the
abundant supply of AMP. Equally unsurprisingly, overexpression ofaprt gene promoted cell growth and α-farnesene production, the
DCW and α-farnesene production of strain X33-37 reached 2.35±0.11 g/L
and 2.94±0.25 g/L (Fig. 6), which were 10.3% and 6.1% higher than
those of strain X33-35 (i.e., 2.13±0.16 g/L and 2.77±0.18 g/L,
respectively), respectively. ATP is a key factor for cell growth and
maintenance and controlling intracellular
environment,48 and thus adequate ATP supply could
increase biomass. In addition, the biosynthesis of 1 molecule of
α-farnesene requires at least 9 molecules of ATP (Fig. 1), thus the
increased intracellular ATP level effectively pulled more carbon flux
into the α-farnesene biosynthetic pathway, resulting in higher
α-farnesene production. These results indicated that overexpression of
endogenous APRT is conducive to increase α-farnesene production because
it facilitates the AMP biosynthesis and thus increasing the ATP supply.
Deletion of NADH-dependent dihydroxyacetone phosphate reductase
elevates the intracellular NADH level and thus elevating the
intracellular ATP level and α-farnesene production in P.
pastoris
NADH plays an important role in maintaining the redox balance and energy
generation NADH,18 it can used as precursor for the
regeneration of NADPH and ATP. In order to maintain the abundant supply
of NADH, we tried to decrease the NADH consumption in shunt pathway. To
do this, we knocked out the
NADH-dependent dihydroxyacetone
phosphate reductase. (GPD1)-coding gene gpd1 in strain X33-37,
resulting in strain X33-38. GPD1 catalyzes the biosynthesis of glycerol
from dihydroxyacetone phosphate and used the NADH as reducing cofactor
(Fig. 1).Previous research indicated that the glycerol biosynthetic
pathway plays an important role in maintaining the intracellular NADH
and NAD+ level.49 So obviously, the
intracellular NADH level and NADH/NAD+ ratio in strain
X33-38 increased by 11.6% and 28.6% as compared with strain X33-37,
respectively (Table 3). Meanwhile, strain X33-38 had certain improvement
in both the intracellular NADPH level and the ATP level (Table 3). The
α-farnesene production of strain X33-38 reached 3.09±0.37 g/L after 72 h
in shake-flask fermentation, which was 5.1% higher than that of strain
X33-37 (i.e., 2.94±0.25 g/L) (Fig. 7a). The DCW of strain X33-38 was
also slightly increased as compared with strain X33-37 (i.e., 2.41±0.23
g/L vs. 2.35±0.11 g/L)(Fig. 7b). It is worth noting that strain X33-38
did not accumulate glycerol throughout the fermentation process, which
was different from the strain X33-37 (Fig. 7c). GPD1 is a key enzyme in
glycerol biosynthesis 50 and He et al.49 also found the similar results, in which the strainS. cerevisiae DRY01 with silencing GPD1 dramatically decreased
the glycerol accumulation. These results indicated that deletion of GPD1
not only improves NADH supply but also decreases the carbon flux in
shunt pathway, and thus increasing the α-farnesene production.