3. Results and Discussion
3.1. Screening and characterization of the
endogenous promoters in Y.
lipolytica
Y. lipolytica has some unique metabolic advantages, such as the
high metabolic flux of acetyl-CoA and citric acid. Therefore, focused on
these metabolic pathways, including glycolysis, tricarboxylic acid cycle
(TCA), pentose phosphate pathway (PPP), and fatty acid synthesis
pathway, we selected 81 promoters for systematic analysis. To obtain
complete promoter sequences, we truncated 1500 bp upstream before the
ATG site of the corresponding gene through literature mining and the
KEGG database.
Noticeably, a stable and reliable reporter system is essential for
accurate analysis of promoter strength.[21]Currently, commonly used reporter systems for promoter analysis mainly
include:[19] fluorescent proteins (generate
fluorescence) and enzymes (generate chromogenic products), such as
X-gluc (5-Bromo-4-chloro-3-indolyl-β-glucuronide). However, Y.
lipolytica could generate severe fluorescence background, which
interferes with the reliabilities of the fluorescent
protein.[19] On the other hand, the reporter
system based on X-gluc also has several defects, such as being
time-consuming, low efficiency, and labor-intensive. Specifically, Wong
et al. developed a luciferase reporter system in Y. lipolyticwith the advantage of being stable, efficient, and
instant.[19] Therefore, we chose the luciferase
reporter system developed by Wong et al.[19] for
this study (Fig. 1).
We next verified the availability of the luciferase reporter system and
characterized the strength of promoter PTEF without
intron (PTEF). Consistent with the previous
report,[19] our results showed that promoter
PTEF is a typical strong constitutive promoter and its
strength reached 4.31×107. For the convenient analysis
of the experimental results, we used the promoter PTEFas control and defined the strength of promoter PTEF as
100, while classifying the endogenous promoters into strong promoters
(that strength is higher than 50), medium-strength promoters (that
strength is from 10 to 50), and weak promoters (that strength is lower
than 10).
3.2. Carbon metabolism
3.2.1 Glycolysis
pathway
In the glycolysis pathway, we analyzed 10 promoters, including
PHXK1 (YALI0B22308g ), PGPI(YALI0F07711g ), PPFK1 (YALI0D16357g ),
PFBA1 (YALI0E26004g ), PTDH1(YALI0C06369g ), PPGK1 (YALI0D12400g ),
PGPM1 (YALI0B02728g ), PENO1(YALI0F16819g ), PPYK1 (YALI0F09185g ), and
PTPI1 (YALI0F05214g ). As shown in Fig. 2, our
experimental results showed that PHXK1,
PGPI, PPFK1, PFBA1,
PTDH1, and PGPM1 are strong promoters,
PENO1, PPYK1, and PTPI1are medium-strength promoters, while PPGK1 is a weak
promoter. Among of these promoters, PTDH1 has the
highest strength and reached 5.47x107, which is
1.27-fold of the PTEF promoter. The promoter
PTDH1 is responsible for the transcription of
glyceraldehyde 3-phosphate dehydrogenase TDH1, which catalyzes the
conversion of glyceraldehyde 3-phosphate to 3-phospho-glyceroyl
phosphate, indicating its important role to maintain glycolysis.
Interestingly, Hapeta et al. identified that the hexokinase HXK1 is a
rate-limiting step for converting glucose to glucose 6-phosphate inY. lipolytica .[22] And, overexpression of
HXK1 by the PTEF promoter could significantly increase
the carbon flux of glycolysis and improved lipid
production.[22] It is reasonable because the
transcriptional activity of PHXK1 is
2.16x107, 50.2% of the PTEF promoter.
Moreover, the strength of other strong promoters PGPI,
PPFK1, PFBA1, and PGPM1reached 3.26x107, 3.00x107,
2.37x107, and 2.46x107,
respectively, which were 75.83%, 69.62%, 55.08% and 57.31% of the
PTEF promoter.
Most notably, the weakest promoter in glycolysis is
PPGK1, reaching 5.74 x105, which is
1.3% of the activity of the PTEF promoter. The promoter
PPGK1 is responsible for the transcription of
phosphoglycerate kinase PGK1, which catalyzes 3-phospho-glyceroyl
phosphate to 3-phospho-glycerate. However, the pathway analysis found
that 3-phospho-glycerate could also be generated in the glyoxylate
metabolism, suggesting that glycolysis is not the primary way for
producing 3-phospho-glycerate in Y. lipolytica . In addition, the
strength of medium-strength promoters PENO1(transcribing enolase ENO1), PPYK1 (transcribing
pyruvate kinase PYK1), and PTPI1 (transcribing
triosephosphate isomerase TIP1) reached 1.22x107,
1.93x107 and 1.65x107, respectively,
which were 28.24%, 44.71% and 38.27% of the PTEFpromoter.
3.2.2 Pentose phosphate
pathway
The PPP pathway is the branch metabolism of glycolysis, which can
completely oxidize glucose into 12 NADPH per
glucose.[5, 23] Specifically, studies have
demonstrated that NADPH supply is a rate-limiting step for fatty acid
synthesis in Y. lipolytica , which affects the electron transfer
efficiency to alter the titer and yield of fatty
acid.[5, 24] However, Y. lipolytica owns
multiple functional NADP+-specific dehydrogenases,
such as malic enzyme, isocitrate dehydrogenase, and glutamate
dehydrogenase, which can complement the PPP
pathway.[18] Nonetheless, Wasylenko et al. used13C Metabolic Flux Analysis to analyze strains with
high/low fatty acid titer, and identified that NADPH for fatty acid
synthesis is mainly supplied by the PPP
pathway.[25] Herein, we investigated 6 promoters
to understand the PPP pathway, including PZWF1(YALI0E22649g ), PGND2 (YALI0B15598g ),
PSOL2 (YALI0C19085g ), PRPE1(YALI0C11880g ), PRKI1 (YALI0B06941g ), and
PTKL2 (YALI0D02277g ).
Concretely, the metabolic reactions with generating NADPH are catalyzed
by glucose-6-phosphate dehydrogenase ZWF1 (transcribed by
PZWF1) and 6-phosphogluconate dehydrogenase GND1
(transcribed by PGND2).[18] Our
results (Fig. 2) showed that the strength of PZWF1 and
PGND2 were 1.30x107 (29.98% of the
PTEF promoter) and 8.27x106 (19.21%
of the PTEF promoter), respectively, indicating that the
intracellular NADPH can be increased by replacing the promoter.
Consistent with our results, Yuzbasheva et al. enhanced the expression
of ZWF1 to increase the carbon flux to the PPP pathway and improved the
fatty acid synthesis.[24] Moreover, promoters
PSOL2, PRPE1, and PTKL2were medium-strength promoters, and their strengths reached
7.88x106, 8.04x106, and
8.74x106, respectively, which were 18.31%, 18.69%,
and 20.3% of the PTEF promoter. Nevertheless,
PRKI1 is a weak promoter with an activity of 2.86
x106, indicating that ribose 5-phosphate isomerase
RKI1 is a rate-limiting step in the PPP pathway.
3.2.3 Pyruvate metabolism and
tricarboxylic acid
cycle
Pyruvate is the end metabolite of glycolysis, which could be oxidized to
acetyl-CoA or converted to byproducts, such as acetate, ethanol, and
lactate.[26] Here, we characterized 8 promoters in
the pyruvate metabolism, including PLPD1(YALI0D20768g ), PLAT1 (YALI0D23683g ),
PPDB1 (YALI0E27005g ), PPDC1(YALI0D06930g ), PPDC2 (YALI0D10131g ),
PADH1 (YALI0A15147g ), PADH2(YALI0A16379g ), and PADH3 (YALI0F09603g ).
As shown in Fig. 2, the promoter PLPD1 is the strongest
promoter, reaching 2.80x107, which is 56.58% of the
PTEF promoter. The promoter PPDB1,
PPDC1, PPDC2, PADH2, and
PADH3 are
medium-strength
promoters, and their strengths are 9.88x106,
5.04x106, 5.57x106,
4.65x106 and 1.07x107, respectively,
which were 22.96%, 11.73%, 12.95%, 10.80% and 24.96% of the
PTEF promoter. Moreover, the promoter
PADH1 is a weak promoter with a strength of
4.63x105, indicating alcohol dehydrogenase ADH1 is not
a primary alcohol dehydrogenase or a condition induced alcohol
dehydrogenase in Y. lipolytica . Surprisingly, the transcriptional
activity of the pyruvate dehydrogenase LAT1 promoter,
PLAT1, displays a low strength, reaching
2.53x106, 5.90% of the PTEF promoter.
This result is beyond our expectations because LAT1 is an indispensable
step in the oxidative reaction of pyruvate to acetyl-CoA.
Undoubtedly, the TCA cycle plays a significant role in cellular
metabolisms, such as maintaining energy metabolism and generating
precursors for cell biomass synthesis.[27] We
investigated the promoters of ATP citrate lyase ACL, citrate synthase
CIT1, aconitate hydratase ACO1, and isocitrate dehydrogenase IDP2. The
promoter PACL (YALI0D24431g ) and
PCIT1 (YALI0E00638g ) are both medium-strength
promoters and are responsible for the transcription of citrate lyase ACL
and citrate synthase CIT1, respectively. Most strikingly, citrate lyase
ACL and citrate synthase CIT1 both catalyze oxaloacetate to citrate.
Specifically, it was reported that cellular AMP levels would decrease
when nitrogen is depleted, further causing the decline of the isocitrate
dehydrogenase activity to accumulate citrate for fatty acid
synthesis.[28] Analogously, we found that the
activity of PACO1 (YALI0D09361g , driving the
expression of aconitate hydratase to produce isocitrate) was
significantly lower than PACL and PCIT1,
indicating the promoter PACO1 is also strongly regulated
by nitrogen starvation. In addition, the promoter PIDP2(YALI0F04095g ), driving isocitrate dehydrogenase, is a
medium-strength promoter and displays an increasing transcriptional
activity at the exponential stage.
3.2.4 Fatty acids
synthesis
The accumulated citrate would be transported from mitochondria into
cytoplasm for cleaving into acetyl-CoA.[29]Acetyl-CoA, a direct precursor, provides the basic building block for
acetyl-ACPs synthesis.[1] Most notably,
acetyl-ACPs are required to be transported into the endoplasmic
reticulum for elongation or desaturation to synthesize fatty
acids.[1, 29] Next, we investigated 20 promoters
(Table 1) in the fatty acids synthesis metabolism. As a result, 1 strong
promoter, 11 medium-strength promoters and 6 weak promoters were
characterized.
Noticeably, acetyl-CoA conversion to malonyl-CoA is a pivotal step in
fatty acids synthesis, catalyzed by acetyl-CoA carboxylase
ACC1.[2] However, our results found that the
transcriptional expression level of PACC1, transcribing
enolase ACC1, is relatively low, only 5.73x105, which
is 1.33% of the PTEF promoter, suggesting acetyl-CoA
carboxylase ACC1 is a rate-limiting step. Therefore, overexpression of
acetyl-CoA carboxylase ACC1 could effectively increase the production of
the malonyl-CoA derivatives.[2, 8] Specifically,
the promoter of fatty acid elongase ELO1, PELO1,
displayed the highest strength, reaching 2.77x107,
64.41% of the PTEF promoter. Moreover, we found that
the transcript level of FAS1 (fatty acid synthase 1) was significantly
lower than that of FAS2 (fatty acid synthase 2) during the whole
fermentation process. Mainly, the transcriptional activities of
desaturases, including POLE1 (transcribing stearoyl-CoA
desaturase OLE1) and PFAD2 (transcribing omega-6 fatty
acid desaturase FAD2), also have been investigated. As shown in Fig. 3,
POLE1 and PFAD2 are both medium-strength
promoters, but the activity of POLE1 is dramatically
higher. These results may be a guideline for directing the engineering
of Y. lipolytica to produce unsaturated and polyunsaturated fatty
acids.
3.2.5 Fatty acids
degradation
In Y. lipolytica , lipogenesis involves the dynamic balance of
fatty acid biosynthesis and degradation.[2,18]Specifically, accumulated fatty acids would be degraded to maintain
cellular metabolism by β-oxidation when carbon substrates are
depleted.[1, 30] For example, two acetyl-CoA
generated from β-oxidation are converted to C4 dicarboxylates (malate,
succinate) for replenishing TCA intermediates by the glyoxylate shunt
pathway in peroxisome.[31] Besides, Dulermo et al.
demonstrated that inactivation of genes POX1-6 in β-oxidation
could improve fatty acids production, increasing to 65-75% of the dry
cell weight.[32] Therefore, β-oxidation is a vital
branch of fatty acids metabolism that cannot be ignored in Y.
lipolytica . At that point, we surveyed 9 promoters (Table 1).
Interestingly, our results showed that five promoters of acyl-CoA
oxidase (POX) are all weak promoters, and their strengths ranged from
7.71x105 to 3.59x106 (Fig. 3).
Notably, it has been demonstrated that the promoter
PPOX2 is induced by fatty acids, and
PPOX1 and PPOX5 are induced by
alkanes.[33] Specifically, the core sequences of a
promoter in fungi are about 200-300 bp.[18]Nevertheless, we truncated 1500 bp sequences before ATG site of the
desired gene as the corresponding promoter, suggesting that promoters
obtained in this study should contain regulatory sequences of
transcription factors. Therefore, it is believable that promoters
PPOX1 (YALI0E32835g ), PPOX2(YALI0F10857g ), and PPOX5 (YALI0C23859g )
still retain the inducible properties and, as a result, show a low
activity under the condition without any additional inducers.
Unexpectedly, the promoter PERG10 (YALI0B08536g ,
transcribing stearoyl-CoA desaturase OLE1) and PPOT1(YALI0E18568g , transcribing stearoyl-CoA desaturase OLE2)
displayed the high transcriptional activities with the strength of
4.95x106 and 1.38x107, respectively.
In addition, the promoter PYAT1 (YALI0F21197g )
transcribes carnitine acetyltransferase YAT1, and its strength reached
1.44x107. The carnitine acetyltransferase YAT1
participates in the carnitine shuttle that transports the peroxisomal
acetyl-CoA into mitochondria.[34, 35] The high
strength of PYAT1 suggests that the carnitine shuttle is
active in Y. lipolytica .
3.2.6 Other carbon
metabolism
Moreover, we also analyzed several promoters in gluconeogenesis and
other carbon utilization pathways, including PFBP1(YALI0A15972g ), PPGM2 (YALI0E02090g ),
PSOU1 (YALI0B16192g ), and PMnDH2(YALI0D18964g ). We found that PFBP1 is a
medium-strength promoter with the strength of 5.80x106and promoters, namely PPGM2 and PSOU1are weak, while PMnDH2 is the strongest promoter in this
study, 1.6-fold of the strength of the PTEF promoter,
reaching 6.87x107.
3.3. Nitrogen metabolism
Nitrogen metabolism and its regulatory pathways play an essential role
in the synthesis and catabolism of amino acids, proteins, and other
nitrogen-containing substances, impacting the overall cellular
metabolism.[36] Here, we investigated 14 promoters
(Table 2), including 1 strong promoter that is PAAT1, 7
medium-strength promoters that include PAAT2,
PAR08, PAR09, PHPD,
PUGA2, PLEU2, and PHPD1,
and 6 weak promoters that include PALT1,
PHIS5, PGAD1, PEHD3,
PGLT1, and PGLN1, revealing the
complicated regulation of the nitrogen metabolism. Notably, studies have
shown that nitrogen metabolism in yeast mainly starts from glutamate and
its derivative glutamine.[37] Specifically,
glutamate could be converted from α-ketoglutarate, a metabolite in the
TCA cycle, by glutamate synthase GLT1, glutamine synthetase GLN1,
alanine transaminase ATL1, and cytoplasmic aspartate aminotransferase
AAT1, and mitochondrial aspartate aminotransferase AAT2 in Y.
lipolytica , which serves as a bridge linking the carbon and nitrogen
metabolism. Our results (Fig. 4) showed that the strength of promoter
PAAT1, PAAT2, PGLT1,
PGLN1, and PATL1 are
2.50x107, 1.45x107,
2.23x106, 1.51x106, and
3.88x106, respectively, which are 58.10%, 33.69%,
5.20%, 3.52%, and 9.01% of the PTEF promoter.
Generally, the muscular strength of PAAT1 and
PAAT2 indicates that the synthetic metabolism of
glutamate is mainly catalyzed by aspartate aminotransferase.
Moreover, aromatic amino acids can be used to synthesize several
high-value compounds, such as p-coumaric acid, violacein, and
flavonoids.[9] The transcriptional analysis showed
that there were 3 medium-strength promoters, namely
PAR08, PAR09, and PHPD,
and 1 weak promoter, namely PHIS5, in the aromatic amino
acid derivatives metabolism. The transcriptional activities of
PAR08, PAR09, PHPD, and
PHIS5 are 6.69x106,
6.03x106, 2.03x107, and
2.27x106, respectively.
3.4. Other metabolisms
Apart from the carbon and nitrogen metabolism, several promoters of
carriers, ribosomes, signaling proteins, and unknown-function proteins
also have been analyzed. As shown in Fig. 4, the promoter
PRSM7 (YALI0D08470g ) transcribes ribosomal
protein, which is a strong promoter with the strength of
2.50x107, 58.18% of the PTEFpromoter. The signaling proteins promoters PSLY1(YALI0D20416g ), PMDR1 (YALI0A18700g ), and
PARP4 (YALI0E18986g ) have the strength of
7.22x106, 2.23x107, and
2.54x107, respectively, which are 16.79%, 58.18%,
and 59.09%. In addition, unknown-function proteins promoters
P2034 (YALI0C12034g ) and P8272(YALI0D08272g ) are strong promoters with the strength of
4.77x107 (110.87% of the PTEFpromoter) and 2.29x107 (55.50% of the
PTEF promoter), while P27533(YALI0F27533g ) is a medium-strength promoter with the strength of
6.45x107 (14.99% of the PTEFpromoter). Particularly, the promoter PPHO89(YALI0E23859g ) is the weakest found in this study, with a 0.06%
strength of the PTEF promoter, which is responsible for
transcribing sodium-dependent phosphate transporter.