DISCUSSION
Over the past 20 years, researchers have made great efforts in enabling
efficient ethanol production from xylose, the second most abundant and
inedible sugar component of lignocellulose biomass, in engineered yeast
as an important step towards a robust second-generation biofuels
industry (Y.-S. Jin, Lee, Choi, Ryu, & Seo, 2000; Kim et al., 2013).
Recently, production of high-value metabolites, such as astaxanthin
(Montanti, Nghiem, & Johnston, 2011), protopanaxadiol (Gao, Caiyin,
Zhao, Wu, & Lu, 2018), squalene, and amorphadiene (Kwak et al., 2017),
from xylose by engineered yeast has gained increasing interest due to
the advantageous traits of xylose metabolism and the attracting economic
profitability of biomass conversion (Kwak et al., 2019).
For the first time, we engineered a yeast S. cerevisiae to
produce β-carotene from xylose. As compared to the conventional sugar
glucose, xylose exhibited superior traits as a carbon source for the
production of β-carotene in engineered S. cerevisiae . When
cultured on xylose, the engineered strain SR8B produced remarkably less
ethanol as compared when glucose was used as a carbon source
(Fig. 3 ). This is attributed to the dysregulation effect of
xylose on the glucose-dependent repression of the respiratory metabolism
(Y.-S. Jin et al., 2004; Matsushika et al., 2014). As such, the
engineered yeast produced β-carotene at a much higher yield from xylose
(2.41 mg β-carotene /g xylose) than from glucose (0.39 mg β-carotene/ g
glucose). As the xylose consumption was slower than glucose, the glucose
cultures were extended to the ethanol consumption phase for a fair
comparison. Nevertheless, the net production of β-carotene from
sequential utilization of glucose and ethanol was still much lower than
that from xylose culture regarding both volumetric titer and specific
content (Fig. 3 ). In addition, a lower cell density was
observed on glucose condition as compared to a corresponding xylose
condition. This might be associated with the energetically high-cost
conversion of ethanol into cytosolic acetyl-CoA in S. cerevisiaewhich restricts the yield of biomass or products that require ATP (Kok
et al., 2012). The higher yield of cell biomass from xylose is another
contributory factor of the enhanced β-carotene titer as cell
concentration is important for the volumetric titers of intracellular
metabolites.
Overexpression of tHMG1 was critical to high-level production of
β-carotene and other isoprenoids by engineered yeast, as described in
previous reports (Verwaal et al., 2007; Xie et al., 2014). As such, we
overexpressed tHMG1 in the SR8B strain in order to further
increase β-carotene production on xylose cultures. As expected, the
newly constructed strain SR8BH produced β-carotene with a higher
specific content than the SR8B strain while cultured on glucose
(Fig. 4 ). However, tHMG1 overexpression did not result
in any improvement of β-carotene production from xylose (Fig.
4 ). More interestingly, the beneficial effects of using xylose instead
of glucose as a carbon source on β-carotene production (a 254%
improvement in β-carotene specific content by SR8B strain) appeared to
be much stronger than that of tHMG1 overexpression on glucose
condition (a 67% improvement in β-carotene specific content by SR8BH
strain as compared to SR8B strain). These results suggested that using
xylose as a carbon source in substitution for glucose is an effective
strategy to increase β-carotene production in S. cerevisiae that
could potentially bypass tHMG1 overexpression and other genetic
manipulations which often resulted in growth defects. Thus, we could not
only avoid the cost of extra genetic perturbations eliciting reduced
growth, but also ensure the stability of engineered strains.
The higher production of β-carotene and accumulation of intermediates
(phytoene and lycopene) (Fig. 2 , Fig. 3 ) suggested a
better supply of precursors for the carotenogenic pathway when xylose
was used as a carbon source as compared to glucose (Verwaal et al.,
2007). To investigate the effects of xylose utilization on metabolic
flux related to β-carotene biosynthesis, the accumulation of endogenous
ergosterol and lipids were monitored as indicators for farnesyl
pyrophosphate (FPP) supply and cytosolic acetyl-CoA pool, respectively
(Fig. 1A ). We observed that the engineered strain produced more
ergosterol on xylose cultures as compared to glucose cultures
(Fig. 5A ). This is indirect evidence of a stronger metabolic
flux through the MVA pathway that provides sufficient supply of FPP as a
common precursor for isoprenoids and sterols. Additionally, cells grown
on xylose were found to accumulate more lipids as compared to those
grown on glucose (Fig. 5B , Fig. S4 ), indicating
increased cytosolic acetyl-CoA pool which is also a key factor for
high-level production of isoprenoids. Moreover, the increased lipids
content might have promoted the accumulation of β-carotene by expanding
cell-storage capacity for β-carotene as a lipophilic end product (Ma et
al., 2019; Wei et al., 2018).
Previous reports demonstrated that xylose utilization in engineeredS. cerevisiae leads to distinct transcriptional patterns of genes
involved in various metabolic pathways as compared to glucose
utilization (Y.-S. Jin et al., 2004; Kwak et al., 2017; Matsushika et
al., 2014). Thus, we investigated the effect of xylose on expression
levels of genes related to cytosolic PDH bypass, lipid synthesis, MVA
pathway and ergosterol pathway via comparative real-time qPCR. Among all
the genes studied, ACS1 and HMG1 were highly expressed
when the cells were grown on xylose as compared to glucose, while others
did not show significant difference in expression levels (Fig.
1C ). It is known that the transcription of ACS1 gene coding for
acetyl-CoA synthase is subject to glucose repression (Berg et al.,
1996). Therefore, we reason that using xylose instead of glucose as
carbon source leads to the alleviation of the glucose-dependent
repression on the transcription of ACS1 , thus resulting in
greater abundance of cytosolic acetyl-CoA as building blocks for lipids,
ergosterol and β-carotene synthesis. As a key rate-limiting gene in the
MVA pathway, HMG1 was an essential target for manipulation in
order to overproduce terpenes and sterols in S. cerevisiae .
Overexpression of native or heterologous HMG1 in engineeredS. cerevisiae was shown to be beneficial for β-carotene
production in previous studies (Li, Sun, Li, & Zhang, 2013; Yan et al.,
2012). Accordingly, the improved transcriptional level of HMG1 by
xylose utilization could have further promoted the conversion of the
abundant cytosolic acetyl-CoA into FPP as a precursor for β-carotene and
ergosterol. This might be the reason why tHMG1 overexpression was
neither necessary nor desirable for β-carotene overproduction while
xylose was used as a carbon source.
Owing to the peculiar physiologic characteristics of xylose
fermentation, including low ethanol production and high cell mass yield,
a high cell density culture of the SR8B strain was achieved through
intermittent xylose feeding instead of further genetic perturbations, or
sophisticated feeding algorithms. Consequently, a final β-carotene titer
of 772.81 mg/L was achieved (Fig. 6 ), which, to our best
knowledge, is one of the highest β-carotene titer reported to date in
engineered S. cerevisiae (López et al., 2019; Xie, Ye, et al.,
2015). However, the final yield (2.22 mg β-carotene/g xylose) and
specific content (11.42 mg β-carotene/g DCW) of β-carotene was
relatively lower than those of the batch fermentation (2.41 mg
β-carotene/g xylose & 13.73 mg β-carotene/g DCW, respectively). This
might be attributed to the large amount of glycerol and acetate
accumulation which consumed noticeable carbon sources and energy. The
considerable accumulation of glycerol and acetate indicates that the
engineered yeast cells might suffer from NADH/NAD+redox and energy imbalance. A previous study also reported high-level
glycerol accumulation in a xylose fed-batch fermentation (Kwak et al.,
2017). The redox imbalance in xylose metabolism was known to be caused
by the different cofactor dependences of XR (xylose reductase) and XDH
(xylitol dehydrogenase) in xylose assimilation pathway (Kwak et al.,
2019). Accordingly, strategies to eliminate the glycerol and acetate
accumulation, such as using a NADH-preferredSpathaspora passalidarum Xyl1.2 in xylose assimilation pathway
(Hou, 2012), replacing native NADPH-specific HMG1 into a
NADH-specific Silicibacter pomeroyi HMG1 in the MVA
pathway (Meadows et al., 2016), or rising the aeration by increasing the
rate of agitation and supply of air, could lead to a further enhanced
capacity of β-carotene production from xylose by our engineered yeast.
In conclusion, we constructed an engineered S. cerevisiae strain
capable of producing β-carotene from xylose—the second most abundant
and non-edible sugar in nature. As compared to the conventional sugar
glucose, xylose displayed superior traits as a carbon source for the
production of β-carotene in engineered S. cerevisiae , including a
lower ethanol production, a higher cell mass yield, a larger cytosolic
acetyl-CoA pool and up-regulated expression levels of rate-limiting
genes. Hence, high-level β-carotene production in engineered S.
cerevisiae was achieved in a fed-batch bioreactor simply through xylose
feeding instead of further genetic perturbations or culture
optimization. Our findings suggest xylose utilization is a promising
strategy for overproduction of carotenoids and other isoprenoids in
engineered S. cerevisiae .