INTRODUCTION
Carotenoids are diverse class of C40 isoprenoids widely
produced by plants, bacteria, fungi and microalgae (Berman et al., 2015;
Henríquez, Escobar, Galarza, & Gimpel, 2016). Of all known carotenoids,
β-carotene is believed to be the most important due to its nutritional
role as pro-vitamin A (Dowling & Wald, 1960) and health-promoting
potential as an antioxidant (Palozza & Krinsky, 1992) and an anti-tumor
agent (Williams, Boileau, Zhou, Clinton, & Erdman, 2000). Its wide
applications in nutraceutical, feed and cosmetic industries lead to a
fast-growing world market (Irwandi Jaswir, 2011). Currently, chemical
synthesis remains the major route of commercial β-carotene production.
Considering the safety concerns of chemical synthesis, and consumer
preferences for natural additives, microbial production of β-carotene
via metabolic engineering gains increasing interests and becomes an
attractive alternative (Yoon et al., 2007; Zhao et al., 2013). The
biological pathway of all isoprenoids use isopentenyl diphosphate (IPP)
as precursor, which is synthesized through either MVA pathway in
eukaryotes, or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in
prokaryotes. Among potential microbial hosts, Saccharomyces
cerevisiae has superior traits in industrial production of isoprenoids
such as the GRAS (generally recognized as safe) status, ease of genetic
manipulation, industrial robustness (Auesukaree et al., 2009), and the
native MVA pathway which is generally considered as an effective
supplier of isoprenoid precursor from acetyl-CoA (Vickers, Williams,
Peng, & Cherry, 2017).
Researchers expend great efforts in heterologous production of
carotenoids using engineered S. cerevisiae . Those efforts have
far involved the optimization of metabolic flux, and balancing necessary
cofactors by manipulating the expression levels of targeted genes (Das
et al., 2007; Peralta-Yahya et al., 2011; Verwaal et al., 2007; Yan,
Wen, & Duan, 2012). Among all the reported manipulation targets,
overexpression of a truncated, soluble form of
3-hydroxy-3-methylglutaryl-coenzyme A reductase (tHMG1 ), a major
rate-limiting enzyme of the MVA pathway, has been consistently
recognized as an essential strategy for high-level production of
carotenoids (Verwaal et al., 2007; Xie, Lv, Ye, Zhou, & Yu, 2015; Zhou
et al., 2017) and other isoprenoids, such as artemisinic acid (Ro et
al., 2006), farnesene (Meadows et al., 2016), squalene and amorphadiene
(Kwak et al., 2017) in S. cerevisiae . In addition to tHMG1overexpression, up-regulation of the MVA pathway related genes such asERG8 , ERG12 , ERG19 , IDI1 , ERG20 (Y.
Sun, Sun, Shang, & Yan, 2016) and down-regulation of the ergosterol
pathway related genes such as ERG9 (Yan et al., 2012) have been
attempted to increase the production of carotenoids. However, extensive
genetic manipulations usually increase metabolic burdens on the host and
thus cause instable performance in industrial-scale fermentation
(Hollinshead, He, & Tang, 2014).
More importantly, despite intensive genetic perturbations for driving
metabolic fluxes towards carotenoids production, ethanol remains a major
product due to the entirely fermentative metabolism of S.
cerevisiae on glucose even in the presence of oxygen (Pfeiffer &
Morley, 2014), which hindered the high-level production of carotenoids.
This well-known metabolic regulation, termed the Crabtree effect, was
not observed while using non-native sugar xylose as a carbon source
(Y.-S. Jin, Laplaza, & Jeffries, 2004; Kwak et al., 2017; Matsushika,
Goshima, & Hoshino, 2014). We, therefore, assumed that xylose
fermentation by engineered S. cerevisiae might facilitate
carotenoids production by alleviating glucose-dependent repression on
respiratory metabolism. Additionally, xylose, comprising up to 30-40 %
of lignocellulosic biomass, is the second most abundant sugar in nature
that derived from non-edible sources (Kim, Ha, Wei, Oh, & Jin, 2012).
Efficient production of value-added chemicals like carotenoids and
vitamin A from xylose is an important step toward economically feasible
and sustainable bioconversion processes of lignocellulosic biomass
(Kwak, Jo, Yun, Jin, & Seo, 2019; L. Sun, Kwak, & Jin, 2019). However,
carotenoids production from xylose in engineered S. cerevisiaehas yet been reported.
As such, in this study, we sought to overproduce β-carotene from xylose
in engineered S. cerevisiae . High-level production of β-carotene
was achieved using xylose as a carbon source without tHMG1overexpression and other genetic perturbations. In order to explore the
advantageous traits of xylose utilization for β-carotene production in
engineered yeast, we assessed the differences in β-carotene production
patterns from glucose and xylose via fermentation profiling, metabolites
analysis and comparative transcriptional studies. To the best of our
knowledge, the titer of β-carotene achieved in this study is among the
highest reported in engineered S. cerevisiae (López et
al., 2019; Xie, Ye, Lv, Xu, & Yu, 2015). This study demonstrated that
using xylose as a carbon source would be a promising strategy
potentially bypassing extensive genetic perturbations for high-level and
stable production of carotenoids and other isoprenoids in S.cerevisiae .