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 .