RESULTS
Construction of a Xylose-fermenting, β-carotene-producingS. cerevisiae. We engineered the GRAS yeast S. cerevisiaeto functionally express the heterologous β-carotene synthetic pathway and produce β-carotene from xylose as well as glucose (Fig. 1 ,Fig. 2 ). Specifically, crtE , crtI , crtYBgenes from X. dendrorhous coding for GGPP synthase, phytoene desaturase, phytoene synthase and lycopene cyclase in the β-carotene biosynthesis pathway were integrated into the genome of the S. cerevisiae SR8, previously engineered to ferment xylose (Kim et al., 2013). The resulted strain was named as the SR8B strain.
Comparison of β-carotene Production Patterns on Glucose and Xylose. When xylose was used as a carbon source, we observed the SR8B cell cultures always exhibited an intense orange color while those using glucose appeared as light yellow (Fig. 1B ). We suspected that carotenoids other than β-carotene might be produced and the composition of carotenoids in the cells grown on glucose and xylose might be different. To identify the carotenoids composition produced by engineered yeast cells grown on different carbon sources, cells cultured either on glucose or on xylose were harvested for carotenoids extraction and HPLC analysis. β-carotene was the predominant carotenoid produced on both conditions according to the chromatographs (Fig. 2 ), while the β-carotene peak in the chromatograph of xylose culture showed much higher intensity than that of glucose culture (Fig. 2 ). The intermediates phytoene and lycopene were also accumulated in xylose cultures, while only phytoene was detected in glucose condition with a lower peak intensity (Fig. 2 ).
The engineered SR8B strain was cultured aerobically on glucose and xylose to compare differences in fermentation profiles and β-carotene production patterns. When cultured on glucose, the engineered strain fermented glucose into a large amount of ethanol, and then started to consume ethanol as carbon source (Fig. 3A ). In contrast, when cultured on xylose, the strain consumed xylose slower with negligible amounts of ethanol production but showed a higher cell mass titer and more glycerol accumulation (Fig. 3B ). In terms of β-carotene production, a rapid fermentation of glucose, and subsequent ethanol consumption led to much less production of β-carotene as compared to xylose culture where xylose is consumed steadily with little ethanol production (Fig. 3C , Fig.3D ). As a result, the SR8B strain accumulated β-carotene intracellularly with a specific content of 13.73 mg/g DCW and a volumetric titer of 96.42 mg/L from 40 g/L xylose. These are approximately two-fold and three-fold higher than 3.88 mg/g DCW and 24.22 mg/L of β-carotene produced from 42 g/L glucose (Fig. 3C , Fig. 3D ).
Effect of tHMG1 Overexpression on β-carotene Production using Xylose as a Carbon Source. To further enhance β-carotene production by engineered strain from xylose, HMG-CoA reductase, a well-known rate-controlling enzyme in the MVA pathway, was selected as the manipulation target (Fig. 1A ). Specifically, an expression cassette containing a truncated HMG1 (tHMG1 ) coding for the catalytic domain of HMG-CoA reductase under the control ofTDH3 promoter was integrated into the genome of the SR8B strain using Cas9-based genome editing. The resulting tHMG1overexpressing strain, named as the SR8BH strain, showed lower cell mass titers as compared to the SR8B strain on both glucose and xylose condition (Fig. S3 ). As expected, the SR8BH strain accumulated more β-carotene (6.49 mg β-carotene/g DCW) than the SR8B strain (3.88 mg β-carotene/g DCW) when glucose was used as a carbon source (Fig. 4 ). While, interestingly, under xylose fermentation, no improvement on β-carotene production was observed in the SR8BH strain (12.01 mg β-carotene/g DCW) compared to the SR8B strain (13.73 mg β-carotene/g DCW) (Fig. 4 ).
Effect of Xylose Utilization on the Production of Ergosterol and Lipids. In order to examine broader impacts of using xylose as a carbon source on other cytosolic acetyl-CoA derived products, cells were taken at the end of fermentation of the SR8B strain for ergosterol and lipid analysis. The SR8B strain accumulated ergosterol with a specific content of 16.99 mg/g DCW from xylose which was 28% more than using glucose, and a higher improvement of 69% in volumetric titer was observed due to the improved cell mass titer in xylose fermentation.
To investigate the effect of xylose utilization on lipids production by the SR8 strain, cells harvested from glucose and xylose cultures were stained and visualized under a confocal microscope. The cells grown on xylose were found to accumulate more lipid bodies (LB) with larger size than cells grown on glucose, leading to a bigger portion of the stained LB to the cell area (Fig. 5A ). The enhanced LB formation suggested a greater lipids production capacity of engineered yeast on xylose fermentation, and this was confirmed by the lipids weight analysis. The engineered SR8B strain produced lipids through xylose utilization with a specific content 58% higher than glucose utilization (43.09 vs. 27.22 mg lipids/g DCW) (Fig. S4 ).
Comparison of Transcription Profiles of the Genes involved in β-carotene Biosynthesis in Engineered Yeast on Glucose and Xylose. To further reveal the potential mechanisms of improved production by xylose utilization, transcriptional analysis was carried out on genes related to cytosolic PDH bypass (ACS1 andACS2 ), lipid biosynthesis pathway (ACC1 ), MVA pathway (ERG10 , ERG13 , HMG1 , HMG2 , ERG12 ,ERG8 , ERG19 , IDI1 , and ERG20 ) and ergosterol pathway (ERG9 ) (Fig. 1C ). Cells for mRNA extraction and quantification were taken at the exponential phase of glucose and xylose fermentation by the SR8B strain, which is 19 hours and 31 hours, respectively. While most of the 13 genes studied did not show significant difference in transcriptional levels between glucose and xylose culture conditions, the expression levels of gene ACS1coding for acetyl-CoA synthase and gene HMG1 coding for HMG-CoA reductase increased significantly in response to xylose substitution, where 2.72 ± 0.14 and 2.21 ± 0.20-fold differences were observed, respectively.
Xylose Fed-batch Fermentation for the Production of β-carotene.The capacity of β-carotene production from xylose by the engineered strain SR8B was assessed in a 1-L bioreactor via a fed-batch fermentation. Cells was inoculated at an initial cell density of OD600 = 3.11, and cultured with 87 g/L of xylose (Fig. 6 ). Upon the depletion of initially added xylose, additional xylose was provided to reach a concentration of 40 ± 5 g/L of xylose. The feeding was repeated seven times until the β-carotene titer ceased to increase. Despite the presence of high concentrations of xylose in the medium, ethanol accumulation was negligible during the fed-batch culture. Finally, cell density reached to OD600 of 165.90 (68.02 g DCW/L) and 772.81 mg/L of β-carotene was produced with a productivity of 5.40 mg/L·h (Fig. 6 ). The final β-carotene yield was 2.22 mg β-carotene/g xylose and specific content was 11.42 mg β-carotene/g DCW. In addition, a large amount of glycerol (30.07 g/L) and acetate (22.46 g/L) were accumulated at the end of fermentation.