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.