Engineering a wild-type diploid Saccharomyces cerevisiae strain for second-generation bioethanol production
- Hongxing Li†1, 2,
- Yu Shen†1,
- Meiling Wu1,
- Jin Hou1,
- Chunlei Jiao1,
- Zailu Li2,
- Xinli Liu2 and
- Xiaoming Bao1, 2Email author
© The Author(s) 2016
Received: 9 October 2016
Accepted: 11 November 2016
Published: 24 November 2016
The cost-effective production of second-generation bioethanol, which is made from lignocellulosic materials, has to face the following two problems: co-fermenting xylose with glucose and enhancing the strain’s tolerance to lignocellulosic inhibitors. Based on our previous study, the wild-type diploid Saccharomyces cerevisiae strain BSIF with robustness and good xylose metabolism genetic background was used as a chassis for constructing efficient xylose-fermenting industrial strains. The performance of the resulting strains in the fermentation of media with sugars and hydrolysates was investigated.
The following two novel heterologous genes were integrated into the genome of the chassis cell: the mutant MGT05196 N360F, which encodes a xylose-specific, glucose-insensitive transporter and is derived from the Meyerozyma guilliermondii transporter gene MGT05196, and Ru-xylA (where Ru represents the rumen), which encodes a xylose isomerase (XI) with higher activity in S. cerevisiae. Additionally, endogenous modifications were also performed, including the overproduction of the xylulokinase Xks1p and the non-oxidative PPP (pentose phosphate pathway), and the inactivation of the aldose reductase Gre3p and the alkaline phosphatase Pho13p. These rationally designed genetic modifications, combined with alternating adaptive evolutions in xylose and SECS liquor (the leach liquor of steam-exploding corn stover), resulted in a final strain, LF1, with excellent xylose fermentation and enhanced inhibitor resistance. The specific xylose consumption rate of LF1 reached as high as 1.089 g g−1 h−1 with xylose as the sole carbon source. Moreover, its highly synchronized utilization of xylose and glucose was particularly significant; 77.6% of xylose was consumed along with glucose within 12 h, and the ethanol yield was 0.475 g g−1, which is more than 93% of the theoretical yield. Additionally, LF1 performed well in fermentations with two different lignocellulosic hydrolysates.
The strain LF1 co-ferments glucose and xylose efficiently and synchronously. This result highlights the great potential of LF1 for the practical production of second-generation bioethanol.
KeywordsXylose-specific transporter Xylose isomerase Synchronous utilization of xylose and glucose Lignocellulosic ethanol Budding yeast
Bioethanol produced from lignocellulose, i.e., second-generation bioethanol, is benefit for a sustainable energy supply. Its cost-effective production process depends on the complete and rapid utilization of all the sugars in the hydrolysates derived from lignocellulosic raw materials (Ko et al. 2016; Moysés et al. 2016; Peng et al. 2012; Zhou et al. 2012). The budding yeast Saccharomyces cerevisiae is a prominent microorganism that has traditionally been used in industrial bioethanol production because of its numerous inherent advantages (Demeke et al. 2013). However, this natural ethanol producer must overcome at least two new challenges when the fermentation substrate is lignocellulosic hydrolysates, rather than starch-based materials. First, natural S. cerevisiae cannot effectively metabolize pentose sugars, particularly d-xylose, the second most abundant sugar in lignocellulosic materials because it lacks an effective initial metabolic pathway (Hahn-Hägerdal et al. 2007; van Maris et al. 2006). Second, the individual and synergistic negative interactions derived from the numerous inhibitory compounds that are formed during the pretreatment process and the hydrolytic release of sugars exert serious negative effects on the fermentation performance of S. cerevisiae (Ko et al. 2016; Palmqvist and Hahn-Hägerdal 2000). Therefore, for the economically viable and sustainable production of lignocellulosic bioethanol, it is necessary to confer the capacity to co-ferment glucose and xylose on an S. cerevisiae strain and to enhance its resistance to harsh production environments (Demeke et al. 2013; Li et al. 2015; Sharma et al. 2016).
The genetic background of the host strain significantly affects the performance of the recombinant strain. Normally, despite strain variation, polyploid, wild-type S. cerevisiae is a better ethanol producer than the haploid strain, which is generally used in the laboratory (Brandberg et al. 2004; Li et al. 2015; Sonderegger et al. 2004; Yamada et al. 2011). Although the genetic background issue is complex, basic selection principles are feasible. By evaluating strains for their glucose-fermenting power, stress tolerance, and the ability to metabolize pentose, we can select a strain suitable for use as the chassis cell in lignocellulosic ethanol production (Li et al. 2015).
To compensate for the shortfall in downstream metabolic flux in ethanol production, the xylulokinase Xks1p and the enzymes in the non-oxidative PPP are normally overexpressed in a xylose-utilizing strain (Bamba et al. 2016; Peng et al. 2012; Sharma et al. 2016). Additionally, the inactivation of aldose reductase Gre3p, which is encoded by GRE3, to decrease the formation of the byproduct xylitol (Bamba et al. 2016; Fujitomi et al. 2012; Hahn-Hägerdal et al. 2007; Kuyper et al. 2005a; Peng et al. 2012), and the inactivation of alkaline phosphatase Pho13p, encoded by PHO13, to reduce ATP waste (Bamba et al. 2016; Lee et al. 2014; Shen et al. 2012; Van Vleet et al. 2008), were also the considered strategies. Normally, xylose is absorbed non-specifically and insufficiently by hexose transporters and is competitively inhibited by glucose in S. cerevisiae (Subtil and Boles 2012). Therefore, to address this problem, both wild-type and mutated endogenous and heterologous transporters were screened (Diao et al. 2013; Farwick et al. 2014; Moon et al. 2013; Nijland et al. 2014; Runquist et al. 2009; Shin et al. 2015; Wang et al. 2015, 2016). Moreover, with higher efficiency and/or specificity for xylose, a transporter lacking glucose inhibition may relieve the glucose repression effect in a xylose absorption node, thereby increasing xylose metabolism. Recently, we found the mutant MGT05196 N360F, which is derived from the Meyerozyma guilliermondii transporter gene MGT05196 and exhibits xylose-specific transport and no glucose inhibition (Wang et al. 2015).
In theory, such rational genetic modifications should endow S. cerevisiae with the capacity to ferment xylose; nonetheless, the xylose-fermentation efficiency of the resulting engineered strain was less than expected, indicating the influence of unknown key factors. Additionally, it remains difficult to rationally enhance the tolerance of a xylose-fermenting strain to the inhibitors present in lignocellulosic hydrolysates. Therefore, non-rational, adaptive evolutions were performed in media that contained xylose as the sole carbon source or toxic lignocellulosic hydrolysates to further select for enhanced characteristics both in xylose metabolism efficiency and robustness (Heer and Sauer 2008; Kuyper et al. 2005b; Moysés et al. 2016; Sharma et al. 2016).
The co-fermentation of hexose and pentose is considered important for the effective industrial production of bioethanol. Normally, the concept of co-fermentation usually encompasses only a strain’s ability to ferment xylose and glucose in the presence of both sugars. Therefore, little attention is given to the synchronous fermentation of both sugars. In fact, the obvious hysteresis of xylose fermentation compared with glucose has been extensively observed, manifestations of which exhibit a delay not only with respect to initial xylose utilization and the xylose consumption rate but also the time to maximum ethanol production; in other words, the ethanol production value increases incrementally after glucose exhaustion (Ko et al. 2016; Peng et al. 2012; Reider Apel et al. 2016; Sharma et al. 2016). Therefore, a yeast strain that co-ferments xylose and glucose with the same consumption rate (i.e., synchronously) would be ideal. We believe that the synchronization of sugar fermentation will reduce the apparent fermentation time, improve equipment utilization and employee effectiveness, and play a vital role in the realization of the scaled-up production of second-generation bioethanol.
In the present work, the diploid S. cerevisiae strain BSIF, which we have previously shown to be robust and to possess a good background for xylose metabolism (Li et al. 2015), was used as the chassis cell. For rational strain design, two novel proprietary heterologous genes, MGT05196 N360F (ZL 2014105263569) (Wang et al. 2015) and the Ru-xylA (US 8,586,336 B2, EP 2679686, ZL 201110042170.2) (Bao et al. 2013; Hou et al. 2016a), were integrated into the genome of diploid strain BSIF. Together with other strategies, including the overexpression of the genes XKS1 (xylulokinase), TAL1 (transaldolase), TKL1 (transketolase), RKI1 (ribose 5-phosphate isomerase) and RPE1 (ribulose 5-phosphate epimerase) in the non-oxidative PPP and the inactivation of GRE3 (aldose reductase) and PHO13 (alkaline phosphatase), a functional xylose metabolic pathway was constructed in BSIF. For non-rational strain design, the recombinant cells were alternately cultured in media using xylose as the sole carbon source or SECS liquor (the leach liquor of steam-exploding corn stover) as stresses for adaptive evolution.
Construction of xylose-fermenting S. cerevisiae strains
Strains and plasmids used in this study
Diploid S. cerevisiae strain isolated from tropical fruit in Thailand
Laboratory preserved (Li et al. 2015)
BSIF derivative; XKS1p:: loxP-TEF1p, gre3:: TPI1p-RKI1-RKI1t-PGK1p-TAL1-TAL1t-FBA1p-TKL1-TKL1t-ADH1p-RPE1-RPE1t-loxP, pho13:: (TEF1p-Ru-xylA-PGK1t) 3 -loxP, three rounds of δ-integration with a fragment containing three tandem expression cassettes of Ru-xylA and the selectable marker loxP-KanMX4-loxP
The present work
Single-colony isolate from adaptive evolution in xylose; based on BSN3
The present work
Single-colony isolate from adaptive evolution in SECS liquor; based on XH7
The present work
XH7 derivative; gre3::MGT05196 N360F
The present work
XHR11 derivative; gre3::MGT05196 N360F
The present work
Single-colony isolate from adaptive evolution in xylose; based on XHR11-N360F
The present work
E.coli plasmid with segment LoxP-KanMX4-LoxP
Güldener et al. (1996)
Bao et al. (2013)
pUC19-based yeast integration plasmid containing the PHO13-targeting recombinant arms PHO13-RA1 and PHO13-RA2, three tandem expression cassettes of Ru-xylA and the selectable marker loxP-KanMX4-loxP
The present work
Similar to pXIP1; the recombinant arms PHO13-RA2 in pXIP1 were replaced with PHO13-RA3 in pXIP2
The present work
pUC19-based yeast integration plasmid containing the δ-sequence-targeting recombinant arms, three tandem expression cassettes of Ru-xylA and the selectable marker loxP-KanMX4-loxP
The present work
pUC19-based yeast integration plasmid containing the GRE3-targeting recombinant arms RA1/2; an expression cassette for Sc-RKI1, Sc-TAL1, Sc-TKL1 and Sc-RPE1; and the selectable marker loxP-KanMX4-loxP
Peng et al. (2012)
The GRE3-targeting recombinant arms GRE3-RA2 in pJPPP3 were replaced with GRE3-RA3 in pJPPP4
The present work
pUC19-based yeast integration plasmid containing GRE3-targeting recombinant arms, an overexpression cassette for MGT05196 N360F, the upstream activating sequence (UAS elements) UASCLB, and the selectable marker loxP-KanMX4-loxP
The present work
YEp24 derivative; GAL1p-Cre-CYC1t, TEF1p-hygB-TEF1t
The concentrations of monosaccharides and inhibitors in SECS liquor and different hydrolysates
SECS hydrolysate (Hy1)a
SPPR hydrolysate (Hy2)b
Main monosaccharides (g L−1)
9.87 ± 0.07
86.60 ± 0.160
54.94 ± 0.50
36.54 ± 0.28
39.09 ± 0.06
23.79 ± 0.19
3.24 ± 0.01
4.13 ± 0.02
Weak acids (g L−1)
4.77 ± 0.14
4.52 ± 0.01
0.00 ± 0.00
Furan aldehydes (g L−1)
0.37 ± 0.01
0.47 ± 0.01
0.71 ± 0.02
0.66 ± 0.03
Total phenolics (mmol L−1)
19.63 ± 0.29
26.68 ± 0.61
To further enhance xylose utilization, the mutant transporter gene MGT05196 N360F, which encodes a xylose-specific, glucose-insensitive transporter (Wang et al. 2015), was introduced into another region of the GRE3 locus in XHR11 (Additional file 1: Fig. S1d). The resulting strain, XHR11-N360F (Fig. 1b), was then evolved in a medium using xylose as the sole carbon source again. After ~200 h of adaptive evolution, the biomass doubling time (T) dropped from ~150 to ~96 min and remained unchanged for several batches. Then, strain LF1 was selected from several isolates due to its faster growth on xylose (Fig. 1b). All the strains used in this study are listed in Table 1.
Combinatorial strategy improved xylose assimilation and strain tolerance for lignocellulosic hydrolysates
Metabolic characteristics of engineered S. cerevisiae strains in batch fermentations of sugars
Initial inoculum (g DCW L−1)
Consumed xylose (g L−1)
Product yield (g g−1 consumed sugars)
Specific consumption or production rate
(g g−1 DCW h−1)f
The genetic basis of the efficient xylose metabolism of the evolved strains
As described above, a series of modified strains were derived from the robust diploid yeast (Fig. 1b), and they exhibited an alternation from increase to decrease in terms of xylose metabolic capacity. Therefore, the genetic changes responsible for the xylose assimilation phenotype were preliminarily analyzed.
The gene expression levels of the engineered strains
Fold change (compared with the chassis strain BSIF)
20.6 ± 5.2
22.0 ± 1.6
12.3 ± 2.9
21.5 ± 0.5
7.7 ± 2.5
10.0 ± 2.0
9.0 ± 0.5
9.4 ± 0.3
7.3 ± 1.8
6.0 ± 0.6
4.9 ± 0.3
4.6 ± 0.2
Ribose 5-phosphate isomerase
14.4 ± 1.3
238.8 ± 15.3
146.78 ± 3.5
144.5 ± 0.6
Ribulose 5-phosphate epimerase
38.0 ± 2.6
51.9 ± 3.6
31.2 ± 0.8
30.0 ± 0.4
Two modifications contributed to the total 55.5% of xylose consumption increase exhibited by LF1 relative to XHR11. The expression of the glucose-insensitive, xylose-specific transporter Mgt05196pN360F (Wang et al. 2015) led directly to an 11.3% increase in the xylose consumption rate of XHR11 (for XH7, a similar increase of 10.5% was observed). The other 44.2% increase exhibited by LF1 was due to adaptive evolution on xylose (Fig. 4a). Because the copy number and transcription level of MGT05196 N360F remained nearly constant (Fig. 4b, c), and strains of XHR11 and XH7 without this transporter were also evolved at the same time but did not exhibit any enhancement in their xylose metabolic capacity in this round of evolution (data not shown), it is evident that this type of transporter was an important positive factor for improving xylose metabolism in S. cerevisiae. The transporter probably not only directly enhanced the absorption of xylose but also affected the overall evolutionary trend.
Recently, strains of S. cerevisiae engineered for second-generation bioethanol production have been continuously improved, inching closer to industrialization. In general, multiple modifications have been used, including the rational establishment of a xylose metabolic pathway and non-rational adaptive evolution to improve xylose metabolic capacity and inhibitor tolerance (Kim et al. 2013; Ko et al. 2016; Kuyper et al. 2005a; Peng et al. 2012; Van Vleet and Jeffries 2009). Moreover, using robust yeast strains as chassis cells has also proved to be an effective strategy (Demeke et al. 2013; Diao et al. 2013; Li et al. 2015). In the present work, based on our previous study, the S. cerevisiae wild-type diploid strain BSIF, which is highly robust and possesses a good genetic background for xylose metabolism, was used as the chassis cell (Li et al. 2015). The following two key novel elements were introduced into the chassis cell: MGT05196 N360F, which encodes a xylose-specific, glucose-insensitive transporter derived from the M. guilliermondii transporter gene MGT05196 (Wang et al. 2015), and Ru-xylA, which encodes a xylose isomerase (XI) with higher activity in S. cerevisiae and was derived from a screen of a bovine rumen metagenomic library (Bao et al. 2013). Another highlight in strain construction was the use of multiple adaptive evolutions under xylose and inhibitor stresses.
Overall, these modifications resulted in strain LF1, which has an excellent xylose fermentation capacity in media prepared from both sugars (Fig. 3) and lignocellulosic hydrolysates (Fig. 5). When xylose was the sole carbon source, LF1 consumed all of the 40 g L−1 xylose in 12 h with initial inoculum size of 0.5 g DCW L−1, and resulted in an ethanol yield of 0.446 g g−1. In contrast, CIBTS0735, a similarly engineered strain harboring the xylose isomerase gene xylA from Piromyces and the transporter gene GXF1 from Candida intermedia (Diao et al. 2013), required 16 h to consume nearly the same amount of xylose with initial inoculum size of 0.63 g DCW L−1, and resulted in a lower ethanol yield of 0.412 g g−1. Moreover, GS1.11-26, another similarly engineered strain that harbors the xylose isomerase gene xylA from Clostridium phytofermentans and overexpresses the endogenous transporter gene HXT7 (Demeke et al. 2013), resulted in a slightly higher ethanol yield of 0.460 g g−1 but also required 17 h to ferment less xylose (35 g L−1) with a larger initial inoculum size (1.3 g DCW L−1). In a glucose–xylose mixture, LF1 consumed 80 g L−1 glucose and 40 g L−1 xylose in 16 h with an ethanol yield of 0.475 g g−1, which was greater than 93% of the theoretical yield. In contrast, the corresponding values for CIBTS0735 (Diao et al. 2013) were 20 h and an ethanol yield of 0.454 g g−1 in a fermentation with the same amount of mixed sugars. Additionally, GS1.11-26 (Demeke et al. 2013) required only 13 h to produce an ethanol yield of 0.46 g g−1 but fermented less sugar (36 g L−1 glucose and 37 g L−1 xylose) with a larger initial inoculum size (1.3 g DCW L−1) compared with the 0.5 and 0.63 g DCW L−1 of LF1 used in the present work (Fig. 5) and CIBTS0735 (Diao et al. 2013), respectively. Other similarly engineered strains did not show improvement in key fermentation parameters, as discussed above (Ko et al. 2016; Romaní et al. 2015; Smith et al. 2014). Additionally, LF1 also performed well in the fermentation of two types of lignocellulosic hydrolysates, even when supplied with the simple nitrogen source urea rather than yeast extract and peptone (Fig. 5). Moreover, LF1 prominently displayed highly synchronized glucose and xylose utilization not only in a mixture of the sugars (with a utilization of 77.6% xylose and glucose depletion at 12 h) but also in SPPR hydrolysate, and ethanol production in LF1 peaked quickly (Fig. 3d vs. f, 5). This characteristic is a significant advantage in second-generation bioethanol production because it will save fermentation time, reduce staff input, and so on.
During approximately 40 years of metabolic engineering in yeast to co-ferment xylose and glucose, researchers have gradually realized that it is easier to establish a xylose metabolic pathway than to improve the efficiency of xylose fermentation and glucose-xylose co-fermentation. Although much research toward this goal has been performed, key factors in this strategy remain unknown. Therefore, non-rational adaptive evolution is generally used for strain engineering (Almeida et al. 2007; Heer and Sauer 2008b; Kuyper et al. 2005b). In the present work, we also focused on the strain breeding, therefore, the contribution for ‘key factors’ exploration maybe not enough.
Increasing the activity of xylose isomerase (XI), which isomerizes xylose directly to xylulose, is considered a good basis for establishing a xylose metabolic pathway in S. cerevisiae (Brat et al. 2009; Kuyper et al. 2003; Madhavan et al. 2009; Walfridsson et al. 1996; Zhou et al. 2012). The specific activity of XI in LF1 (0.46 U mg−1) was ~31.0% lower than in XH7 (which exhibited the highest activity of the strains in this study, 0.67 U mg−1) but was higher than in BSN3 (0.30 U mg−1) (Fig. 6a). However, the xylose fermentation capacity of LF1 was much improved compared with that of XH7 and BSN3 (Fig. 3; Table 3). This result implied that maintaining a moderate XI activity has a positive effect on the enhancement of xylose fermentation. The overexpression of Xks1p and four enzymes in the non-oxidative PPP is a common strategy to strengthen xylose metabolism (Bamba et al. 2016; Kuyper et al. 2005a; Peng et al. 2012; Sharma et al. 2016). Previous reports have shown that moderate, rather than extremely high, Xks1p activity is more beneficial for growth and ethanol production from xylose because this enzyme catalyzes an ATP-consuming reaction (Jin et al. 2003; Peng et al. 2011). In the present work (Table 4), the Xks1p (encoded by XKS1) expression level fluctuated noticeably. However, the expression level remained relatively high and constant in both improved xylose-fermenting strains, XH7 and LF1. Therefore, such a Xks1p expression level is apparently suitable for xylose metabolism. However, BSN3 also exhibited a similar Xks1p expression level (Table 4), even though this strain has not undergone any adaptive evolution and utilizes xylose poorly (Table 3, Line 1). Obviously, then, a suitable Xks1p activity is not the sole factor governing xylose metabolic capacity. Except for that of ribose 5-phosphate isomerase (encoded by RKI1), the expression levels of the enzymes in the non-oxidative PPP showed no significant change (Table 4). After the first round of adaptive evolution under xylose stress, the expression of RKI1 was upregulated from 14.4 ± 1.3 in BSN3 to 238.8 ± 15.3 in XH7 (in fold change relative to the original chassis, BSIF). Then, inhibitor stress dropped its expression to 146.78 ± 3.5 in XHR11 and remained at 144.5 ± 0.6 in LF1 (Table 4). As discussed above, XH7 and LF1 (but not BSN3 and XHR11) showed improved xylose fermentation, which indicates that ribose 5-phosphate isomerase, rather than the other three enzymes in the non-oxidative PPP, are important for xylose fermentation, as well as other key factors.
The transport issue is another limiting factor for xylose fermentation. Mgt05196pN360F, a xylose-specific transporter with no glucose inhibition (Wang et al. 2015), was therefore introduced into the chassis cells. As expected, the transporter enhanced xylose utilization to some extent by simple expression. Moreover, after the Mgt05196pN360F-containing strain was evolved in the presence of xylose (Fig. 1b), glucose and xylose utilization efficiency and synchronicity improved (Fig. 3; Table 3). Strain XHR11, which lacked this transporter, did not show any change in its xylose metabolic capacity during the same adaptive evolution, and the copy numbers and transcription levels of MGT05196 N360F remained constant in XHR11-360F and LF1. Therefore, we deduced that Mgt05196pN360F absorbed significantly more intracellular xylose, which resulted in stronger xylose stress tolerance in these cells and drove evolution beneficially toward xylose metabolism. Therefore, Xks1p expression recovered in LF1 relative to its parent. However, ribose 5-phosphate isomerase expression did not recover in LF1 (Table 4), which indicates that non-rational adaptive evolution led to random changes in other genes, and the key factors contributing to xylose metabolism must be numerous. An exploration of these unknown factors and their function via inverse metabolic engineering would be a significant undertaking. S. cerevisiae maintains high levels of glycolysis and PPP enzymes when glucose is present, and it shifts to respiratory metabolism after glucose is exhausted (Salusjarvi et al. 2008). Because xylose is metabolized through the PPP and glycolysis, its utilization will obviously drop as well. Furthermore, in respiratory metabolism, pyruvate prefers to enter the tricarboxylic acid cycle instead of producing ethanol. Therefore, using a specific transporter to facilitate the synchronized co-fermentation of glucose and xylose is beneficial for their co-utilization.
It was observed that xylose consumption was inhibited by the presence of glucose; therefore, the specific xylose consumption rates in the sugar mixture were lower than when xylose was the sole carbon source (Table 3, line 9 vs. 12, 10 vs. 13, 11 vs. 14) (Demeke et al. 2013; Diao et al. 2013; Peng et al. 2012; Shen et al. 2012), However, the same parameter was still kept lower (Fig. 3g) in the xylose consumption phase (Hou et al. 2009b) after the depletion of glucose from the sugar mixture. We defined such a phenotype—the continued impact of glucose on xylose metabolism after glucose depletion—as the “post-glucose effect” in the present work, which is an underappreciated phenomenon frequently evident in data shown in previous publications (Demeke et al. 2013; Diao et al. 2013; Peng et al. 2012; Shen et al. 2012). Exploration of the mechanisms underlying the post-glucose effect and the discovery of elements to overcome this phenotype will be the focus of future research.
In the present work, two novel proprietary heterologous genes (MGT05196 N360F and Ru-xylA) were combined with other multiple genetic modifications and three rounds of adaptive evolution to engineer the diploid S. cerevisiae chassis strain BSIF. The final resulting strain of this effort, LF1, consumed all xylose (40 g L−1) and glucose (80 g L−1) in 16 h with an ethanol yield that was over 93% of the theoretical yield. LF1 also performed well in lignocellulosic hydrolysates, with ethanol yields of over 80.0% of the theoretical yields. Moreover, LF1 exhibited highly synchronous utilization of glucose and xylose. These results highlight the great potential for the practical use of LF1 in the production of second-generation bioethanol. Despite the directed genetic changes detailed in this work, some unknown factors derived from adaptive evolution were also responsible for the superior fermentation performance of LF1. We also outlined the “post-glucose effect” phenomenon. An exploration of its underlying mechanisms remains key.
In the present work, YP was composed of 10 g L−1 yeast extract and 20 g L−1 peptone. The YPD and YPX were composed of YP amended with glucose and xylose, respectively, in varying concentrations (g L−1). For example, YPX40 indicated that 40 g L−1 xylose was added to YP. Hy1 and Hy2 refer to the hydrolysates from SECS supplied by Novozymes and SPPR supplied by the Shandong Tranlin Group, respectively. The main components of both hydrolysates are listed in Table 2. Hy1(2)-YP was a mixture of the hydrolysate Hy1 with YP. Hy1(2)-Urea included an additional 5 g L−1 urea. SECS liquor referred to the liquid fraction of the steam-exploding corn stover without enzymolysis, which therefore contained less glucose (Table 2).
The chassis cell BSIF was diploid; therefore, to destroy two alleles of PHO13, which would benefit xylose metabolism (Bamba et al. 2016; Lee et al. 2014; Shen et al. 2012; Van Vleet et al. 2008), two plasmids were assembled in the plasmid pUC19 as follows: pXIP1/2 (Additional file 1: Fig. S2a) containing two pairs of PHO13-targeted recombinant arms, PHO13- RA1 vs. PHO13- RA2 and PHO13- RA1 vs. PHO13- RA3. Three tandem TEF1p-Ru-xylA-PGK1t cassettes, amplified from the plasmid pJX7 (Hou et al. 2014), and the loxP-KanMX4-loxP cassette, amplified from the plasmid pUG6 (Güldener et al. 1996; Peng et al. 2012), were also ligated into the plasmid. Given that the multiple repeats of the δ-sequence in the chromosome could lead to multiple integrated copies of Ru-xylA in the genome (Cho et al. 1999; Yamada et al. 2010), a new pair of δ-targeted recombinant arms, δ-RA1 and δ-RA2, were introduced into pXIP1 instead of the PHO13-targeted recombinant arms, resulting in pXIδ (Additional file 1: Fig. S2b). Similarly, inactivating the two alleles of GRE3 should also benefit xylose metabolism (Kuyper et al. 2005a). The plasmids pJPPP3 (Peng et al. 2012) (which contains individual expression cassettes for four non-oxidative PPP genes and a pair of GRE3-targeted recombinant arms, GRE3-RA1/AR2) and pJPPP4 (which was constructed by replacing the GRE3-RA2 fragment of pJPPP3 with GRE3-RA3) (Additional file 1: Fig. S2c) were used for this aim.
The specific transporter gene MGT05196 N360F (Wang et al. 2015) was inserted into another region of the GRE3 locus. To achieve this aim, a new pair of GRE3-targeted recombinant arms, GRE3-RA4/AR5, together with the transporter expression cassette TDH3p-MGT05196 N360F-CYC1t and the selectable marker loxP-KanMX4-loxP, were ligated into pUC19. Additionally, to heighten expression, a fragment including three tandem sequences of the upstream activating sequence UASCLB (Blazeck et al. 2012) was also ligated in front of the promoter TDH3p. The resulting plasmid, pUC-N360F, is shown in Additional file 1: Fig. S2d.
The relative positions of the introduced gene loci are shown in Additional file 1: Fig. S1. All of the plasmids and primers (with their relevant restriction enzyme sites) used in the present work are listed in Table 1 and Additional file 1: Table S1. respectively.
All S. cerevisiae strains used in this work are listed in Table 1. The strain parentage is shown in Fig. 1b, and diagrams of the relative locations of each genetic manipulation are detailed in Additional file 1: Fig. S1.
Use of the Cre-loxP recombination system, which allows repeated use of a selection marker (Güldener et al. 1996), facilitated construction of the wild-type chassis cell. Accordingly, a new plasmid, YEp-CH, was constructed by the ligation of two fragments, an inducible GAL1p-controlled CreA recombinase expression cassette and a dominant hygromycin resistance gene expression cassette, into YEp24. The integration of each fragment into the genome involved several operational steps. The chassis cell was transformed with a loxP-KanMX4-loxP-containing fragment and grown on YPD20 agar plates supplemented with G418 (400 mg L−1). After evaluation and verification, the best-growing colony was then transformed with YEp-CH and grown on YPD20 agar plates supplemented with hygromycin (200 mg L−1). Then, the transformant was cultured in YP supplemented with galactose (20 g L−1) to induce the expression of the CreA recombinase and remove the KanMX4 cassette. Finally, the YEp-CH plasmid was lost on YPD20 without hygromycin selection. Such steps were cycled (Fig. 1b) in the wild-type chassis cell BSIF (Li et al. 2015).
The loxP-KanMX4-loxP-containing integration fragments were obtained in a different way. The Ru-xylA segments were recovered from pXIP1/2 and pXIδ double-digested with EcoRI/SphI. The four genes in the PPP segments were recovered from pJPPP3/4 digested with SmiI. The MGT05196 N360F segments were linearized from pUC-N360F digested with SmiI. The TEF1p segments with two pairs of XKS1-targeted recombinant arms, XKS1-RA1/XKS1-RA3 and XKS1-RA2/XKS1-RA3, upstream of XKS1 were amplified by overlap PCR, which was used to moderately strengthen the expression of XKS1 (Peng et al. 2011) by replacing its native promoter with a stronger one in two alleles (Additional file 1: Fig. S1c).
Dozens of colonies were isolated from the evolved strain suspension. Evaluations were performed under the same stress using the BioScreen system (Oy Growth Curves Ab Ltd, Helsinki, Finland) and further verified in shake flasks. Then, the single colony that grew fastest was selected for further use.
The batch fermentations were performed in either a 1.4-L fermenter with a 1000 mL working volume (Infors AG, Switzerland) or in 250-mL shake flasks with a 50-mL working volume. The different media described above were used as needed. The fermentations were performed at 30 °C and 200 rpm agitation rate, with other parameters of either 0.04 vvm ventilation (the oxygen-limited condition) (Zhang et al. 2010), pH 5.5 in the fermenters, or an initial pH of 6 in the shake flasks with a rubber stopper plug in a syringe needle (the oxygen-limited condition) (Walfridsson et al. 1996). Overnight cultures of a single colony were transferred to 100 mL YPD20 in 250-mL shake flasks at an initial OD600 of 0.2 and incubated at 30 °C for another 12 h for inoculations (Li et al. 2015). The data from the independent triplicate cultivations were compared using Student’s t-test whenever indicated. A P < 0.05 was considered statistically significant.
Compounds Analysis and Calculation
Biomass was determined by measuring OD600 (Eppendorf AG, 22331, Hamburg, Germany) and DCW. Different strains exhibited different coefficients between OD600 and DCW (data not shown). The maximum growth rates (μmax) are the linear regression coefficients of the ln(OD600) versus time during the exponential growth phase.
The concentrations of glucose, xylose, xylitol, glycerol, acetate, and ethanol were determined by HPLC (Shimadzu, Japan) with a BIO-RAD Aminex HPX-87H ion exclusion column (300 × 7.8 mm) as previously described (Diao et al. 2013; Hou et al. 2016b; Peng et al. 2012). The mobile phase was 5 mmol L−1 H2SO4 with a flow rate of 0.6 mL min−1. The temperature of the column oven was 45 °C. The specific consumption or production rates of xylose, xylitol, glycerol, acetate, and ethanol were calculated as previously described (Peng et al. 2012).
The monosaccharides, weak acid, furfural and HMF in the hydrolysates were measured using an HPX-87H column as described above (Li et al. 2015). The total phenolics were determined using the Folin phenol method, and vanillin was used to prepare the standard curve (Singleton et al. 1999). Solubilized lignin was determined by measuring the absorbance at 320 nm (Li et al. 2015; Tan et al. 2013).
Measurement of enzyme activities
Overnight cultures were transferred into fresh YPD20 medium with 0.2 initial OD600 and cultured at 30 °C. The cells were harvested at OD600 4.0 and washed twice with sterile water and resuspended in 100 mmol L−1 Tris–HCl (pH 7.5) with a proteinase inhibitor cocktail (for fungal/yeast cells; Sangon Biotech Co., Ltd., Shanghai, China). Then, the cell-free extract was prepared as the crude enzyme using a Precellys 24 cell homogenizer (Bertin Technologies, France), as previously described (Shen et al. 2012). Protein concentration was measured using an Enhanced BCA Protein Assay Kit (Beyotime Biotechnology, China). The xylose isomerase activity of the crude enzyme was determined at 30 °C by measuring the absorbance change of the coenzymes at 340 nm with a spectrophotometer (Helios Gamma, Thermo Fisher Scientific, Waltham, MA). The 1-mL reaction mixture contained 100 mmol L−1 Tris–HCl buffer (pH 7.5), 10 mmol L−1 MgCl2, 500 mmol L−1 xylose, 2 U of sorbitol dehydrogenase (Roche, Boulder, CO), 0.15 mmol L−1 NADH, and crude enzyme (Kuyper et al. 2003). One unit of xylose isomerase activity was defined as the amount of enzyme required to oxidize 1 μmol of coenzyme per minute under the conditions of the assay, and the specific activity was expressed in units per milligram of protein (Shen et al. 2012).
Gene transcription level analysis
Overnight cultures were transferred into fresh YPD20 medium with 0.2 initial OD600 and cultured at 30 °C. Cells were harvested at OD600 0.8–1.0. Total RNA was isolated using Trizol reagent (Takara, Japan) and purified using a NucleoSpin® Extract II Kit (Machery-Nagel Corp., Germany). The total RNA (0.25 μg) was used to synthesize the first strand of cDNA in a 10-μL reverse transcription (RT) reaction. Real-time quantitative PCR was performed using the Light Cycle PCR System (Roche Molecular Biochemicals, Germany) and SYBR Green Real-time PCR Master Mix (TOYOBO, Japan). The normalization reference was ACT1 (Peng et al. 2012). The primers for the quantitative real-time PCR are listed in Additional file 1: Table S1. Real-time quantitative PCR data were analyzed according to the 2−ΔΔCT method (Livak and Schmittgen 2001). All data were the average of values from three separate cultures, and each culture was tested in triplicate. The replicates were compared using Student’s t-test whenever indicated. A P < 0.05 was considered statistically significant.
Determination of gene copy number
Cells were washed and resuspended in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.5) and lysed with a Precellys 24 cell homogenizer (Bertin Technologies, France). The protein was removed using the phenol–chloroform method (Ellington & Jack D. Pollard, in Short Protocols in Molecular Biology, Volume 1, 5th Edition. Edited by Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J. G. Seidman, John A. Smith, and Kevin Struhl. John Wiley & Sons, Inc.). The genomic DNA was precipitated with ethanol, resuspended in water, and then used to analyze the gene copy number via quantitative real-time PCR. The normalization reference was ACT1. The copy number was calculated according to the following equation: plasmid copy number = 2CT(act1)/2CT(xylA) (Lee et al. 2006; Shen et al. 2012).
pentose phosphate pathway
steam-exploding corn stover
sulfite pretreatment papermaking residue
- SECS liquor:
the leach liquor of steam-exploding corn stover
yeast extract and peptone
dry cell weight
- OD or OD600 :
optical density at 600 nm
quantitative reverse-transcription PCR
quantitative real-time PCR
HXL, YS, JH and XMB conceived of the study. HXL and MLW performed the experiments. CLJ, ZLL and XLL participated in the design of the study and helped with data analysis. HXL, YS and XMB analyzed the results and drafted the manuscript. All authors read and approved the final manuscript.
The authors wish to acknowledge the financial support from Project of National Energy Administration of China (NY20130402), National High-Technology Research and Development Program of China under Grant (2014AA021903, 2012AA022106), National Key Technology R&D Program of China (2014BAD02B07), National Natural Science Foundation of China (31470166, 31270151), the State Key Laboratory of Motor Vehicle Biofuel Technology (2013004, KFKT2013002). The work also supported by Shandong Key Laboratory of Straw Biorefinement Technologies and Taishan Scholar Construction Project.
The authors declare that they have no competing interests.
Availability of supporting data
All data generated or analyzed during this study are included in this article and its Additional files.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Almeida JRM, Modig T, Petersson A, Hähn-Hägerdal B, Lidén G, Gorwa-Grauslund MF (2007) Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol 82:340–349. doi:10.1002/jctb.1676 View ArticleGoogle Scholar
- Bamba T, Hasunuma T, Kondo A (2016) Disruption of PHO13 improves ethanol production via the xylose isomerase pathway. AMB Expres 6:4. doi:10.1186/s13568-015-0175-7 View ArticleGoogle Scholar
- Bao X, Shen Y, GE R (2013) Nucleic acid molecule for encoding xylose isomerase and xylose isomerase encoded by the nucleic acid molecule. US Patent 8,586,336 B2, 19 Nov 2013. http://xs1.hntvchina.com/patents/US8586336
- Blazeck J, Garg R, Reed B, Alper HS (2012) Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters. Biotechnol Bioeng 109:2884–2895. doi:10.1002/bit.24552 View ArticleGoogle Scholar
- Brandberg T, FranzéN CJ, Gustafsson L (2004) The fermentation performance of nine strains of Saccharomyces cerevisiae in batch and fed-batch cultures in dilute-acid wood hydrolysate. J Biosci Bioeng 98:122–125. doi:10.1016/S1389-1723(04)70252-2 View ArticleGoogle Scholar
- Brat D, Boles E, Wiedemann B (2009) Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae. Appl Environ Microbiol 75:2304–2311View ArticleGoogle Scholar
- Cho KM, Yoo YJ, Kang HS (1999) δ-Integration of endo/exo-glucanase and β-glucosidase genes into the yeast chromosomes for direct conversion of cellulose to ethanol. Enzyme Microb Technol 25:23–30. doi:10.1016/s0141-0229(99)00011-3 View ArticleGoogle Scholar
- Demeke M, Dietz H, Li Y, Foulquie-Moreno M, Mutturi S, Deprez S, Den Abt T, Bonini B, Liden G, Dumortier F et al (2013) Development of a d-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering. Biotechnol Biofuels 6:89. doi:10.1186/1754-6834-6-89 View ArticleGoogle Scholar
- Diao L, Liu Y, Qian F, Yang J, Jiang Y, Yang S (2013) Construction of fast xylose-fermenting yeast based on industrial ethanol-producing diploid Saccharomyces cerevisiae by rational design and adaptive evolution. BMC Biotechnol 13:110View ArticleGoogle Scholar
- Farwick A, Bruder S, Schadeweg V, Oreb M, Boles E (2014) Engineering of yeast hexose transporters to transport d-xylose without inhibition by d-glucose. Proc Natl Acad Sci USA 111:5159–5164. doi:10.1073/pnas.1323464111 View ArticleGoogle Scholar
- Fujitomi K, Sanda T, Hasunuma T, Kondo A (2012) Deletion of the PHO13 gene in Saccharomyces cerevisiae improves ethanol production from lignocellulosic hydrolysate in the presence of acetic and formic acids, and furfural. Bioresour Technol 111:161–166. doi:10.1016/j.biortech.2012.01.161 View ArticleGoogle Scholar
- Güldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524. doi:10.1093/nar/24.13.2519 View ArticleGoogle Scholar
- Hahn-Hägerdal B, Karhumaa K, Fonseca C, Spencer-Martins I, Gorwa-Grauslund M (2007) Towards industrial pentose-fermenting yeast strains. Appl Microbiol Biotechnol 74:937–953. doi:10.1007/s00253-006-0827-2 View ArticleGoogle Scholar
- Heer D, Sauer U (2008) Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain. Microb Biotechnol 1:497–506. doi:10.1111/j.1751-7915.2008.00050.x View ArticleGoogle Scholar
- Ho NW, Chen Z, Brainard AP, Sedlak M (1999) Successful design and development of genetically engineered Saccharomyces yeasts for effective cofermentation of glucose and xylose from cellulosic biomass to fuel ethanol. Adv Biochem Eng Biotechnol 65:163–192Google Scholar
- Hou J, Lages NF, Oldiges M, Vemuri GN (2009a) Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae. Metab Eng 11:253–261. doi:10.1016/j.ymben.2009.05.001 View ArticleGoogle Scholar
- Hou J, Vemuri GN, Bao X, Olsson L (2009b) Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae. Appl Microbiol Biotechnol 82:909–919. doi:10.1007/s00253-009-1900-4 View ArticleGoogle Scholar
- Hou J, Suo F, Wang CQ, Li XW, Shen Y, Bao XM (2014) Fine-tuning of NADH oxidase decreases byproduct accumulation in respiration deficient xylose metabolic Saccharomyces cerevisiae. BMC Biotechnol. doi:10.1186/1472-6750-14-13 Google Scholar
- Hou J, Shen Y, Jiao C, Ge R, Zhang X, Bao X (2016a) Characterization and evolution of xylose isomerase screened from the bovine rumen metagenome in Saccharomyces cerevisiae. J Biosci Bioeng 121:160–165. doi:10.1016/j.jbiosc.2015.05.014 View ArticleGoogle Scholar
- Hou J, Jiao C, Peng B, Shen Y, Bao X (2016b) Mutation of a regulator Ask10p improves xylose isomerase activity through up-regulation of molecular chaperones in Saccharomyces cerevisiae. Metab Eng 38:241–250. doi:10.1016/j.ymben.2016.08.001 View ArticleGoogle Scholar
- Jin Y-S, Ni H, Laplaza J, Jeffries T (2003) Optimal growth and ethanol production from xylose by recombinant Saccharomyces cerevisiae require moderate d-xylulokinase activity. Appl Environ Microbiol 69:495–503. doi:10.1128/AEM.69.1.495-503.2003 View ArticleGoogle Scholar
- Kim SR, Park YC, Jin YS, Seo JH (2013) Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism. Biotechnol Adv 31:851–861. doi:10.1016/j.biotechadv.2013.03.004 View ArticleGoogle Scholar
- Ko JK, Um Y, Woo HM, Kim KH, Lee S-M (2016) Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway. Bioresour Technol 209:290–296. doi:10.1016/j.biortech.2016.02.124 View ArticleGoogle Scholar
- Kuyper M, Harhangi HR, Stave AK, Winkler AA, Jetten MSM, de Laat WTAM, den Ridder JJJ, Op den Camp HJM, van Dijken JP, Pronk JT (2003) High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae? FEMS Yeast Res 4:69–78. doi:10.1016/S1567-1356(03)00141-7 View ArticleGoogle Scholar
- Kuyper M, Winkler AA, van Dijken JP, Pronk JT (2004) Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle. FEMS Yeast Res 4:655–664. doi:10.1016/j.femsyr.2004.01.003 View ArticleGoogle Scholar
- Kuyper M, Hartog MMP, Toirkens MJ, Almering MJH, Winkler AA, van Dijken JP, Pronk JT (2005a) Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. FEMS Yeast Res 5:399–409. doi:10.1016/j.femsyr.2004.09.010 View ArticleGoogle Scholar
- Kuyper M, Toirkens MJ, Diderich JA, Winkler AA, van Dijken JP, Pronk JT (2005b) Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain. FEMS Yeast Res 5:925–934. doi:10.1016/j.femsyr.2005.04.004 View ArticleGoogle Scholar
- Lee C, Kim J, Shin SG, Hwang S (2006) Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol 123:273–280. doi:10.1016/j.jbiotec.2005.11.014 View ArticleGoogle Scholar
- Lee S-M, Jellison T, Alper H (2014) Systematic and evolutionary engineering of a xylose isomerase-based pathway in Saccharomyces cerevisiae for efficient conversion yields. Biotechnol Biofuels 7:122. doi:10.1186/s13068-014-0122-x Google Scholar
- Li H, Wu M, Xu L, Hou J, Guo T, Bao X, Shen Y (2015) Evaluation of industrial Saccharomyces cerevisiae strains as the chassis cell for second-generation bioethanol production. Microb Biotechnol 8:266–274. doi:10.1111/1751-7915.12245 View ArticleGoogle Scholar
- Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. doi:10.1006/meth.2001.1262 View ArticleGoogle Scholar
- Madhavan A, Tamalampudi S, Ushida K, Kanai D, Katahira S, Srivastava A, Fukuda H, Bisaria VS, Kondo A (2009) Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol. Appl Microbiol Biotechnol 82:1067–1078. doi:10.1007/s00253-008-1794-6 View ArticleGoogle Scholar
- Moon J, Lewis Liu Z, Ma M, Slininger PJ (2013) New genotypes of industrial yeast Saccharomyces cerevisiae engineered with YXI and heterologous xylose transporters improve xylose utilization and ethanol production. Bio catal Agric Biotechnol 2:247–254. doi:10.1016/j.bcab.2013.03.005 Google Scholar
- Moysés DN, Reis VCB, Almeida JRMd, Moraes LMPd, Torres FAG (2016) Xylose Fermentation by Saccharomyces cerevisiae: challenges and prospects. Int J Mol Sci 17:207. doi:10.3390/ijms17030207 View ArticleGoogle Scholar
- Nijland JG, Shin HY, de Jong RM, de Waal PP, Klaassen P, Driessen AJ (2014) Engineering of an endogenous hexose transporter into a specific d-xylose transporter facilitates glucose-xylose co-consumption in Saccharomyces cerevisiae. Biotechnol Biofuels 7:1–11. doi:10.1186/s13068-014-0168-9 View ArticleGoogle Scholar
- Palmqvist E, Hahn-Hägerdal B (2000) Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour Technol 74:25–33. doi:10.1016/s0960-8524(99)00161-3 View ArticleGoogle Scholar
- Pannunzio NR, Manthey GM, Liddell LC, Fu BXH, Roberts CM, Bailis AM (2012) Rad59 regulates association of Rad52 with DNA double-strand breaks. Microbiol Open 1:285–297. doi:10.1002/mbo3.31 View ArticleGoogle Scholar
- Peng B, Chen X, Shen Y, Bao X (2011) Effect of controlled overexpression of xylulokinase by different promoters on xylose metabolism in Saccharomyces cerevisiae. Acta Microbiol Sin. 51(7):914–922Google Scholar
- Peng B, Shen Y, Li X, Chen X, Hou J, Bao X (2012) Improvement of xylose fermentation in respiratory-deficient xylose-fermenting Saccharomyces cerevisiae. Metab Eng 14:9–18. doi:10.1016/j.ymben.2011.12.001 View ArticleGoogle Scholar
- Reider Apel A, Ouellet M, Szmidt-Middleton H, Keasling JD, Mukhopadhyay A (2016) Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae. Sci Rep 6:19512. doi:10.1038/srep19512 View ArticleGoogle Scholar
- Robberecht C, Voet T, Esteki MZ, Nowakowska BA, Vermeesch JR (2013) Nonallelic homologous recombination between retrotransposable elements is a driver of de novo unbalanced translocations. Genome Res 23:411–418. doi:10.1101/gr.145631.112 View ArticleGoogle Scholar
- Romaní A, Pereira F, Johansson B, Domingues L (2015) Metabolic engineering of Saccharomyces cerevisiae ethanol strains PE-2 and CAT-1 for efficient lignocellulosic fermentation. Bioresour Technol 179:150–158. doi:10.1016/j.biortech.2014.12.020 View ArticleGoogle Scholar
- Runquist D, Fonseca C, Rådström P, Spencer-Martins I, Hahn-Hägerdal B (2009) Expression of the Gxf1 transporter from Candida intermedia improves fermentation performance in recombinant xylose-utilizing Saccharomyces cerevisiae. Appl Microbiol Biotechnol 82:123–130. doi:10.1007/s00253-008-1773-y View ArticleGoogle Scholar
- Salusjarvi L, Kankainen M, Soliymani R, Pitkanen J-P, Penttila M, Ruohonen L (2008) Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae. Microb Cell Fact 7:18. doi:10.1186/1475-2859-7-18 View ArticleGoogle Scholar
- Sharma S, Sharma S, Singh S, Lata Arora A (2016) Improving yeast strains for pentose hexose co-fermentation: successes and hurdles. In: Kumar S, Khanal KS, Yadav KY (eds) Proceedings of the first international conference on recent advances in bioenergy research. Springer Proceedings in Energy. Springer, New DelhiGoogle Scholar
- Shen Y, Chen X, Peng B, Chen L, Hou J, Bao X (2012) An efficient xylose-fermenting recombinant Saccharomyces cerevisiae strain obtained through adaptive evolution and its global transcription profile. Appl Microbiol Biotechnol 96:1079–1091. doi:10.1007/s00253-012-4418-0 View ArticleGoogle Scholar
- Shin HY, Nijland JG, de Waal PP, de Jong RM, Klaassen P, Driessen AJ (2015) An engineered cryptic Hxt11 sugar transporter facilitates glucose–xylose co-consumption in Saccharomyces cerevisiae. Biotechnol Biofuels 8:176. doi:10.1186/s13068-015-0360-6 View ArticleGoogle Scholar
- Singleton VL, Orthofer R, Lamuela-Raventós RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. In: Pecoraro Vincent L (ed) Oxidants and antioxidants Part A methods in enzymology, vol 299. Academic Press, New York, pp 152–178View ArticleGoogle Scholar
- Smith J, van Rensburg E, Goergens JF (2014) Simultaneously improving xylose fermentation and tolerance to lignocellulosic inhibitors through evolutionary engineering of recombinant Saccharomyces cerevisiae harbouring xylose isomerase. BMC Biotechnol 14:41. doi:10.1186/1472-6750-14-41 View ArticleGoogle Scholar
- Sonderegger M, Jeppsson M, Larsson C, Gorwa-Grauslund MF, Boles E, Olsson L, Spencer-Martins I, Hahn-Hagerdal B, Sauer U (2004) Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains. Biotechnol Bioeng 87:90–98. doi:10.1002/bit.20094 View ArticleGoogle Scholar
- Subtil T, Boles E (2012) Competition between pentoses and glucose during uptake and catabolism in recombinant Saccharomyces cerevisiae. Biotechnol Biofuels 5:1. doi:10.1186/1754-6834-5-14 View ArticleGoogle Scholar
- Tan L, Yu Y, Li X, Zhao J, Qu Y, Choo YM, Loh SK (2013) Pretreatment of empty fruit bunch from oil palm for fuel ethanol production and proposed biorefinery process. Bioresour Technol 135:275–282. doi:10.1016/j.biortech.2012.10.134 View ArticleGoogle Scholar
- van Maris AJ, Abbott DA, Bellissimi E, van den Brink J, Kuyper M, Luttik MA, Wisselink HW, Scheffers WA, van Dijken JP, Pronk JT (2006) Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Antonie Van Leeuwenhoek 90:391–418. doi:10.1007/s10482-006-9085-7 View ArticleGoogle Scholar
- Van Vleet J, Jeffries T (2009) Yeast metabolic engineering for hemicellulosic ethanol production. Curr Opin Biotechnol 20:300–306. doi:10.1016/j.copbio.2009.06.001 View ArticleGoogle Scholar
- Van Vleet J, Jeffries T, Olsson L (2008) Deleting the para-nitrophenyl phosphatase (pNPPase), PHO13, in recombinant Saccharomyces cerevisiae improves growth and ethanol production on d-xylose. Metab Eng 10:360–369. doi:10.1016/j.ymben.2007.12.002 View ArticleGoogle Scholar
- Walfridsson M, Bao X, Anderlund M, Lilius G, Bülow L, Hahn-Hägerdal B (1996) Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene, which expresses an active xylose (glucose) isomerase. Appl Environ Microbiol 62:4648–4651Google Scholar
- Walfridsson M, Anderlund M, Bao X, Hahn-Hagerdal B (1997) Expression of different levels of enzymes from the Pichia stipitis XYL1 and XYL2 genes in Saccharomyces cerevisiae and its effects on product formation during xylose utilisation. Appl Microbiol Biotechnol 48:218–224. doi:10.1007/s002530051041 View ArticleGoogle Scholar
- Wang Y, Shi W-L, Liu X-Y, Shen Y, Bao X-M, Bai F-W, Qu Y-B (2004) Establishment of a xylose metabolic pathway in an industrial strain of Saccharomyces cerevisiae. Biotechnol Lett 26:885–890. doi:10.1023/B:bile.0000025897.21106.92 View ArticleGoogle Scholar
- Wang C, Bao X, Li Y, Jiao C, Hou J, Zhang Q, Zhang W, Liu W, Shen Y (2015) Cloning and characterization of heterologous transporters in Saccharomyces cerevisiae and identification of important amino acids for xylose utilization. Metab Eng 30:79–88. doi:10.1016/j.ymben.2015.04.007 View ArticleGoogle Scholar
- Wang M, Yu C, Zhao H (2016) Directed evolution of xylose specific transporters to facilitate glucose–xylose co-utilization. Biotechnol Bioeng 113:484–491. doi:10.1002/bit.25724 View ArticleGoogle Scholar
- Xu H, Kim S, Sorek H, Lee Y, Jeong D, Kim J, Oh EJ, Yun EJ, Wemmer DE, Kim KH (2016) PHO13 deletion-induced transcriptional activation prevents sedoheptulose accumulation during xylose metabolism in engineered Saccharomyces cerevisiae. Metab Eng 34:88–96. doi:10.1016/j.ymben.2015.12.007 View ArticleGoogle Scholar
- Yamada R, Tanaka T, Ogino C, Fukuda H, Kondo A (2010) Novel strategy for yeast construction using δ-integration and cell fusion to efficiently produce ethanol from raw starch. Appl Microbiol Biotechnol 85:1491–1498. doi:10.1007/s00253-009-2198-y View ArticleGoogle Scholar
- Yamada R, Taniguchi N, Tanaka T, Ogino C, Fukuda H, Kondo A (2011) Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression. Biotechnol Biofuels 4:8. doi:10.1186/1754-6834-4-8 View ArticleGoogle Scholar
- Zha J, Hu ML, Shen MH, Li BZ, Wang JY, Yuan YJ (2012) Balance of XYL1 and XYL2 expression in different yeast chassis for improved xylose fermentation. Front Microbiol 3:355. doi:10.3389/fmicb.2012.00355 View ArticleGoogle Scholar
- Zhang X, Shen Y, Shi W, Bao X (2010) Ethanolic cofermentation with glucose and xylose by the recombinant industrial strain Saccharomyces cerevisiae NAN-127 and the effect of furfural on xylitol production. Bioresour Technol 101:7104–7110. doi:10.1016/j.biortech.2010.03.129 Google Scholar
- Zhou H, J-s Cheng, Wang BL, Fink GR, Stephanopoulos G (2012) Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae. Metab Eng 14:611–622. doi:10.1016/j.ymben.2012.07.011 View ArticleGoogle Scholar