Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two β-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both β-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.


Cas9-based integration of CDT-2 expression cassette into the SOR1 locus
Although xylitol has a variety of uses in the food, cosmetic, nutraceutical, and pharmaceutical industries 34 , this metabolite may face competition from an available carbon source, reducing the efficiency of ethanol production.S. cerevisiae strains possess genes that encode enzymes capable of xylose reduction, such as GRE3, GCY1, YPR1, YDL124W, YJR096W, and xylitol oxidation such as SOR1, SOR2, XYL2, XDH1; which can result in xylitol formation during xylose fermentation 35 .To reduce xylitol production and divert the carbon towards ethanol production, two distinct genes, namely GRE3 and SOR1, were selected for knockout.First, SOR1 was replaced by a CDT-2 expression cassette in the genomic DNA of strain SR8A6S3, yielding SR8A6S3-CDT-2 (sor1Δ).The required integration of the CDT-2 cassette was confirmed through PCR analysis.Colony PCR was performed directly from 27 colonies of the positive-control plate (Additional file 1; Fig. S1).Once the desired integration was confirmed, both the SR8A6S3-CDT-2 and SR8A6S3 strains were compared in anaerobic and micro-aerobic batch cultures (Figs.2A, B and 3) in YPDXA containing: 20 g L −1 glucose, 80 g L −1 xylose, and 8 g L −1 acetate, with an initial OD 600 of 1.
Deletion of SOR1 led to a reduced rate of xylose and acetate consumption under both anaerobic and microaerobic conditions (Figs. 2A, B and 3).Under anaerobic batch cultivation, 75% of the initial xylose was consumed by the SR8A6S3 strain, while SR8A6S3-CDT-2 was only able to consume 53% of the original concentration within 72 h (Additional file 2: Fig. 2A).The xylose consumption rate of SR8A6S3 was also higher after 24 h of anaerobic cultivation in comparison to SR8A6S3-CDT-2 (Table 1).
Concerning acetate metabolism, the control strain consumed 71% of the initial acetate in the medium, while SR8A6S3-CDT-2 consumed only 43% in 72 h of cultivation (Fig. 2A, B, and Additional file 2: Fig. S2E).For glucose metabolism, no difference was observed between the two strains (Fig. 2).However, despite the greater consumption of xylose and acetate by the control strain (83.08 ± 1.46 versus 70.58 ± 2.91 g L −1 ), SR8A6S3-CDT-2 had a slightly higher ethanol yield (Table 1) and produced 66% less xylitol and 12% less glycerol as a by-product (Additional file 2: Fig. S2).We observed that glycerol was primarily coming from glucose for both cultivations.A great amount of total glycerol was produced after 24 h of cultivation, 66% for SR8A6S3 and 60% for SR8A6S3-CDT-2.Considering these results, it is possible to conclude that in the control strain cultivation, the carbon source was channelled towards metabolites whose pathways allowed for the balance of redox cofactors, such as xylitol and glycerol.Thereby, SOR1 is responsible for a significant amount of xylitol production, but sor1Δ primarily slows down xylose metabolism.However, sor1Δ enabled the engineered strain to drive more carbon toward the desired product (ethanol).Presumably, this is because the NADH / NAD + balance changed, while the ethanol yield had marginally increased via the pyruvate decarboxylase route as, relative to xylose, acetate metabolism is proportionally lower when the two strains are compared.
Other metabolites were also measured to compare the fermentation profiles of SR8A6S3-CDT-2 and SR8A6S3 (Additional file 2: Fig. S2).Elimination of xylitol production through sor1Δ increases the availability of intracellular NADH, which enabled the recombinant cell to produce more ethanol per gram of consumed sugar (ethanol yield).Deletion of the sor1 gene does not eliminate xylitol production, since other genes encode enzymes capable of xylose reduction or xylitol oxidation, resulting in xylitol production.However, under strict anaerobic cultivation, the xylitol amount was reduced from 1.9 to 0.69 g L −1 , when comparing the parental SR8A6S3 and recombinant SR8A6S3-CDT-2 strains, respectively (Additional file 2: Fig. S2C).In principle, NAD + should be available to drive the xylitol to xylulose reaction.Under strict anaerobic conditions, ethanol is the most important primary metabolite produced, in terms of re-oxidation of excess NADH and redox balancing, followed by the production of glycerol 36 , which is important to support xylulose production from xylitol.When oxygen is available in the flask, redox balancing of NADH/ NAD + can also occur through the electron transport chain, which should result in less xylitol accumulation in the medium.We corroborated this hypothesis during batch cultivations under micro-aerobic conditions, where lower xylitol production was observed for both strains (Fig. 3 and Additional file 3: S3C).Micro-aerobic batch fermentations were performed in complex YP media supplemented with 20 g L −1 glucose, 80 g L −1 xylose, and 8 g L −1 acetate with an initial OD 600 of 1 (Fig. 3 and Additional file 3: S3).
Under micro-aerobic conditions the ethanol yields of SR8A6S3-CDT-2 and SR8A6S3 were 0.39 g ethanol g consumed sugars −1 and 0.37 g ethanol g consumed sugars −1 , respectively.As expected, xylitol yield was lower in SR8A6S3-CDT-2
To investigate the engineered strain, a YP-based medium was used to cultivate SR8A6S3-CDT-2-GH43-2/7 and measure xylose and acetate fermentation performance; in comparison to SR8A6S3-CDT-2 and their parental strain, SR8A6S3.Anaerobic batch cultivation was carried out for xylose and acetate consumption evaluation; and ethanol and xylitol production in high sugar content media (20 g L −1 glucose, 80 g L −1 xylose, and 8 g L −1 acetate), with an initial OD 600 of 1 (Fig. 2 and Additional file 5: S5).
In the first 24 h of cultivation, SR8A6S3-CDT-2-GH43-2/7 had an increased xylose consumption profile, compared with the SR8A6S3-CDT-2 strain.The latest engineered strain consumed 13.65 ± 0.53 g L −1 of xylose, which represents 18% of the initial xylose concentration; and the immediate parent consumed 10.73 ± 0.53 g L −1 (15% of the initial xylose concentration).During the same period, the acetate consumption profile was slightly higher for SR8A6S3-CDT-2-GH43-2/7 than SR8A6S3-CDT-2, 17% against 15% of the initial acetate concentration, respectively (Fig. 2B and C).Following a similar trend, the glycerol production profile within the first 24 h was higher for SR8A6S3-CDT-2-GH43-2/7 than for the SR8A6S3-CDT-2 strain.The former produced 1.47 ± 0.04 g L −1 , and the latter achieved 0.98 ± 0.13 g L −1 of glycerol.Conversely, within 24 and 72 h of cultivation, SR8A6S3-CDT-2-GH43-2/7 consumed lesser amounts of xylose and acetate than its immediate parent strain, 28.84 ± 0.66 g L −1 against 34.61 ± 3.45 g L −1 for xylose, and 1.81 ± 0.14 g L −1 against 2.38 ± 0.14 g L −1 for acetate, respectively; as well as produced lesser amounts of glycerol, 0.34 ± 0.04 g L −1 against 0.66 ± 0.12 g L −1 , respectively for SR8A6S3-CDT-2-GH43-2/7 and SR8A6S3-CDT-2 (Fig. 2B and C).The change in the xylose, acetate, and glycerol profile for both strains in the first 24 h of cultivation and after, is presumed to be because of the change in the NADH / NAD + balance.Deletion of GRE3 and increased production of glycerol (within 24 h of cultivation) might result in the higher availability of cofactors required for xylose metabolism (Additional file 6: Fig. S6), which reflected a better xylose consumption profile for SR8A6S3-CDT-2-GH43-2/7 in the first 24 h of cultivation.After the depletion of glucose, the glycerol production profile decreased for both strains (Fig. 2B and  C), however, gre3Δ slows down xylose metabolism.
Instead, compared with SR8A6S3 (Fig. 2A and B), the latest engineered strain had impaired xylose and acetate consumption profiles at all times during cultivation.Within the first 24 h of cultivation, SR8A6S3 consumed 22.53 ± 1.24 g L −1 of xylose, which represents 28% of the initial concentration, and 2.28 ± 0.10 g L −1 of acetate.The SR8A6S3-CDT-2-GH43-2/7 strain after 72 h consumed only 56% and 40% of the initial xylose and acetate concentration, respectively.Although, intriguingly, the rate of xylose consumption was marginally higher than the parent strains after 24 h.However, SR8A6S3-CDT 2 -GH43 2/7 had a slightly higher ethanol yield compared to both SR8A6S3-CDT 2 and SR8A6S3 (Table 1).Therefore, although xylitol production after 72 h was similar for SR8A6S3-CDT-2-GH43-2/7 and SR8A6S3-CDT-2, the deletion of both sor1Δ and gre3Δ, should have increased the availability of reduced cofactors, and enabled cells to produce more ethanol per gram of consumed sugar (ethanol yield) than just the sor1Δ single deletion (Additional file 6: Fig. S6).Moreover, the biomass production profile, which was analysed by the measurement of OD 600 , was similar until 24 h for SR8A6S3-CDT-2-GH43-2/7 and SR8A6S3-CDT-2, but was lower for the reference strain (SR8A6S3).The double-engineered strain and its immediate parent (SR8A6S3-CDT-2) presented an increase in biomass content of 2.95 ± 0.01 and 2.81 ± 0.01, representing an increase of 376% and 384% of OD 600 within the first 24 h of cultivation.In the meantime, SR8A6S3 achieved a growth of 584% of initial cell concentration, and achieved an OD 600 of 3.90 ± 0.03 at 24 h of cultivation.The sor1Δ decreased the xylose consumption rate, while gre3Δ increased the rate at 24 h, but was still lower than SR8A6S3 (Table 1).

At 24 h
At 72 h www.nature.com/scientificreports/wild, industrial, and laboratory backgrounds to determine the xylose-positive phenotype.Of 647 studied strains, some wine strains appeared to be able to grow modestly on xylose.Through the application of high-throughput sequencing to bulk segregant analysis, they were able to identify a novel XDH gene, homologous to SOR1 (which was called XDH1), responsible for this phenotype.Next, the authors performed a comprehensive analysis of the involvement of the genes GCY1, GRE3, YDL124W, YJR096W, YPR1, SOR1, SOR2, XDH1, XYL2, and XKS1 in the XDH1 background strain (which has a xylose-positive phenotype) through single or combined deletion of the target genes.Single deletion of putative xylitol dehydrogenases (SOR1, SOR2 and XYL2) increased the xylose utilization rate relative to the positive control; this phenotype was further enhanced when all three genes were deleted (sor1Δ sor2Δ xyl2Δ) 35 .
To assess the effect of endogenous xylitol-assimilating pathway genes on the xylitol production profile by an engineered S. cerevisiae industrial strain, CK17, overexpressing Candida tropicalis XYL1 (encoding xylose reductase) in both batch and fed-batch fermentation with xylose and glucose as carbon sources.Yang et al. 37 performed single deletion of the following genes: XYL2 (yielding the strain CK17Δxyl2), SOR1/SOR2 (yielding the strain CK17Δsor), and XKS1 (yielding the strain CK17Δxks1) 37 .According to the authors, the mutant sorΔ had a reduced xylose consumption rate (12.4%) and xylitol production rate (4.7%), compared with its parental CK17 strain, which is consistent with our findings for SR8A6S3-CDT-2.The strain, CK17Δxks1, had the highest xylose consumption (0.65 g L −1 h −1 ) and xylitol production rate (0.644 g L −1 h −1 ), while the control strain consumed xylose and xylitol at 0.598 g L −1 h −1 and 0.549 g L −1 h −1 , respectively 37 .
Träff et al. 32 conducted a study where they deleted the GRE3 gene to enhance xylose metabolism in S. cerevisiae CEN.PK2-1C, which expressed the xylose isomerase encoding gene xylA sourced from Thermus thermophilus 32 .The recombinant gre3Δ strains produced less xylitol than the parental strain 32 .According to the authors, deletion of GRE3 in S. cerevisiae decreased xylitol formation two-to threefold but not completely, as xylitol may also be formed by the products of other genes, such as XDH (homologous to SOR1 gene), or through the reduction of xylulose or putative XR enzyme 35,38,39 .Similarly, in the construction of a S. cerevisiae strain expressing the isomerase pathway (xylA) from the anaerobic fungus Orpinomyces sp.(GenBank No. MK335957), several genetic modifications were made.Specifically, the gre3Δ, sor1Δ deletions were introduced along with the overexpression of XYL3 and TAL1 genes.These genetic modifications aimed to reduce xylitol accumulation and increase the growth rate 30 .
On the other hand, overexpression of the endogenous genes GRE3 and XYL2 under endogenous promoters, coding for nonspecific aldose reductase and xylitol dehydrogenase, respectively, enhanced the growth of S. cerevisiae on xylose in the presence of glucose in aerobic shake flask cultivation 31 .However, significantly more xylitol was formed by the CEN.PK2 strain overexpressing the S. cerevisiae enzymes in comparison to the strain that carries XR and XDH from S. stipitis.Furthermore, transcriptional analysis of xylose and glucose grown cultures shows that the expression of SOR1, which encodes sorbitol dehydrogenase, was elevated in transformed cultures.Therefore, the presence of xylose resulted in higher XDH activity and induced the expression of the SOR1 gene, which also has XDH activity 31 .
GRE3 and SOR1 genes were considered for improving xylose fermentation based on these previous studies.In some of them, sor1Δ increased xylose utilization, and gre3Δ plus sor1Δ decreased xylitol accumulation.Similarly, we have observed that gre3Δ plus sor1Δ in S. cerevisiae SR8A6S3 decreased xylitol formation (Table 1).However, in contrast, sor1Δ alone did not increase the xylose consumption rate of SR8A6S3 (Table 1) as reported by 35 .

Fermentation of hydrolysed xylan by the engineered SR8A6S3-CDT-2-GH43-2/7 strain
To evaluate XOS utilization, strain SR8A6S3-CDT-2-GH43-2/7 and the parental (control) strain SR8A6S3 were cultivated under micro-aerobic conditions at 30 °C, in a YP medium supplemented with hydrolysed xylan (YPXyl) and a mix of hydrolysed xylan plus acetate (YPAXyl) media.These media were designed to mimic a hemicellulosic hydrolysate, without the presence of inhibitory compounds, which can negatively influence yeast fermentations 13,44 .The engineered SR8A6S3-CDT-2-GH43-2/7 strain and its parental SR8A6S3 strain were cultivated in YPXy (Fig. 5B and D), and in YPAXyl (Fig. 5A and C) with an initial OD 600 of 10.As expected, the engineered SR8A6S3-CDT 2 -GH43 2/7 strain produced higher titres of ethanol than the parental SR8A6S3 strain under all test conditions.In the medium, both X2 and X3 were the main carbon sources provided.X2 concentrations decreased during the growth of both the SR8A6S3-CDT-2-GH43-2/7 and SR8A6S3 strains (Fig. 5); however, SR8A6S3 did not express either heterologous xylanolytic enzymes or a XOS-transporter.One explanation could be that X2 entered the cell through a natural transport system in S. cerevisiae and was converted into the non-metabolizable xylosylxylitol compound by XR (xylose reductase), as observed previously by Li and colleagues 25 .It is important to note that S. cerevisiae can consume disaccharides such as maltose, sucrose, and trehalose, which are up taken through the action of membrane transporters 45 .The uptake of sucrose (disaccharide composed of glucose and fructose) can occur via the proton-symport (Mal11p) 46 .While trehalose (disaccharide composed of two glucose) can be taken up via Agt1p-mediated trehalose transport, followed by intracellular hydrolysis catalysed by trehalase Nth1.Furthermore, the AGT1/MAL11 gene is controlled by the MAL system.Maltose is transported to the cytosol by an energy-dependent process coupled to the electrochemical proton gradient (Lagunas 45 ).
Within the first 24 h of cultivation, SR8A6S3 depleted all xylose present in the medium (Fig. 5C and D) while SR8A6S3-CDT-2-GH43-2/7 spent more time fermenting xylose completely (Fig. 5A and B).At the same time, the doubly-engineered strain consumed 6.77 ± 0.03 g L −1 of X2, which represents 30% of the initial X2 concentration and 1.79 ± 1.08 g L −1 of X2 (8% of the initial X2 concentration) respectively, for the cultivations in YPXyl and YPAXyl.The presence of acetate changed the X2 consumption profile of SR8A6S3-CDT 2 -GH43 2/7 (Fig. 5A).Intriguingly, the presence of X2 changed the acetate consumption profile of the parent strain, which consumed 4% of the initial acetate concentration up to 24 h, and 10% of the initial acetate concentration within first 48 h of cultivation (Fig. 5C).The slight acetate reduction between 24 and 48 h of cultivation might be affected by the oxidation of ethanol 47 , which peaked at 24 h (Fig. 5C).Concerning the X3 consumption profile, the SR8A6S3 parent strain barely metabolized X3 in either medium (Fig. 5C and D).Conversely, the engineered strain started to metabolize X3 after 24 h.The higher initial concentration of X2 than X3 probably interfered with X3 transportation.Instead, in 24-48 h, SR8A6S3-CDT-2-GH43-2/7 consumed 2.67 ± 0.85 g L −1 and 1.73 ± 1.32 g L −1 of X3 from YPXyl (Fig. 5B) and YPAXyl (Fig. 5A) cultivations, respectively.After 72 h of cultivation, no substantial decrease in X3 amount was observed for XOS-consuming strain cultivations.

Fermentation of hemicellulosic hydrolysate by the engineered SR8A6S3-CDT-2-GH43-2/7 strain
Following successful cultivation in a simulated hemicellulose hydrolysate, strain SR8A6S3-CDT 2 -GH43 2/7 was cultivated under micro-aerobic conditions in a YP medium supplemented with an authentic XOS-rich hemicellulosic hydrolysate 48 (Fig. 6A and B).This mimics the context of a lignocellulosic biorefinery, which makes full use of hemicellulose.The breakdown of hemicellulose, which is acetylated 13 , releases highly toxic acetate; reducing the fermentative performance of S. cerevisiae 21,25 .The SR8A6S3 strain was previously engineered through an optimized expression of AADH and ACS in the acetate reduction pathway, enabling acetate conversion into ethanol by the optimized strain 22 .Therefore, the current study tested whether the acetate reduction pathway could operate simultaneously with XOS fermentation, as a means to augment ethanol yield from the lignocellulosic hydrolysate.www.nature.com/scientificreports/Under this condition, we observed that xylose, X2, and X3 presented similar consumption profiles in the XOS-consuming strain cultivation.These carbon sources were primarily consumed before 24 h of cultivation.The latest engineered strain consumed 6.57 ± 0.28 g L −1 of xylose, which represents 92% of the initial xylose concentration, and 7.57 ± 0.08 g L −1 of X2, which represents 97% of the initial X2 concentration, and 2.76 ± 0.14 g L −1 of X3 (69% of the initial X3 concentration).Conversely, during the same period, the parent strain consumed only 3.73 ± 0.94 g L −1 of xylose (55% of the initial xylose concentration), and 16% and 15.5% of the initial X2 and X3 concentrations, respectively.It is worth pointing out that, as aforementioned, SR8A6S3 did not express either  ).www.nature.com/scientificreports/heterologous xylanolytic enzymes or an XOS-transporter; therefore, the uptake of XOS probably occurs through the action of membrane transporters that carry out disaccharide transport.
Acetate consumption was not observed in both SR8A6S3 and SR8A6S3-CDT-2-GH43-2/7 cultivations within the first 24 h of cultivation.These results might indicate that transportation of X2 and X3 might result in changes of the NADH / NAD + and ATP balance, which impaired the acetate consumption profile.In a previous study, Zhang et al. 22 highlighted that three major factors might limit the metabolic fluxes of the acetate reduction pathway, these include the intracellular ATP levels, NADH levels, and the activities of key enzymes (ACS and AADH) being the last major limiting factor among them.In this study, the expression of key enzymes was not modified through genetic intervention.Between 24 and 96 h of cultivation, acetate was reduced by 25% and 53% for the XOS-consuming and control strains, respectively.The acetate profile seems to be a combination of acetate consumption and acetate production, resulting from ethanol oxidation 47 .The lesser change in acetate consumption profile for SR8A6S3-CDT-2-GH43-2/7 than SR8A6S3 might have resulted from the higher amount of ethanol produced by this strain, which could be converted into acetate after exhaustion of the sugars 47 .Hence, it appears to use less acetate.Regarding xylitol production, the SR8A6S3-CDT-2-GH43-2/7 strain produced a lower xylitol yield, 0.041 ± 0.01 g xylitol g consumed xylose −1 , than the control cultivation, in which the yield was 0.083 ± 0.01 g xylitol g consumed xylose −1 .To evaluate the improvement obtained through the introduction of the XOS-consumption pathway in the SR8A6S3 strain, ethanol yield based on consumed monomeric xylose was calculated for each condition (Table 2).This was calculated to highlight that ethanol was also produced by consumption of XOS in addition to monomeric xylose consumption, since stochiometric maximal conversion of xylose into ethanol is around 0.51 g ethanol g xylose -1 .The ethanol yield of the SR8A6S3-CDT-2-GH43-2/7 strain increased substantially as compared to the SR8A6S3 strain.This substantial yield increase is very likely due to the additional conversion of XOS to ethanol.
Lignocellulose-derived ethanol provides environmental and economic benefits besides being a promising industry in the expected transition from fossil fuels to renewable energy 13 .Hemicellulosic-derived sugar comprises 15-35% of lignocellulosic biomass, representing a large source of renewable material that is available at a low cost 6,13,49 .Engineered strains able to consume XOS derived from hemicellulose via intracellular hydrolysis represent a potential benefit for bioethanol production; since these strains would have a competitive advantage concerning other microorganisms, such as contaminating bacteria and wild Saccharomyces and non-Saccharomyces species that are expected to be unable to utilize XOS as a carbon source 7,50 .

Conclusions
Xylose metabolism into ethanol in S cerevisiae SR8A6S3 is metabolically inefficient due to the production of xylitol.In this study we have integrated genes necessary to create a XOS-consumption pathway into two xylitol-production-related genes, SOR1 and GRE3.The resulting strains, SR8A6S3-CDT-2 and SR8A6S3-CDT-2-GH43-2/7, which are sor1Δ and sor1Δ-gre3Δ, respectively, showed a reduction in xylitol production and www.nature.com/scientificreports/improvement in ethanol yield when compared with their parental strain SR8A6S3 in YPDXA cultivations under both micro-aerobic and anaerobic conditions.However, this coincided with a reduced rate of xylose metabolism, implying that there is scope for improvement in overall flux from xylose to ethanol.SR8A6S3-CDT 2 -GH43 2/7 was able to ferment X2 and X3 efficiently for ethanol production, and achieved the highest apparent ethanol yield (based only on the content of monomeric xylose) of 1.43 ± 0.05 g ethanol g xylose -1 (64% higher than theoretical ethanol yield) in YP supplemented with hydrolysed xylan and acetate.When grown on a medium containing hemicellulosic hydrolysate with low monomeric xylose content, fermentation of X2 and X3 was poor, but this was dramatically improved with the addition of monomeric xylose.This, and other evidence which shows that X2 and X3 metabolism slows down once the monomeric carbohydrates have been depleted, suggests that the latter are required to provide the energy demands of the former (e.g. for enzyme biosynthesis).While there is clearly room for further improvement, this demonstrates that a XOS fraction generated by simple hydrothermal/steam explosion pre-treatment of lignocellulosic agricultural residues, without any subsequent enzymatic hydrolysis, is a potential resource for renewable biofuel production using a XOS-utilising yeast.

Strains and media
E. coli strain DH5α was used for the construction and propagation of plasmids.E. coli was cultured in Lysogeny Broth (LB) medium (5 g L −1 yeast extract, 10 g L −1 tryptone, and 10 g L −1 NaCl) at 37 °C, and 100 µg mL −1 ampicillin (LBA) was added for selection when required.All engineered S. cerevisiae strains used and constructed in this work are summarized in Table 3. Yeast strains transformed with plasmids containing antibiotics were propagated on YPD plates supplemented with the plasmid corresponding antibiotics, such as clonNAT (100 µg mL −1 ), geneticin G418 (200 µg mL −1 ), and hygromycin B (200 µg mL −1 ).The SR8A6S3-CDT 2 strain was generated by integrating the CDT-2 transporter overexpressing gene cassette into the SOR1 locus of the SR8A6S3 genome.

Plasmids and strain construction
All plasmids and primers in this work are summarized in Tables 4 and 5, respectively.The guide RNA (gRNA) plasmids (Table 6), gRNA-sor-K and gRNA-gre-K, were amplified from Cas9-NAT by using primer pairs DPO_089 and DPO-090, DPO_087 and DPO_088 carrying a 20 bp PAM sequence for SOR1 and GRE3 loci, respectively.The gRNAs were predicted by the website 53 .All gRNA sequences are listed in Table 5.
For genomic integration of CDT-2 through CRISPR-Cas9-based integration in the SOR1 gene site of SR8A6S3, CDT-2 donor DNA was amplified from plasmid pRS426-CDT2 using a primer pair DPO_081 and DPO_082.Transformants with CDT-2 integration were identified by PCR using primers DPO_083 and DPO_084, and the resulting strain was designated as the SR8A6S3-CDT-2 (Table 3).The PCR reaction was performed using 1.25 µL forward primer, 1.25 µL reverse primer, 1 µL DNA sample, 12.5 µL Phusion high-fidelity DNA polymerase master mix with HF buffer (New England BioLabs), and 9 µL of nuclease-free water.
β-xylosidase activity was measured according to Tramontina et al. 58 .Briefly, 30 µL of the clarified supernatant and 50 µL of 5 mM ρ-Nitrophenyl-β-d-xylopyranoside (pNPX) solution were added to 20 µL of reaction buffer (250 mM MES, and 5 mM CaCl 2 , pH 7); which was then incubated at 30 °C for 60 min for the enzyme reaction.The reaction was stopped by adding 100 µL of 2 M Na 2 CO 3 and the amount of ρ-Nitrophenol produced was estimated spectrophotometrically at a wavelength of 405 nm; and the absorbance converted to concentration using a standard curve.One unit of enzyme activity was defined as the amount of enzyme catalysing the hydrolysis of 1 µmol pNPX per minute in 1 mL of yeast intracellular lysate (μmol mL −1 min −1 ) "U mL −1 ", or per mg of total lysate protein (μmol mg −1 min −1 ) "U mg -1 ", or per gram of cells (μmol g CDW −1 min −1 ) under the described assay conditions.The protein concentrations of each sample were determined using the Bradford dye method 59 .www.nature.com/scientificreports/

Fermentation and analytical methods
Anaerobic batch fermentation experiments were performed in 100 mL serum bottles with 30 mL fermentation media.Serum bottles were sealed with a butyl rubber stopper and then flushed with nitrogen gas, which had been passed through a heated, reduced copper column to remove traces of oxygen.Micro-aerobic batch fermentation experiments were performed in a 125 mL Erlenmeyer flask with 30 mL of fermentation media.Both anaerobic and micro-aerobic cultures were incubated in a rotary shaker at 100 rpm at 30 °C.For all cultivations, yeasts were pre-grown in yeast extract-peptone (YP) medium (10 g L −1 yeast extract, 20 g L −1 peptone) supplemented with 20 g L −1 glucose, and harvested by centrifugation at 3,134 × g, at 4 °C for 5 min, and washed three times with sterile distilled water.Washed yeast cells were inoculated in serum bottles or Erlenmeyer flasks containing either: YP supplemented with a mixture of glucose, xylose, and acetate (YPDXA); hemicellulosic hydrolysate (YPH); hemicellulosic hydrolysate, xylose and acetate (YPXAH); hydrolysed xylan (YPXy); and hydrolysed xylan and acetate (YPAXy).Initial cell concentration varied according to the cultivation, OD 600 was 1 or 10.Xylan hydrolysis was carried out according to 60 .The hemicellulosic hydrolysate from sugar cane straw was obtained by a two-stage procedure: mild acetylation at 60 °C, 30 min, 0.8% (w w −1 ) of NaOH and 10% (w w -1 ) of solids, followed by hydrothermal pre-treatment at 190 °C, 20 min, 10% (w w −1 ) of solids.The hydrolysate obtained after the second step was enzymatically treated with a GH11 from Neocallimastix patriciarum (Megazyme® Ireland), as detailed elsewhere 48 .Afterwards, the hemicellulosic hydrolysate rich in XOS was concentrated, approximately five-fold, in a rotary vacuum evaporator.Table 7 shows the chemical composition of the XOS-rich hemicellulosic hydrolysate.
Samples were taken using a syringe and needle from serum bottles, or manual single-channel pipette (Gilson, USA) from Erlenmeyer flasks, at appropriate intervals to measure cell growth and metabolites concentrations.Cell growth was monitored as the optical density at 600 nm (OD 600 ), measured using a UV-visible Spectrophotometer (Biomate 5).The samples were centrifuged at 14,000 × g for 10 min and supernatants diluted appropriately for the determination of glucose, xylose, xylitol, glycerol, succinate, acetic acid, and ethanol by high-performance liquid chromatography (HPLC, Agilent Technologies 1200 Series); equipped with a refractive index detector (RID).Chromatography was done on a Rezex ROA-Organic Acid H+ (8%) column (Phenomenex Inc., Torrance, CA) maintained at 60 °C, with 0.005 N H 2 SO 4 as eluent at a flow rate of 0.6 mL min −1 .Analyte concentrations were determined by using the RID detector.

Xylo-oligosaccharide quantification
The enzymatic products were analysed by high-performance anion-exchange chromatography with pulsed amperometry detection (HPAEC-PAD), to detect xylose and XOS produced by the xylanase enzymes.Separation was performed using a Dionex ICS-3000 instrument (Thermo Fisher Scientific, Sunnyvale, CA, USA) with a CarboPac PA100 column (4 × 250 mm) and CarboPac PA100 guard column (4 × 50 mm), and eluted with a linear gradient of A (NaOH 500 mM) and B (NaOAc 500 mM, and NaOH 80 mM).The gradient program was 15% of A and 2% of B for 0-10 min, followed by 15-50% of A and 2-20% of B from 10-20 min, with a flow rate of 1.0 mL min −1 .The integrated peak areas were converted to concentrations based on standards (× 1 to × 6).

Figure 1 .
Figure 1.Expected routes of XOS metabolism after expression of the XOS-transporter (CTD-2) and betaxylosidases (GH43-2 and GH43-7) from N. crassa in SR8A6S3, including xylose metabolism by xylose reductase (XR) and xylitol dehydrogenase (XDH) from S. stipitis.The surplus NADH produced during xylose fermentation can be exploited to detoxify acetate, reducing it to ethanol through the exogenous acetate reduction pathway, involving conversion of acetate into acetyl-CoA by ACS, production of acetaldehyde from the acetyl-CoA by AADH, and ethanol production from acetaldehyde through the action of alcohol dehydrogenase (ADH).Adapted from 22 .

Figure 3 .
Figure 3. Fermentation profiles of SR8A6S3-CDT-2 (A), and SR8A6S3 (B) when fermenting 20 g L −1 glucose, 80 g L −1 xylose, and 8 g L −1 acetate under micro-aerobic conditions.Data are presented as mean values and standard deviations of three independent biological replicates.

Figure 4 .
Figure 4. Intracellular β-xylosidase activity of SR8A6S3 and SR8A6S3-CDT-2-GH43-2/7 pellet extracts.The strains were cultured in YP-medium supplemented with 20 g L −1 glucose, 80 g L −1 xylose, and 8 g L −1 acetate) under microaerobic conditions until the late log phase.The intracellular β-xylosidase activity of GH43-2 and GH43-7 with pNPX as substrate were calculated relative to mg of protein and g of cell dry weight.

Figure 5 .
Figure 5. Fermentation profiles of SR8A6S3-CDT-2-GH43-2/7 (A and B) and SR8A6S3 (C and D) during batch cultivation in YPAXyl (YP medium containing hydrolysed xylan and acetate), (A) and (C), and YPXyl (YP medium containing hydrolysed xylan), (B) and (D).Cultivations were performed at 30 °C and 100 rpm with an initial OD 600 of 10.Data are presented as the mean value and standard deviation of two independent biological replicates.

Figure 6 .
Figure 6.Fermentation profiles of SR8A6S3-CDT-2-GH43-2/7 (A) and SR8A6S3 (B) during batch cultivation in YPXAH (YP medium containing xylose, acetate, and hydrolysed hemicellulose).Cultivations were performed at 30 °C and 100 rpm with an initial OD 600 of 1. Data are presented as mean values and standard deviations of two independent biological replicates.
GTC AAG AAC CCC ATC CTC CCC GGC TTC AATC DPO_063 TTA CTT CCC AGC CGG CTG CTT TTC CCC ACA AAT CTT CCC CTC TTCA DPO_064 CCG GGC TGC AGG AAT TCG AT DPO_065 GGG ATC CAC TAG TTC TAG AA DPO_062 CCA GAA CTT AGT TTC GAC GGA TTC TAG AAC TAG TGG ATC CCA TGC CCC TCG TCA AGA ACC DPO_074 GAC GGT ATC GAT AAG CTT GAT ATC GAA TTC CTG CAG CCC GGT TAC TTC CCA GCC GGC TGC DPO_057 GTA ATA TAA ATC GTA AAG GAA AAT TGG AAA TTT TTT AAA GGT AAT ACG ACT CAC TAT AGG DPO_058 TTG TTC ATA TCG TCG TTG AGT ATG GAT TTT ACT GGC TGG AAA TTA ACC CTC ACT AAA GGG DPO_081 AAT CAA CAA GAA AAA ATA CTA AAA AAA AAA ATT GAA AAA TGT AAA ACG ACG GCC AGT DPO_082 TAT ATA TGG ACA TGA ACC AGT GCC GAA AAG TAT TCA CTT TAC AGG AAA CAG CTA TGAC DPO_089 TGT GTC GAA CCC TTA TCA GTG TTT TAG AGC TAG AAA TAG CAA G DPO_090 ACT GAT AAG GGT TCG ACA CAG ATC ATT TAT CTT TCA CTG CGG A DPO_083 CCG GTC TCG TAT CTC CTT T DPO_084 CTA TCA ACT GGA AGT AAT GCG DPO_069 GGG GGC CTA TCA AGT AAA TTA CTC CTGGT DPO_070 GTT CAG ATT CAC TTC TTG ATA TTT CC DPO_087 TCC TCA ATC ATT CAT TGA GAG TTT TAG AGC TAG AAA TAG CAA G DPO_088 TCT CAA TGA ATG ATT GAG GAG ATC ATT TAT CTT TCA CTG CGG A

Table 3 .
The yeast strains used in this study.

Table 4 .
Plasmids used in this study.

Table 5 .
Primers used in this study.

Table 6 .
gRNA used in this study.