Strain Improvement of Native Saccharomyces cerevisiae LN ITCC 8246 Strain Through Protoplast Fusion To Enhance Its Xylose Uptake

Co-utilization of xylose and glucose and subsequent fermentation using Saccharomyces cerevisiae could enhance ethanol productivity. Directed engineering approaches have met with limited success due to interconnectivity of xylose metabolism with other intrinsic, hidden pathways. Therefore, random approaches like protoplast fusion were used to reprogram unidentified mechanisms. Saccharomyces cerevisiae LN, the best hexose fermenter, was fused with xylose fermenting Pichia stipitis NCIM 3498. Protoplasts prepared using glucanex were fused under electric impulse and fusants were selected using 10% ethanol and cycloheximide (50 ppm) markers. Two fusants, 1a.23 and 1a.30 showing fast growth on xylose and tolerance to 10% ethanol, were selected. Higher extracellular protein expression observed in fusants as compared to parents was corroborated by higher number of bands resolved by two-dimensional analysis. Overexpression of XYL1, XYL2, XKS, and XUT4 in fusants as compared to S. cerevisiae LN as observed by RT-PCR analysis was substantiated by higher specific activities of XR, XDH, and XKS enzymes in fusants. During lignocellulosic hydrolysate fermentation, fusants could utilize glucose faster than the parent P. stipitis NCIM 3498 and xylose consumption in fusants was higher than S. cerevisiae LN.


Introduction
To enhance ethanol productivity, xylose utilization is imperative since it forms 20-30% of sugar present in the lignocellulosic biomass hydrolysates. Saccharomyces cerevisiae is the work horse of bio-ethanol industry because of its robust traits like ethanol tolerance, osmotolerance, inhibitor tolerance, and resistance towards bacteriophage infection [1,2]. However, it is unable to efficiently utilize and ferment xylose.
For industrial production of cellulosic ethanol from lignocellulosic biomass, effective coutilization of pentoses and hexoses by a fast fermenting, ethanol tolerant, sugar tolerant, and inhibitor tolerant yeast strain is required [3]. It is because, initially, xylose is converted to xylitol by the enzyme xylose reductase which requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) for its functioning. Then, xylitol is converted to xylulose by the enzyme xylitol dehydrogenase which requires nicotinamide adenine dinucleotide (NAD) (Fig.  1). Heterologous expression of xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Scheffersomyces sp. to Saccharomyces sp. improved its xylose fermentation abilities. Nevertheless, differences in the co-factor preferences of these enzymes resulted in redox imbalance and hence the production of several byproducts rather than ethanol [4]. Therefore, as a new strategy, xylose isomerase (XI) from the bacteria which directly converts xylose to xylulose was expressed in Saccharomyces strain. However, ethanol production was still lower because of xylitol accumulation as Gre3 (aldose reductase produced by S. cerevisiae) reduces xylose to xylitol and XI is inhibited by xylitol [5,6].
Many metabolic engineering strategies were applied but with limited success due to other interlinked pathways and genes which include pentose phosphate pathway genes like transaldolase (TAL), transketolase (TKL), and transporter genes [7]. Various efforts to Fig. 1 Schematic representation of xylose metabolic pathway in yeasts construct a recombinant strain of S. cerevisiae yielded strains with lower fermentation rates of xylose [8][9][10][11]. This may be due to the fact that xylose is not recognized as a fermentative substrate by the yeast regulatory network for its complete fermentation to ethanol. Therefore, in addition to the directed genetic manipulations, random mutational approaches would provide better insights into complex network linked with xylose metabolism. This would assist in the development of an improved strain with mixed sugar utilization/fermentation potential [12]. Protoplast fusion is one such technique to develop an effective strain with improved capability. It causes random mutations in the genomes of microorganisms without requiring any pre-targeted approach. This is applicable for developing inter-specific, intraspecific, and inter-generic, intra-generic supra hybrids with higher capability. It is a significant tool for genetic manipulation as it resolves the barrier to genetic exchange imposed by conventional mating systems. It is particularly useful for industrially important microorganisms [13][14][15]. Random mutagenesis by protoplast fusion of intergeneric yeast strains has been carried out earlier [16][17][18]. A fusant strain constructed by Pasha et al. [3], F11, showed 0.459 g L −1 ethanol yield with 90% fermentation efficiency.
Consequently, in the current study, protoplast fusion was applied to fuse two intergeneric yeast strains, Saccharomyces cerevisiae LN ITCC 8246 and Pichia stipitis NCIM 3498. Saccharomyces being an efficient glucose fermenter and Pichia being xylose assimilator were selected to improve mix sugar utilization and fermentation of native S. cerevisiae strain

Strain Selection
The main objective of the study is to produce a strain that is capable of co-utilizing xylose and glucose present in the lignocellulosic biomass hydrolysates. Saccharomyces cerevisiae LN ITCC 8246, hexose fermenter isolated from fruit juices along with P. stipitis NCIM 3498, a xylose assimilator and fermenter, was selected for protoplast fusion. Both the cultures were grown on MGYP medium (3 g L −1 malt extract, 10 g L −1 glucose, 3 g L −1 yeast extract, and 5 g L −1 peptone) at 30°C and stored at 4°C as slants.

Screening Markers and Enzyme Used for Protoplast Fusion
Cycloheximide and ethanol were chosen as selection markers. Both the parent strains were grown on MGYP medium plates with different concentrations of cycloheximide (50-200 ppm). For ethanol tolerance, both strains were grown on MGYP broth with different concentrations of ethanol (8-11%). Growth was monitored by measuring absorbance at 600 nm after every 24 h interval.
Glucanex enzyme (Sigma Aldrich) was used for formation of protoplasts of both the cultures. It is a cost-effective, yeast lytic enzyme obtained from the fungus Trichoderma harzianum [19].

Protoplast Formation
Protoplasts of both the yeast strains were generated by following protocol of Krishnamoorthy et al. [19]. Overnight grown cultures with 1 OD600nm were used; 5 mL culture was taken in tubes and centrifuged at 8000 rpm for 10 min. Culture supernatant was discarded and pellet was washed with protoplast solution (0.6 M KCl, 10 mM β-mercaptoethanol, 50 mM phosphate buffer). Different concentrations of enzyme (10 to 50 mg mL −1 ) prepared in protoplast solution were added to the culture pellets and incubated for 72 h at 30°C and 150 rpm. Protoplast formation was monitored after 24 h interval by staining cells with lactophenol cotton blue dye under bright field microscope.

Protoplast Fusion and Regeneration of Fusant Cell Walls
Protoplasts of both the strains were subjected to fusion under electric impulse following yeast transformation protocol (University of Michigan, Mapp Lab). Briefly, protoplast suspensions were mixed together in equal volumes and immediately subjected to electric impulse using electroporator (Bio-Rad). After electrofusion, the fusion mix was immediately added to the 50 mL regeneration medium (1 g L −1 KH2PO4, 5 g L −1 MgSO4, 5 g L −1 (NH4)2SO4, 1 g L −1 yeast extract, and 1% xylose). Selection of regenerated fusants was done using screening markers in stepwise manner; 1 mL of regeneration medium was used to inoculate 10 mL vial of minimal medium (same composition as regeneration medium) with 1% xylose and 10% ethanol. After 24 h, growth was distinctly visible in the vials. In the second step, this culture was plated on to minimal medium with xylose 5% and cycloheximide (50 ppm). Fast growing colonies were selected as fusants after 24 h of growth.

Validation and Evaluation of Fusants
The selected fusant strains were characterized and validated on the basis of properties including ethanol tolerance, sugar utilization and fermentation potential, enzymatic activities, proteomic analysis, and gene validation through RT-PCR.

Ethanol Tolerance of Fusants
Fusants and parent cultures were grown in 50 mL minimal medium in 100 mL conical flasks. Minimal medium was supplemented with 1% xylose and 10% ethanol and inoculated with overnight grown cultures at 10% level. Growth was monitored for 72 h at 30°C, 150 rpm.
Enzyme Activities of XR, XDH, and XKS Genes Parent and fusant strains were grown for 72 h on 1% xylose in MXYP medium with shaking at 150 rpm at 28°C. After 48 h, cultures were centrifuged at 8000 rpm for 10 min and supernatants were discarded. Pellet was processed for determining XR (xylose reductase), XDH (xylitol dehydrogenase) [20], and XKS activities in intracellular milieu.
For determining XKS activity, protocol from Eliasson et al. [21] was followed. Pellet was washed with Tris-Cl buffer (pH 7.5) twice and finally suspended in the same buffer.
Reaction cocktail consisted of 2 mM MgCl2, 0.2 mM NADH, 8.5 mM Xylulose, 0.2 mM Phosphoenolpyruvate, 10 U Pyruvate Kinase, 10 U Lactate Dehydrogenase, and 2 mM ATP (adenosine triphosphate). To 490 μL of reaction mix, 10 μL enzymatic lysate was added. Prior to this, reaction mix is pre-incubated for 2 min to allow the conversion of traces of ADP present in the commercial ATP preparation. Enzyme activity was measured in the absence and presence of xylulose substrate. Difference in the activity of the enzyme in the presence and absence of substrate was calculated as enzyme activity. Specific activities were calculated as enzyme activities per mg of protein [21].

Proteomic Analysis
Since proteins are the functional molecules of a cell, proteome studies of the fusants would help us better understand the functionality of these strains. For extraction of proteome, cultures were grown in 500 mL MGYP medium, pH 5.0 and incubated for 24 h at 28°C, 150 rpm. Cultures were harvested through centrifugation at 8000 rpm and 4°C. Pellets were stored at − 20°C. Liquid nitrogen (LN2) was used to crush the pellet into powdered form. This powder was further used to extract protein [22]. To the ground culture pellet, extraction buffer was added. Suspension was vortexed and 15 mL phenol was added to this solution. Incubated with shaking for 45 min and centrifuged at 8000 rpm, 4°C for 15 min. Upper layer was taken in new centrifuge tube to which precipitation buffer was added in the ratio of 1:4 and incubated overnight. Suspension was again centrifuged under similar conditions and pellet was taken and washed thrice with washing buffer. Pellet was finally dried and solubilized in solubilizing buffer.
Solubilized pellet was used for isoelectric focusing using 11 inch IPG strips (3-10 pI). Strips were overlaid with the protein samples (quantified through Bradford reagent). Before running IEF, strips were rehydrated in rehydration buffer (7 mM Urea, 2 M Thio-Urea, 2% CHAPS, 0.225% Ampholyte, and 50 mM Dithiothreitol). IEF was run overnight, and the strips stored for 2D gel analysis. Strips were treated with DTT and iodoacetamide for 30 min each. Treated strips were then loaded onto the 12% gel and run until the dye passes into the buffer.

Image Analysis
Image analysis of the gels was carried out using Image Master 2D Platinum 7 Software (Sandor Lifesciences).

Genomic Validation Through Real Time PCR
To investigate the impact on the metabolic pathway of xylose uptake and utilization, expression of genes involved in xylose metabolism was selected for validating fusants.

Genes
Genes chosen for the analysis were transporter genes and genes corresponding to enzymes in the xylose metabolic pathway. These genes were XYL1 for xylose reductase, XYL2 (xylitol dehydrogenase), XKS1 (xylulokinase), XUT4, XUT5, and XUT7 (xylose transporter genes). Primers were designed using online primer designing tool (IDT primerquest) (Additional file 1: Table S1). Reference genome used for primer designing was P. stipitis CB6054. To check ethanologenic property, ADH gene sequence was selected from the S. cerevisiae genome.

RNA Isolation
For RNA isolation, TriZol method was used. Yeast cultures were grown in minimal medium with 1% glucose and 1% xylose for 24 h and then centrifuged at 8000 rpm for 10 min in 50 mL falcon tubes. Supernatant was discarded and pellet was used for RNA isolation; 500 μL sample was taken in an eppendorf tube with a glass bead and homogenized in a homogenizer (20 Hz frequency for 7 min).
After homogenization, eppendorfs were immediately immersed in liquid nitrogen for 10 min. To this, 1 mL TriZol was added and mixed by inverting/vortexing tubes. Then, the tubes were centrifuged for 10 min, 10,000 rpm at 4°C. Upper aqueous layer was taken (~400 μL), to which equal amount of chloroform was added and mixed by inverting. Centrifugation was again done at 12,000×g for 10 min at 4°C. To the upper aqueous layer (extracted to a new tubẽ 200 μL), isopropanol was added (~200 μL), properly mixed, and incubated for 10 min at − 20°C. Supernatant was removed, and pellet was washed with 70% ethanol (100 μL) and centrifuged for 5 min. Again, the pellet was recovered and after the ethanol was completely evaporated, 25 μL nuclease-free water was added to each vial.

DNase Treatment
DNase treatment was done using Thermo Scientific DNase I, RNase free kit. To the above tubes, 1 μL 10× Buffer (with MgCl2), 1 μL DNase, and 10 μL DEPC water were added and incubated at 37°C (water bath) for 30 min. After incubation, tubes were incubated at 65°C for 10 min and the concentration of RNA was determined using nano-drop. Quantification was done through nano-drop (Thermo Fisher Scientific).

c-DNA Preparation
c-DNA synthesis was carried out using Invitrogen superscript III one step qRT-PCR kit. Reaction mixture for c-DNA synthesis has been provided (Additional file 1: Table S2). PCR program for the synthesis was as mentioned on the kit manual. One cycle of c-DNA synthesis at 45-60°C for 15-30 min was followed by 1 cycle of denaturation at 94°C for 2 min. This is trailed by amplification wherein there are 40 cycles of denaturation at 94°C for 15 s, 30 s annealing at 65°C, and extension at 68°C for 1 min. It was finally followed by 1 cycle of final extension at 68°C for 5 min. c-DNA was run on agarose gel and quantified using nano-drop.

RT-PCR
Using primers, RT-PCR reaction was set up as given in Table S3 (Additional file 1); 31.5 μL of above master mix was taken and 3.5 μL of c-DNA (1:10 dilution) was added to it. It was placed in three wells (10 μL each); 18S was taken as reference gene. Finally, results were normalized and fold change in expression was calculated with respect to both the parents individually. Results (fold change in expression) were statistically verified by calculating p value (p < 0.05) and standard deviation; 2^-ΔΔCt was calculated as: fold change expression = 2^-ΔΔCt [23] Roche light cycler 96 was used for this purpose.

Preparation of Rice Straw Hydrolysates
Ten grams of rice straw hydrolysate with 5% moisture content was taken and pretreated with 1% NaOH. Saccharification of the pretreated biomass was done using Accellerase®1500 and Celluclast (to be published somewhere else). Saccharification was carried out using 3 and 6% glucan loading by mixing with 150 μL and 300 μL enzyme respectively in 0.05 M Sodium citrate buffer (pH 4.8). Total volume of each reaction mixture was 10 mL in 30 mL bottles. The reaction mixture was kept at 50°C and 150 rpm in shaker water bath 96 h post incubation. After every 24 h interval, 0.5 mL sample was withdrawn. Sugars were estimated both by DNS assay [24] and HPLC. Fusant strains were also grown on 2% xylose supplemented in minimal medium in 100 mL Erlenmeyer flasks with 50 mL medium volume. Samples were taken periodically after 24 h interval till 72 h.

Results and Discussions
Saccharomyces cerevisiae LN ITCC 8246 was selected for fusion as Saccharomyces is the preferred microorganism for fermentation industry. Its robustness and tolerance to various stresses makes it suitable for commercial fermentation processes. It displays traits superior to bacteria and other yeasts like pH tolerance with acidic optimum making the fermentation process less prone to infections unlike bacteria, better ethanol tolerance makes it superior to other fermenting yeasts. It can ferment glucose very efficiently and its strains are used on industrial level for LCB fermentation [25]. P. stipitis strains are well known for their xylose utilization and fermentation abilities [26]; therefore, this strain was found suitable for fusion with S. cerevisiae.

Protoplast Formation and Intergeneric Protoplast Fusion for Random Mutagenesis
Protoplasts were prepared using glucanex enzyme (possessing cellulase, protease, and chitinase activities to ease the process of cell wall degradation of yeast cell) after 72 h incubation period when it showed highest protoplast formation frequency as observed under microscope (Fig. S1).
The protoplasts of both the strains were fused under electric impulse. Electroporation makes the process of fusion fast and easier as compared to chemical fusion done through polyethylene glycol (PEG). Cells were completely regenerated after 72 h of incubation in the regeneration medium and fusants were screened using two selection markers, i.e., tolerance to 10% ethanol and resistance to cycloheximide 50 ppm in a stepwise manner. Krishnamoorthy et al. [19] used five different substrates as selection markers to screen fusants. At 10% ethanol concentration, growth of P. stipitis NCIM 3498 was completely inhibited while S. cerevisiae LN was growing very well. However, cycloheximide (50 ppm) was lethal for the growth of S. cerevisiae LN but P. stipitis NCIM 3498 could grow well in its presence (Additional file 1: Table S4, Fig. S2). Eighty fast growing colonies on medium containing xylose were selected for further characterization as fusants. Two putative fusants showing higher xylose utilization were selected and designated as 1a.23 and 1a.30.

Validation of Fusants
Cells are inherently robust and resist the modifications made in their metabolic system and try to preserve the native functional state. Therefore, metabolic engineering of such robust strains leads to unwanted byproducts, other than the main metabolic product. Expression profiling of such changes, e.g., protein expression, gene expression, and physiological expression, could help perceive the alteration and improvement occurred in the strain. Xylose metabolism affects carbon flux and also influences gene expression, protein expression, and other catabolic pathways [12]. Therefore, fusant strains were validated for their fermentation properties on physiological basis, gene expression, and protein expression with respect to the parent strains.

Ethanol Tolerance
Till date, S. cerevisiae strains have been favored for ethanol production from lignocellulosic biomass [27,28] due to its higher ethanol tolerance trait. The two fusants obtained in this study showed growth at par with S. cerevisiae LN parent strain in presence of 10% ethanol concentration. They showed higher tolerance to 10% ethanol than P. stipitis (Fig. 2). At 48 h interval, 0.46 absorbance was exhibited by P. stipitis NCIM 3498. However, at the same interval, fusants 1a.23 and 1a.30 showed 1.37 and 1.09 absorbance, respectively. Ethanol tolerance is a significant parameter as only the ethanol tolerant yeasts can be considered for large-scale ethanol production.

Sugar Utilization
Biomass hydrolysates possess mixture of sugars with glucose, xylose, and arabinose being the predominant. Therefore, the improved strains utilizing mixed sugars efficiently were preferred. Both fusant strains were found to utilize sugars like, xylose, adonitol, rhamnose, cellobiose, melibiose, saccharose, raffinose, trehalose, glucose, and lactose similar to both the parent strains exhibiting same sugar utilization trend. Studies indicate that extended sugar utilization range of the yeast strains contributes to the effective xylose fermentation capabilities [29]. This makes the fusant strains (1a.23 and 1a.30) more potent for xylose utilization and fermentation with diverse sugar utilization properties.

Enzyme Activities and Protein Expression in Fusants as Compared to the Parent Strains
Higher sp. activities of enzymes XR and XKS in fusant strains than S. cerevisiae LN and higher sp. activity for XDH in 1a.30 showed that fusant strains were better equipped for xylose utilization and produced ethanol from it as compared to S. cerevisiae LN. XKS activities of fusants were higher while XDH activities were lower with respect to P. stipitis NCIM 3498. Higher XKS activity suggested formation of xylulose-5-phosphate and thereby formation of ethanol as a fermentation product. But XDH activities were low and XR activities were comparable, which shows that the xylose utilization was lower in fusants as compared to the parent P. stipitis NCIM 3498 (Table 1). Metabolic flux of sugars is affected by the activities of the enzymes. Studies have demonstrated that flux is distributed equally over all the steps of a pathway rather than being dependent on one enzyme [12]. In this study, XKS activities were higher in fusants than P. stipitis NCIM 3498 and ethanol production was at par with parent strains. Higher XKS activities can hamper cell growth leading to lower ethanol productivity [27]. In a similar study, fusants developed through protoplast fusion were evaluated for their enhanced killer activities [28]. Fusants exhibited intermediate traits between the parent strains such as enzyme activities, ethanol production, and sugar consumption [27,30].
Proteomics offers a global strategy to study the effects of xylose pathway or other processes. Lately, it has gained attention in resolving metabolic problem in yeast [31]. In the present study, there was upregulation and proteins spots were showing more than two-fold increase, indicating higher overall protein expression.
A higher number of protein spots were resolved using 2D SDS PAGE for both fusant strains than parent strains showing higher expression of diverse proteins in the fusants. There was differential expression of proteins and some proteins showed more abundance while a few proteins were downregulated. The data showed that the number of differentially expressed spots in the fusants with respect to P. stipitis NCIM 3498 is 29 and 34 for fusant 1a.23 and 1a.30, respectively. The number of upregulated proteins was higher as compared to the downregulated ones (23 and 26), suggesting higher expression of proteins in the fusants. In addition, the number of protein spots resolved for the fusant 1a.23 (246) was higher as compared to that of the parent Pichia strain (Additional file 1: Table S5a). Similarly, in case of S. cerevisiae LN, a total of 184 spots were resolved which was lower than both the fusants (246 and 201 for 1a.23 and 1a.30, respectively). Furthermore, in this case, also the upregulated proteins were higher in fusants (24 and 13) than the downregulated proteins (Additional file 1: Table S5b). Proteome studies carried out by Huang and Lefsrud [32], at different time points during co-culture fermentation process using S. cerevisiae and Scheffersomyces stipitis concluded that there was induction of XR and XDH before the depletion of glucose, suggesting co-utilization and fermentation of both the sugars.

Gene Expression of Xylose Metabolizing Genes and Xylose Transporter Genes Using qRT-PCR
Since xylose metabolism is grossly interconnected with other complex pathways and simple manipulation of the xylose, metabolizing genes would not create any inexplicable improvements in yeast strains. Presence of genes does not suffice the metabolism and fermentation of xylose unless the genes are being expressed for the production of desired metabolites. Therefore, gene expression using RT-PCR was monitored.
For studying the levels of upregulation and downregulation of genes involved in xylose metabolism, quantitative analysis was carried out. Since uptake of sugars plays a vital role, therefore, transporters were also included in the study. P. stipitis CB6054 was taken as a reference genome. It was observed that xylitol accumulation occurred in the process as XYL1 was overexpressed but XYL2 was not expressed in case of fusants with respect to S. cerevisiae LN, which was clear from the HPLC chromatogram peak (data not shown). It has been shown that increasing the expression of XDH with respect to XR activity results in higher ethanol production and lower xylitol accumulation [33,34].
As evident from the graph (Fig. 3a), XUT7, XKS1, and XYL1 genes were expressed to a much higher level (i.e., 1244 and 1061 for XYL1, 282 and 911 for XKS1, each respectively for 1a.30 and 1a.23), whereas XUT5, XUT4, XYL2, and Sc-Adh genes were least expressed in the fusants as compared to S. cerevisiae LN. However, expression of XUT7 was higher in case of fusant 1a.30 only. This was also evident from the specific activities of enzymes XR and XKS showing higher values as compared to S. cerevisiae LN.
Sc-ADH gene corresponds to the alcohol dehydrogenase enzyme responsible for ethanol production. In S. cerevisiae, there are two copies of ADH and it can switch between respiratory mode and fermentation mode and shows biphasic growth pattern [35]. It has been observed that ADH overexpression does not induce ethanol production in fusants [28]. This coincides with our study, where ethanol productivities are at par with the parent strains with higher ADH expression as compared to P. stipitis NCIM 3498. Glycolytic genes, an integral part of fermentation process, impact the glycolytic flux, which further drives the uptake of available carbon sources. Additionally, when glucose concentrations are low, xylose uptake rate is increased by S. cerevisiae due to induction of its HXT genes responsible for xylose transport [12]. This substantiates current data observations where fusants exhibit higher xylose utilization as compared to parent strain, S. cerevisiae LN. Figure 3b suggested that XUT4 gene was highly overexpressed in both the fusants as compared to P. stipitis NCIM 3498. Also, expression of Sc-ADH gene was higher in fusants. Fusants show higher expression of Sc-ADH; however, low ethanol was produced by the fusants as compared to P. stipitis NCIM 3498. ADH has been overexpressed as compared to parent 2 (P. stipitis NCIM 3498) while not as compared to parent 1, i.e., S. cerevisiae LN. XUT7 also showed fairly higher expression in fusants than in the parent strain indicating higher xylose uptake and assimilation by the fusants. Xylose uptake is also limited by the interspersed pathways and genes. There was higher XKS gene expression in fusants. Expression of XYL2, XUT5, and XYL1 genes was very less and hence considered insignificant as compared to the parent strain.
Amplification of genomic DNA of fusants and parent strains using cDNA primers designed for RT-PCR analyses showed amplified bands for XR, XDH, and transporter genes (XUT4 and XUT5) (Additional file 1: Fig. S3). This advocates the existence of xylose metabolizing machinery in the fusant strains. Hence, it is worthwhile to mention that both fusants strains carry improved genetic traits as compared to their parent strains in terms of co-utilization of sugars.

Utilization and Fermentation Competences of Fusants
On synthetic sugars, with 2% xylose in minimal medium, fusant strains exhibited ethanol production of~2% (1a.30) after 72 h ( Table 2). Both the fusant strains showed~40% xylose utilization. However, S. cerevisiae LN strain showed~20% utilization on 2% xylose a b Fig. 3 a Fold induction of genes in fusants with respect to S. cerevisiae LN. In the graph, each bar represents mean of three replicates and error bars represent standard deviation. b Fold induction of genes in fusants with respect to P. stipitis NCIM 3498 fermentation as demonstrated by Sharma et al. [20]. This suggests two-fold increase in xylose utilization by the fusants. A table showing comparison of the fusant strains of this study with other improved strains has been provided ( Table 3). It depicts the fusant strains could be potentially significant candidates for further modifications through targeted approach or through adaptive laboratory evolution.
In a fermentation experiment carried out on rice straw hydrolysates containing~4.5% total sugars, it was estimated that S. cerevisiae LN could not utilize xylose while glucose was consumed completely within 24 h [40]. However, P. stipitis NCIM 3498 and fusant strains were shown to consume both the sugars after 96 h. Furthermore, Pichia could not metabolize glucose before 72 h, while fusants could metabolize it within 24 h. Several reports suggest the expression of transporter genes in S. cerevisiae enhances the xylose utilization and fermentation process [35,41]. Investigations on S. cerevisiae demonstrated complete glucose utilization in both glucose and xylose chemostat cultures [12].
Thus, for exploitation at industrial level, S. cerevisiae is still the preferred strain for its better stress tolerance properties like high ethanol productivity from mixed sugars, inhibitor tolerance, and ethanol tolerance. Fusants in this study also showed faster glucose utilization and xylose utilization. Salusjärvi [12] also observed complete glucose utilization in S. cerevisiae in mixed sugar fermentations.
Fermentation time is crucial at industrial level fermentations. Prolonged fermentations raise several other issues, such as ethanol toxicity and toxicity to inhibitors present in the hydrolysates [42]. Selim et al. [5] have stated that P. stipitis maneuvers most of its metabolic flux into ethanol production with very less xylitol accumulation. Also, rate of xylose fermentation is much slower as compared to glucose fermentation rate in S. cerevisiae. The regulation of fermentation is different in S. cerevisiae and P. stipitis. S. cerevisiae regulates fermentation in response to glucose while P. stipitis promotes fermentation in response to oxygen limitation. Reference

Conclusion
Genetic engineering approaches require prior knowledge of metabolic pathways, contrary to the randomly targeted approaches. Therefore, as a result of protoplast fusion of S. cerevisiae LN and P. stipitis NCIM 3498, two mutant fusants were obtained (1a.23 and 1a.30). After validation, they showed higher ethanol tolerance (10%), enhanced XR, XDH, and XKS activities, and differential expression of proteins in the fusant strains with higher number of upregulated proteins as compared to the parent strains. There was two-fold increase in xylose utilization than Saccharomyces and 100% glucose utilization within 24 h of fermentation unlike Pichia. Overall, the fusants possessed mixed sugar fermentation abilities which could further be enhanced through adaptive evolution