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Synergistic effect of thioredoxin and its reductase from Kluyveromyces marxianus on enhanced tolerance to multiple lignocellulose-derived inhibitors



Multiple lignocellulose-derived inhibitors represent great challenges for bioethanol production from lignocellulosic materials. These inhibitors that are related to the levels of intracellular reactive oxidative species (ROS) make oxidoreductases a potential target for an enhanced tolerance in yeasts.


In this study, the thioredoxin and its reductase from Kluyveromyces marxianus Y179 was identified, which was subsequently achieved over-expression in Saccharomyces cerevisiae 280. In spite of the negative effects by expression of thioredoxin gene (KmTRX), the thioredoxin reductase (KmTrxR) helped to enhance tolerance to multiple lignocellulose-derived inhibitors, such as formic acid and acetic acid. In particular, compared with each gene expression, the double over-expression of KmTRX2 and KmTrxR achieved a better ethanol fermentative profiles under a mixture of formic acid, acetic acid, and furfural (FAF) with a shorter lag period. At last, the mechanism that improves the tolerance depended on a normal level of intracellular ROS for cell survival under stress.


The synergistic effect of KmTrxR and KmTRX2 provided the potential possibility for ethanol production from lignocellulosic materials, and give a general insight into the possible toxicity mechanisms for further theoretical research.


More recently, lignocellulose, as the most typical representative of non-grain feedstock, attracts increasing attentions due to low costs and rich sources [1]. Lignocellulose is mainly composed of cellulose, hemicellulose and lignin to form the crystalline microfiber structure, which requires a pretreatment step to produce fermentable monosaccharides for yeasts [2]. Destroying the complex structure of lignocellulose mainly depends on some physicochemical methods like steam explosion, acids or alkali, which may generate inhibitors [3], such as weak acids (formic acid and acetic acid), furan derivatives (furfural and 5-hydroxymethyl furfural), and phenolic compounds (phenol and O-methoxyphenol) [4]. In spite of the emerging pretreatment approaches by rot fungi [2], supercritical fluids [5], and ionic liquids [6], the low efficiency makes them to only play a supporting role at the current stage.

Lignocellulose-derived inhibitors are supposed to be inevitable in the hydrolysates, and every single step to reveal resistance mechanism seems to be crucial. Though inhibitors may be removed by detoxification of lignocellulosic hydrolysates [7], loss of sugars and increased costs greatly prevent its wide applications [8]. Therefore, exploring the toxicity of typical inhibitors to cells and its mechanism to achieve excellent strains with enhanced tolerance, is becoming a more vital component of ethanol production from lignocellulosic materials. However, mechanisms of toxicities of these inhibitors in yeasts are very complex and greatly variable depending on strains [9]. Fortunately, inhibitors like acetic acid, furfural and phenol have been reported to be related to the redox state inside cells, inducing reactive oxidative species (ROS) generation [10,11,12].

Acetic acid generally affects cell metabolism and stabilities of proteins by a drop in intracellular pH and potential, leading to a net negative effect on cell growth and proliferation [13, 14]. Acetic acid diffusing across plasma membrane damages cells by accumulating ROS [10]. Unlike acetic acid, the toxicity of furfural results from the inhibition of glycolytic and fermentative enzymes essential to central metabolic pathways, which reduces cell growth rates eventually [11]. Interestingly, furfural also induces the accumulation of ROS inside cells by lowering activities of intracellular oxidoreductases [15]. Phenolic compounds are supposed to be even more toxic than furfural by generating ROS like peroxides and super oxides inside cells [11]. But beyond that, ionic liquids and hydrogen peroxide have become a new kind of inhibitors as the pretreatment technologies evolve. Hence, oxidoreductases have shown a perfect application in the enhancement of tolerance to multiple inhibitors from pretreatment of lignocellulose.

To reduce levels of intracellular ROS that is induced by multiple lignocellulose-derived inhibitors, the overexpression of oxidoreductases might be a reasonable strategy [16], which may correspondingly contribute to an enhanced tolerance. Actually, expression of some oxidoreductases like mitochondrial cytochrome C oxidase chaperone gene (encoded by COX20) improves tolerance to weak acids, especially acetic acid in S. cerevisiae [17]. Moreover, an enhancement of intracellular proline concentration by addition of proline or overexpression of a proline synthesis related gene (PRO1) decreased the ROS level in yeast cells, which eventually led to an obvious increase in tolerance to both acetic acid and furfural [18]. Beyond that, oxidoreductases like alcohol dehydrogenase ADH1 [19] and ADH6 [20], 3-methylglyoxal reductase GRE2 [21], aldehyde reductase ARI1 [22] and xylose reductase XYL1 [23] could be quite effective in enhancing tolerance of yeast cells to inhibitors in lignocellulosic hydrolysates. But, at present, studies have been reported to be applied in increased tolerance to a single inhibitor, few are focused on a mixture of inhibitors above in lignocellulosic hydrolysates [18].

High-throughput sequencing is a powerful tool to gain insight in new genes and their new functions, and helps to reveal the mechanisms of toxicities of lignocellulose-dereived inhibitors [16, 24,25,26]. Recently, the non-conventional yeast, Kluyveromyces marxianus, attracts increasing attentions in the field of industrial biotechnology for its advantages in high-temperature resistance, rapid growth rate and diversity of substrates. With the improvement of technologies of genomics and transcriptomics, more and more new genes or enzymes from K. marxianus have been reported for its further theoretical research [27].

Our previous study on transcriptional analysis of K. marxianus obtained lots of differentially expressed genes (DEGs) [28], among which oxidoreductases have been proved to be greatly involved in stress tolerance of yeasts [29]. Therefore, an essential defender for ROS in yeasts, thioredoxin (TRX) and its reductase (TrxR), was further investigated in this study, evaluating the possible applications in stress tolerance under harshest conditions. To test tolerance to multiple inhibitors or stressors, both single expression of KmTRX gene, or KmTrxR gene, and the synergistic effect of these two genes were achieved in the recombinant S. cerevisiae.


The potential targets related to stress tolerance in K. marxianus

The over-expression of thioredoxin family from K. marxianus, composed of thioredoxin and its reductase, under harshest conditions (high ROS and ethanol) may be regarded as a potential target for an enhanced tolerance to multiple inhibitors during ethanol fermentation. Thioredoxin reductase belongs to the nucleotide pyridine disulfide oxidoreductase family [30]. The nucleotide sequence of thioredoxin reductase from K. marxianus (KmTrxR) has an open reading frame of 960 nucleotides, which encodes 319 amino acids. Alignment of KmTrxR with other related TrxR sequences revealed that active site is highly conserved from bacteria to fungi. KmTrxR showed about 50–90% amino acid sequence identities to our selected sequences (Fig. 1a; Additional file 1: Figure S1). Besides, as shown in Fig. 1b, KmTrxR is a typical homodimeric protein with the conserved disulfide motifs of Cys142-X-X-Cys145 from a homology modeling of TrxR in S. cerevisiae [30]. Two additional cysteine residues (Cys167 and Cys305) close to the active site are identified in KmTrxR.

Fig. 1
figure 1

Typical homodimeric proteins, thioredoxin and its reductase, in common yeasts. a, c Evolutionary trees of thioredoxin reductase and thioredoxin from Y179 and its allied species using MEGA 4 software. b, d Predicted 3D structure of thioredoxin reductase and thioredoxin from K. marxianus, respectively

Two kinds of thioredoxins were detected in K. marxianus, which is homologous to TRX2 and TRX3 in S. cerevisiae, respectively (Fig. 1c). KmTrx2 encodes 104 amino acids, with an approximate molecular weight of 11.2 kDa, and KmTrx3 encodes 149 amino acids, with an approximate molecular weight of 16.3 kDa. Both thioredoxins from K. marxianus possess a redox-active dithiol/disulfide within the conserved active site sequence, which lies in Cys30-Gly-Pro-Cys33 and Cys67-Gly-Pro-Cys70, respectively (Fig. 1d; Additional file 1: Figure S2).

Functional analysis of single KmTRX overexpression in S. cerevisiae

Saccharomyces cerevisiae 280 cells overexpressing KmTRX2 or KmTRX3 genes were adopted to evaluate the possible functions in an enhanced tolerance to multiple inhibitors (Fig. 2). The growth behavior of overexpressed strain Trx2 and control strain 423 displayed no obvious differences without, or with multiple inhibitors. Unfortunately, inhibitors like formic acid and acetic acid even repressed the growth of strain Trx3. These findings indicated that the single overexpression of gene KmTRX2, or KmTRX3, contributed little to the increase of stress tolerance in S. cerevisiae.

Fig. 2
figure 2

Stress response of single KmTRX over-expression to multiple inhibitors by serial dilution assay. Cells in log phase with OD620 of 10 were serially diluted to 10−5, and then spotted onto SC-His plates containing various inhibitors. Cells were cultivated at 30 °C for 3 days and then photographed

Functional analysis of single KmTrxR overexpression in S. cerevisiae

To test the functions of KmTrxR in increasing tolerance to lignocellulose-derived inhibitors, weak acids (formic acid and acetic acid) and furfural were firstly selected as the representative inhibitors in hydrolysates. As shown in Fig. 3, gene KmTrxR played a positive role in the enhanced tolerance to formic acid and acetic acid. Over-expression of KmTrxR would not affect the cell growth, while the growth of control strain 425 had been greatly repressed on plates with 0.4 g/L of formic acid and 3 g/L of acetic acid, which was 1–2 gradients less than strain TrxR in the serial dilution assay. However, the effect of gene KmTrxR in promoting the tolerance to furfural, another typical lignocellulose-derived inhibitor, was not very obvious.

Fig. 3
figure 3

Stress response of KmTrxR over-expression to the presence of multiple lignocellulose-derived inhibitors by serial dilution assay. Cells in log phase with OD620 of 10 were serially diluted to 10−5, and then spotted onto SC-Leu plates containing various inhibitors. Cells were cultivated at 30 or 42 °C for 3 days and then photographed

Batch fermentation was conducted in flask levels containing 50 g/L of glucose and FAF inhibitors (0.3 g/L of formic acid, 1.2 g/L of acetic acid, and 0.5 g/L of furfural) to simulate real lignocellulosic hydrolysates. As shown in Fig. 4 and Table 1, the over-expression of KmTrxR gene in cells helped to accelerate the process of fermentation under inhibitors, and to reduce the lag phase. The greatest differences of these two strains were between 24 and 48 h, when glucose consumption rate and ethanol production rate of TrxR achieved up to 1.50 and 0.68 g/L/h, respectively, both of which were about 15% more than those in control strain. Particularly, there were significant differences in fermentative parameters between two strains, such as residual glucose concentrations, ethanol concentrations, and productivities within 48 h because of an accelerated fermentative process (Table 1), which may make great differences to industrial-scale ethanol production.

Fig. 4
figure 4

Fermentation profile in KmTrxR-expressing S. cerevisiae cells during batch ethanol production process under FAF stress. a Growth behavior of two strains within 60 h. b Glucose consumption and ethanol production under FAF stress. Cells were pre-cultured in SC-Leu medium containing 1 mM H2O2 for 16–18 h. 1% of seed culture was inoculated into a 250 mL flask with a 100 mL working volume. Data are given as mean ± SD, n = 4

Table 1 Fermentative performance under FAF inhibitors within 48 h

Synergistic effect of KmTrxR and KmTRX in S. cerevisiae

To verify the synergistic effect of KmTrxR and KmTRX to stress tolerance, the engineered strains with double gene expressions were constructed. Stress tolerance assay as above was subsequently conducted.

As is shown in Fig. 5, in serial dilution assay, no significant differences in growth were discovered under no inhibitors, of the double expression strains (Trx2-TrxR and Trx3-TrxR) and control strain (423–425). There were no obvious differences in cell growth under conditions of ethanol, acetic acid, and furfural in strains of Trx2-TrxR and 423–425. In particular, the tolerance of Trx2-TrxR strain to formic acid was greatly increased, which was 1–2 gradients more than control strains. More importantly, the enhancement of the tolerance to a mixed FAF inhibitors in overexpressed strain (Trx2-TrxR) was detected, which was also increased by at least 2 gradients. However, the synergistic effect of KmTRX3 and KmTrxR was barely observed under the same conditions (Fig. 5).

Fig. 5
figure 5

Synergistic effect of KmTrxR and KmTRX over-expression to multiple inhibitors by serial dilution assay. Cells in log phase with OD620 of 10 were serially diluted to 10−5, and then spotted onto SC-Leu-His plates containing various inhibitors. Cells were cultivated at 30 °C for 3 days and then photographed

Furthermore, batch ethanol fermentation was performed in media containing 50 g/L glucose and FAF inhibitors, to further evaluate the possible synergistic effect of KmTRX2 and KmTrxR. Coupled with KmTrxR gene, the overexpression of KmTRX2 gene accelerated the fermentative process, and shortened the lag phase. Strain Trx2-TrxR showed no lag phase, which was superior to those in TrxR and 425. More importantly, the glucose consumption rate of Trx2-TrxR increased by over 30%, achieving 1.43 g/L/h, compared to that of 13% between TrxR and 425 (Fig. 6 and Table 1).

Fig. 6
figure 6

Fermentation profile in Trx2-TrxR strain during batch ethanol production process under FAF stress. a Growth behavior of two strains within 60 h. b Glucose consumption and ethanol production under FAF stress. Cells were pre-cultured in SC-Leu-His medium containing 1 mM H2O2 for 16–18 h. 1% of seed culture was inoculated into a 250 mL flask with a 100 mL working volume. Data are given as mean ± SD, n = 4

Mechanisms of an enhanced tolerance related to intracellular ROS levels

According to existing theories and our results, the possible mechanisms of oxidordeuctases enhancing tolerance to inhibitors is inferred to be related to the levels intracellular ROS. When cells were exposed to inhibitors, ROS like OH·, H2O2, and O ·−2 might be generated, which may cause molecular damages and cellar effects to the normal cells, and eventually lead to cell death [10]. However, some oxidordeuctases achieved functional dimer proteins after transcription and translation under peroxides [31]. The activated dimers removed excess ROS inside cells to maintain normal cell metabolism and to ensure a high rates of cell viabilities (Fig. 7).

Fig. 7
figure 7

Oxidative defense pathways and essential antioxidant genes in yeast cells

Fortunately, the measurement of intracellular ROS provided strong supports for our inference (Fig. 8). Without inhibitors FAF, the levels of intracellular ROS in strains with KmTRX, or KmTrxR genes almost remained consistent, and the double expression strains (Trx2-TrxR and Trx3-TrxR) contained much lower ROS than the control strain (423–425). However, the very significant differences occurred when cells were exposed to FAF inhibitors. The levels of intracellular ROS in overexpressed strains decreased dramatically, especially strain with both KmTRX2 and KmTrxR with a 40% reduction. Consequently, we concluded that a more effective remove of ROS in over-expressed strains was an important guarantee of growth, metabolism and multiplication of yeasts.

Fig. 8
figure 8

Intracellular ROS levels in recombinant strains with different target genes with, or without FAF inhibitors by DCFH-DA (n = 4, *P < 0.05, **P < 0.01)


Under certain conditions, ROS generated from cell metabolism causes more cell damages, and ROS generally includes super oxide, peroxide, and hydroxyl radical et al. [32]. In order to prevent from ROS damages, microorganisms build a perfect defense system, which involves some essential oxidoreductases and compounds, such as peroxiredoxins (Prxs), thioredoxin (TRX) and its reductase (TrxR), glutathione and its reductase (GLR) and peroxidase (GPX), catalase (CTT1), and superoxide dismutase (SOD) (Fig. 7a). These oxidoreductases might be also related to the tolerance to some specific inhibitors [32, 33]. Actually, the peroxiredoxin, encoded by KmTPX1, has been proved to contribute to an obvious enhancement of tolerance to both oxidative stress and lignocellulose-derived inhibitors [29]. Therefore, to further explore the potential functions of oxidoreductases on cells survival under tough conditions, the thioredoxin system from K. marxianus, composed of thioredoxin (KmTrx) and its reductase (KmTrxR), was selected as the potential targets to increase the stress tolerance in yeasts [28].

The thioredoxin system is widely present in most yeast mitochondria and acts as a defense against ROS damages. Thioredoxins are thiol-disulfide oxidoreductases that involve in a variety of cellular functions depending on its conserved active sites WCXXC [34, 35]. Both thioredoxin genes detected in K. marxianus (KmTRX2 and KmTRX3) possess the conserved domain (–CGPC–, Fig. 1d; Additional file 1: Figure S2). In spite of the shortest distance with the TRX1 gene in S. cerevisiae (Fig. 1c), KmTRX2 was defined as previously described [27]. Active thioredoxin system requires that thioredoxins are maintained at reduced form by its reductase (Fig. 7a). Thioredoxin reductase, as a kind of flavoenzyme, also contains one or two dithiol-disulfide motifs [36]. Except for the typical dithiol-disulfide motifs, two additional cysteine residues may possess their potential structural and functional roles in KmTrxR (Fig. 1b; Additional file 1: Figure S1), considering that TrxR catalyzes reactions using NADPH via FAD molecular and the redox-active cysteine residues [37].

Trx-TrxR system in Archaea, bacteria, plants, and lower eukaryotes (such as yeasts) contributes to regulating the intracellular redox balance [16, 30, 34, 35] In particular, a thioredoxin from Endocarpon pusillum showed its distinctive role in stress tolerance [34], which strikes us that the gene KmTRX and KmTrxR may possess the potential applications in increasing tolerance of yeasts to some specific inhibitors during ethanol fermentation. To test the possible functions of KmTrxR and KmTRX2/3, the preculture of yeast cells were incubated with low concentration of peroxide (1 mM) to turn on the specific reactions, and the possible influence of indigenous redox system in S. cerevisiae by the addition of H2O2 may be excluded by our experimental settings based on the same background, and by the facts that similar growth behaviors were achieved among all strains without inhibitors (Figs. 2, 3, 5). In this case, our results indicated that single overexpression of both Trx genes from K. marxianus showed a weak association with an enhanced stress tolerance (Fig. 2). Actually, the repressive effect of KmTRX3 in tolerance to formate makes it distinguished from other Trxs in cells. Compared to Trx1 and Trx2, specific location and sequence of Trx3 in yeast was reported [38], which might be related to the negative responses. In this case, the possible functions of gene KmTrxR and the synergistic effect of KmTRX and KmTrxR were further explored by serial dilution assay and batch ethanol fermentation.

On the one hand, single expression of gene KmTrxR was achieved in S. cerevisiae cells (Figs. 3, 4). First, considering lignocellulose-derived inhibitors, inducing the generation of intracellular ROS that is closely related to the redox state in cells [10,11,12], an enhanced tolerance to multiple lignocellulose-derived inhibitors, especially to formic acid and acetic acid, were observed in KmTrxR-overexpressing strain by serial dilution assay (Fig. 3). These observations have also been supported by a variety of similar oxidoreductases that might be contributed to the increased tolerance of S. cerevisiae to the inhibitors [17, 19,20,21,22,23, 39]; Besides, different from previous results achieved, such as a 2-Cys Prx from Oryza sativa [39] and a thioredoxin from Endocarpon pusillum [347], the principal advantage of KmTrxR gene tends to increase a comprehensively enhanced tolerance of yeasts to a mixture of lignocellulose-derived inhibitors (FAF), which may provide more references and potential applications for ethanol production from lignocellulosic materials (Fig. 3). Surprisingly, an obviously enhanced tolerance of strain TrxR to high concentration of salt was detected (data not shown). These finding is supposed to validate our previous inferences, and makes KmTrxR a promising gene in ethanol production from lignocellulosic materials. Finally, a better fermentative profile was achieved when cells were exposed to FAF mixtures (Fig. 4) during batch ethanol production, which was manifested as the shorter lag period and accelerated process in the fermentation, just as expected previously [18, 39].

On the other hand, the possible synergistic effect of KmTRX and KmTrxR was evaluated to strengthen the functions by overexpressing single KmTrxR gene (Figs. 5, 6). In spite of the contribution to an enhanced stress tolerance of KmTrxR gene, the poor effect, compared to the previous KmTPX1 gene [29] makes little sense. Fortunately, the double overexpression strain (Trx2-TrxR) showed the greatly enhanced tolerance to the mixed FAF inhibitors, especially for the formic acid among it (Fig. 6). Considering the negative effect on acetic acid and furfural, the enhancement was supposed to be consequences of amplification effect on formic acid.

The reasonable growth behavior under conditions of FAF by the synergistic effect of KmTRX2 and KmTrxR was superior to those by single overexpression of KmTRX and KmTrxR. The fermentative process of TrxR and 425 (SC-Leu) was much faster than that of Trx2-TrxR and 423–425 (SC-Leu-His), considering the differences in nutrition. Even so, we found that the differences of Trx-TrxR and 423–425 in glucose consumption rate and productivity were very significant (P < 0.01), compared with that were only significant (P < 0.05) between TrxR and 425 (Table 1; Additional file 1: Figure S4). In particular, the Trx2-TrxR strain achieved a greatly enhanced ethanol fermentation profile, compared with that of TrxR strain (Figs. 4, 6, Table 1). The increase of glucose consumption (by 30% vs 13%) with shorter lag phase from strain Trx2-TrxR under nutrition limited conditions (Table 1; Additional file 1: Figure S4), has all proved that KmTrxR gene coupled with KmTRX gene plays a more significant role in promoting stress tolerance to multiple lignocellulose-derived inhibitors than each single gene. Therefore, these findings under existing conditions further confirmed the synergistic effect of KmTRX and KmTrxR, which may lay a good foundation for its applications in the future.

In a word, the recombination S. cerevisiae with KmTRX and KmTrxR overexpressing possesses an enhanced tolerance to multiple lignocellulose-derived inhibitors, such as formic acid, acetic acid, and even the FAF mixtures (Figs. 3, 6). However, considering the complicated mechanisms that these inhibitors are toxic to cells, they may, as what we all know, induce cells to generate intracellular ROS [10,11,12], which has also been proved by our results in Fig. 8. Therefore, oxidoreductases help to remove the excess ROS, maintaining it at a normal level, to achieve an elevated tolerance and to reduce damages to cells. Intracellular ROS in cells with an increased tolerance has been reported to decrease under inhibitors, no matter what strategies were adopted [18, 39, 40].

An obviously decreased levels of intracellular ROS in overexpressed strains have proved that the overexpression of KmTRX and KmTrxR gene helps cells to maintain a normal amount of ROS (Fig. 8). Single expression of KmTRX2 and KmTRX3 contributed little to the reduction of ROS levels, which was in line with their negative functions in tolerance to inhibitors. In spite of a 20% reduction of ROS in KmTrxR-overexpressing strain, the promotion on an enhanced tolerance to FAF inhibitors was so limited. On this basis, the double expression of KmTRX2 and KmTrxR played a more positive role in removing the intracellular ROS levels (with an over 40% reduction) with, or without inhibitors, which further confirmed the synergistic effect of these two genes. Above all, our results and some previous reports all confirmed that oxidoreductases maintained a normal level of intracellular ROS, which eventually manifested as an apparently enhanced tolerance of S. cerevisiae to multiple lignocellulose-derived inhibitors [18, 39, 40].


An enhanced tolerance of yeasts to multiple lignocellulose-derived inhibitors contributes to the process of lignocellulosic ethanol production. Here we reported that the overexpression of both thioredoxin and its reductase from K. marxianus can enhanced tolerance to multiple lignocellulose-derived inhibitors and achieved the remarkable ethanol fermentative profiles under inhibitors. Actually, compared with single gene expression, the synergistic effect of KmTRX2 and KmTrxR was further confirmed under a mixture of formic acid, acetic acid, and furfural (FAF) with a shorter lag period. At last, the mechanism that improves the tolerance depended on a normal level of intracellular ROS for cell survival under stress. Consequently, all these findings provided the potential possibility for ethanol production from lignocellulosic materials, and give a general insight into the possible toxicity mechanisms for further theoretical research.


Strains, media and growth conditions

All strains used in this study are listed in Table 2. Yeast cells were grown at 30 °C in YPD medium (2% glucose, 1% yeast extract and 2% peptone) or in synthetic complete medium (SC) containing 2% glucose and 0.67% yeast nitrogen base, supplemented with the appropriate amino acids (Additional file 1: Table S1). Escherichia Coli DH5α was cultivated at 37 °C in LB medium (0.5% yeast extract 1% peptone and 1% sodium chloride).

Table 2 Strains, plasmids and primers used in this study

Plasmids construction and transformation

To construct an overexpression vector with the KmTrxR gene and resistance gene, plasmid pRS425 [41] was used for DNA manipulation and cloning. DNA manipulation was performed by standard procedures [42]. KmTrxR gene fragment, amplified from genome of K. marxianus Y179 with primers KmTrxR-F and KmTrxR-R. The obtained fragment with a coding region and a native promoter of KmTrxR gene was inserted into pRS425 to generate pRS425-TrxR after digestion with SacI and SacII. The constructions were verified by both digestion of restriction enzyme and sequencing. Similarly, KmTRX2 and KmTRX3 gene with their own promoters, amplified with the corresponding primers in Table 2, were inserted into plasmid pRS423 with restriction enzymes of SacI and SpeI.

Transformation of E. coli cells was referred to CaCl2 method [43]. For screening the transformants, ampicillin (100 μg/mL) was added into LB selective plates. Yeast transformation was conducted by LiAc/PEG method described by Gietz et al. [44], and transformants were screened by SC medium without leucine (SC-Leu), or Histidine (SC-His), or both (SC-Leu-His). Transformants with the recombinant plasmids were marked as described in Table 2 in this study. All transformants were verified by PCR to prove the presence of the constructs. To test the normal functions of all overexpressed genes under the control their native promoters, the qPCR analysis of all recombinant strains with corresponding target genes were performed (Additional file 1: Tables S2, S3). A detailed description could be found in Additional file 1.

Stress tolerance assay

To test functions of KmTrxR gene and KmTRX gene, serial dilution assay were performed using recombinant strains. Yeast cells were cultivated in corresponding selective medium with 1 mM H2O2 at 30 °C and 150 rpm for 16–18 h (OD620 = 2), and then collected by centrifugation at 3000×g for 5 min. Concentrated cells (final OD620 = 10) were serially diluted with distilled water, after which 10 μL of the diluted cells was loaded onto SC selective agar plates containing a single or mixed lignocellulose-derived inhibitors. The inhibitors applied in this section involved in formic acid, acetic acid, furfural, and their mixture FAF. All plates were incubated for 2–3 days at 30 °C and then photographed. For high-temperature test, plates were incubated at 42 °C.

Laboratory-scale batch fermentation

To evaluate fermentative performance of strain TrxR and strain Trx2-TrxR, batch fermentations were conducted in a flask level for around 60 h. Pre-cultures of yeast cells were carried out in SC selective medium with 1 mM H2O2, and then cells was inoculated in corresponding SC medium containing 50 g/L of glucose and FAF mixture (0.3 g/L formic acid, 1.2 g/L acetic acid and 0.5 g/L furfural) at 30 °C and 150 rpm. The initial OD620 was adjusted to 0.03, and samples were taken every 12 h to test cell growth, sugar and by-products.

Bioinformatics analysis

Evolutionary tree of some KmTRX genes and KmTrxR genes from both Y179 and its allied species using MEGA 4 software [45], and amino acid sequences applied are listed as follows: KmTrxR, thioredoxin reductase from K. marxianus Y179 in this study; KlTrxR, thioredoxin reductase from K. lactis (Accession No. XP_454928.1); ScTrxR, thioredoxin reductase from S. cerevisiae (Accession No. NP_010640.1); CtTrxR, thioredoxin reductase from Candida tropicalis (Accession No. XP_002546509.1); SsTSA1, thioredoxin reductase from Scheffersomyces stipitis (Accession No. XP_001384831.1); AnTrxR, thioredoxin reductase from Aspergillus niger (Accession No. XP_001389279.1); EcTrxR, thioredoxin reductase from E. coli (Accession No. ADX51538.1); EpTrxR, thioredoxin reductase from Endocarpon pusillum (Accession No. AEH41540.1); KmTRX2 and KmTRX3, thioredoxins from Y179; ScTRX1, thioredoxin 1 from S. cerevisiae (Accession No. NP_013144.1); ScTRX2, thioredoxin 2 from S. cerevisiae (Accession No. NP_011725.3); ScTRX3, thioredoxin 3 from S. cerevisiae (Accession No. NC_001135.5); KlTRX, thioredoxin from K. lactis (Accession No. XP_454686.1); CtTRX2, thioredoxin 2 from C. tropicalis (Accession No. XP_002546341.1). Alignment was conducted by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) software (

The three dimensional (3D) structure of KmTrxR was predicted by SWISS-MODEL ( and the 3D structure of TrxR from S. cerevisiae (PDB Accession No. 3ITJ) was adopted as the structure template. Similarly, the 3D structure template of KmTRX gene was also from the homologous ScTRX2 from S. cerevisiae (PDB Accession No. 2hsy.1.A). Software RasMol was used to view and analyze the predicted 3D structure of KmTrxR, as was shown in Fig. 1.

Expression pattern analysis of differentially expressed genes related to oxidative stress from transcriptome of K. marxianus Y179 [28] was clustered using Cluster software [46] and Java Treeview software [47]. The hierarchical clustering of the chosen experimental conditions and genes was carried out by using Euclidean Distance as the formula of the distance matrix.

ROS level analysis

Levels of ROS inside cells were tested by a common method using 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma-35845, dissolved in absolute ethanol) as an indicator. Cells after pre-culture were cultivated for 16–18 h in corresponding selective medium with FAF (0.3 g/L formic acid, 1.2 g/L acetic acid and 0.5 g/L furfural) at 30 °C and 150 rpm (OD620 1.0). Cell pellets were washed twice with distilled water, and then re-suspended in 0.5 mL 10 mM PBS (pH 7.0) containing 10 μM DCFH-DA. After incubation at 37 °C for 60 min, fluorescence was measured by a Multiskan spectrum microplate spectrophotometer (PerkinElmer, USA) with λEX 485 nm and λEM 525 nm [40].

Analytical methods

The cell concentration was measured using the optical density at 620 nm. Concentrations of glucose, ethanol and glycerol were analyzed by Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad, Hercules) in HPLC system, which used 0.01 mol/L H2SO4 as mobile phase and eluted at 50 °C with a flow rate of 0.5 mL/min. All analysis in this study was done in quadruplicate except indicated, and the mean values are shown in “Results” and “Discussion” section.



reactive oxidative species


thioredoxin gene from K. marxianus

KmTrxR :

thioredoxin reductase gene from K. marxianus


a mixed inhibitors of formic acid, acetic acid and furfural


synthetic complete medium


SC medium without Leucine


SC medium without Leucine and Histidine


2′,7′-dichlorofluorescein diacetate


  1. Choudhary J, Singh S, Nain L. Thermotolerant fermenting yeasts for simultaneous saccharification fermentation of lignocellulosic biomass. Electron J Biotechnol. 2016;21:82–92.

    Article  CAS  Google Scholar 

  2. Sarkar N, Ghosh SK, Bannerjee S, Aikat K. Bioethanol production from agricultural wastes: an overview. Renew Energy. 2012;37:19–27.

    Article  CAS  Google Scholar 

  3. Alfenore S, Molina-Jouve C. Current status and future prospects of conversion of lignocellulosic resources to biofuels using yeasts and bacteria. Process Biochem. 2016;51:1747–56.

    Article  CAS  Google Scholar 

  4. Palmqvist E, Hahn-Hägerdal B. Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour Technol. 2000;74:25–33.

    Article  CAS  Google Scholar 

  5. Serna LVD, Alzate CEO, Alzate CAC. Supercritical fluids as a green technology for the pretreatment of lignocellulosic biomass. Bioresour Technol. 2016;199:113–20.

    Article  Google Scholar 

  6. Gu T, Held MA, Faik A. Supercritical CO2 and ionic liquids for the pretreatment of lignocellulosic biomass in bioethanol production. Environ Technol. 2013;34:1735–49.

    Article  CAS  Google Scholar 

  7. Lee JE, Guragain YN, Bastola KP, Vadlani PV. Innovative methods to generate clean sugar stream from biomass feedstocks for efficient fermentation. Bioprocess Biosyst Eng. 2017;40:633–41.

    Article  CAS  Google Scholar 

  8. Lee HJ, Lim WS, Lee JW. Improvement of ethanol fermentation from lignocellulosic hydrolysates by the removal of inhibitors. J Ind Eng Chem. 2013;19:2010–5.

    Article  CAS  Google Scholar 

  9. Chen R, Dou J. Biofuels and bio-based chemicals from lignocellulose: metabolic engineering strategies in strain development. Biotechnol Lett. 2016;38:213–21.

    Article  CAS  Google Scholar 

  10. Giannattasio S, Guaragnella N, Ždralević M, Marra E. Molecular mechanisms of Saccharomyces cerevisiae stress adaptation and programmed cell death in response to acetic acid. Front Microbiol. 2013;4:33.

    Article  CAS  Google Scholar 

  11. Ibraheem O, Ndimba BK. Molecular adaptation mechanisms employed by ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds. Int J Biol Sci. 2013;9:598–612.

    Article  Google Scholar 

  12. Kim SK, Jin YS, Choi IG, Park YC, Seo JH. Enhanced tolerance of Saccharomyces cerevisiae to multiple lignocellulose-derived inhibitors through modulation of spermidine contents. Metab Eng. 2015;29:46–55.

    Article  CAS  Google Scholar 

  13. Roe AJ, McLaggan D, Davidson I, O’Byrne C, Booth IR. Perturbation of anion balance during inhibition of growth of Escherichia coli by weak acids. J Bacteriol. 1998;180:767–72.

    CAS  Google Scholar 

  14. Zhao J, Wang M, Yang Z, Gong Q, Lu Y, Yang Z. Mediated electrochemical measurement of the inhibitory effects of furfural and acetic acid on Saccharomyces cerevisiae and Candida shehatae. Biotechnol Lett. 2005;2005(27):207–11.

    Article  Google Scholar 

  15. Allen SA, Clark W, McCaffery JM, Cai Z, Lanctot A, Slininger PJ, et al. Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae. Biotechnol Biofuels. 2010;3:2.

    Article  Google Scholar 

  16. Grant CM. Role of the glutathione/glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions. Mol Microbiol. 2001;39:533–41.

    Article  CAS  Google Scholar 

  17. Kumar V, Hart AJ, Keerthiraju ER, Waldron PR, Tucker GA, Greetham D. Expression of mitochondrial cytochrome C oxidase chaperone gene (COX20) improves tolerance to weak acid and oxidative stress during yeast fermentation. PLoS ONE. 2015;10:e0139129.

    Article  Google Scholar 

  18. Wang X, Bai X, Chen DF, Chen FZ, Li BZ, Yuan YJ. Increasing proline and myo-inositol improves tolerance of Saccharomyces cerevisiae to the mixture of multiple lignocellulose-derived inhibitors. Biotechnol Biofuels. 2015;8:142.

    Article  Google Scholar 

  19. Almeida JR, Röder A, Modig T, Laadan B, Lidén G, Gorwa-Grauslund MF. NADH-vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2008;78:939–45.

    Article  CAS  Google Scholar 

  20. Petersson A, Almeida JR, Modig T, Karhumaa K, Hahn-Hägerdal B, Gorwa-Grauslund MF, Lidén G. A 5-hydroxymethyl furfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance. Yeast. 2006;23:455–64.

    Article  CAS  Google Scholar 

  21. Moon J, Liu ZL. Engineered NADH-dependent GRE2 from Saccharomyces cerevisiae by directed enzyme evolution enhances HMF reduction using additional cofactor NADPH. Enzyme Microb Technol. 2012;50:115–20.

    Article  CAS  Google Scholar 

  22. Liu ZL, Moon J. A novel NADPH-dependent aldehyde reductase gene from Saccharomyces cerevisiae NRRL Y-12632 involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion. Gene. 2009;446:1–10.

    Article  CAS  Google Scholar 

  23. Almeida JR, Modig T, Petersson A, Hähn-Hägerdal B, Lidén G, Gorwa-Grauslund MF. Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol. 2007;82:340–9.

    Article  CAS  Google Scholar 

  24. Ma M, Liu ZL. Comparative transcriptome profiling analyses during the lag phase uncover YAP1, PDR1, PDR3, RPN4, and HSF1 as key regulatory genes in genomic adaptation to the lignocellulose derived inhibitor HMF for Saccharomyces cerevisiae. BMC Genom. 2010;11:660.

    Article  CAS  Google Scholar 

  25. Yang J, Ding MZ, Li BZ, Liu ZL, Wang X, Yuan YJ. Integrated phospholipidomics and transcriptomics analysis of Saccharomyces cerevisiae with enhanced tolerance to a mixture of acetic acid, furfural, and phenol. OMICS. 2012;16:374–86.

    Article  CAS  Google Scholar 

  26. Zhou Q, Liu ZL, Ning K, Wang A, Zeng X, Xu J. Genomic and transcriptome analyses reveal that MAPK-and phosphatidylinositol-signaling pathways mediate tolerance to 5-hydroxymethyl-2-furaldehyde for industrial yeast Saccharomyces cerevisiae. Sci Rep. 2014;4:6556.

    Article  CAS  Google Scholar 

  27. Lertwattanasakul N, Kosaka T, Hosoyama A, Suzuki Y, Rodrussamee N, Matsutani M, et al. Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses. Biotechnol Biofuels. 2015;8:47.

    Article  Google Scholar 

  28. Gao JQ, Yuan WJ, Li YM, Xiang RJ, Hou SB, Zhong SJ, et al. Transcriptional analysis of Kluyveromyces marxianus for ethanol production from inulin using consolidated bioprocessing technology. Biotechnol Biofuels. 2015;8:115.

    Article  Google Scholar 

  29. Gao JQ, Feng HL, Yuan WJ, Li YM, Hou SB, Zhong SJ, et al. Enhanced fermentative performance under stresses of multiple lignocellulose-derived inhibitors by overexpression of a typical 2-Cys peroxiredoxin from Kluyveromyces marxianus. Biotechnol Biofuels. 2017;10:79.

    Article  Google Scholar 

  30. Oliveira MA, Discola KF, Alves SV, Medrano FJ, Guimaraes BG, Netto LE. Insights into the specificity of thioredoxin reductase—thioredoxin interactions: a structural and functional investigation of the yeast thioredoxin system. Biochemistry. 2010;49:3317–26.

    Article  CAS  Google Scholar 

  31. Rhee SG. Overview on peroxiredoxin. Mol Cells. 2016;39:1–5.

    Article  CAS  Google Scholar 

  32. Arellano-Plaza M, Gschaedler-Mathis A, Noriega-Cisneros R, Clemente-Guerrero M, Manzo-Ávalos S, González-Hernández JC, et al. Respiratory capacity of the Kluyveromyces marxianus yeast isolated from the mezcal process during oxidative stress. World J Microbiol Biotechnol. 2013;29:1279–87.

    Article  CAS  Google Scholar 

  33. Miura T, Abe F, Inoue A, Usami R, Horikoshi K. Superoxide dismutase is involved in high tolerance to copper in the deep-sea yeast, Cryptococcus sp. N6. Biotechnol Lett. 2002;24:1069–74.

    Article  CAS  Google Scholar 

  34. Li H, Wei JC. Functional analysis of thioredoxin from the desert lichen-forming fungus, Endocarpon pusillum Hedwig, reveals its role in stress tolerance. Sci Rep. 2016;6:27184.

    Article  CAS  Google Scholar 

  35. Miranda-Vizuete A, Damdimopoulos AE, Spyrou G. The mitochondrial thioredoxin system. Antioxid Redox Signal. 2000;2:801–10.

    Article  CAS  Google Scholar 

  36. Rajashankar RK, Bryk R, Kniewel R, Buglino JA, Nathan CF, Lima CD. Crystal structure e functional analysis of lipoamide dehydrogenase from Mycobacterium tuberculosis. J Biol Chem. 2005;280:33977–83.

    Article  CAS  Google Scholar 

  37. Waksman G, Krishna TSR, Williams CH, Kuriyan J. Crystal structure of Escherichia coli thioredoxin reductase refined at 2 Å resolution. Implications for a large conformational change during catalysis. J Mol Biol. 1994;236:800–16.

    Article  CAS  Google Scholar 

  38. Kim SJ, Jung EM, Jung HJ, Song YS, Park EH, Lim CJ. Cellular functions and transcriptional regulation of a third thioredoxin from Schizosaccharomyces pombe. Can J Microbiol. 2007;53:775–83.

    Article  CAS  Google Scholar 

  39. Kim IS, Kim YS, Yoon HS. Expression of salt-induced 2-Cys peroxiredoxin from Oryza sativa increases stress tolerance and fermentation capacity in genetically engineered yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2013;97:3519–33.

    Article  CAS  Google Scholar 

  40. Zhang MM, Zhao XQ, Cheng C, Bai FW. Improved growth and ethanol fermentation of Saccharomyces cerevisiae in the presence of acetic acid by overexpression of SET5 and PPR1. Biotechnol J. 2015;10:1903–11.

    Article  CAS  Google Scholar 

  41. Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989;122:19–27.

    CAS  Google Scholar 

  42. Green MR, Sambrook J. Molecular cloning: a laboratory manual, vol. 1. New York: Cold Spring Harbor Laboratory Press; 2012.

    Google Scholar 

  43. Cohen SN, Chang AC, Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci. 1972;69:2110–4.

    Article  CAS  Google Scholar 

  44. Gietz RD, Schiestl RH. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2007;2:31–4.

    Article  CAS  Google Scholar 

  45. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24:1596–9.

    Article  CAS  Google Scholar 

  46. de Hoon MJL, Imoto S, Nolan J, Miyano S. Open source clustering software. Bioinformatics. 2004;20:1453–4.

    Article  Google Scholar 

  47. Saldanha J. Java Treeview-extensible visualization of microarray data. Bioinformatics. 2004;20:3246–8.

    Article  CAS  Google Scholar 

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Authors’ contributions

JQG designed the research, performed most of the experiments, collected data and prepared the manuscript. WJY conceived the concept and design of the experiment. YML carried out microbiological sampling and conducted analyses. FWB and YJ participated in the design of the study and assisted in drafting the manuscript. All authors read and approved the final manuscript.


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This study was supported by projects of International Cooperation and Exchanges NSFC (51561145014), the China Postdoctoral Science Foundation (2015M571316), and the National Key Laboratory project of Microbial Metabolism (MMLKF16-04).

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Gao, J., Yuan, W., Li, Y. et al. Synergistic effect of thioredoxin and its reductase from Kluyveromyces marxianus on enhanced tolerance to multiple lignocellulose-derived inhibitors. Microb Cell Fact 16, 181 (2017).

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