Chain link fences. Existing site. Within the sand. Explorations were monitored by a GEI engineer. Sand (SW), and the thickness ranged from 2 to 9 feet. The size, mass and speed of the moving ferromagnetic object.
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- (SysML) to create a linkage for deriving information from a model to fill in an. Index Terms— Software standards, Testing, Test equipment. Gorringe is with Cassidian Test Engineering Services Ltd. Missing or degraded. Fielded test assets at Preliminary Design Review (PDR) 3.
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ResultsTo increase alkane tolerance in S. Cerevisiae, we sought to exploit the pleiotropic drug resistance (Pdr) transcription factors Pdr1p and Pdr3p, which are master regulators of genes with pleiotropic drug resistance elements (PDREs)-containing upstream sequences. Wild-type and site-mutated Pdr1p and Pdr3p were expressed in S. Cerevisiae BY4741 pdr1Δ pdr3Δ (BYL13).
The point mutations of PDR1 (F815S) and PDR3 (Y276H) in BYL13 resulted in the highest tolerance to C10 alkane, and the expression of wild-type PDR3 in BYL13 led to the highest tolerance to C11 alkane. To identify and verify the correlation between the Pdr transcription factors and tolerance improvement, we analyzed the expression patterns of genes regulated by the Pdr transcription factors in the most tolerant strains against C10 and C11 alkanes.
Quantitative PCR results showed that the Pdr transcription factors differentially regulated genes associated with multi-drug resistance, stress responses, and membrane modifications, suggesting different extents of intracellular alkane levels, reactive oxygen species (ROS) production and membrane integrity. We further showed that (i) the expression of Pdr1 mt1 + Pdr3 mt reduced intracellular C10 alkane by 67% and ROS by 53%, and significantly alleviated membrane damage; and (ii) the expression of the Pdr3 wt reduced intracellular C11 alkane by 72% and ROS by 21%. Alkane transport assays also revealed that the reduction of alkane accumulation was due to higher export (C10 and C11 alkanes) and lower import (C11 alkane).
Biologically synthesized alkanes can be used as ‘drop in’ to existing transportation infrastructure as alkanes are important components of gasoline and jet fuels. Even though alkanes have been successfully produced in microbes , , the yields and titers should be a key consideration for industrial-scale production, and the toxicity of alkanes to microbial hosts could eventually be a bottleneck for high productivity ,.Our previous transcriptome analyses suggested that alkanes induce a range of cellular mechanisms such as efflux pumps, membrane modification, radical detoxification, and energy supply in yeast. Indeed, the mechanisms underlying cell responses to toxic molecules can provide useful strategies to improve cell tolerance and viability. Such strategies include engineering efflux pumps and transcription factors , , and modifying cellular membrane.
Transcription factors regulate multiple and simultaneous perturbations of the transcriptome towards a global phenotype of tolerance. By knockout or overexpression of transcription factors involved in genetic regulatory networks of isooctane response in Escherichia coli, Kang et al.
improved E. Coli’s tolerance to isooctane. In addition, Matsui et al. Discovered a modified transcription factor endowing Saccharomyces cerevisiae with organic-solvent tolerance.Towards the development of alkane-tolerant S. Cerevisiae, a well-studied model eukaryote with wide industrial applications, we sought to exploit its transcription factors Pdr1p and Pdr3p, which are master regulators of genes with pleiotropic drug resistance elements (PDREs)-containing upstream sequences.
Currently, a thorough investigation of the roles of Pdr1p and Pdr3p in cellular tolerance to alkanes is lacking. In this study, we demonstrated a significant improvement in yeast’s tolerance to n-decane (C10) and n-undecane (C11) by modulating the expression of wild-type and site-mutated Pdr1p and Pdr3p. The correlation between Pdr transcription factors and tolerance improvement was confirmed by analyzing gene patterns, alkane transport, reactive oxygen species (ROS) levels, and membrane integrity. Results and discussion. Transcription factor engineering is widely used to improve microbial strain tolerance against toxic molecules ,.
Cerevisiae, transcription factors Pdr1p and Pdr3p have a DNA-binding domain, an inhibitory domain, and a transcription activation domain. The inhibitory domain in a locked conformation interacts with the transcription activation domain , , which is associated with Pdr-DNA or Pdr–Pdr interactions and pleiotropic drug resistance. Amino acid substitutions in the inhibitory domains could alter the actions of the transcription activation domain, leading to changes in Pdr1 and Pdr3 activity and the pleiotropic drug resistance. Recently, a series of site mutations in the inhibitory domains have been shown to improve pleiotropic drug resistance, and three site mutations (F815S and R821S in Pdr1p, and Y276H in Pdr3p) are most effective to improve the tolerance against various toxic molecules ,. Currently, a thorough investigation of the roles of these mutations in cellular tolerance to alkanes is lacking.
In this study, we chose F815S and R821S in Pdr1p and Y276H in Pdr3p for improving the tolerance to alkane biofuels in S. Figure shows the chosen mutation sites and cloning of the wild-type and site-mutated PDR1 and PDR3 into pESC-Ura. We induced the expression of the wild-type and site-mutated PDR1 and PDR3 in a double gene-deletion mutant S. Cerevisiae BYL13 ( pdr1Δ pdr3Δ). Fig. 1Site mutagenesis of PDR1 and PDR3, and plasmid construction. Plasmid pESC-Ura ( ) was used as a vector to express the transcription factors. Wild-type and mutant alleles of PDR1 and PDR3 were cloned into MCS2 and MCS1, respectively.
Asterisks mutation sites Conditions for protein induction and alkane exposureTo test yeast cell tolerance towards alkanes, we determined suitable conditions for protein induction and alkane exposure. Additional file: Figure S1A shows that the growth of BYL13 expressing the site-mutated Pdr transcription factor genes was inhibited, suggesting that lower induction of the Pdr transcription factors might lead to lower growth inhibition. To determine suitable induction conditions, we added various amounts of galactose (0.5 g/l, 5 g/l, and 20 g/l) and compared the resulting cell densities. Additional file: Figure S1B shows that the growth inhibition was lower (one-tailed Student t test, p.
We then investigated the tolerance of BYL13 expressing Pdr transcription factors against C8, C9, C10, and C11 alkanes. Figure a and Additional file: Figure S3 show that in the presence of (i) 1% C10 alkane, BYL13 expressing the site-mutated Pdr transcription factors (particularly Pdr1 mt1 + Pdr3 mt) had significantly higher cell densities than the control cells (with pESC-Ura); and (ii) 5% C11 alkane, BYL13 expressing the wild-type Pdr transcription factors (particularly Pdr3 wt) had significantly higher cell densities, whilst BYL13 expressing the site-mutated Pdr transcription factors had modestly higher cell densities than the controls. The enhanced cell densities correspond with increased cell viability (Fig. However, there was no improvement in tolerance in BYL13 expressing the wild-type Pdr transcription factors against C10 alkane, or in BYL13 expressing any Pdr transcription factors against C8 or C9 alkanes. Expression of the representative transcription factors (Pdr1 mt1 + Pdr3 mt, and Pdr3 wt) was confirmed by Western blots (Additional file: Figure S4).
Furthermore, we performed quantitative PCR (qPCR) to understand the roles of PDR expression levels and site mutations in the tolerance improvement. Additional file: Tables S1 and S2 show that the C10 tolerance was attributed to the site mutations (Pdr1 F815S, and Pdr3 Y276H) regardless of the PDR1 mt1 and PDR3 mt expression levels. The results of the growth assays and expression analyses indicate that the expression of Pdr transcription factors improved yeast tolerance to C10 and C11 alkanes. Fig. 2Tolerance of BYL13 + Pdr (Pdr1 mt1 + Pdr3 mt and Pdr3 wt) against C10 and C11 alkanes. A Cell density (OD 600) of BYL13 expressing Pdr1 mt1 + Pdr3 mt against 1% C10 (PDR1 mt1 + PDR3 mt + C10), and BYL13 expressing Pdr3 wt against 5% C11 (PDR3 wt + C11) was measured every 12 h. Error bars SD from three biological replicates.
B Ten microliters of serially diluted cells (24 h) were spotted onto YPD agar plates for cell viability assays. Control, BYL13 with pESC-Ura Gene patterns in BYL13 expressing Pdr1 mt1 + Pdr3 mt and Pdr3 wt in the presence of C10 and C11 alkanesThe improved alkane tolerance of yeast, conferred by the expression of Pdr1 mt1 + Pdr3 mt or Pdr3 wt, might be contributed to by perturbations made to the expression levels of the genes regulated by the Pdr transcription factors, such as ABC efflux pump genes, stress responsive genes, and genes involved in membrane modifications. To examine this possibility, we studied the expression patterns of those target genes in the presence of C10 and C11 alkanes by qPCR.First, to choose a suitable reference gene, we evaluated expression stability of five reference genes ( ACT1, ALG9, TAF10, UBC6, and TFC1) by comparing their M values under the above conditions. Here, a lower M value stands for higher stability of gene expression ,.
Additional file: Table S1 shows that UBC6 gene had the lowest M value out of the five reference gene candidates under the conditions of Pdr expression and alkane exposure. Hence, UBC6 was chosen as the reference gene for qPCR analyses. Second, we compared expression levels of the target genes in BYL13 expressing Pdr1 mt1 + Pdr3 mt (in the presence of C10 alkane) or Pdr3 wt (in the presence of C11 alkane) to those in BYL13 under exposure to C10 or C11 alkane. Figure shows that, in BYL13 expressing either Pdr1 mt1 + Pdr3 mt (C10) or Pdr3 wt (C11), (i) ABC efflux pump genes (i.e., YOR1, SNQ2, PDR5, and PDR15) were up-regulated by 4.2 to 46.6-fold (C10) and 1.6 to 17.1-fold (C11); (ii) cytosolic catalase gene CTT1 was up-regulated by 2.1-fold (C10) and 2.6-fold (C11); and (iii) lysophosphatidic acid acyltransferase gene ICT1 was up-regulated by 5.8-fold (C10) and 3.4-fold (C11). Considering the roles of the efflux pump genes in multi-drug resistance (MDR) , CTT1 in ROS detoxification , and ICT1 in membrane modifications , we hypothesized that intracellular alkane amount, ROS levels, the efficiency of alkane transport, and membrane damage might be involved in the increased alkane tolerance of BYL13 expressing Pdr1 mt1 + Pdr3 mt or Pdr3 wt. This hypothesis was investigated, as explained in the following sections. Fig. 3Relative gene expression levels under the regulation of Pdr transcription factors in the presence of C10 and C11 alkanes.
In the induction medium containing 0.5 g/l galactose, BYL13 + Pdr1 mt1 + Pdr3 mt cells were exposed to 1% C10 alkane (PDR1 mt1 + PDR3 mt + C10), and BYL13 + Pdr3 wt cells were exposed to 5% C11 alkane (PDR3 wt + C11). The expression levels of genes at 24 h were normalized to those in controls (BYL13 + pESC-Ura + C10, BYL13 + pESC-Ura + C11) and the reference gene UBC6. Error bars SD from three biological replicates Alkane levels in BYL13 expressing Pdr1 mt1 + Pdr3 mt and Pdr3 wt. Fig. 4Analyses of intracellular alkane levels in BYL13 expressing Pdr transcription factors. A Upon exposure to C10 and C11 alkanes, respectively, intracellular alkanes were extracted, measured by GC, and quantified by normalizing areas of GC peaks to those of an internal standard (IS) n-dodecane as well as corresponding protein amount. B To identify involvement of ABC efflux pumps in reducing intracellular alkanes, intracellular alkanes were compared between BYL13 expressing Pdr transcription factors with and without NaN 3. C To verify lower import of alkanes, intracellular alkanes were compared between BYL13 with pESC-Ura and expressing Pdr transcription factors in the presence of NaN 3.
Alkane amount in cells with pESC-Ura, and with NaN 3 were set as 1. Asterisks significant difference (one-tailed Student t test, p. ROS levels were quantified to investigate the effect of Pdr transcription factor expression on ROS production in the presence of alkanes. Figure a, b shows that C10 alkane enhanced ROS levels by more than fourfold whereas C11 alkane increased ROS levels by 1.5-fold. Further, in comparison to BYL13 carrying pESC-Ura, intracellular ROS was reduced by 53% in BYL13 expressing Pdr1 mt1 + Pdr3 mt in the presence of C10 alkane, and reduced by 21% in BYL13 expressing Pdr3 wt in the presence of C11 alkane.
The reduction of ROS in BYL13 expressing the Pdr transcription factors was further supported by our microscopy results. Figure c shows that, upon exposure to C10 alkane, over 90% of the cells with pESC-Ura fluoresced in green, and only about 30% of the cells with Pdr1 mt1 + Pdr3 mt fluoresced in green. On the other hand, upon exposure to C11 alkane, 15% of the cells with pESC-Ura fluoresced in green, and no cells with Pdr3 wt fluoresced in green.
Here, more green cells and higher fluorescence intensities represent more ROS. The results of ROS quantification and microscopy suggest significant reduction of ROS in BYL13 expressing the Pdr transcription factors in the presence of C10 and C11 alkanes.
Fig. 5Quantification of ROS levels in BYL13 expressing Pdr transcription factors. A and b ROS levels upon exposure to C10 and C11 alkanes. The relative ROS levels of BYL13 without alkane were set to 1. C Comparison of fluorescence images, where stronger green fluorescence indicates higher ROS levels.
AU arbitrary unit. Asterisk significant difference (one-tailed Student t test, p. To this end, we exposed the cells to C10 and C11 alkanes and stained the exposed cells with fluorescence nucleic acid stains PI and SYTO 9. Subsequently, we measured fluorescence signals and observed the cells under microscope. Figure a shows that relative fluorescence unit (RFU) ratios of PI to SYTO 9 were enhanced by 16.7-fold in BYL13 with pESC-Ura, and enhanced by 6.4-fold in BYL13 expressing Pdr1 mt1 + Pdr3 mt, upon exposure to C10 alkane as compared with those without alkane exposure. Moreover, in the presence of C10 alkane, the RFU ratio in BYL13 expressing Pdr1 mt1 + Pdr3 mt was about 62% lower than that with pESC-Ura, likely due to Ict1p-mediated membrane modifications in the presence of C10 alkane. However, Fig.
B shows that, in the presence of C11 alkane, both BYL13 with Pdr3 wt and the control cells had comparable RFU ratios, suggesting intact cell membrane in the presence of C11 alkane. Fig. 6Assays of membrane integrity in BYL13 expressing Pdr transcription factors. A and b RFU ratios of PI and SYTO 9 upon exposure to C10 and C11 alkanes. The relative RFU ratios of BYL13 without alkane were set to 1.
C Representative fluorescent images. Error bars SD from three biological replicatesThe low RFU ratio suggests that BYL13 expressing Pdr1 mt + Pdr3 mt had less membrane damage than the control cells in the presence of C10 alkane, and the comparable ratios suggest no membrane damage to both BYL13 expressing Pdr3 wt and the control cells in the presence of C11 alkane, in line with the fluorescence microscopy images in Fig. C.In this study, we improved yeast’s alkane tolerance by expressing wild-type or site-mutated Pdr transcription factors in S. Cerevisiae pdr1Δ pdr3Δ, and provided the evidence that, in the most tolerant strains expressing Pdr transcription factors, (i) a series of genes (e.g., ABC efflux pump genes, CTT1, and ICT1) were up-regulated by C10 and C11 alkanes; (ii) intracellular alkane levels were reduced over 67% due to alkane efflux and/or low import; and (iii) ROS levels were reduced over 21%; and (iv) cell membrane damage was also reduced. However, expression of any Pdr transcription factors did not improve tolerance to C8 or C9 alkanes at toxic levels. The susceptibility of yeast to alkanes is associated with multiple factors such as alkane carbon-chain length, alkane concentration, and strain background.
Additional file: Figure S2 shows that, more C8 and C9 alkanes were required to inhibit BYL13 with pESC-Ura than C10 alkane, although C8 and C9 alkanes are more toxic than C10 and C11 alkanes. This could be because C8 and C9 alkanes are more volatile than longer-chain alkanes. Furthermore, although Pdr1p and Pdr3p improved tolerance to C10 and C11 alkanes in BYL13, we found that Pdr1 R821S (Pdr1 mt2) could not improve tolerance to C9 alkane, which is inconsistent with a previous study.
This discrepancy is likely due to difference of strain background between BYL13 and KK-211 used in the previous study. To demonstrate that strain background can affect cell tolerance towards alkanes, we expressed either Pdr1 mt1 + Pdr3 mt or Pdr3 wt in BY4741, a parental strain of BYL13, and evaluated the cell growth in the presence of alkanes. Figure and Additional file: Figure S5 show that BY4741 expressing Pdr transcription factors grew better than BYL13 expressing the same Pdr transcription factors upon exposure to C10 and C11 alkanes.According to Mamnun and coworkers , Pdr1p and Pdr3p form homo- and hetero-dimers to mediate pleiotropic drug resistance in S. Cerevisiae, and these homo- and hetero-dimers could show diverse transcriptional activity to their target genes involved in tolerance to alkanes.
In line with the diversity of Pdr dimers and their transcriptional activity, Fig. And Additional file: Figure S3 indicate discrepant tolerance conferred by the individual and co-expressed Pdr transcription factors.Future efforts could be made to identify DNA-binding efficacy of Pdr1p- and Pdr3p-dimers as well as influences of various Pdr dimers on the transcriptome in response to alkanes, and to discriminate significance of each mechanism (alkanes efflux, membrane modifications, ROS reduction, and alleviation of membrane damage) in the Pdr transcription factors involving tolerance improvement towards alkane biofuels. In addition, a tool of global transcription machinery engineering (gTME) can be applied to construct Pdr transcription factor libraries and obtain phenotypes of resistance against a wide spectrum of biochemical molecules.
The site mutants Pdr1 F815S + Pdr3 Y276H (Pdr1 mt1 + Pdr3 mt) and the wild-type Pdr3p (Pdr3 wt) improved the tolerance of BYL13 and BY4741 to C10 and C11 alkanes, respectively. We found that the mechanisms underlying Pdr1p- and Pdr3p-mediated tolerance are multilayered.
As depicted in Fig., we hypothesize that Pdr1p and Pdr3p regulate genes involved in alkane efflux (e.g., SNQ2, and PDR5), stress responses (e.g., CTT1) and membrane modifications (e.g., ICT1) in the presence of C10 and C11 alkanes. The tolerance to alkanes was improved through (i) reduced intracellular alkanes contributed by alkane efflux (C10 and C11) and lower alkane import (C11 alkane), (ii) decreased ROS production probably contributed by lower alkane accumulation (C10 and C11 alkanes) and Ctt1p-mediated ROS decomposition (C10 and C11 alkanes), and (iii) alleviated membrane damage contributed to by membrane modifications (C10 alkane). These findings provide valuable insights into engineering alkane-tolerant yeast for improved alkane productivity. Fig. 7A schematic of proposed mechanisms on tolerance improvement towards C10 ( shaded in light blue) and C11 ( shaded in light orange) alkanes conferred by Pdr1p and Pdr3p.
Pdr1p and Pdr3p improve tolerance to C10 alkane likely through reducing membrane damage ( A, blue), alkane efflux ( B, green), and reducing ROS production ( C, orange); the tolerance to C11 alkane is improved likely through reducing C11 import due to potential membrane modifications ( D, purple), as well as ( B) and ( C) Methods. Strains and plasmids used in this study are listed in Table. Yeast cells were grown at 30 °C in YPD (10 g/l Yeast Extract, 20 g/l Peptone, and 20 g/l Dextrose), minimal medium (6.7 g/l Yeast Nitrogen Base, 20 g/l Dextrose, and 1.92 g/l Yeast synthetic dropout medium supplements without uracil), or induction medium (6.7 g/l Yeast Nitrogen Base, 10 g/l D-raffinose, 1.92 g/l Yeast synthetic dropout medium supplements without uracil, and appropriate amount of D-(+)-galactose).
Coli was grown at 37 °C in Luria–Bertani (LB) broth. Antibiotics (200 μg/ml G418, or 100 μg/ml Ampicillin) and appropriate amount of n-alkanes were added if necessary. Strains, plasmids, and genesDescriptionSourcesS.
Authors’ contributionsHL and MWC conceived the project and designed the experiments. HL carried out gene disruption, ROS and membrane integrity analyses, and participated in alkane detection and tolerance tests. NKPJ carried out Western blotting and participated in tolerance tests. WST carried out gene cloning and participated in alkane detection. RL carried out RNA extraction and qPCR analyses.
HL, SSJL and MWC wrote the manuscript. MWC supervised the project. All authors read and approved the final manuscript.
ResultsIn the presence of complex mixtures of toxic substances from spruce wood, transformants overexpressing YAP1 and STB5, TFs involved in oxidative stress response, exhibited enhanced relative growth rates amounting to 4.589 ± 0.261 and 1.455 ± 0.185, respectively. Other TFs identified as important for resistance included DAL81, GZF3, LEU3, PUT3, and WAR1. Potential overlapping functions of YAP1 and STB5 were investigated in experiments with permutations of deletions and overexpression of the two genes. YAP1 complemented STB5 with respect to resistance to 5-hydroxymethylfurfural, but had a distinct role with regard to resistance to coniferyl aldehyde as deletion of YAP1 rendered the cell incapable of resisting coniferyl aldehyde even if STB5 was overexpressed.
ConclusionsWe have investigated 30 deletion mutants and eight transformants overexpressing MDR transcription factors with regard to the roles the transcription factors play in the resistance to toxic concentrations of lignocellulose-derived substances. This work provides an overview of the involvement of thirty transcription factors in the resistance to lignocellulose-derived substances, shows distinct and complementary roles played by YAP1 and STB5, and offers directions for the engineering of robust yeast strains for fermentation processes based on lignocellulosic feedstocks. BackgroundLignocellulosic biomass is an abundant renewable resource for production of advanced biofuels and other products that can be obtained through microbial fermentation processes. Prior to saccharification and fermentation, the lignocellulose is typically pretreated to make the cellulose more accessible to cellulolytic enzymes.
During pretreatment a wide range of fermentation inhibitors are generated together with the fermentable sugars. The fermentation inhibitors, which include phenolic compounds, furan aldehydes, and aliphatic acids, are toxic to yeast cells and can cause various stress conditions during the ethanolic fermentation. Coniferyl aldehyde, furfural and HMF (5-hydroxymethyl-2-furaldehyde) are examples of lignocellulose-derived inhibitors the effects of which have been much studied in model experiments. The response to inhibitors includes transcriptional reprogramming of gene expression to provide a proteome that is better suited to cope with the induced stress.Multidrug resistance (MDR) is the ability to acquire simultaneous resistance to distinct drugs or chemicals of a wide variety of structures and function.
Saccharomyces cerevisiae has been extensively studied as a model organism with regard to MDR. In yeast, MDR membrane proteins are divided into two superfamilies: the ATP-binding cassette (ABC) superfamily and the major facilitator superfamily (MFS). They are regulated by various transcription factors (TFs) and are responsible for yeast cell survival under many kinds of stress conditions ,.TFs bind to unique DNA elements and cause either transcriptional activation or repression. Many transcriptional activators and repressors are involved in the regulation of the expression of MDR proteins, and they are classified into different families due to their structural or functional similarity.In our previous work, we found by deoxyribonucleic acid microarray analysis that the MDR TF Yap1p of S. Cerevisiae was related to resistance to lignocellulose-derived inhibitors.
We found that the overexpression of the gene resulted in enhanced resistance to coniferyl aldehyde, HMF and spruce wood hydrolysate. Yap1p is involved in MDR and is the main regulator in response to oxidative stress. We have studied the mechanism of transcriptional activation by Yap1p and the target genes of the TF.
However, the control of gene expression in response to stress conditions is mediated by several TFs. The exact roles of the TFs and their relationships to each other during stress response are still unclear.In this study, we selected 30 TFs that regulate MDR proteins from either the ATP-binding cassette (ABC) superfamily or the major facilitator superfamily (MFS), and investigated their involvement in the resistance to chemical stress induced by lignocellulose-derived inhibitors. The deletion mutants of the 30 TFs were screened in experiments covering simple inhibition scenarios (separate compounds including coniferyl aldehyde, furfural and HMF) and complex inhibition scenarios (using the pretreatment liquid from pretreated sugarcane bagasse and Norway spruce).
The involvements of the TFs in the response to the simple inhibition scenarios, which represent partially the inhibition effects of the pretreatment liquids, were compared with that of the pretreatment liquids. Eight TFs were selected and overexpressed, and the resistance of the transformants to the two pretreatment liquids was evaluated. Furthermore, the two TFs encoded by YAP1 and STB5 were studied in detail in experiments with permutations of deletions and overexpression of the two genes to investigate if they have distinct or overlapping roles.
This investigation elucidated the adaptation of yeast to harsh conditions prevailing during fermentation of pretreated lignocellulose to desired products, and is helpful for the design of genetically engineered yeast with improved performance in biomass conversion. This investigation is also helpful for understanding the roles of specific MDR-related transcription factors in the response to toxic compounds. Plasmid constructionThe vectors used in this study were derived from the episomal yeast shuttle vector pAJ401. The pAJ401 plasmid contains the ColE1 origin of replication, the β-lactamase gene, the 2 μ origin of replication, the URA3 selection marker and the PGK1 expression cassette. As a negative control, a plasmid denoted PB (plasmid backbone) was used (pAJ401 without any insert between the PGK1 promoter and the terminator ).
The pAJ401-derived plasmid with the YAP1 gene was constructed previously. The synthesis of seven other genes and the construction of the corresponding pAJ401 expression plasmids were performed by Gene Oracle (Santa Clara, CA, USA).
The integrity of the synthesized gene and the construction was checked by DNA sequencing. Preparation of plasmid DNA and transformation of S. CerevisiaeTo amplify the plasmids, competent E. Coli DH5α cells were transformed with the plasmids by using calcium chloride and heat-shock treatment.The E. Coli transformants were selected on LB agar plates with ampicillin. Transformants from the selective agar plates were grown at 37 °C in shake flasks with 50 ml of LB medium with ampicillin.
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Purification of plasmids was carried out with a Plasmid Purification Kit (Qiagen).To obtain the same strain background as in the experiments with deletion mutants, the auxotrophic S. Cerevisiae strain BY4741 was transformed with the three plasmids (negative control plasmid denoted PB, and expression plasmids of YAP1 and STB5) by electroporation. Electroporation was carried out as previously described. The auxotrophic S. Cerevisiae deletion mutant of YAP1 was transformed with the STB5 overexpression plasmid (the resulting transformant was named SIY), and the deletion mutant of STB5 was transformed with the YAP1 overexpression plasmid (the resulting transformant was named YIS), using the same electroporation method as described above.Three different colonies were picked for each of the transformants and evaluated in the microtitre plate experiments and flask experiments. Experiments with microtitre platesFive sets of experiments were carried out with the deletion mutants, and one set of experiments was carried out with the BY4741 transformants of the 8 transcription factors. Another set of experiments was for comparing STB5 and YAP1, and the YAP1 deletion mutant (M-Y), the STB5 deletion mutant (M-S), the YAP1 transformant of BY4741 (T-Y), the STB5 transformant of BY4741 (T-S), the SIY transformant and the YIS transformant were included in this set of experiment, as well as the two controls (BY4741 and the BY4741 transformant carrying the plasmid backbone, PB, without insert).
The resistance of the deletion mutants and the transformants was evaluated by comparing the relative growth rate, which was calculated based on cell growth (OD 620). For each deletion mutant or transformant, all sets of experiments were performed in triplicates. For the deletion mutants, technical triplicates were used with the same mutant. For the transformants, biological triplicates were used with three different colonies picked for each transformant. The average OD values of triplicates were used in the evaluation.
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Preparation of preculturesThe deletion mutants were inoculated in 50 ml Falcon tubes containing 10 ml of SC medium (with 200 mg/l uracil). Three different colonies of each transformant were selected and inoculated in different 50 ml Falcon tubes containing 10 ml of SC-Ura medium. The Falcon tubes were incubated at 30 °C with agitation. The cultures were harvested after an overnight cultivation, and inoculated again into 50 ml Falcon tubes containing 10 ml of SC. The cultures were incubated for 4–8 h, and were harvested in the exponential growth phase by centrifugation (Eppendorf 5810R, Eppendorf AG, Hamburg, Germany) at 8000 rpm for 2 min. The cells were then resuspended in an appropriate volume of sterilized deionized water to give a start inoculum with a biomass concentration of 0.12 g/l DW (dry weight).Hundred microliters of triple concentrated SC or SC-Ura medium and 100 ml of cell suspension were added to each well of a microtitre plate (Nunc, Roskilde, Denmark). In the experiments with the deletion mutants, 100 μl of inhibitor solution or diluted pretreatment liquid were added to the culture to a final concentration of 1.0 mM coniferyl aldehyde, 10.4 mM (1.0 g/l) furfural, and 14.0 mM HMF.
In the experiments with the transformants, final concentrations of 1.2 mM coniferyl aldehyde, 17.0 mM furfural and 24.0 mM HMF were used. Since coniferyl aldehyde is hard to dissolve in pure water, it was first dissolved in a few milliliters of ethanol before the addition of water. The final concentration of the ethanol added was below 0.1% (v/v), and its effect on yeast cell growth was negligible. In the experiments with microtitre plates, 15% (v/v) spruce pretreatment liquid or 10% (v/v) bagasse pretreatment liquid were used, and the pH of the pretreatment liquids was adjusted to 6.0 before adding them into the wells of the microtiter plates. The pH of the cultures was adjusted to 6.0 at the start of the experiment, and the final pH of the cultures was measured. The final volume in each well in all the microtitre plate experiments was 300 μl out of the maximum volume 330 μl.The cultivation was anaerobic as the oxygen was depleted shortly after the start of the cultivation.
Airproof adhesive film was used to seal the microtitre plates to avoid well-to-well contamination and sample evaporation, and to keep the anaerobic environment. Control wells filled with only culture medium were included to confirm there was no cross contamination. A start OD (optical density) was measured at 620 nm (Victor 2 1420 Multilabel Counter, Perkin Elmer, Waltham, MA, USA). The plate was then incubated at 30 °C in a shaker incubator (Ecotron, Infors AG, Bottmingen, Switzerland) with a shaking speed of 180 rpm. The OD was measured after 12 and 24 h, and extra time points (4, 9, 18, 36, 38, 40, 42, 48 and 60 h) in some sets of the experiments.
CalculationsIn the screening experiments with microtiter plates, while measuring the OD 620 value, the samples were not always within the optimal reading range of the plate reader. Therefore, a standard curve was used to establish an equation for correction of the OD 620 values.A yeast cell culture was diluted to seven different concentrations, and the diluted samples were measured with the plate reader. The standard curve was made for OD 620 value correction.
Calculations were carried out by using Matlab and the polynomial fitting method. The following equation was used for corrections. p1, p2, p3, p4, p5 = - 7.766, 23.749, - 3.345, 44.811, - 0.025The OD 620 values of the cultures were measured after 0, 12, and 24 h, and extra time points (4, 9, 18, 36, 38, 40, 42, 48 and 60 h) in some sets of experiments. After the corrections of the OD 620 values as described above, fitting curves of growth curves were made for each independent culture in each well of the microtitre plate. The fitting curves were made with Piecewise Cubic Hermite Interpolation (PCHI) in Matlab.
For each independent culture, a piecewise cubic function (monotone function) was made between t (time) and y (the corrected OD 620 value). Heatmap of relative growth rate in the screening experiments with deletion mutants. The deletion mutants were cultivated with 1.0 mM coniferyl aldehyde (CFA), 10.4 mM furfural (FUR), 14.0 mM HMF, sugarcane bagasse pretreatment liquid (BPL), and spruce pretreatment liquid (SPL). The data indicate: relative growth rate 1.5. The result with the NGG1 deletion mutant was not included in the figure, since the growth of the NGG1 deletion mutant in the SC medium was slower than that of BY4741.
The numerical data for relative growth are included in Additional file: Table S1Looking at the whole set of mutants, there were in total 12 deletion mutants (of STB5, YAP1, WAR1, PDR1, PDR8, RDR1, YRR1, YRM1, CAT8, GAL4, PUT3 and GZF3) that were more sensitive than the control to all the three specific compounds and to the two pretreatment liquids. There was one deletion mutant, that of MSN2, which was resistant to all the three specific compounds and to the two pretreatment liquids. The two furan aldehydes typically gave similar response in the screening experiment (for 27 out of the 29 TFs), while the response invoked by coniferyl aldehyde was frequently different from that of the two furan aldehydes (for 12 out of the 29 TFs) (Fig. ). The two PLs gave similar response (23 out of the 29 TFs). In some cases ( CRZ1, LEU3, DAL81), the response of the pretreatment liquids was similar to that of coniferyl aldehyde but not to that of the two furan aldehydes, while in other cases ( CIN5, MSN4, ADR1, ECM22, UPC2), the response of the pretreatment liquids was more similar to that of the furan aldehydes than to that of coniferyl aldehyde (Fig. ).The relative growth rates of the deletion mutants of STB5 and YAP1, which encode TFs directly related to oxidative stress, were lower than 0.9 when cultivated with the three specific inhibitors and the two pretreatment liquids (Fig. ).
In the presence of inhibitors and pretreatment liquids, the relative growth rates of the STB5 deletion mutant was always 1.1 when cultivated with the pretreatment liquids (Fig. ). The relative growth rates of the other five deletion mutants in this group were 1.1 with coniferyl aldehyde and pretreatment liquids, but. Microtitre plate experiment with transformantsEight of the TFs ( STB5, YAP1, WAR1, RPN4, CAT8, PDR8, PUT3 and GZF3), whose deletion mutants had relative growth rates under 0.7 (very sensitive) with both the sugarcane bagasse and the spruce pretreatment liquids (Additional file: Table S1), were overexpressed in S. Cerevisiae BY4741 under the control of the potent PGK1 promoter.
The tolerance of the transformants to the sugarcane bagasse pretreatment liquid and the spruce pretreatment liquid were examined with microtiter plates. Except for the transformants of RPN4, all the transformants showed enhanced tolerance to the pretreatment liquids compared with the control transformants (Fig. ). The relative growth rate of the transformants of YAP1 were the highest among all the transformants, and were 2.848 ± 0.153 and 2.746 ± 0.209 with the bagasse pretreatment liquid and the spruce pretreatment liquid, respectively. The transformants of STB5, the other transcription factor directly related to oxidative stress, also had relative growth rates 2.0 with the two pretreatment liquids. The overexpression of the transcription factors from other categories, such as WAR1 (acid stress adaption), PDR8 (pleiotropic drug resistance), CAT1 (carbon source responsive), PUT3 (amino-acid biosynthesis) and GZF3 (nitrogen catabolism), were found to enhance the tolerance of the yeast strains to the two pretreatment liquids (Fig. ). Microtitre-plate experiments and flask experiments with permutations of deletion and overexpression of STB5 and YAP1Deletion mutations of STB5 and YAP1, the two TFs related to oxidative stress adaption, were consistently more sensitive to specific inhibitors and to pretreatment liquids.
Further experiments were carried out to study their relationship with regard to inhibitor resistance and to see if it was possible to distinguish differences that the deletion mutant screening experiment (Fig. ) did not reveal. STB5 was overexpressed in the deletion mutant of YAP1 (transformant denoted SIY), and YAP1 was overexpressed in the deletion mutant of STB5 (transformant denoted YIS). In addition, the deletion mutants of YAP1 (M-Y) and STB5 (M-S), the control strain for mutants (BY4741), the control for transformants (BY4741-PB), and the transformants overexpressing YAP1 (T-Y) and STB5 (T-S) were included in the experiment. The cell growth (OD 620) of the deletion mutants and transformants was measured after 30 and 156 h of incubation (Table ). ABY4741, BY4741 host strain; BY4741-PB, BY4741 control transformant with plasmid backbone (PB) without insert; M-Y, deletion mutant of YAP1; M-S, deletion mutant of STB5; SIY, STB5 overexpressed in deletion mutant of YAP1; YIS, YAP1 overexpressed in deletion mutant of STB5; T-S, BY4741 transformant of STB5; T-Y, BY4741 transformant of YAP1bThe values were corrected as described in “” sectionAfter 30 h of incubation, the control transformant BY4741-PB did not exhibit more resistance than the host strain BY4741. Thus, as expected, transformation with the plasmid backbone did not lead to elevated resistance to inhibitors.
M-Y and M-S were more sensitive than the BY4741 control to coniferyl aldehyde, furfural, HMF and to the two pretreatment liquids after 30 h incubation. After 156 h incubation, M-Y did not grow in coniferyl aldehyde, and was more sensitive than M-S, which grew to the relatively high OD 620 of 0.68 (Table ). After 156 h of incubation in medium with HMF, M-S grew to an (OD 620) of only 0.16, and was more sensitive than M-Y, which grew to 0.90. This indicated that STB5 was more important than YAP1 for the adaptation of yeast to HMF.
After 30 h incubation, M-S was more sensitive to furfural than M-Y, but the two deletion mutants grew to a similar level after 156 h incubation. The sensitivity of SIY to the inhibitors and the pretreatment liquids were similar to that of M-Y. Neither M-Y nor SIY grew with 1.1 mM coniferyl aldehyde after 156 h incubation, indicating that YAP1 is vital for the resistance of yeast to coniferyl aldehyde, and it was not possible to substitute STB5 for YAP1 with regard to coniferyl aldehyde resistance. After 30 h incubation with inhibitors and pretreatment liquids, YIS grew poorer than BY4741 and BY4741-PB, but better than M-S (Table ). That indicated that YAP1 could only partially compensate for STB5 with regard to resistance against the inhibitors and the pretreatment liquids. After 156 h incubation with coniferyl aldehyde and HMF, the growth differences between YIS and M-S became larger.
With coniferyl aldehyde, YIS grew to OD 620 1.14, while M-S grew to 0.68. With HMF, YIS grew to OD 620 0.67, while M-S grew to only 0.16. The result indicated that overexpression of YAP1 in M-S could partially relieve the sensitivity of M-S to coniferyl aldehyde and HMF, and that YAP1 partially compensated for STB5 with regard to resistance against the two inhibitors.After 30 h incubation, T-S and T-Y were more resistant than BY4741-PB in most of the cases (except for that T-Y was slightly more sensitive than BY4741-PB to furfural) (Table ). Comparing T-S and SIY, T-S was more resistant than SIY after 30 h incubation with all the inhibitors and pretreatment liquids. T-Y was also more resistant than YIS in all the cases after 30 h incubation. The results indicated that even though either of YAP1 or STB5 was overexpressed, the deletion of the other TF of those two TFs would impair the resistance of yeast to the inhibitors and pretreatment liquids.
That meant that either YAP1 or STB5 was indispensable for good resistance.BY4741, M-Y, M-S, SIY and YIS were selected for cultivation in flasks. Coniferyl aldehyde and HMF were used to further investigate the different involvements of STB5 and YAP1 in the resistance of yeast to the two compounds. Cell growth, glucose consumption, ethanol production and cell viability were measured during the fermentation. BY4741, the deletion mutants, and the transformants grew similarly in culture medium without inhibitors (control medium). After 8 h cultivation, all mutants and transformants had entered the exponential phase, and after 16 h they had entered the stationary phase. Accordingly, after 16 h of cultivation the glucose in the control medium was below 2 g/l for all mutants and transformants (Fig. a). The resistance of the yeast cells to 1.1 mM coniferyl aldehyde was in an order BY4741 YIS M-S M-Y = SIY (Fig. b).
All five mutants and transformants were still in the lag phase after 16 h cultivation (Fig. b). BY4741 started to grow between 16 and 32 h, and reached the stationary phase before 32 h.
In accordance with this, the glucose was almost depleted by BY4741 between 16 and 32 h (Fig. b). YIS started to grow after 32 h cultivation, and reached the stationary phase before 64 h.
In agreement with that, YIS consumed almost all the glucose between 32 and 64 h (Fig. b). M-S entered the exponential phase after 88 h cultivation, and reached the stationary phase before 112 h.
The glucose was accordingly consumed during this period. M-Y and SIY could not grow with 1.1 mM coniferyl aldehyde after 136 h incubation, and the glucose was not consumed by M-Y and SIY (Fig. b). The resistance of the yeast cells to 24.0 mM HMF was in an order BY4741 M-Y SIY YIS M-S (Fig. c).
BY4741 and M-Y grew similarly until 64 h cultivation, and both reached an OD of around 0.6 at 64 h (Fig. c). However, BY4741 grew faster than M-Y after 64 h, and reached OD 0.87 at 136 h, while M-Y reached OD 0.65 at the same time. The glucose in culture medium with HMF was consumed before 88 h by both BY4741 and M-Y. SIY grew slower than M-Y.
The OD of the SIY culture was 0.52 at 64 h, and 0.56 at 136 h. The glucose concentration of SIY in medium with HMF was 4.56 g/l at 64 h, and 2.72 g/l glucose was left at 136 h. YIS grew slower than SIY.
The OD of YIS reached 0.41 after 136 h cultivation, and 8.71 g/l glucose was left in the culture medium at 136 h. Even after 136 h of cultivation, M-S did not grow with 24.0 mM HMF, and, accordingly, almost no glucose was consumed (Fig. c). Cell growth and glucose consumption during flask experiments. BY4741, M-Y, M-S, SIY and YIS were cultivated in control medium ( a), medium with 1.1 mM coniferyl aldehyde ( b), and medium with 24 mM HMF ( c). The data indicate: OD of BY4741 (filled black square), M-Y (filled red circle), M-S (filled orange triangle), SIY (filled inverted blue triangle), YIS (filled green rhombus), and glucose concentration of BY4741 (open black square), M-Y (open red circle), M-S (open orange triangle), SIY (open inverted blue triangle), YIS (open green rhombus)With the control medium, the volumetric ethanol productivity ( Q 16h) of the five mutants and transformants reached about 0.55 g/l/h (Table ).
With coniferyl aldehyde in the culture medium, none of the five mutants and transformants produced any ethanol before 16 h. After 136 h fermentation with coniferyl aldehyde, the ethanol yields ( Y E/G) of BY4741, M-S and YIS were 0.349, 0.375, and 0.317 g/g, respectively, while the ethanol yields of the M-Y and the SIY cultures were not even detectable.
When cultivated with HMF, Q 16h of BY4741, M-Y and SIY were about 0.1 g/l/h, which was much higher than that of YIS. M-S did not produce any ethanol in the 136 h fermentation with HMF. NDTD none detectedaBY4741, BY4741 host strain; M-Y, deletion mutant of YAP1; M-S, deletion mutant of STB5; SIY, STB5 overexpressed in deletion mutant of YAP1; YIS, YAP1 overexpressed in deletion mutant of STB5bCalculations based on the results within the first 16 h of fermentation. Q is the volumetric ethanol productivity, and q is the specific ethanol productivityc Y E/G is the ethanol yield on the initial amount of fermentable sugar (glucose).
The calculations are based on the maximum ethanol concentrations obtained within 136 h of fermentationdN/A not applicable; no inhibitor was added to the culture mediumThe cell viability was measured after 8 h of cultivation with coniferyl aldehyde, HMF and the control medium. In all the cultures, less than 10% of the cells were dead and had no intact cell membranes.
However, the portion of cells with intact cell membrane was lower in cultures with coniferyl aldehyde than in cultures with control medium, especially with regard to M-Y. DiscussionThe engineering of microbial strains has been an important technique for production of biofuels and bioproducts –. Knowledge of resistance of microorganisms to stress conditions is important for engineering robust microbial strains. Besides the development of novel carbohydrate-utilization pathways and the overexpression of cellulase in S.
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Cerevisiae , development of robust yeast strains with greater resistance to fermentation inhibitors is important for efficient conversion of lignocellulosic feedstocks to cellulosic ethanol and other commodities. A better understanding of the regulation of the resistance of yeast to lignocellulose-derived inhibitors, as provided by this investigation, facilitates characterization and engineering of hyper-resistant strains.Using deletion mutants we screened the involvement of 29 MDR-related TFs with respect to resistance to three model inhibitors, coniferyl aldehyde, furfural, and HMF, and two pretreatment liquids, one from sugarcane bagasse and the other from spruce. All deletion mutants investigated showed increased sensitivity or increased resistance to at least one compound or pretreatment liquid, indicating that all those MDR-related TFs in some way were involved in the resistance to lignocellulose-derived inhibitors.As 13 out of 29 TFs gave the same response for all the three model inhibitors as for the two pretreatment liquids, the set of model inhibitors chosen was well connected with the inhibitory effects of complex lignocellulosic hydrolysates. However, the relative growth rate of the deletion mutant of CAD1 was 1.1 when cultivated with the pretreatment liquids, but between 0.9 and 1.1 (not resistant) when cultivated with the three specific inhibitors.
The results therefore also indicate that the three model inhibitors do not cover all inhibitory effects of the pretreatment liquids, which is expected as there are many other inhibitors that can affect yeast cell growth ,.Using comparative transcriptome analysis, YAP1, PDR1, PDR3, RPN4, and HSF1 were proposed to be key TFs of yeast in response to stress induced by HMF. The TFs included in our study cover four of the five proposed key TFs for HMF resistance, and the results show that it was none of them but instead STB5 that was most important with regard to HMF resistance. The discrepancy is probably due to the different approaches taken.
We evaluated the involvement of the TFs in the response to the inhibitors through the relative growth rates of deletion mutants and transformants, not through transcriptomics as Ma and Liu. The approach taken in our study seems more advantageous for finding proteins that are truly important for resistance, as products of genes that are not much induced in microarray analysis studies (e.g. STB5) might be very important for the adaption to the inhibitor. Furthermore, transcripts differ in stability and their abundance may not directly reflect the abundance of the corresponding proteins. The effect of different TFs on transcription levels may also differ.The RPN4 transcription factor stimulates expression of proteasome genes, and is rapidly degraded by the 26S proteasome. Disruption of the Rpn4-induced proteasome expression in S. Cerevisiae reduces cell viability under stressed conditions, and proteasomal degradation of Rpn4p in S.
Cerevisiae is critical for cell viability under stressed conditions. ConclusionsIn this study, we have profiled a set of 29 deletion mutants of TFs related to MDR with regard to their roles in the resistance to toxic concentrations of lignocellulose-derived substances. All of the TFs studied were found to be involved in the resistance of yeast to coniferyl aldehyde, furfural, HMF, sugarcane bagasse pretreatment liquid or spruce pretreatment liquid. The overexpression of seven of the transcription factors improved the tolerance of yeast cells to the inhibitors in both sugarcane bagasse and spruce hydrolysates. Some deletion mutants (of e.g. MSN2 and LEU3) seemed to be more resistant to the lignocellulose-derived inhibitors.
Further experiments are needed to investigate the potential significance of this phenomenon. Moreover, the roles of STB5 and YAP1, genes encoding TFs involved in oxidative stress response, were elucidated in detail with regard to resistance to coniferyl aldehyde and HMF. STB5 was most important for yeast adaption to HMF, while YAP1 was most important for the adaption to coniferyl aldehyde. The roles of the two TFs were complementary with regard to HMF resistance, but distinct with regard to coniferyl aldehyde resistance. The complementarity may be attributed to the roles played by the TFs encoded by STB5 and YAP1 in the regulation of the pentose phosphate pathway, while the distinct role played by the YAP1 transcription factor in the resistance to coniferyl aldehyde may be attributed to its regulation of MDR proteins not affected by STB5. Our study clearly shows the important roles played by the MDR transcription factors in resistance to the multiple lignocellulose-derived inhibitors in hydrolysates. The transcription factors which are involved in the resistance of yeast to the inhibitors can be overexpressed to construct robust strain in biofuels and biochemicals production from lignocellulosic biomass.
We predict that genes belonging to two functional categories (as defined by MIPS Functional Categories) are potentially important for the resistance of yeast to the inhibitors. Those genes can be further studied with regard to engineering the resistance of yeast. We also show the important role of STB5 in the resistance to furan aldehydes, something that has been overlooked previously in studies based on transcriptomics. The information on the complementary and distinct roles of the transcription factors with regard to the resistance to lignocellulose-derived inhibitors is useful in future investigations on engineering hyper-resistant yeast for biomass conversion.