Comparative transcriptomics analysis of Zygosaccharomyces mellis under high-glucose stress

Gongling Liu,Xinyu Bi,Chngli To,Yongto Fei,?,Sujun Go,Jinglong Ling,Weidong Bi,?College of Light Industry nd Food Science,Zhongki University of Agriculture nd Engineering,Gungzhou,510225,Chin

bGuangdong Province Key Laboratory for Biotechnology Drug Candidates,School of Biosciences and Biopharmaceutics,Guangdong Pharmaceutical University,Guangzhou,510006,China

Keywords:

Zygosaccharomyces mellis

Transcriptomics

High-glucose tolerance

HOG-MAPK

Trehalose

ABSTRACT

The high-glucose tolerance of yeast is the main factor determining the efficiency of high-density alcohol fermentation.Zygosaccharomyces mellis LGL-1 isolated from honey could survive under 700 g/L highglucose stress and its tolerant characteristics were identified in our previous study.This study was performed to explore and clarify the high-glucose tolerance mechanism of Z.mellis LGL-1.Comparative transcriptomic analysis was used to analyze the genes with differential expression in Z.mellis under high-glucose conditions of 300,500 and 700 g/L.With 300 g/L samples as reference,there were 937 and 2380 differentially expressed genes(DEGs)in the 500 and 700 g/L samples,respectively.Meanwhile,there was 825 significant DEGs in the 700 g/L samples compared with that of the 500 g/L samples.The result revealed that transcriptional changes in multiple metabolic pathways occur in response to highglucose stress.q-RT PCR analysis further confirmed that several stress response pathways,such as the high osmolarity glycerol mitogen-activated protein kinase(HOG-MAPK)signal transduction pathway,trehalose synthesis pathway and oxidative stress response are closely related to high-glucose tolerance in Z.mellis.This study clarifies mechanisms of Z.mellis in response to high-glucose osmotic stress,providing theoretical basis for the process control of high-density alcohol fermentation.

1.Introduction

High-density fermentation of yeast has many advantages in field of alcohol production in comparison with traditional methods[1].This technology could not only increase alcohol production and equipment utilization[2],but also reduce the consumption of labor and energy[3].The resistance of yeast likeSaccharomyces cerevisiaeto high osmotic pressure caused by high substrate concentration directly affected the yield of alcohol[4,5].Under high-glucose stress,some yeasts likeS.cerevisiaeandZygosaccharomyces rouxiican tolerate the harsh condition by adjusting reproduction modes and producing protective metabolites,such as synthetic glycerol,trehalose,and the related oxidases[6,7].In respects of transcriptomic level,several studies in terms of yeast response to high-sugar stress have been reported.The microarray analysis indicated thatS.cerevisiaePSY316 had an upregulation of trehalose and glycerol biosynthetic genes when exposed to 20%(m/V)glucose[8].Recent study also revealed that when cultured under hypertonic conditions,S.cerevisiaecould protect cells by accumulating intracellular trehalose and regulating expression of genes involved in the integrity of the recombinant yeast,as well as the expression of catalase and superoxide dismutase genes,which could increase the fermentation capacity in concentrated mash[9].Furthermore,high-glucose stress was found to up-regulate genes of the glycolytic and pentose phosphate pathway and down-regulate the genes involved in de novo biosynthesis of pyrimidines,purines,lysine and histidine inS.cerevisiae[10].Genes ofS.cerevisiaeinvolved in respiration and HOG pathway have important transcriptomic changes under high-sugar stress[11].Besides,it was reported thatZygosaccharomyces rouxiistrains with a more rigid cell wall tends to be less osmotolerant than those having a more flexible cell wall[12].

In our previous study,strainZygosaccharomyces mellisLGL-1(also namedZ.mellis6–7431)isolated from honey was found to produce maximum concentration of ethanol at 500 g/L glucose concentration and tolerate 700 g/L high-glucose stress,indicating its potential application in high-density fermentation[13].Meanwhile,the metabolic characteristics ofZ.mellisLGL-1 in response to high-glucose stress were investigated, finding that this strain could change reproduction pattern, produce glycerol and trehalose under the osmotic stress[14].Currently,research on the high-glucose tolerance mechanism ofZ.mellisis limited to the metabolic level,and there is no comprehensive recognition of related metabolic pathways and crucial genes[15].In this paper,transcriptomic analysis was used to analyze the differences of gene expression under high-glucose stress conditions.The accuracy of transcriptomic sequencing was verified by real-time quantitative PCR(q-RT PCR) to elucidate the osmotolerant mechanism ofZ.mellistolerance to high concentrations of glucose.

2.Materials and Methods

2.1.Sample preparation

Z.mellisLGL-1was preserved in the China Type Culture Collection under the accession number of CCTCC M2015545.The strain stored at?80?C refrigerator was taken out and placed at room temperature for half an hour.Z.mellisLGL-1 was streaked in Potato Dextrose Agar(PDA)with glucose concentration of 100 g/L and then cultured at 28?C for 48 h.Then the single colony was picked up and inoculated in the 100 mL medium with glucose concentration of 300,500 and 700 g/L,respectively.The inoculated medium was cultured in a shake incubator at 28?C,100 r/min until the mid-log phase.The cells were washed with sterile water and subjected to RNA extraction according to the instructions of the fungal transcriptome RNA extraction kit.

2.2.Transcriptome sequencing

The medium with glucose concentration of 300,500 and 700 g/L were prepared.Each glucose concentration gradient has three parallel samples. A total of 9 samples were marked by following abbreviations:L-30-1,L-30-2,L-30-3,L-50-1,L-50-2,L-50-3,L-70-1,L-70-2,and L-70-3.Total RNA ofZ.melliscollected at midlog phase were extracted.The enrichment of mRNA was carried out with magnetic beads with oligo(dT).The first cDNA strand was synthesized using 6-nucleotide random primers with short fragments of mRNA as template.Then the second cDNA strand was synthesized using buffer A,dNTPs,RNase H,and DNA polymerase I.The cDNA was then purified using the QIAquick PCR Kit(Qiagen,Germany Hilden).The resulting cDNA end was repaired and added with base A,then recovered with agarose gel electrophoresis. PCR amplification was performed to construct DNA library.After that,the library sequencing was performed using Illumina HiSeqTM2000(Illumina,San Diego,US).The transcriptome sequencing raw data have been deposited in Sequence Read Archive(SRA)under accession number PRJNA523100.

2.3.Real-time PCR

The target gene sequences(30)were selected,using three internal reference genes of aspartyl protease,adenosylmethionine-8-amino-7-oxononanoate transaminase and alpha-trehalase for quantitative analysis.The corresponding primers were designed and synthesized based on the above sequences(Table S1).The realtime PCR was performed with ExTaq RT PCR Kit and SYBR green dye(TaKaRa,China).The reverse transcript was diluted as template to amplify the internal reference genes and the target genes according to the requirements of the fluorescence quantitative system for the concentration of cDNA.The gene expression levels were analyzed using a Bio-Rad CFX96 real-time PCR detection system(Bio-Rad,Hercules,USA).Real-time PCR protocols were as follows:preheating with 2 min at 94?C;45 cycles with denaturing time of 5 s at 94?C,annealing time of 15 s at 60?C,and an elongation time of 10 s at 72?C.The changes in gene expression were calculated and compared with the sequencing results of the transcriptome[16].Each gene was sequenced three times in each sample.

3.Results

3.1.Sequencing,assembly and annotation of the LGL-1 transcriptome

A total of 523 million raw reads were obtained,and 517 million clean reads were remained.After low-mass sequences were removed,the resulting reads with high quality were assembled to obtian 6257 unigenes with GC content of 39%.The normal distribution of N50 was 3003,indicating that the sequencing data was highly readable.The average length of each unigene was 1719 bp.The assembled sequences were annotated in several databases,including NR database,Swiss-Prot database,KOG database,and KEGG database.There were 2525 unigenes identified in the four databases among the 6257 annotated unigenes,accounting for 40.35% of the reference transcript genes.The reference transcriptome was annotated to 6810 genes of cellular components,3734 genes of molecular functions,and 7733 genes of biological processes by GO analysis(Fig.1).Under the cellular component classification,genes were mainly distributed in cell part,organelle and cell.Under the molecular function classification,genes were mainly distributed in binding and catalytic activity.Under the classification of biological process,genes were mainly concentrated in cellular process,metabolic process and single-organism process(Fig.1).

3.2.DEGs in LGL-1 under different high-glucose stresses

The up-regulated or down-regulated genes were identified with FDR＜0.05 and an absolute value of log2Ratio＞1 as filtering thresholds[17].Using the 300 g/L glucose concentration as reference,937 DEGs were identified under the 500 g/L high-glucose stress condition,of which 780 genes were significantly up-regulated and 157 genes were down-regulated.Under the 700 g/L high-glucose stress,2380 DEGs were detected with 1918 up-regulated genes and 462 down-regulated genes.Using the 500 g/L glucose concentration as control,825 DEGs were identified under the 700 g/L high-glucose stress condition with 495 up-regulated genes and 462 down-regulated genes(Table 1).

3.3.DEG analysis in GO and KEGG

Compared with the gene expression of LGL-1under 300 g/L glucose concentration,DEGs under 500 g/L high-glucose stress were mainly annotated to “cell”,“cell part”and “organelle”under the cell component classification, “binding”and “catalytic activity”under the molecular function classification,and “cellular process”,“metabolic process”,and “single-organism process”under the biological process classification(Fig.2c).Compared with the gene expression under 500 g/L glucose concentration,DEGs of LGL-1 under 700 g/L high-glucose stress were also annotated to the same eight parts under three different classification(Fig.2b).Compared with the gene expression under 300 g/L glucose concentration,DEGs of LGL-1 under 700 g/L high-glucose stress were mainly annotated to the same parts as the above groups under cellular component classification. But the DEGs in the following items were different from that of previous groups,such as “ingredients”and“macromolecular compounds”under the molecular function classification,and“response to stimulus”,“l(fā)ocalization”,and“biological regulation”under the biological process classification(Fig.2a).

Fig.1.Reference Functional Group Classification of GO Functional Groups.

Table 1DEGs statistics of Z.mellis LGL-1 under high glucose conditions.

The most amount of DEGs in the L-30-VS-L-50,L-50-VS-L-70 and L-30-VS-L-70 groups were classified in the part of“metabolic pathways”.There are 2380 DEGs that identified in the above three glucose concentration groups,which were annotated to 75,75,and 108 metabolic pathways,respectively.As the glucose concentration increased,the number of DEGs annotated to the “metabolic pathways”significantly improved.In addition,the DEGs of“response to stimulus”increased significantly to 143 in 700 g/L glucose compared with that of 300 g/L glucose while DEGs of other two groups were only 38 and 50,respectively(Fig.2).Furthermore,the DEGs involved in cell growth and biosynthesis of LGL-1 changed evidently under high-glucose stress,including“ribosome biogenesis in eukaryotes”, “cell cycle yeast”,“meiosis yeast”,and “biosynthesis of secondary metabolites”.Besides,highglucose stress also influences the amino acid and protein synthesis of LGL-1,including “biosynthesis of amino acids”,“protein processing in endoplasmic reticulum”,“biosynthesis of antibiotics”and“microbial metabolism in diverse environments”.

When the glucose concentration increased from 300 g/L to 700 g/L,the number of DEGs(2380)including up-and downregulated genes in LGL-1 were higher than that of other groups(Table 2).The L-30-VS-L-70 group has the largest number of DEGs in both of up-regulated and down-regulated genes.In respects ofup-regulated genes,the number was the least(495)when glucose concentration increased from 500 g/L to 700 g/L.In terms of downregulated genes,the least number of gene expression changes(157)was in the group of L-30-VS-L-50.This result indicated that the number of DEGs was in direct proportion to osmotic stress caused by glucose concentration,suggesting that LGL-1 can improve the expression of multiple genes to make the cell adapt to a highglucose,or converting the glucose of medium into other protective substances(glycerol and trehalose),thereby reducing the influence of high osmotic pressure on the cells[18].

Table 2The number of up-and down-regulated in different metabolic pathway by KEGG analysis.

Table 2(Continued)

3.4.Confirmation of unigene expression using q-PCR analysis

Based on the analysis of transcriptome,a total of 30 DEGs involved in the HOG-MAPK signal transduction pathway,purine metabolism,trehalose metabolism and other biological processes were determined.There are 30 genes with significant changes in expression,which consisted 25 clearly up-regulated and 5 significantly down-regulated genes.The q-PCR analysis was displayed in Table 3,which indicated that the results analyzed with the two methods were basically consistent.

4.Discussion

When exposed to hyperosmotic stress,a series of complex adaptive procedures are initiated in yeast,including extension of cell-cycle progression,changes of transcription and translation,and the synthesis of osmolyte glycerol[19].These responses are mainly controlled by HOG-MAPK pathway which is evolutionarily conserved signaling units in diverse eukaryotic organisms.The synthesis and accumulation of the intracellular compatible osmolyte glycerol is an important adaptive way of HOG-MAPK activated by hyperosmotic stress[18].The key genes in HOG pathway were identified(Fig.3),which includeshog(mitogen-activated protein kinase),gpd(glycerol-3-phosphate dehydrogenase),sln(osmosensing histidine protein kinase),sho(osmosensor),ssk(mitogen-activated protein kinase),pck(phosphoenolpyruvate carboxykinase),andmsb(signaling mucin).With 300 g/L glucose concentration as reference,the expression levels ofsho1,ssk2andgpdgenes in LGL-1 under 700 g/L high-glucose stress were significantly improved with 3.51,2.66,7.33 times,respectively(Fig.3),which lead to the accumulation of glycerol to protect the cell of LGL-1.Besides,Hog1 was able to control gene expression by regulating transcription factors,such asmsn1,msn4,msn5(stress-responsive transcriptional activator),andhot(highosmolarity-induced transcription protein)which are responsible for controlling the expression of a series of osmotic responsive genes[20,21].With the glucose concentrations of 300and500g/Las references,themsn5gene expression level increased significantly under the 700 g/L high-glucose stress but there is no significant change in the expression level ofmsn1andmsn4genes.The similar mechanisms were also found inZ.rouxiiandS.cerevisiaewhen resisting the high osmotic environment by upregulating expression of a series of genes such asHog1,gpdandmsn2in HOG-MAPK pathway[22].Therefore,the HOG pathway of LGL-1 was activated as an important way to protect the cell from high-glucose osmotic stress.

Trehalose is common used as energy substance that provides energy for the anabolic metabolism of organisms[23].Besides,it also plays an important role in protecting the integrity of yeast cells from various injuries caused by osmotic stress due to its special physical properties[24].There are many genes participating in the metabolism of trehalose,which includestps(trehalose-phosphate synthase),otsB(trehalose 6-phosphate phosphatase complex),nth(alpha-trehalase),tsl(trehalose synthase complex regulatory sub-unit)andath(alpha-trehalase)[25].It has been reported that knockout of theathgene and overexpression of thetpsgene could increase the intracellular concentration of trehalose,and then improving the hyperosmolar tolerance[26].Thetps,tsl,ath,andnthgenes were identified in the LGL-1,andtps1andotsBgenes were significantly up-regulated with 4.41 and 2.96 times respectively,under 700 g/L high-glucose stress with 300 g/L glucose concentrations as references(Fig.4).Our previous study showed thatZ.mellisLGL-1 could synthesize trehalose under high sugar conditions following the same trend of the cell growth[14],the phenomenon of which were also found in several yeast species likeS.cerevisiaeandTorulaspora delbrueckii[27].It is demonstrated thatS.cerevisiaeis more tolerant to osmotic conditions during stationary than exponential phase when it mainlyproduces trehalose[28].Therefore,the LGL-1 activated the trehalose synthesis pathway by increasing the expression of key genes such astps1andotsBunder the high-glucose stress of 700g/L,leading to the accumulation of intracellular trehalose to tolerate the high-glucose stress.

Table 3Verification of the RNA-seq results by qPCR.

High osmotic pressure brought damage to genetic materials,which promote the synthesis of DNA in the fission yeast to offset the deficiency of DNA[29].Several genes involved in the DNA synthesis were identified in honey-associated yeast,includingamd 1(adenylosuccinate synthetase),ade 12,ade 13(adenylosuccinate synthetase),apt 1,andpnp 1(purine nucleoside phosphorylase)(Fig.5).The protein coded by geneamd1participated in the first pathway of IMP biosynthesis from AMP[30].With 300 g/L glucose concentration as a reference,theamd1,ade12andade13genes under 500 and 700 g/L high-glucose stress were both upregulated with the increase of glucose concentration to respond to the destruction of DNA caused by high osmotic pressure.The expression level ofade12andade13were significantly increased under700g/L high-glucose stress(Table3).These genes are responsible for catalyzing the first committed step in the biosynthesis of AMP from IMP,which plays an important role in the de novo pathway and in the salvage pathway of purine nucleotide biosynthesis[31].The stress inducible DNA repair system was found in many yeast such asS.cerevisiae,Z.rouxiiandHortaea werneckii[32–34].These genes involved in the synthesis of DNA were activated to counteract with damage of DNA when LGL-1 was exposed to high osmotic pressure.

Fig.2.DEGs in LGL-1at different concentration of glucose based on the GO analysis.(a)DEGs in Z.mellis LGL-1 at the 300 and 700 g/L glucose concentrations,(b)DEGs in Z.mellis LGL-1 at the 500 and 700 g/L glucose concentrations,(c)DEGs in Z.mellis LGL-1 at the 300 and 500 g/L glucose concentrations.

Fig.3.qPCR results in the HOG-MAPK signal transduction pathway.The red font means up-regulation genes.The green font means down-regulation gene.

Fig.4.qPCR results in trehalose metabolism.The red font means up-regulation genes.The green font means down-regulation genes.

Fig.5.qPCR results in purine metabolism.The red font means up-regulation genes.The green font means down-regulation genes.

The comparative transcriptomic analysis ofZ.mellisLGL-1 confirmed that HOG-MAPK signal transduction pathway,purine metabolism pathway and trehalose synthesis pathway are closely related to high-glucose tolerance.The organic osmolytes mentioned in the above pathways,including glycerol and trehalose,protect yeast from osmostress,not only by impeding water ef flux and retaining osmotic balance,but also by playing important roles in antioxidation and stabilization of intracellular proteins[35].A number of genes that protect cells from oxidative damage are up-regulated under osmotic stress,which have similar protective functions as these osmolytes[36].Multiple genes that involved in oxidative stress were identified,includingyap3(DNA-binding transcription factor),hsp(heat shock protein),sod(plasma membrane sodium ion/proton antiporter),gre2(NADPH-dependent methylglyoxal reductase),cat(carnitine O-acetyltransferase),ctt(catalase T),andaft(DNA-binding transcription factor).Geneyap3is involved in oxidative stress response and redox homeostasis as a transcription activator,regulating the transcription of genes encoding antioxidant enzymes and components of the cellular thiol-reducing pathways.Fouryap(DNA-binding transcription factor)genes were identified,which includes oneyap1gene,twoyap3genes,and oneyap1801gene.The genesyap3regulating the expression of other hyperosmotic stress genes such ashsp,sod2andgrein LGL-1 is significantly up-regulated under 700g/L glucose stress compared with that of 300 g/L glucose concentration(Table 2).The expression levels of thehsp104gene andsod2gene increased significantly while the expression levels of other genes did not display evident fluctuations.Genesod2has function of destroying superoxide anion radicals which are toxic to biological systems but normally produced within the cells[37].The up-regulation ofsod2gene can improve biosynthesis of antioxidative compounds to protect cells from oxidative damage under high-glucose stress.With 300 g/L glucose concentration as reference,the expression level ofhsp104gene increased approximately 1.5 times under 700g/L high-glucose stress,andhsp26gene also increased evidently under 500 and 700 g/L high-glucose stress(Table 2).Up-regulation of geneshsp26andhsp104that encode the corresponding chaperones can protect the yeast cells from damage by protein denaturation under high-glucose osmotic pressure.Besides,13gregenes including 12gre2 genes and onegre3gene were identified in LGL-1.Using the 300 g/L glucose concentration as reference,the expression level ofgre2gene was up-regulated at the 700 g/L high-glucose stress(P＜0.05),and that ofgre3did not fluctuated significantly.The genegre2coding for NADPH-dependent methylglyoxal reductase was mainly involved in the redox metabolism of irreversibly reducing the cytotoxic compound methylglyoxal(MG,2-oxopropanal)to(S)-lactaldehyde as an alternative to detoxification of MG under stress conditions,such as oxidative stress,osmotic stress,and heat stress[38].Under high-glucose osmotic stress,LGL-1 was forced to activate the general stress responses like oxidative stress to protect the cells from osmotic stress.

The Cell Wall Integrity(CWI)pathway is a conserved pathway in yeast[39].This pathway amplifies cell membrane signaling,then prompting the cell to respond as soon as possible when experiencing the external stress environment[40].In LGL-1,genes related to the CWI pathway were identified,such asslt(mitogen-activated serine/threonine-protein kinase),kre(cell wall biosynthesis protein),bre(proliferating cell nuclear)andsap(synaptosomal-associated protein).Using the 300 g/L glucose concentration as reference,the expression level of thekre5gene increased exponentially under 500 and 700 g/L high-glucose stress(Table2).Genekre5is indispensable in the synthesis of(1→6)-β-DGlucan in the cell wall[41].The integrity of cell wall was broken for rapid loss of water under high-glucose stress,which promote the expression ofkre5gene to synthesize(1→6)-β-D-Glucan,repairing the aberrant cell wall.Besides,the expression level ofslt2gene that was involved in a signal transduction pathway important for morphogenesis in yeast cell[42]increased significantly under the 700 g/L high-glucose stress(Table 2).Thesapgenes were reported to be involved in chromosome organization and integrity where it is involved in chromosome segregation[43].The related expression level of genessap1,sap190,andsap155also increased exponentially to make sure the chromosome integrity in the reproduction under high-glucose stress(Table 2).As a result,the overexpression of these genes in the CWI pathway of honey-joint yeast could made this strain respond rapidly,further keeping the integrity of chromosome and cell wall,enhancing the tolerance of the LGL-1 in high-glucose stress environment.

The CCR4-NOT complex is an evolutionarily conserved multisubunit complex, which is responsible for regulating gene expression at various levels,including transcription initiation,mRNA elongation,deadenylation and subsequent degradation of mRNA in the level of transcription, and even translation and protein degradation at translation level[44].In this study,threecaf(CCR4-NOT core subunit)genes includingcaf40,caf130,andcaf120,were identified,which act as general transcription factor in the nucleus and the major mRNA deadenylase involved in mRNA turnover in the cytoplasm.In the differential expression statistics,the expression levels ofcaf40andcaf130were increased exponentially under high-glucose stress at 700 g/L,using the 300 g/L glucose concentration as a reference(Table 2).The result suggested that CCR4-NOT in the cell of LGL-1 can act as general stress response by regulating expression of genes in the transcriptional and translational level.

Proline,glutamic acid and its derivatives can be used as intracellular compatible substances,which were conducive to maintain cell activity of microorganisms in hyperosmotic stress environment[45,46].The key genes responsible for amino acid absorption and conversion areput(1-pyrroline-5-carboxylate dehydrogenase)andgap(amino acid permease)[47].Eightputgenes,such asput1,put2,put3,andput4,and onegap1gene were identified in LGL-1.Using the 300 g/L glucose concentration as a reference,theput1,put2,put3andput4genes expression under 500 and 700 g/L highglucose stress were multiple-fold up-regulated with the increase of glucose concentration(Table 2).The genes ofputwere involved in subpathway that convertsL-proline intoL-glutamate,a more effective compatible substance under osmotic stress[48].However,the expression level of genegap1coding for amino acid permease was down-regulated,the similar result of which was reported inS.cerevisiaeunder high-salt concentration[49].Although amino acid was compatible osmolytes that contribute to adaptation to osmotic stress,glycerol and trehalose seems to be more important compatible osmolyte forS.cerevisiaein the presence of high osmolarity[20].As a result, except the confirmed pathways of HOGMAPK,trehalose synthesis,and purine metabolism,the pathways of oxidative stress response,CCR4–NOT complex,cell wall integrity(CWI)pathway also played important role in the response to highglucose osmotic stress.However,the most contributing pathway in response to high-glucose stress remains unsolved and need to be further determined in the following study.The results of this study illustrates the mechanisms ofZ.mellisin response to high-glucose osmotic stress,providing theoretical basis for the process control of high-density alcohol fermentation.

Funding

This work was supported by Key-Area Research and Development Program of Guangdong Province(2018B020206001);Guangdong Provincial Agricultural Science and Technology Innovation and Extension Project in 2019(2019KJ101);National key research and development plan(2018YFC1604105);National Natural Science Foundation of China(81703053).

Appendix A.Supplementary data

Supplementary material related to this article can be found,in the online version,at doi:https://doi.org/10.1016/j.fshw.2020.05.006.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.