APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY

Cellodextrin transporters play important roles in cellulaseinduction in the cellulolytic fungus Penicillium oxalicum

Jie Li & Guodong Liu & Mei Chen & Zhonghai Li &Yuqi Qin & Yinbo Qu

Received: 2 July 2013 /Revised: 25 September 2013 /Accepted: 27 September 2013 /Published online: 17 October 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Cellodextrin transporters (cellodextrin permeases)have been identified in fungi in recent years. However, thefunctions of these transporters in cellulose utilization andcellulase expression have not been well studied. In this study,three cellodextrin transporters, namely, CdtC, CdtD, andCdtG, in the cellulolytic fungus Penicillium oxalicum (for-mally was classified as P. decumbens ) were identified, andtheir functions were analyzed. The deletion of a singlecellodextrin transporter gene slightly decreased cellobioseconsumption, but no observable effect on cellulase expressionwas observed, which was attributed to the overlapping activityof isozymes. Further simultaneous deletion of cdtC and cdtDresulted in significantly decreased cellobiose consumptionand poor growth on cellulose. The extracellular activity andtranscription level of cellulases in the mutant without cdtCand cdtD were significantly lower than those in the wild-type

strain when grown on cellulose. This result provides directevidence of the crucial function of cellodextrin transporters inthe induction of cellulase expression by insoluble cellulose.

Keywords Cellodextrin transporter . Cellobiose . Cellulase .

Penicillium oxalicum

Introduction

The β-(1,4)-linked glucose polymer cellulose is the most abun-dant biomass on earth. Cellulosic fuels can displace a signifi-cant portion of the current demand for fossil fuels (Rubin et al.2008). However, the high cost of cellulase hinders commercial-ization of cellulosic fuels (Klein−Marcuschamer et al. 2012).Industrial cellulases are mainly produced by filamentous fungi,in which cellulase gene expression is induced only in thepresence of cellulose or some other inducers (Lynd et al.2002; Suto and Tomita 2001). Several positive or negativetranscription factors have been proven to have critical functionsin the transcriptional regulation of cellulase expression in fungi(Aro et al. 2005; Coradetti et al. 2012). However, the upstreamregulatory mechanism by which extracellular cellulose triggersthe signaling pathway for cellulase induction remains unclear.

Soluble cellodextrins as the intermediates of cellulose hy-drolysis are important in cellulase induction by insolublecellulose. This argument has been supported by the significantinduction of cellulase expression by exogenous cellodextrinsin β-glucosidase (which hydrolyzes cellodextrins to glucose)-deficient strains (Znameroski et al. 2012; Zhou et al. 2012).The remarkable negative effect of intracellular β-glucosidaseon cellulase induction indicated that cellodextrins possiblyinduce cellulase gene expression after transport into the cell(Znameroski et al. 2012; Chen et al. 2013). Membrane pro-teins transporting cellodextrins in filamentous fungi were firstbiochemically reported in Trichoderma reesei (Kubicek et al.

Electronic supplementary material The online version of this article(doi:10.1007/s00253-013-5301-3) contains supplementary material,which is available to authorized users.

J. Li :G. Liu (*) :M. Chen : Z. Li :Y. Qin :Y. QuState Key Laboratory of Microbial Technology, School of LifeScience, Shandong University, 27 Shanda South Road, Jinan,Shandong 250100, People’s Republic of Chinae-mail: liu-gd@hotmail.com

J. Lie-mail: ljley@163.com

M. Chene-mail: chenmei1305@gmail.com

Z. Lie-mail: lzh7801@gmail.com

Y. Qine-mail: qinyuqi@sdu.edu.cn

Y. Qin :Y. Qu (*)National Glycoengineering Research Center, Shandong University,Jinan, Shandong 250100, People’s Republic of Chinae-mail: quyinbo@sdu.edu.cn

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1993), and the genes that encode these proteins were cloned inNeurospora crassa (Galazka et al. 2010). Lactose permeaseencoded by LAC12 in Kluyveromyces lactis can also uptakecellobiose when expressed in Saccharomyces cerevisiae(Sadie et al. 2011). All these transporters are members of themajor facilitator superfamily, which is an ancient and wide-spread family of secondary active transporters (Pao et al.1998). Gene deletion of cellodextrin transporter CDT-2 in N .crassa results in impaired growth on cellulose (Galazka et al.2010). However, the relationship between cellodextrin trans-porters and cellulase induction remains unclear.

Penicillium oxalicum 114-2, formerly classified asPenicillium decumbens (Liu et al. 2013a), is a fast-growingfilamentous fungus that secretes various lignocellulolytic en-zymes (Qu et al. 1984). Hyperproducing mutants of P.oxalicum have been used to produce industrial-scale prepara-tions of cellulase (Liu et al. 2013b). The genome of strain 114-2was recently sequenced and annotated (Liu et al. 2013c). In thepresent study, we first pursued a functional survey of nineputative cellodextrin transporters in P. oxalicum 114-2 usinga recombinant S . cerevisiae host expressing intracellular β-glucosidase. The functions of three functionally activecellodextrin transporters in cellobiose transport and cellulose-induced cellulase expression were then studied through geneknockout approaches.

Materials and methods

Strains and plasmids

The P. oxalicum wild-type strain 114-2 (CGMCC 5302) werestored in our laboratory. All P. oxalicum strains used in thisstudy are described in Table 1. Recombinant S . cerevisiaestrain YGH1-1 and modified plasmid vector pRS426(ATCC® 77107) containing ADH1 promoter and terminator

were kindly provided by Professor Weifeng Liu at ShandongUniversity. YGH1-1 was constructed by transforming strainW303 (ATCC® 208352) (MATa leu2-3 , 112 trp1-1 can1-100 ura3 -1 ade2 -1 his3 -11 ,15 ) with plasmid pRS425(ATCC® 77106) carrying intracellular β-glucosidase genegh1 -1 from N . crassa (NCBI Gene ID: 3872338).Escherichia coli DH5α (TransGen Biotech, Beijing, China)was used for routine gene cloning and vector construction.The vectors were isolated using a Plasmid Miniprep Kit(Bioteke, Beijing, China).

Media and culture conditions

Yeasts, bacterial strains, and fungal conidiawere stored at−80 °Cin 15% glycerol. Yeasts were cultivated at 30 °C in YEPD broth(2 % peptone, 1 % yeast extract, 2 % glucose, w/v). To selecttransformants, yeast synthetic complete medium composed of6.7 g/L yeast nitrogen base, 20 g/L glucose, 20 g/L agar, 20 μg/mL histidine, 40 μg/mL tryptophan, and 1.4 g/L yeast syntheticdrop-out medium supplement (Sigma, St. Louis, MO, USA; cat.no. Y2001) was used. The E . coli strains were grown in LB–Miller broth supplementedwith 50μg/mL ampicillin for plasmidpropagation when necessary. The P. oxalicum wild-type strain114-2 was cultured on wheat bran extract agar (10 %wheat branextract, 2 % agar, w/v) plates at 30 °C for 2 days for sporecollection. For transcription level analysis, the spores were inoc-ulated into Vogel's medium (Vogel 1956) with 2 % glucose for24 h, and then mycelia were harvested by filtration and trans-ferred to Vogel's salts plus an appropriate carbon source forfurther growth. ForP. oxalicum transformant screening, minimalmedium (Mandels and Andreotti 1978) containing glucose (2%,w/v), 1.2 M D-sorbitol, and 0.2 μg/mL pyrithiamine (Sigma, St.Louis, MO, USA) or 400 μg/mL hygromycin B (Roche,Mannheim, Germany) was used as the selective medium. Allstrains were cultivated through 200 rpm orbital shaking.

Prediction of candidate cellodextrin transporters

The candidate cellodextrin transporters were predicted basedon protein Basic Local Alignment Search Tool (BLASTP)search of the P. oxalicum 114-2 proteins (Liu et al. 2013c)against a local transporter sequence database. The databasecomprises downloaded Transporter Classification Database(Saier et al. 2009) FASTA sequences (last modified, 15 July2011), and sequences of two reported cellodextrin transportersCDT-1 and CDT-2 from N . crassa (Galazka et al. 2010).Proteins with E values ≤1.0×10−5 and best hits toCDT-1, CDT-2, or K . lactis Lac12 were considered ascandidate cellodextrin transporters. The same methodwas used to predict candidate cellodextrin transporters inN . crassa and T. reesei .

Table 1 P. oxalicum strains constructed and used in this study

Strain Parent Genotype Reference

114-2 – Wild type Qu et al. (1984)

ΔcdtC 114-2 ΔcdtC ::ptra This study

ΔcdtD 114-2 ΔcdtD ::ptra This study

ΔcdtG 114-2 ΔcdtG ::ptra This study

ΔcdtDC ΔcdtD ΔcdtD ::ptra; ΔcdtC::hph This study

ΔcdtGC ΔcdtG ΔcdtG ::ptra; ΔcdtC::hph This study

ΔcdtGD ΔcdtG ΔcdtG ::ptra; ΔcdtD::hph This study

OcdtC 114-2 cbh1(p)::cdtC ::hph This study

OcdtD 114-2 cbh1(p)::cdtD ::hph This study

OcdtG 114-2 cbh1(p)::cdtG ::hph This study

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Yeast transformation and growth on cellobioseand cellodextrins

For the expression of candidate cellodextrin transporters in S .cerevisiae YGH1-1, the coding sequences of the candidategenes were amplified from the cDNA of P. oxalicum withprimers harboring appropriate restriction enzyme sites andligated into modified pRS426 vector after digestion with thesame enzyme to obtain the recombinant plasmids. Theinserted genes were then sequenced prior to yeast transforma-tions, which were conducted using the protocol described byGietz and Schiestl (2007). The transformants were analyzedby PCR with the corresponding primer pairs. To monitorgrowth on cellobiose, recombinant yeasts were grown inYEPD broth overnight. The cells were then harvested andwashedwith ddH2O, and inoculated to the medium containing6.7 g/L yeast nitrogen base, 1.4 g/L yeast synthetic drop-outmedium supplement (Sigma, St. Louis, MO, USA; cat. no.Y2001), 20 μg/mL histidine, 60 μg/mL leucine, 40 μg/mLtryptophan, 20 μg/mL uracil, and 1 % (w /v ) cellobiose withan initial O.D. (600 nm) of 0.1. Culture samples were obtainedat regular intervals, and the change in O.D. was measured at600 nm using a monochromator spectrophotometer UV-2550(Shimadzu, Kyoto, Japan). Cellodextrins were prepared bymixed-acid hydrolysis (Zhang and Lynd 2003). To monitorwhether S . cerevisiae expressing cdtC , cdtD , or cdtG coulduse cellodextrins, cells grown in YEPD overnight werewashed and inoculated into a medium containing 0.5 %cellodextrins, 2 % peptone, and 1 % yeast extract for furthergrowth.

Phylogenetic analysis of transporters

Amino acid sequences of transporters were aligned usingClustalX 2.0 (Thompson et al. 1997), and the phylogenetictree was generated by MEGA 5.10 (Tamura et al. 2011) usingbootstrap maximum likelihood method with 1,000 replicates.

Deletion and overexpression of cdt genes in P. oxalicum

Selectable markers, including E . coli hygromycin Bphosphotransferase gene (hph ) and Aspergillus oryzaepyrithiamine resistance gene (ptrA) (Li et al. 2010), were usedin the construction of deletion cassettes, as shown inElectronic supplementary material (ESM) Figs. S1 and S2.The cassettes were constructed using double-joint PCR (Yuet al. 2004). For construction of overexpression vectors, theselectable marker gene hph expression cassette was firstcloned into pMD18-T (TaKaRa, Tokyo, Japan). The P.oxalicum cbh1 promoter was then inserted into the EcoRIsite of the recombinant plasmid. Finally, cdt genes and theirown terminator sequences were separately inserted down-stream the cbh1 promoter.

Determination of cellobiose consumption

The P. oxalicum strains were grown in 50 mL of Vogel's saltswith 2 % (w /v ) glucose at 30 °C for 24 h, starting with aninoculum of conidia at a final concentration of 106/mL. Themycelia were harvested by filtration, washed with Vogel'ssalts, and transferred to Vogel's salts with 1 % (w /v ) purecellulose (Sangon, Shanghai, China) for further growth of 3 hto induce the expression of cellodextrin transporters. Then,0.5 g (wet weight) of mycelia were harvested by filtration,washed with Vogel's salts, and resuspended in 2 mL of Vogel'ssalts with 1.5 mM cellobiose. The amounts of cellobioseremaining in the supernatant at different times were deter-mined by LC-10 AD HPLC (Shimadzu, Japan) using a Bio-Rad Aminex HPX-42A carbohydrate column as previouslydescribed (Liu et al. 2010).

Real-time quantitative PCR

The total RNA was extracted using the RNAisoTM reagent(TaKaRa, Tokyo, Japan) according to the manufacturer's pro-tocol. Then, mRNAwas purified, and first-strand cDNAwassynthesized using the PrimeScript RT Reagent Kit with cDNAEraser (TaKaRa, Tokyo, Japan) according to the manufac-turer's instructions. The primer sequences used are shown inESM Table S1. Real-time quantitative PCR was run on aLightCycler instrument with software Version 4.0 (Roche,Mannheim, Germany) as previously described (Wei et al.2011). The reactions were performed in triplicate with a totalreaction volume of 20 μL, which included 7.4 μL of water,0.8 μL of each primer (10 μM), 10 μL of SYBR1 Premix ExTaqTM (Perfect Real Time) (TaKaRa, Tokyo, Japan), and 1μLof template cDNA. The copy numbers of gene transcriptswere calculated by comparing their Cp values in real-timequantitative PCR to the standard curve of each gene (Leeet al. 2008). To compare gene expression levels betweendifferent conditions, the ratios of copy numbers of transcriptsto those of actin gene in the same sample were calculated andshown (Cho et al. 2006; Wang et al. 2012). It should bepointed out that the ratios do not represent accurate ratios ofmRNA abundances because different reverse transcriptionefficiencies between different genes were not considered.

Enzyme assays

Filter paper activity was measured usingWhatman No. 1 filterpaper (Whatman, Maidstone, UK) as the substrate for 60 min(Ghose 1987), and the amount of reducing sugar released wasdetermined using the dinitrosalicylic acid (DNS) method withglucose as the standard (Cheng et al. 2009). Cellobiohydrolaseactivity was measured using p -nitrophenyl β-D-cellobioside(Sigma, St. Louis, MO, USA) as the substrate for 30 min(Deshpande et al. 1984). One unit of enzyme activity was

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defined as the amount of enzyme that liberates 1 μmol glucoseequivalent or p -nitrophenol per minute under the assayconditions.

Accession numbers

The GenBank accession numbers for the three cellodextrintransporters are as follows: CdtC, EPS25673; CdtD,EPS25817; CdtG, EPS34431.

Results

Three cellodextrin transporters were identifiedthrough a functional survey of nine candidates

A total of 11 putative cellodextrin transporters (named fromCdtA to CdtK) were predicted in P. oxalicum (ESMTable S2). A previous transcriptome analysis (NCBI GEOaccession NO. GSE34288) showed that the transcription levelof cdtD is the highest among the 11 genes, and the transcrip-tion level of cdtC showed a remarkable increase on cellulosethan that on glucose (ESM Table S2). The genes encodingnine of the candidate cellodextrin transporters were separatelytransformed into a recombinant S . cerevisiae host YGH1-1expressing intracellular β-glucosidase. The other two candi-dates, cdtA and cdtI , were not successfully amplified from thecDNA of P. oxalicum possibly because of their very lowexpression levels (ESM Table S2). To characterize cellobioseutilization abilities, the growth rates of S . cerevisiae strainsharboring these nine transporters were measured using cello-biose as the sole carbon source. Of the nine strains evaluated,three exhibited significant growth phenotypes (Fig. 1), whichindicates that the three expressed transporters CdtC, CdtD,and CdtG could transport cellobiose. The S . cerevisiae straincarrying CdtD (named YCdtD) or CdtG (named YCdtG)

showed a longer lag phase than that harboring CdtC (namedYCdtC). As shown in ESM Fig. S3, the three recombinantyeasts could also use cellodextrins with a degree of polymer-ization higher than two. In phylogenetic analysis (Fig. 2),CdtD and CDT-2 from N . crassa were grouped into onecluster, whereas CdtC, CdtG, cellodextrin transporter CDT-1from N . crassa , and three lactose permeases from K . lactis ,Aspergillus nidulans (Fekete et al. 2012), and T. reesei(Tre3405) (Ivanova et al. 2013) were grouped into anothercluster.

Transcriptions of cdtC and cdtD are repressed by glucoseand induced by cellulose

The two cdt genes in N . crassa are induced by celluloseaccording to transcriptome analysis (Galazka et al. 2010). Toexamine the effects of different carbon sources (glucose,cellulose, and cellobiose) on cdt gene expression in P.oxalicum , the transcript abundances of cdtC , cdtD , andcdtG were monitored by real-time quantitative PCR. Whenthe mycelia were transferred into and grown in a mediumcontaining 0.5 % of each carbon source for 8 h, residual sugarswere detected (for glucose and cellobiose) or visualized (forcellulose) in the cultures (data not shown). Figure 3a, b showthat glucose clearly repressed the expression of the three cdtgenes with reductions of 82, 74, and 45 % at 3 h and 94, 94,and 93 % at 8 h, respectively, than those in the mediumcontaining no carbon source. The expression levels of cdtCand cdtD were significantly upregulated (>490-fold at 3 h and>50-fold at 8 h, respectively, compared with those in themedium containing no carbon source) when cultured on cel-lulose. However, cellulose did not efficiently induce cdtGgene transcription. Cellobiose at 0.5 % efficiently inducedcdtC transcription, but had no obvious inductive effect onthe transcription of cdtD or cdtG . Cellobiose at this concen-tration could not induce cellulase expression (data not shown).

Fig. 1 Cellobiose-mediated growth of recombinant yeast strains express-ing cdt genes. a Growth of recombinant yeast strains on cellobiose as thesole carbon source. Of the nine recombinant strains evaluated, the strainsexpressing the genes cdtC (named YCdtC), cdtD (named YCdtD) andcdtG (named YCdtG) could grow on cellobiose. All O.D. values are the

mean of two measurements with SD <5 %. b Cellobiose consumption ofYCdtC, YCdtD, and YCdtG. The amount of residual cellobiose wasdetermined using the DNS method. All values are the mean of threemeasurements with SD<6 %

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The difference between the effects of cellobiose and celluloseon the expression of cdt genes was attributed to the differentconcentrations of cellodextrins (high initial amounts vs.sustained release) in the media.

Single deletion of cdt genes did not affect cellulase productionin P. oxalicum

To study the function of cellodextrin transporters during cellulaseinduction, cdt genes were first deleted individually from the P.oxalicum 114-2 genome (ESMFig. S1a). TransformantsΔcdtC ,ΔcdtD , and ΔcdtG were confirmed to have a deletion cassetteinserted into the corresponding cdt gene locus by Southern blotanalysis (ESM Fig. S1b–d). The strains showed a slight decreasein cellobiose consumption than the parental strain 114-2 (ESMFig. S4a). In addition, no significant difference in cellulaseproduction on cellulose was detected between the mutants and114-2 (ESM Fig. S4b). The transcription of cdtC was signifi-cantly upregulated in ΔcdtD (by fourfold at 4 and 8 h) andΔcdtG (by sixfold at 3 h and twelvefold at 8 h) than that in 114-2(Fig. 3c−d).

Crucial functions of cellodextrin transporters in cellulaseinduction revealed by double gene deletion

Given that single deletion of cdt genes only slightly reducescellodextrin transport in P. oxalicum , we constructed cdt

double deletion strains, including ΔcdtDC , ΔcdtGC , andΔcdtGD (ESM Fig. S2). One of the strains, namely,ΔcdtDC , showed a decrease of about 50 % in cellobioseconsumption than the wild-type strain 114-2 (Fig. 4a). Thismeans the lack of both cdtC and cdtD results in a poorcapacity to transport cellobiose. ΔcdtDC could hardly growon cellulose as the sole carbon source, and a decreased extra-cellular cellulase activity (measured as filter paper activity,FPase) was observed in ΔcdtDC than that in 114-2(Fig. 4b–d). Growth and cellulase production of ΔcdtGCand ΔcdtGD on cellulose had no significant difference fromthose of the wild-type strain (data not shown). To determinewhether the decreased lignocellulolytic activities in ΔcdtDCwere due to decreased expression levels of lignocellulolyticgenes, the transcript abundances of the major cellulase genecbh1 (cel7A -2 ) and the major xylanase gene xyn1 (xyn10A)(Liu et al. 2013c; Wei et al. 2011) were monitored aftercultivation under cellulose-inducing conditions for 3 and24 h. The transcriptional changes in the two genes wereexpected to represent those of other cellulase and hemicellulasegenes because these genes are co-expressed under differentenvironmental and genetic conditions (Liu et al. 2013b; Weiet al. 2011). Figure 4e, f show that the transcription levels ofcbh1 and xyn1 in mutant ΔcdtDC decreased by 91 and 84 %at 3 h and by 99 and 97% at 24 h, respectively, compared withthose in the wild-type strain 114-2. The significant decrease incellulase gene transcription at 3 h after cell transfer suggests

Fig. 2 Phylogenetic analysis ofputative cellodextrin transportersand several previouslyfunctionally analyzed transportersin other fungi. The threefunctional cellodextrintransporters identified in P.oxalicum are labeled in bold. Thesubstrates of functionaltransporters are shown (C forcellodextrins and L for lactose).Note that the functions ofTre77517 and Tre79202(Porciuncula et al. 2013) inlactose uptake by T. reesei are notas remarkable as that of Tre3405(Ivanova et al. 2013)

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that the impaired cellulase production should be the cause ofpoor mutant growth on cellulose, rather than vice versa. Nosignificant differences were observed for the transcriptionlevels of cbh1 and xyn1 between ΔcdtGC and 114-2. Inmutant ΔcdtGD , the expression levels of xyn1 decreased by71 and 66 % at 3 and 24 h, respectively, compared with thosein 114-2. The transcription levels of cbh1 increased at 3 h butnot at 24 h in ΔcdtGD , compared with those in 114-2.

Overexpression of cellodextrin transporters improvedcellulase production in P. oxalicum 114-2

The deletion of cellodextrin transporter genes causes a decreasein cellulase production. Thus, we investigated whether overex-pression of cellodextrin transporter genes could improve cellu-lase production in P. oxalicum 114-2. The three cellodextrintransporter genes were separately over-expressed under the con-trol of the cbh1 promoter. As expected, the expression levels ofcdtC , cdtD , and cdtG increased in the corresponding over-expressing mutants, especially for cdtG with low-expression in114-2 (Fig. 5a). The expression levels of major (hemi-)cellulasegenes cbh1 , egl1 (cel7B), and xyn1 (Liu et al. 2013c) signifi-cantly increased in the threemutants comparedwith those in 114-2, with the highest fold changes in cdtG overexpressing mutant(Fig. 5b). Consistently, the extracellular cellobiohydrolase activ-ities of the mutants were elevated (17–55 % higher at 72 h)compared with those in 114-2 (Fig. 5c).

Discussion

The presence of intracellular β-glucosidases in many fungalspecies (Inglin et al. 1980; Kurasawa 1992) implies the exis-tence of cellodextrin transport system in filamentous fungi.Gene cloning of cellodextrin transporters was first reported inN . crassa , and their biological functions have been prelimi-narily studied (Galazka et al. 2010). Three cellodextrin trans-porters were identified in P. oxalicum in this study.Subsequent studies provided deeper understanding of thisfamily of membrane transporters in fungi regarding theirsequence conservation, functional redundancy, and physio-logical functions. These three issues will be discussed asfollows.

First, three functionally active cellodextrin transporters wereidentified from nine predicted candidates based on proteinhomology analysis. Among the three transporters, CdtC andCdtG were the best BLAST hits of cellobiose-transporting N .crassa CDT-1 and K . lactis Lac12, respectively (ESMTable S2). Bidirectional BLASTP analysis indicated they areortholog pairs (data not shown). This result suggests in somedegree of sequence and functional conservation of cellodextrintransporters among fungal species. However, another functionalcellodextrin transporter, CdtD, has no ortholog in N . crassa orT. reesei . The whole phylogenetic tree of all the predictedcandidate cellodextrin transporters in P. oxalicum , N . crassa ,and T. reesei (Fig. 2) poorly recapitulates the species tree (Liuet al. 2013c), indicating fast evolution of this group of

Fig. 3 Transcription levels of cdt genes in wild-type strain 114-2 and cdtknockout strains. a–b Relative expression levels of cdtC , cdtD , and cdtGin 114-2 grown on different carbon sources. Strain 114-2 was first grownon 2 % (w /v) glucose for 24 h, and the mycelia were transferred into newmedia containing no carbon source, 0.5 % (w /v) glucose, 0.5 % (w /v)

cellulose, or 0.5 % (w /v) cellobiose for further growth of 3 h (a) or 8 h(b). c–d Relative expression levels of cdtC, cdtD, and cdtG in 114-2 andcdt single gene deletion mutants. The strains were first grown on 2 % (w /v) glucose for 24 h, and then transferred to 0.5 % (w /v) cellulose for 3 h(c) or 8 h (d). Error bars indicate SDs of triplicate measurements

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Fig. 4 Effects of cdt doubledeletions on cellulase production inP. oxalicum. a Cellobioseconsumption by P. oxalicum wild-type and cdt double knockoutstrains. The strains were incubatedwith 1.5 mM cellobiose. Errorbars indicate SDs of threeindependent incubations. bErlenmeyer flasks of wild-typestrain 114-2 (left) and two cdtCand cdtD double-deletedtransformantsΔcdtDC (middle)andΔcdtDC-2 (right). The strainswere first grown on 2 % (w/v)glucose for 24 h, and the myceliawere transferred into new mediacontaining 1 % (w/v) cellulose forfurther growth of 3 days. cBiomass protein concentration(representing biomass) of wild-type strain 114-2 andΔcdtDC.Mycelia were grown in 1 % (w/v)cellulose medium. Data are meansof results from three independentcultures. Error bars indicate SDs.d Extracellular filter paperhydrolyase (FPase) activity inwild-type strain 114-2 andΔcdtDC.Data are means of results fromthree independent cultures. Errorbars indicate SDs. e–fTranscription levels of cbh1 andxyn1 in wild-type and cdt doubleknockout strains. The strains werefirst grown on 2 % (w/v) glucosefor 24 h and then transferred to 1%(w/v) cellulose for 3 h (e) and 24 h(f). Error bars indicate SDs oftriplicate measurements

Fig. 5 Effects of cdt overexpression on cellulase production in P.oxalicum . The three mutants with overexpression of cdtC, cdtD , andcdtG were named OcdtC , OcdtD , and OcdtG , respectively. a Transcrip-tion levels of cdtC, cdtD , and cdtG in wild-type 114-2 and cdt overex-pressing mutants. b Transcription levels of cbh1 , eg1 , and xyn1 in wild-type 114-2 and cdt overexpressing mutants. For transcriptional level

determination in a and b , the strains were first grown on 2 % (w /v)glucose for 24 h, and then transferred to 1 % (w /v) cellulose for furthergrowth of 24 h. Error bars indicate SDs of triplicate measurements. cExtracellular cellobiohydrolase activities in wild-type strain 114-2 andcdt overexpressing mutants. Data are means of results from three inde-pendent cultures. Error bars indicate SDs

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membrane transporters. CdtB, CdtE, CdtF, CdtH, CdtJ, andCdtK belonging to the same clusters as the functionalcellodextrin transporters in phylogenetic analysis (Fig. 2) didnot confer S . cerevisiae the ability to transport cellobiose.These proteins possibly function in the transport of other sub-strates despite showing high sequence identities withcellodextrin transporters. There is also the possibility that theproteins were not actively expressed in the yeast background inour experiment, which needs to be examined for conclusiveevidence. Transporters active on cellodextrin and lactose (bothare β-1,4-linked oligosaccharides) could not be distinguishedby phylogenetic analysis (Fig. 2), which indicates the possibleside activity on lactose by the three cellodextrin transportersthat were found.

Second, P. oxalicum possessed two highly expressedcellodextrin transporters with overlapping activities.Analysis of cdt single and double deletion mutants sup-ported that CdtC and CdtD, but not CdtG, could compen-sate for the loss of other cellodextrin transporters (Fig. 4;ESM Fig. S4). This result differs from that in N . crassa ,in which single deletion of the gene encoding CDT-2(relatively closer to CdtD in P. oxalicum ) significantlydecreases cellobiose uptake and growth on cellulose(Galazka et al. 2010). Similar compensating effects werealso observed for other proteins, such as the paralogoustranscription factors in yeast (Gitter et al. 2009), andpossibly provide robustness to environmental disturbancesfor cell functions. The expression of cdtC remarkablyincreased in mutants lacking cdtD or cdtG (Fig. 3c–d),which might be caused by some metabolite changes aftersingle deletion of the later two genes. However, elevatedcdtD transcription in ΔcdtC was only observed at 8 h,suggesting the compensatory function of CdtD in thismutant mainly occurs at the level of protein activity butnot gene expression. The remaining cellobiose transportand cellulase induction in mutant ΔcdtDC were ascribedto the existence of CdtG or other unidentified cellodextrintransporters.

Finally, cellodextrin transporters exhibited critical func-tions in cellulase induction by cellulose. Given the previouslywell-documented inductive effect of cellodextrins on cellulaseexpression (Znameroski et al. 2012; Zhou et al. 2012), twopossible mechanisms for cellodextrin (or their further metab-olites) signal transduction possibly exist (Fig. 6): (a)cellodextrins transported into cells activate intracellular sen-sors, similar to galactose sensing (Bhat and Murthy 2001) andglucose sensing (Mayordomo and Sanz 2001) in yeast, and (b)extracellular cellodextrins activate plasma membrane sensors,such as transporter-like proteins or G protein-coupled recep-tors, similar to glucose sensing (Özcan et al. 1996; Rollandet al. 2000) and amino acid sensing (Klasson et al. 1999) inyeast. The second hypothesis cannot be ruled out by thepreviously reported negative function of intracellular β-

glucosidases in cellulase induction (Znameroski et al. 2012;Zhou et al. 2012) because the deletion of intracellular β-glucosidase may only release intracellular carbon cataboliterepression (Fig. 6). In our previous study, we found thatdeletion of the major extracellular β-glucosidase gene bgl1in P. oxalicum does not increase cellulase expression regard-less of the accumulation of extracellular cellobiose (Chen et al.2013), which indicated that cellobiose cannot be sensed onplasma membrane. In T. reesei mutants, the positive correla-tion between cellobiose transport activity and cellulase pro-duction levels implies the existence of the first mechanismstated above (Kubicek et al. 1993). In the present study, thedramatic decrease in cellulase expression in mutant ΔcdtDCexperimentally validated the assumption that transport ofcellodextrin (or their derivates) into cells is important for thecellulase induction by insoluble cellulose. The function wasconfirmed by the increased cellulase expression in cdt over-expressing mutants.

Overall, this study identified the main cellodextrin trans-porters in P. oxalicum and demonstrated their crucial func-tions in cellulase induction by cellulose. Further research isexpected to identify the predicted intracellular cellodextrinsensors, and to clarify the mechanisms for transduction ofcellodextrin signals. Also, the enzymatic properties (e.g., ki-netic parameters) and structures of the identified transportersare to be studied to deepen our understanding of the catalyticmechanisms of cellodextrin transporters.

Fig. 6 Two possible cellodextrin-sensing mechanisms in fungi.Cellodextrins (or their further metabolites) may be recognized by intra-cellular sensor after transport into a cell (a), or recognized by transmem-brane sensor (b). Both kinds of putative cellodextrin sensors have notbeen identified. Black arrows indicate mass flows and gray arrowsindicate regulatory interactions. Dashed arrows indicate possible multi-ple steps of signal transduction. CDTs refer to cellodextrin transporters.BGL refers to β-glucosidase

10486 Appl Microbiol Biotechnol (2013) 97:10479–10488

Acknowledgments This study was supported by grants from NationalBasic Research Program of China (grant no. 2011CB707403) and NationalNatural Sciences Foundation of China (grant no.31030001).

We acknowledge the generosity ofWeifeng Liu for the contribution ofrecombinant S . cerevisiae YGH1-1.

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