IMPROVED CELLULOLYTIC EFFICACY IN PeniCilliuM DeCuMBens VIA HETEROLOGOUS EXPRESSION OF hYPoCRea

Hypocrea jecorina endoglucanase II (Hjegl2) was heterologously expressed in Penicillium decumbens (yielding strain Pd::Hjegl2). After induction in cellulose containing media, strain Pd::Hjeg2 displayed increased carboxymethylcellulase activity (CMCase, 5.77 IU/ml, representing a 21% increase) and cellulose degradation determined with a filter paper assay (FPA, 0.40 IU/ml, 67% increase), as compared to the parent strain. In media supplemented with glucose (2%), Pd::Hjegl2, displayed 51.2-fold and 3-fold higher CMCase and FPA activities, respectively, as compared to the parent strain. No changes in the expression levels of the four main native cellulase genes of P. decumbens (Pdegl1, Pdegl2, Pdcbh1, and Pdcbh2) were noted between the transformant and wild-type strains. These data support the idea that Hjegl2 cleaves both internal and terminal glycosidic residues, in a relatively random and processive manner. In situ polyacrylamide gelactivity staining of extracts derived from wild-type and Pd::Hjegl2 revealed two additional active fractions in the latter strain; one with a molecular mass ~50-65 KDa and another ~80-116 kDa.


INTRODUCTION
Lignocellulosic biomass represents an abundant, renewable, and exploitable global carbon resource.Cellulose can be efficiently degraded through the synergistic action of cellulolytic enzymes that contain three major groups based on their amino acid sequence and substrate specificity: endoglucanases (EC 3.2.1.4),cellobiohydrolases (EC 3.2.1.91),and β-glucosidases (EC 3.2.1.21).Endoglucanases act randomly against the amorphous structure of cellulose chains producing both reducing and nonreducing ends, substrates for cellobiohydrolases that produce cellobiose.Cellulose chains are thus efficiently degraded to soluble cellobiose and cellooligosaccharides by the endo-exo synergism of these two enzymes.In the last step of enzymatic cellulose degradation, cello-oligosaccharides are hydrolyzed to glucose by β-glucosidases (Lynd et al., 2002).
Among the cellulolytic bacteria and fungi studied, Hypocrea jecorina (previous Trichoderma reesei) represents one of the best-studied cellulase-producing organisms (Martinez et al., 2008).In H. jecorina, two main endoglucanases (Hjegl1 and Hjegl2) have been characterized.Hjegl2 appears to be the preponderant endoglucanase accounting for 5-10% of the total cellulase and >60% of the endoglucanase activity produced by H. jecorina (Medve et al., 1998a, Fujita et al., 2004).Lack of Hjegl2 production reduces the endoglucanase activity in the culture supernatant by as much as 55% (Karlsson et al., 2002).Recently, Hjegl2 in combination with other cellulolytic enzymes was used for saccharification and fermentation of cellulose to ethanol (du Plessis et al., 2010;Apiwatanapiwat et al., 2011).
Several Penicillium species have been reported in recent years with the ability to produce high cellulase and hemicellulase specific activities and are therefore being considered as valuable additional strains for industrial applications (Krogh et al., 2004;Jørgensen et al., 2005;Dutta et al., 2008;Singhvi et al., 2011;Dillon et al., 2011).Penicillium decumbens 114-2 isolated in our laboratory is a fast-growing filamentous fungus that secretes a complete extracellular cellulolytic enzyme system capable of breaking down cellulose to glucose (Sun et al., 2008).A modified P. decumbens mutant, resulting in a strain with 24-fold higher cellulase activity, has been used in industrial applications for cellulase production and biomass hydrolysis (Cheng et al., 2009, Liu, 2010).As in H. jecorina, P. decumbens also possesses two main endoglucanases (Pdegl1 and Pdegl2).However, while the orthologous enzymes from these two fungi are similar in amino acid sequence, Hjegl2 displays a 12-fold higher specific activity than Pdegl2, using amorphous cellulose as a substrate (Wei et al., 2010).We sought, therefore, to test the hypothesis that heterologous expression of Hjeg2 in P. decumbens would be an effective approach to improve the cellulolytic ability of P. decumbens.
In this report, we show that heterologous expression of Hjegl2 in P decumbens increases the cellulytic potential of P decumbens as examined using both carboxymethylcellulase (CMCase) and filter paper assays (FPA).

Construction of Hjegl2 expression vector and transformation into P. decumbens
Escherichia coli DH5α (supE44,ΔlacU169 (φ80lacZΔM15),hsdR17,recA1,endA1,gyrA96,relA1) was used as the host strain for the recombinant DNA manipulations.Plasmid ANIp8 harboring the pyrG gene (kind gift from Adrian Tsang, Concordia University, Canada) was used as the expression vector.Full-length Hjegl2 derived from fun-gal cDNA was amplified by PCR using primers Peg-F and Peg-R (Table 1), designed to add a six amino acid His-tag to the carboxy terminus of the protein.
The resultant product was digested with NheI and ligated into the NheI site of ANIp8 to yield ANIp8-Hjegl2, where the Pki promoter from Aspergillus niger present on ANIp8 would drive expression of the insert.The ANIp8-Hjegl2 expression cassette and the empty vector (ANIp8, control) were transformed into the uracil-auxotrophic mutant of P. decumbens (M12) as described previously (Koukaki et al., 2003).
Transformants were selected on SDC medium without uracil and were purified through single spore isolation and culturing for further analysis.
Genomic DNA of putative transformants was isolated and used as a template for PCR reactions using primer designed to the expression cassettes (Pz-F and Pz-R, Table 1) under the following reaction conditions: 1 cycle at 94ºC (2 min), 30 cycles of 94ºC (1 min), 56ºC (1 min), 72ºC (2 min) and 1 cycle at 72ºC (10 min).The PCR products were purified by E.Z.N.A. Gel Extraction Kit (OMEGA, U.S.A.) and cloned into the pEASY-T1 cloning vector (Trans-Gene Biotech, China).The integrity of all constructs was verified by sequencing.
Enzyme activity measurements P. decumbens M12::ANIp8-Hjegl2 (Pd::Hjegl2) and an empty vector transformant (Pd::ANIp8) were grown in modified Mandels' solution containing (per liter): 3 g KH 2 PO 4 , 2.6 g NaNO 3 , 0.5 g MgSO 4 •7H 2 O, 0.5 g CaCl 2 , 0.5 g urea, 7.5 mg FeSO 4 •7H 2 O, 2.5 mg MnSO 4 •H 2 O, 3.6 mg ZnSO 4 •7H 2 O, 3.7 mg CoCl 2 •6H 2 O and 1 g peptone, supplemented with either 2% w/v of glucose, OR 1% w/v of wheat bran + 1% w/v of microcrystalline cellulose.The pH of all media was adjusted to 5.5.Cultures were cultivated in 1 l baffled shaker-flasks containing 200 ml medium and were grown at 29°C, 180 rpm.Cultures were harvested after 1 day, and the cells removed by centrifugation (10000g, 10 min).Aliquots of supernatants were diluted to assay the enzyme activities.One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 μmol of product per minute under the standard assay conditions.The reaction system was 0.2 mol l -1 acetate buffer (pH 4.8) with different substrates at the reaction temperature of 50°C.Endoglucanase activity (CMCase activity) was measured using 1% (w ⁄ v) sodium carboxymethyl cellulose for 30 min.Filter paper activity (FPase activity) was measured using Whatman No.1 filter *: with an NheI restriction site is underlined and a six histidine tag is shown in bold.
paper (1 × 6 cm, 50 mg) for 60 min; the amount of reducing sugar released from the enzyme reaction was estimated by the DNS method with glucose as the standard (Sun et al., 2008;Cheng et al., 2009)

Quantitative real time PCR
The expression levels of Pdegl1, Pdegl2, Pdcbh1, and Pdcbh2 were determined using quantitative real-time PCR.Mycelia derived from Pd::Hjegl2 and Pd::ANIp8 were harvested after 48 h growth in the modified Mandels' glucose media as described above.Total RNA was prepared from the mycelia by grinding under liquid nitrogen with a mortar and pestle, followed by extraction using the RNAiso TM reagent (TaKaRa, Japan) according to manufacturer's protocol.Total RNA was treated with DNase I (RNase free) in order to remove any contaminating genome DNA.First-strand cDNA was synthesized using the PrimeScript TM RT reagent Kit (TaKaRa, Japan) using oligo-dT and random 6-mers as primers.PCR amplification was performed using SYBR ® Premix Ex Taq TM Kit (TaKaRa) with primer sets for actin (accession no.EU855739), Pdegl1 (accession no.EU339127), Pdegl2 (accession no.EU315320), Pdcbh1 (accession no.EF397602), and Pdcbh2 (accession no.HQ286637) (Table 1).Gene expression copy numbers were calculated using standard curves constructed for each gene, and the data were then normalized to the expression levels of the actin gene.Data processing and statistical analyses were performed using Microsoft Excel.The average values of at least three determinations for each gene were considered as its expression level, with standard deviations below 10%.

SDS-PAGE with activity staining and Western blot
Proteins in the culture supernatant were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by activity staining.Carboxymethylcellulase (CMCase) activity was visualized in the polyacrylamide gels after renaturation as described (Medve et al., 1998b) with the following modifications.Proteins were electrophoretically separated on a 10% polyacrylamide gel containing 0.15% sodium carboxymethyl cellulose (CMC-Na).After electrophoresis, the gel was washed at room temperature in two changes of washing solution: 0.5 mol l -1 NaAc and 25% isopropanol, to remove the SDS.Proteins were renaturated in a 50 mmol l -1 acetate buffer (pH 5.0) containing 5 mmol l -1 β-mercaptoethanol by rocking the gel overnight at 4°C.The gel was then placed in 50 mmol l -1 NaAc-HAc buffer (pH 5.0) for 2 h and incubated at 50°C for another 2 h.For detection of activity, the gel was stained in 0.2% Congo Red for 1 h and destained in 1 mol l -1 NaCl.Clear bands on the red background indicated the CMC digestion.
For Western blot analyses, culture supernatants derived from Pd::Hjegl2 and Pd::ANIp8 were separated by SDS-PAGE and electroblotted onto PVDF membranes.Membranes were blocked for 1 h with 5% nonfat dry milk powder in Tris-buffered saline containing 0.05% Tween 20 and then immunoblotted with a mouse monoclonal His antibody (Beyotime, China) for 2 h.After washing the membranes six times with TBST, they were incubated with mouse peroxidase-conjugated secondary antibody for 1 h (Beyotime, China).Finally, the blots were washed and developed by enhanced chemiluminescent staining using ECL reagents (Amersham Buchler).

Isolation and selection of pyrG-auxotrophic mutants
In order to use the ANIp8 vector that carries the pryG selection marker, a P. decumbens uracil auxotroph was isolated.Approximately 1x10 8 spores of P. decumbens were spread on SDC medium supplemented with uracil and 5-FOA as described in the Methods section.A total of 44 5-FOA resistant colonies were isolated and further evaluated via replica plating on SDC and SDC supplemented with uracil and/or 5-FOA.A collection of eighteen mutants displaying a consistent phenotype was further tested for the ability to maintain uracil auxotrophy in rich medium (wheat bran agar supplemented with glucose and uracil) over several generations.Of these, thirteen of the mutants reverted to uracil prototrophy, whereas the remaining five were unable to grow on the minimal-medium plates containing either orotic acid or orotidine.These latter mutants represented a class of uracil auxotrophs likely due to deficiencies in either OMP decarboxylase (pyrG) and/or OPRTase.One such mutant designated as P. decumbens M12 was chosen for further study.

DNA analysis of uracil auxotroph P. decumbens-M12 and subsequent transformation of the M12
In order to verify the nature of the mutation in M12 that resulted in uracil auxotrophy, the nucleotide sequence the pyrG gene was isolated and sequenced.
Comparison of the mutant (M12) sequence to the wild-type P. decumbens pyrG indicated the presence of a nonsense mutation, most likely as a consequence of a C-T transversion at nucleotide 676 that resulted in the conversion of the codon for glutamine 226 to a stop codon.These data supported the conclusion that P. decumbens M12 could be defined as an OMP decarboxylase deficient (pyrG -) mutant.
P. decumbens strain M12 was then used as the host for transformation via complementation of the pyrG mutation by the appropriate selection marker on plasmid ANIp8.A construct bearing the H. jecorina egl2 gene driven by an A. nidulans Pki promoter and containing a 6-His tag carboxy terminus fusion to the protein product (ANIp8-Hjegl2), was made and transformed into M12 as described in the Methods section, yielding strain Pd::Hjegl2.A control strain transformed with the empty vector, Pd::ANIp8, was also constructed.Transformants bearing the ANIp8-Hjegl2 construct were verified by PCR using primers designed to Hjegl2.PCR products corresponding to the predicted (898 bp) fragment were obtained from Pd::Hjegl2 strains and not from any wild-type parent or Pd::ANIp8 strains.Sequencing of the PCR products obtained from the former cells confirmed its identity to Hjegl2.

Increased cellulase activity in strain Pd::Hjegl2
Two assays were used to measure overall cellulytic activity: (1) endoglucanase activity using carboxymethylcellulose as the substrate (CMCase), and (2) broad cellulase measurement using a filter paper activity assay (FPA).The CMCase and FPA of strains Pd::Hjegl2 and Pd::ANIp8 were measured after cultivation for 1 to 5 days.In culture media supplemented with glucose, the glucose was rapidly taken up by the cells (Fig 1a,open symbols).Under these growth conditions, little to no CMCase activity was observed in Pd::ANIp8, whereas 0.4-0.5 IU/ml of CMCase activity was produced by Pd::Hjegl2 on day 1: this increased two fold (representing a >50-fold higher level of CMCase activity than the control) and remained constant for the rest of the time period examined (up to five days, Fig. 1a, closed symbols).These data confirmed the catabolite repressive effect of glucose on CMCase activity and the constitutive nature of CMCase activity in the Hjegl2 transformant.FPA activity was also higher in Pd::Hjegl2 than in Pd::ANIp8, reaching a maximum difference of ~ 3-fold on day 3 (Fig. 1 b).
In culture media supplemented with cellulose, CMCase activity increased to ~4 and 5.5 IU/ml for Pd::ANIp8 and Pd::Hjegl2, respectively, representing a 21% increase for Pd::Hjegl2 as compared to the control strain (Fig. 2a).Maximum FPA (0.40 IU/ml) was seen for Pd::Hjegl2 on day 3, representing a 67% increase in activity as compared to the control strain, Pd::ANIp8 (Fig. 2 b).In all cases, both CMCase and FPA were elevated in Pd::Hjegl2 as compared to the control strain when grown in either glucose or cellulose containing media.
Real-time quantitative PCR was used to examine the expression patterns of the four major P. decumbens cellulose genes, Pdegl1, Pdegl2, Pdcbh1, and Pdcbh2, in strains Pd::Hjegl2 and Pd::ANIp8.Transcript levels of the four genes were quantified in cells grown in glucose or cellulose containing media for 24 and 48 h (Fig. 3).A small increase in Pdegl2 transcript levels was seen after growth in glucose for 48 h as compared to the 24 h time-point, but a dramatic increase was seen (>100-fold) when the cells were grown on cellulose.No significant differences, however, were observed between strains Pd::Hjegl2 and Pd::ANIp8 in transcript levels of Pdegl2 or any of the other genes (Pdegl1, Pdchb1, and Pdchb2) in cells grown on either glucose or cellulose for either 24 or 48 h.Pdegl2 levels (in both Pd::Hjegl2 and Pd::ANIp8) increased ~10-fold after 48 h of growth in glucose and was approximately another 10-fold higher in cells grown on cellulose for 24 h, remaining level after 48 h.Pdchb1.Pdchb2 levels also increased ~10-fold from 24 to 48 h of growth in glucose, but the transcript levels of these two genes was more than 1,000-fold higher when grown on cellulose (whether at 24 or 48 h) as compared to the glucose 48 h levels.

The molecular mass and in situ activity staining of P. decumbens expressed Hjegl2
Cell-free culture supernatants derived from Pd::Hjegl2 and Pd::ANIp8 grown in either wheat-bran microcrystalline cellulose or glucose-containing media were analyzed by SDS-PAGE, followed by in-gel renaturation of the proteins and in situ CMCase activity staining as described in the Methods section.At least five bands of CMCase activity, of approximate molecular weights of 25, 31, 35, 45, and 60 kDa, with the latter two displaying the largest zones of clearing of the substrate, could be detected in the control strain when grown in the cellulose-containing media  (Pd::ANIp8, Fig. 4, lane 1).These same bands could be seen in the Pd::Hjegl2 strain as well as two additional somewhat diffuse bands between ~50-65 and ~80-116 kDa (Fig. 4, lane 2).The two diffuse sets of CMCase activity bands were evident in the culture extracts derived from Pd::Hjegl2 grown in glucose, whereas no CMCase activity could be seen in extracts from Pd::ANIp8 grown under identical conditions (Fig. 4, lanes 3 and 4).Western blots probed with an anti-His tag monoclonal antibody revealed the presence of two proteins in strain Pd::Hjegl2, one with a molecular mass of approximately 22 kDa and another ~60-65 kDa (Fig. 4, lane 5).

DISCUSSION
Improvements in the catalytic efficiency of cellulose degradation by microbes can have tremendous impact in increasing the potential of industrial applications.Our data show that cellulose degradation, as determined by both CMCase activity and FPA of  the host strain P. decumbens, could be improved by heterologous expression of endoglucanase II from H. jecorina.Expression of Hjegl2 in P. decumbens was constitutive.The transformed strain, Pd::Hjegl2, showed significant CMCase and FPA activity levels, even in glucose-containing catabolite-repressed culture conditions, an important industrial problem leading to the inhibition of the synthesis of native cellulases (Calza 1991;Alriksson et al., 2009).Expression analysis of the four main cellulose genes of P. decumbens revealed no changes in their transcript levels between the wild-type and Hjegl2-transformed strains, supporting the conclusion that the observed increase in cellulytic activity was due to the heterologous expression of the Hjegl2 gene.The expression studies did, however, confirm the inductive nature of the presence of cellulose on the transcript levels of the genes that comprise the endogenous cellulytic pathway of P. decumbens.
As expected, expression of Hjegl2 in P. decumbens led to increased CMCase activity.However, since the filter paper assay is an indication of what can be considered total cellulase activity in the sense that it requires the functions of three kinds of enzyme activities (endoglucanases, cellobiohydrolases and β-glucosidases), the question arises as to why the expression of Hjegl2 would lead to an increase in this activity.There are several possible explanations for this result.First, it is conceivable that the endoglucanases activity is the rate-limiting step, thus an increase in this enzyme and its products would lead to an increase in apparent total cellulose activity.Alternatively, according to the most widely accepted hypothesis, a strict endoglucanase cleaves the cellulose substrate internally, thus disrupting the crystalline structure of the cellulose and generating new free ends (Beguin and Aubert, 1994).However, although endoglucanase II has been labeled as an endo-glucanase, its enzymatic reactivity could be broader, producing glucose, cellobiose, and/or cellotriose during its hydrolysis of cellulose.The production of cellobiose in particular would be important since this is not an expected product of a typical endoglucanase (Nakazawa et al., 2008;Qin et al., 2008a).Indeed, it has been hypothesized that H. jecorina endoglu-canase II may be partially processive due to its extended substrate-binding site and the presence of a cellulose-binding domain (Medve et al., 1998a, Qin et al., 2008b).Similarly, in some organisms, such as Gloeophyllum trabeum, Fomitopsis pinicola, and Clostridium thermocellum, processive endoglucanases are produced that cleave cellulose internally but also release soluble oligosaccharides before detaching from the polysaccharide substrate (Cohen et al., 2005;Zverlov et al., 2005;Yoon et al., 2008).
In situ activity staining of the heterologous-expressed Hjegl2 in P. decumbens revealed the presence of two rather diffuse activity bands (between ~50-65 and ~80-116 kDa), not found in the control strain.These were especially evident in extracts derived from cells grown in glucose-containing media.Unexpectedly however, probing of the extract in Western blots using an anti-His tag antibody revealed the presence of two bands not found in controls corresponding to 25 and 60-65 kDa, respectively.Although the latter His-antibody identified band matches the corresponding activity bands, it is unclear why the higher molecular weight activity bands (80-116 kDa) did not react with the antibody, or what the 25 kDa Hisantibody reactive band may correspond to.The possibility exists that the lower band may correspond to a proteolytic cleavage product of Hjegl2, and the upper bands failed to react with the antibody due to steric hindrance derived from a post-translational modification (e.g.glycosylation or phosphorylation).Further experiments examining these possibilities may shed light on this issue, and precedent for such modification occurring exists.Homologous expression of the H. jecorina Cel7B (endoglucanase I) catalytic module revealed the production of several isoforms of the protein in the recombinant strain, and that this heterogeneity was considerably reduced after enzymatic N-deglycosylation (Eriksson et al., 2004;Hui et al., 2002).Besides N-glycan heterogeneity, Olinked glycosylation and/or protein oligomerization have also been shown to result in protein production heterogeneity.The linker region of CBH I from H. jecorina was shown to be extensively glycosylated with di-, and tri-saccharides at Thr and Ser residues (Hui et al., 2001), and furthermore, a band around 20 kDa (apparently inactive) was also observed in this case via the Western blot analysis; the authors suggest that this band may be due to proteolytic cleavage of the heterologous product.
In conclusion, Hjegl2 was expressed in P. decumbens, increasing its cellulose degrading potential.Expression of the enzyme was constitutive and two diffuse bands of activity could be visualized.Western blot analysis matched one of the activity band regions with the predicted product, although the other active band did not appear to react with the antibody, and an additional smaller molecular-weight (inactive) band anti-His antibody reactive band was visualized.Future work aimed at further maximizing the cellulytic potential of P. decumbens is underway.

Fig. 1 .
Fig. 1.Glucose consumption and endoglucanase activity (a) and filter paper activity (b) of Pd::Hjegl2 and Pd::ANIp8 cultured in media containing glucose as the main carbon source.

Fig. 2 .
Fig. 2. Endoglucanase activity (a) and filter paper activity (b) of Pd::Hjegl2 and Pd::ANIp8 cultured in media containing microcrystalline cellulose as the main carbon source.

Fig. 3 .
Fig.3.Gene expression of the four native P. decumbens cellulases genes (Pdegl1, Pdegl2, Pdcbh1, and Pdcbh2) in strains Pd::Hjegl2 and Pd::ANIp8 cultured in media containing glucose or wheat bran-microcrystalline cellulose as carbon sources, respectively.Expression of the actin gene was analyzed as a control.Copy number was normalized using actin expression for the same sample.

Table 1 .
Primers used in this work.