Heterologous expression of Melanocarpus albomyces cellobiohydrolase Cel7B, and random mutagenesis to improve its thermostability

Enzyme and Microbial Technology 41 (2007) 234–243

Heterologous expression of Melanocarpus albomyces cellobiohydrolase Cel7B, and random mutagenesis to improve its thermostability Sanni P. Voutilainen a , Harry Boer a , Markus B. Linder a , Terhi Puranen b , Juha Rouvinen c , Jari Vehmaanper¨a b , Anu Koivula a,∗ a

VTT Technical Research Centre of Finland, P.O. Box 10500, FI-02044 VTT, Finland b ROAL Oy, P.O. Box 57, FI-05201 Rajam¨ aki, Finland c Department of Chemistry, University of Joensuu, P.O. Box 111, FIN-80101 Joensuu, Finland Received 17 October 2006; received in revised form 11 January 2007; accepted 17 January 2007

Abstract Fungal cellobiohydrolases from the glycosyl hydrolase family 7 are key enzymes in crystalline cellulose hydrolysis. Difficulties in heterologous expression in a bacterial or yeast host have hampered engineering of these cellulases for industrial application. We report here a successful expression of the single-module cellobiohydrolase Cel7B from a thermophilic fungus Melanocarpus albomyces in Saccharomyces cerevisiae (Sc Cel7B). An automated, robotic thermostability screening method, based on residual activity measurements on a small soluble substrate methylumbelliferyllactoside (MULac), was then set-up to screen the first generation random mutant libraries. Out of the nine positive thermostable mutants, we picked three based on structural considerations, each containing a single amino acid change (A30T, G184D or S290T). Cel7B A30T and S290T mutants showed improved unfolding temperature (Tm ) by 1.5 and 3.5 ◦ C, respectively. In addition, the temperature optimum (Topt ) on a soluble substrate had improved by 5 ◦ C for the A30T mutant. Interestingly, the best enzyme variant on microcrystalline cellulose (Avicel) hydrolysis was the Cel7B S290T, which could hydrolyse Avicel at 70 ◦ C two times more effectively than the Sc Cel7B. Overall the consensus mutation S290T, located in the hydrophobic core of Cel7B, led to a cellobiohydrolase variant having also application potential in hydrolysis of polymeric substrates at elevated temperatures. © 2007 Elsevier Inc. All rights reserved. Keywords: Cellulase; Heterologous expression; High-throughput screening; Random mutagenesis; Thermostability; Saccharomyces cerevisiae; Melanocarpus albomyces

1. Introduction Cellulose is an important industrial raw material and a source of renewable energy in the biosphere. In nature cellulose exists mainly in plant cell walls as an insoluble, highly-ordered crystalline form. Cellulose is composed of d-glucose residues always linked by ␤-1,4-glycosidic bonds to form linear polymers with average chain length of 10,000 glycosidic residues, or even

Abbreviations: CBM, cellulose-binding module; CD, circular dichroism; GH, glycosyl hydrolase; Glc2 , cellobiose; Ma Cel7B, Melanocarpus albomyces cellobiohydrolase belonging to the glycosyl hydrolase family 7; MU, 4-Methylumbelliferone; MULac, 4-Methylumbelliferyl-␤-d-lactoside; PAHBAH, p-hydroxylbenzoic acid hydrazine; SC, synthetic complete; Sc Cel7B, Melanocarpus albomyces Cel7B produced in Saccharomyces cerevisiae; Tm , melting temperature; TPI, triose phosphate isomerase; Ura, uracil ∗ Corresponding author. Tel.: +358 20 7225110; fax: +358 20 7227071. E-mail address: [email protected] (A. Koivula). 0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2007.01.015

more. The smallest repetitive unit in cellulose is cellobiose, a disaccharide. Cellulolytic enzymes, produced mainly by bacteria and fungi living in plant litter and soil, cleave the ␤-1,4 linkages of a cellulose chain. An efficient, complete hydrolysis of insoluble cellulose requires several enzymes acting cooperatively (for a review, see [1]). Cellobiohydrolases (EC 3.2.1.91) are the key enzymes for the degradation of the highly ordered crystalline cellulose. In most cases, cellobiohydrolases are modular enzymes consisting of a minimum of one catalytic module and one carbohydrate-binding module (CBM) [2,3]. The CBM is responsible for bringing the catalytic module near the substrate and improving the action on polymeric substrates while not affecting the activity on small soluble substrates [3]. Both the catalytic and the carbohydrate-binding modules can be divided into different families based on sequence similarities and protein folds (http://afmb.cnrs-mrs.fr/CAZY/citing.html; [4]). The glycosyl hydrolase (GH) family of the catalytic modules relates also to the stereochemistry of the O-glycosidic bond cleavage, which

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can occur either through a retaining or inverting acid hydrolysis mechanism [5]. Enzymatic cellulose degradation has considerable interest due to its evident ecological and industrial importance. Cellulases can be used in pulp and paper, textile, detergent, food and feed industries, as well as in total hydrolysis of biomass to sugars for production of transport fuels (bioethanol) (for a review, see [1]). For many applications it would be desirable to have enzymes that are thermostable. Thermophilic organisms, particularly thermophilic eubacteria and archae, offer a potential source of thermostable enzymes for industrial applications, the drawback often being a low production level in the host organism, or in the standard recombinant production hosts. Another approach is to engineer an existing, well produced cellulase either by structure based rational mutagenesis, or by directed evolution approaches. Using directed evolution as a tool, protein engineering is not limited by lack of a known three-dimensional structure for the protein. Moreover, detailed structural information does not necessarily guarantee a successful outcome. In directed molecular evolution approaches, genetic diversity is created by random mutagenesis and/or DNA shuffling, which can be targeted to certain regions, or to the whole gene sequence. Prerequisites for directed evolution approaches are a functional gene expression system in a suitable host organism, such as a bacterium or yeast, and a screening method that works in a high-throughput fashion. A thermophilic ascomycete fungus Melanocarpus albomyces, formerly known also as Myriococcum albomyces or Thielavia albomyces, has been reported to produce xylanases and cellulases with pronounced thermal stability and activity in the neutral to alkaline pH range [6–8]. Three neutral cellulases (pH optima in the range 6–8) belonging to the GH families 7 and 45 have been recently produced and purified from the original Melanocarpus host, and subsequently cloned and expressed at high levels in an industrial production host Trichoderma reesei [9,10]. These cellulases have industrial applications where improved activity at higher temperatures would be beneficial. Single amino acid changes usually only cause relative small increases in thermostability (0.5–3 ◦ C) [11,12]. However, small improvements can have considerable economic benefits and the mutated proteins are potential starting points for further mutagenesis, because thermostability mutations are frequently additive [11,12]. We describe here the construction and use of a system for random mutagenesis of the single-module cellobiohydrolase Cel7B from M. albomyces (Ma Cel7B) by heterologous expression of its gene in the yeast Saccharomyces cerevisiae (Sc Cel7B). First generation random mutagenesis libraries of Sc Cel7B were screened by a novel automated highthroughput selection for improved thermostability and the three most interesting mutants were characterised in more detail. 2. Materials and methods 2.1. DNA manipulations in E. coli Unless specified otherwise, standard recombinant DNA techniques were carried out as described in [13]. Plasmid isolation from E. coli XL1-blue

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cells was performed using the Nucleospin plasmid kit (Macherey-Nagel, Germany). All sequencing steps were performed with the Big Dye Terminator Cycle Sequencing kit and using an ABI Prism 3100 Genetic Analyzer automated DNA sequencer (Applied Biosystems). PCR products were purified from a 1% (w/v) agarose gel using the Qiagen gel extract kit (Qiagen).

2.2. Cloning of the M. albomyces Cel7B cDNA and construction of its expression plasmid for Saccharomyces cerevisiae A Trichoderma reesei ALKO3620 derived strain, carrying multiple copies of the M. albomyces cel7B gene under the endogenous cel7A promoter, was grown for 64 h in the shake flask cultivation for mRNA isolation from mycelia with QuickPrep® Micro mRNA Purification Kit (Pharmacia Biotech) [14]. Synthesis of the M. albomyces Cel7B cDNA was performed by using the Universal RiboClone® cDNA Synthesis System (Promega) at 42 ◦ C for 2 h without sodium pyrophosphate. Thereafter, the first strand cDNA pool was used as a template to PCR the M. albomyces cel7B cDNA with primers 5 -TATATGAATTCCAGCACTCTGCAACCATGAT-3 (forward) and 5 -ATATTGAATTCGTGCATGGACCGGCTTAGAA-3 (reverse). The PCR reaction mixture contained 10 mM Tris–HCl, pH 8.8, 1.5 mM MgCl2 , 50 mM KCl, 0.1% (v/v) Triton X-100, 0.2 mM dNTPs, 1 ␮M each primer and 2 units of Dynazyme II DNA polymerase (Finnzymes, Finland). The thermal cycling parameters were 98 ◦ C for 5 min (1 cycle); 95 ◦ C for 1 min, 58 ◦ C for 1 min, 72 ◦ C for 2 min (30 cycles); 72 ◦ C for 10 min (1 cycle). The synthesized M. albomyces cel7B cDNA was ligated to EcoRI cut pUC19 vector and the resulting plasmid was designated as pALK1523. The cDNA encoding the M. albomyces Cel7B was isolated from the pALK1523 plasmid as NdeI-EcoRI fragment, and ligated into the yeast expression plasmid pYX212 (R&D Systems, USA), and the resulting Cel7B expression vector was called pSV7. The plasmid was characterised with restriction analysis and the nucleotide sequence of the whole insert was confirmed. For Cel7B expression, the plasmid pSV7 was transformed into S. cerevisiae strain NY179 (leu 2-3,112, ura 3–52) (received from Peter J. Novick, Yale University School of Medicine, USA), using the Gietz Lab Yeast Transformation kit (Tetra-Link, USA), and the transformation mixture was plated on SC-Ura selective agar plates [15] containing 2% (w/v) glucose. The corresponding control vector, pYX212, was also transformed into yeast. After 3 days growth at 30 ◦ C, transformants were picked and grown in 10 ml SC-Ura media buffered to pH 6 with 170 mM succinate and supplemented with 2% (w/v) glucose, at 30 ◦ C for 3 days. Extracellular cellulase activity was monitored daily by measuring in a microtiter plate the hydrolysis of 1.8 mM MULac in 50 mM NaOAc buffer pH 5, at 37 ◦ C. The reaction was stopped with 1 M Na2 CO3 and the fluorescence (i.e. production of MU) in each well was measured with a Victor2 V MTP (Wallac) reader (ex. 355 nm and em. 460 nm). The cellulase (MULac) positive transformants were further assayed for the presence of Sc Cel7B in yeast culture supernatants by Western blotting (see below).

2.3. Generation of the random mutant libraries of M. albomyces Cel7B Two random mutant libraries (A and B) of Cel7B were generated by errorprone PCR using MutazymeTM DNA polymerase according to GeneMorph PCR mutagenesis kit (Stratagene) instructions using the following conditions: 1 ␮M of each primer 5 -CAGTGGCATGTGAGATTCTCCG-3 (forward) and 5 -CCAGTGAATTGTAATACGAC-3 (reverse), 100 pg of template (in both libraries), 0.2 mM of each of the nucleotides (dNTPs), Mutazyme buffer (1×) and 2.5 U of Mutazyme polymerase. The thermal cycling parameters were 95 ◦ C for 5 min (1 cycle); 94 ◦ C for 1 min, 55 ◦ C for 1 min, 72 ◦ C for 2 min (30 cycles); 72 ◦ C for 5 min (1 cycle). For Library A, both the purified PCR product and the plasmid pYX212 were cut with EcoRI and XhoI, and ligated (T4 DNA ligase, Promega), and the ligation mixture was transformed into yeast (see above). For Library B, the mutagenized PCR product was transformed together with a linearised pYX212 plasmid (cut with EcoRI and XhoI) into S. cerevisiae strain NY179 making use of the homologous recombination in yeast [16]. The PCR product contained homologous flanking regions with the linearised vector at the 5 end (300 bp) and at the 3 end (150 bp). The yeast transformants were plated on large (22 × 22 cm)

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SC-Ura plates and incubated at 30 ◦ C for 3 days to recover the transformants. The colonies were counted, collected and resuspended into 0.9% (w/v) NaCl, 15% (v/v) glycerol, and stored at −80 ◦ C. About 30 colonies from the mutant Library B were randomly picked, and the plasmids isolated from yeast and transformed into E. coli by first breaking the yeast cells with glass beads (Sigma), and then using modified Qiagen’s alkaline lysis method for plasmid isolation. The plasmid DNA isolated from E. coli was used for further analysis by restriction enzyme digestions and DNA sequencing.

2.4. Robotic screening procedure Screening of the thermostability was performed in 96-well plates, with a soluble cellulase substrate, MULac (Biokemis, Russia). Thermostability was assessed based on residual activity measurement after a heat inactivation step. Robotic screening consisted of two rounds (screens 1 and 2). For the robotic Screen 1, single colonies were picked with a QPix colony picker (Genetix, UK), using yeast colony picking pins, into 96-well microtiter plates containing 200 ␮l of SC-Ura medium, pH 6. The microtiter plates were incubated at 30 ◦ C for 3 days with shaking and analysed using a robotic work station. The yeast growth in each microtiter plate, i.e. mother plate, was first verified at λ = 490 nm. From each well in the mother plates 50 ␮l of the culture supernatant was transferred to two new 96-well enzymatic assay plates (black polypropylene PCR type plates, MJ Research, USA) using a Multimek 96 channel pipettor (Beckman, USA). Then 50 ␮l of 50 mM NaOAc buffer, pH 5.5 was added. The final pH in the assay mixture was 5.8. The other plate went through a heat inactivation treatment at 76 ◦ C for 4 min in a PCR heater block, and then both plates were pre-incubated at 37 ◦ C for 15 min before adding the substrate with a Multidrop dispenser (10 ␮l of 5 mM MULac, in 50 mM NaOAc pH 5.5, 10% (v/v) DMSO). The reaction time for both plates was 30 min at 37 ◦ C. The reaction was stopped by adding 100 ␮l of 1 M Na2 CO2 , and the fluorescence in each well was measured with a Victor2 V MTP reader (ex. 355 nm and em. 460 nm). Screen 2 was done in a similar manner with the positive clones from Screen 1, except that every positive clone was now analysed in duplicate. After screen 2 the clones which were better than Sc Cel7B clones, were again picked and an activity screen was performed manually (3rd screen). Yeast clones were cultivated for 3 days at 30 ◦ C in test tubes (3 ml). MULac activity of the supernatants at pH 5.8, 22 ◦ C, was measured after 30 min reaction time, prior and after heat treatments (for 5 and 10 min) at 70 ◦ C.

2.5. Production of the Sc Cel7B and the selected mutants in shake flasks and in fermentor scale For larger scale production of the Sc Cel7B and the mutated variants, 2 l of SC-Ura medium, pH 6 was inoculated with a 10% volume of an overnight preculture. The cultures were grown under shaking (210 rpm) at 30 ◦ C. After 3 days of cultivation the supernatant was harvested by removing the cells by centrifugation for 15 min at 4000 × g. The same growth medium supplemented with 4% (w/v) glucose was also used for the 10 or 30 l bioreactor cultivations (Braun Biostat ED or C, Germany) of the Sc Cel7B and mutant enzyme producing S. cerevisiae strains. The temperature was 30 ◦ C, the pH range 5.8 ± 0.3 (NaOH/H2 SO4 ), aeration 0.5 vvm, stirring rate 400 rpm and the cultivation time 72 h. The supernatant was recovered by centrifugation and filtering through Seitz-K 150 and EK filters (Pall SeitzSchenk Filtersystems GmbH, Germany).

2.6. Protein purification The culture supernatant from the fermentation or shake flask cultivation was concentrated, and the buffer exchanged to 25 mM sodium phosphate buffer pH 6.1 by ultrafiltration with a 10 kDa cut-off Pellicon PTGC membrane (Millipore, USA). The sample was loaded onto a DEAE Sepharose FF (Pharmacia) anion exchange column equilibrated with 25 mM sodium phosphate buffer, pH 6.1. The column was washed with the equilibration buffer until the A280 of the effluent reached the value of the equilibration buffer, and then eluted with a linear gradient of 0–0.6 M NaCl in the equilibration buffer. Fractions were collected and screened for the presence of Sc Cel7B by measuring the activity against MULac

and analysing them on SDS-polyacrylamide gel electrophoresis (SDS–PAGE) as described below. Fractions containing a single band of Sc Cel7B on SDS-PAGE were pooled, and the samples were concentrated and the buffer was changed to 50 mM sodium phosphate buffer, pH 6.0 using 20 ml spin concentrators, cut-off 10,000 Da (Vivaspin, Vivascience GmbH, Germany).

2.7. Protein analysis Protein samples were characterised by SDS-PAGE performed as described in [17], and the gels were stained with Gel Code Blue Staining Reagent (Pierce, USA). For Western blot analysis rabbit polyclonal antibody raised against M. albomyces Cel7B enzyme was used to detect the different yeast expressed Cel7B variants [10,18]. The secondary antibody used in Western blots was alkaline phosphatase conjugated goat anti-rabbit immunoglobulin G (Bio-Rad, USA). The correct processing of the N-terminus of the Sc Cel7B protein was verified by Edman degradation. N-terminal amino acid sequence analysis was done with Procise 494A sequencer (PerkinElmer Applied Biosystems Division) from the concentrated yeast culture supernatant after gel electrophoresis. Differences in the N-glycosylation pattern of the heterologously expressed Cel7B were studied by EndoH treatment. The concentrated culture supernatant samples or purified Sc Cel7B and mutant proteins were incubated first in 0.2% (w/v) SDS, 1% (v/v) ␤-mercaptoethanol and 0.05 M EDTA, pH 8 at 94 ◦ C for 10 min. 0.5% (w/v) octylglucoside and 0.025 U of EndoH (Boehringer, Germany) was then added to the cooled samples and the reaction was allowed to proceed for 15 h at 21 ◦ C. The reaction samples were analysed on SDS-PAGE and Western blotting as described above. The isoelectric points of the purified Sc Cel7B and mutant S290T enzymes were determined by isoelectric focusing in the pH range of 3.5–9.5 (Ampholine PAGplate for IEF, Pharmacia) on an LKB 2117 Multiphor II Electrophoresis System (LKB Pharmacia) according to the manufacturer’s instructions. Bands containing cellobiohydrolase activity were visualised by staining the gel with 1 mM MULac in 50 mM sodium phosphate buffer (pH 6.0) and the Cel7B activity was detected under UV light (366 nm) as fluorescenting bands. Bands containing proteins were visualised with Gel Code Blue Staining Reagent. Protein concentrations of purified enzymes were measured by their absorption at 280 nm using a theoretical molar extinction coefficient, ε = 85,000 M−1 cm−1 , which was calculated from the primary amino acid sequence [19].

2.8. Kinetic measurements on soluble and insoluble substrates The specific activity of the purified yeast expressed Cel7B proteins as a function of temperature (45–75 ◦ C) was measured in 50 mM sodium phosphate buffer, pH 6.0 using MULac. The substrate concentration for each experiment was 3.2 mM and enzyme concentration 0.12 ␮M. At each temperature the measurements were repeated for four times, except at temperature 45 ◦ C where the measurements were done as duplicates. In each case 100 ␮l of purified protein sample was incubated with 400 ␮l of substrate. For each time point (2.5, 5, 7.5 and 10 min) 100 ␮l of this reaction mixture was transferred into a 1.5 ml eppendorf tube containing 200 ␮l 1 M Na2 CO3 to stop the reaction. Two hundred microliters of this mixture was transferred into a black microtiter plate and the liberation of MU was detected as described above. The microcrystalline cellulose (Avicel) hydrolysis assays were performed in 1.5 ml tube scale. Avicel (PH 101, Fluka, Switzerland) suspension (50 mg/ml) in 50 mM sodium phosphate, pH 6.0 was shaken at 55, 60, 65, 70, or 75 ◦ C, with the enzyme solution (80–90 ␮g/ml) in the final volume of 325 ␮l. The Avicel hydrolysis was followed up to 17 h by taking samples at four different time points, and stopping the reaction by adding 163 ␮l of stop reagent containing 9 vol of 94% (w/v) ethanol and 1 vol of 1 M glycine (pH 11). The solution was filtered through a Millex GV13 0.22 ␮m filtration unit (Millipore, USA). All experiments were performed as duplicates. The formation of soluble reducing sugars in the filtrate was determined by the para-hydroxybenzoic acid hydrazide (PAHBAH) method [20] using a cellobiose standard curve (50–800 ␮M cellobiose). A freshly made 0.1 M PAHBAH (Sigma) in 0.5 M NaOH (100 ␮l) solution was added to 150 ␮l of the filtered sample and boiled for 10 min. The absorbance of the cooled samples was measured at 405 nm.

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2.9. Enzyme stability measurements Initial unfolding studies were performed by monitoring the intrinsic tryptophan fluorescence of Sc Cel7B and the mutant proteins using a Varian Gary Eclipse spectrofluorometer [21]. Temperature-induced unfolding was monitored by heating samples gradually (approximately 1 ◦ C/min) from 25 ◦ C up to 78 ◦ C and measuring the fluorescence intensity. The temperature of sample solution was measured continuously using a thermocouple that was immersed in the solution. Intrinsic fluorescence of the samples was recorded after every 2 ◦ C by measuring emission at 340 nm using an excitation wavelength of 280 nm. All the experiments were performed in 50 mM sodium phosphate pH 6.0 using a protein concentration of 1.0 ␮M. CD measurements of the Sc Cel7B and four mutant enzymes were performed with a Jasco (model J-720) circular dichroism spectrometer equipped with a PTC-348 WI Peltier-type temperature control system [22]. Spectra were recorded from 240 to 190 nm using a 1 mm cell and a bandwidth of 1 nm. The unfolding curves were measured at 202 nm using the temperature scan mode with a gradient of 2 ◦ C/min until a temperature of 80 ◦ C was reached. The measurements were performed in 10 mM sodium phosphate buffer at pH 6.0 using a protein concentration of 3 ␮M.

2.10. Structural analysis The preliminary crystal structure of Ma Cel7B enzyme has been determined ˚ diffraction resolution (T. Parkkinen, A. Koivula, J. Vehmaanper¨a and J. at 1.9 A Rouvinen, unpublished work). The structural consequences of mutations were analyzed by mutating corresponding amino acids in the program XtalView [23].

3. Results 3.1. Heterologous expression of Cel7B in the yeast S. cerevisiae The single-module cellobiohydrolase (Ma Cel7B; Mw = 47.5 kDa) from a thermophilic fungus Melanocarpus albomyces was expressed in S. cerevisiae strain NY179. The heterologous expression was detected by measuring the activity on a soluble substrate, MULac, and by SDS-PAGE (Fig. 1A, lane 3). As can be seen from the Western blot analysis (Fig. 1B, lane 3), there is one major and one minor band of the Sc Cel7B protein produced in yeast (both bands located at around 50 kDa). The Ma Cel7B amino acid sequence contains two putative N-glycosylation sites at positions N5 and N320, which might cause multiple protein bands in the Western analysis. The EndoH treatment revealed indeed, that the upper minor band, visible in the Western blot, is due to hyperglycosylation in yeast, while no shift in the mobility of the major lower band could be detected on SDS-PAGE gel upon EndoH treatment (Fig. 1B, lane 1). The mobility of the lower band is virtually the same as the Ma Cel7B produced in a heterologous host T. reesei or in the original host M. albomyces [10]. A correct processing of the N-terminus after secretion of the Sc Cel7B protein (both protein bands) was verified from the concentrated yeast supernatant samples. The transformants secreted Sc Cel7B protein up to 3–5 mg/l of culture supernatant (in a test tube scale). Sc Cel7B enzyme could be purified with a one-step anion exchange column, resulting in a single Cel7B band devoid of the overglycosylated form (Fig. 1A and B, lane 5). Isoelectric focusing of the purified Sc Cel7B showed one band around pH 4 (theoretical pI = 4.4) upon activity staining with MULac (data not shown).

Fig. 1. SDS-PAGE analysis (A) and Western blot (B) of the S. cerevisiae supernatants and the purified Cel7B proteins. On both gels (A and B), the order of the samples is the same. Lanes 1 and 2: 20 times concentrated and EndoH treated culture supernatants of the Sc Cel7B and S290T mutant, respectively; lanes 3 and 4: 20 times concentrated culture supernatants of Sc Cel7B and S290T mutant without Endo H treatment, respectively; lanes 5 and 6: purified Sc Cel7B and S290T mutant protein, respectively. The positions of the molecular weight markers (94, 66, 45 and 30 kDa) are shown on the left side of the picture.

3.2. Creation of random mutant libraries The whole gene (cDNA) sequence coding for the Ma Cel7B was subjected to random mutagenesis by error-prone PCR. Both random mutant libraries (Library A and B) made in this study, were created with the Mutazyme polymerase and aiming in both cases at 3–4 nucleotide mutations per 1000 nucleotides. The total amount of transformants obtained was 2.6 × 105 clones in Library A, and 7.7 × 105 clones in Library B. The background i.e. the amount of transformants obtained with linearised vector DNA only was 8.8% and 1.5%, respectively. Homologous recombination of two partially overlapping DNA fragments in yeast produced thus a slightly higher yield of mutant clones, and also a lower vector background (Library B), as compared to transformation of a ligation mixture (Library A). Sequence analysis of 31 randomly picked clones showed that Library B contained on average 4.5 nucleotide mutations per gene, which corresponded with 2.2 amino acid changes per Cel7B mutant protein. The mutations were distributed over the whole gene sequence. Mutazyme polymerase can generate all possible nucleotide substitutions, but the frequency of incorporating each

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type of mutations differs: the likelihood of incorporating G → N and C → N mutations is three times higher than that of A → N and T → N mutations, where N is any of the four nucleotides. Our sequencing results corresponded well with these properties reported for the Mutazyme polymerase (GeneMorphTM PCR Mutagenesis Kit, Instruction manual, Stratagene). 3.3. Screening of the random mutant libraries The mutant libraries were screened for a cellulase with higher temperature stability using an activity assay based on the soluble fluorogenic substrate MULac, performed in 96-well plates. The residual activity of each clone was assayed after a heat inactivation step. For the heat inactivation step, several different temperatures and heat inactivation times were initially tested using the yeast clones expressing the Sc Cel7B. Due to evaporation in the microtiter plate wells, the heat inactivation time was set relatively short. The samples were incubated at 76 ◦ C for 4 min and after this the activity was measured and compared to the activity of a non-heat treated control plate. We used the ratio of the activity in the heat inactivated plate to the control plate as a measure for the thermostability of a mutant clone. By comparing the calculated ratios, the effect of variation in the expression levels of different mutant clones was controlled. As a measure for the variability in the readouts and ratios, the coefficient of variation (CV) was computed for the Sc Cel7B clone. For the final optimised robotic screen, a CV of 10–12% of the value for the ratio was achieved, and the residual activity ratio for the Sc Cel7B clone was 0.5–0.6. The mutant clones having higher activity ratio than Sc Cel7B were qualified for the next screening round. Altogether 380 colonies from the Library A and 14,200 colonies from Library B were screened with the robotic assay (screen 1), and 900 colonies out of these were picked and cul-

tivated again in a microtiter plate as duplicates, and analysed with the same robotic assay (screen 2). 200 positive clones were picked for a third, manual thermostability screening round at 70 ◦ C (see Materials and Methods), and 49 clones were finally found to be more thermostable than the Sc Cel7B. The complete gene sequences of the 49 clones from screen 3 were determined, and showed a minimum of one and maximum of four amino acid changes. 3.4. Purification and initial characterisation of the Sc Cel7B and mutant enzyme variants Out of the 49 positive mutant clones from the third screen, nine clones having the highest activity ratio were selected for further studies. These mutant clones were grown either in shakeflasks or in fermentor, and the excreted proteins were purified to homogeneity as shown by SDS-PAGE. In the case of fermentation, yield of the purified enzyme protein was 10–12 mg/l, and in the case of shake flask cultivations, 3–5 mg/l. The effect of the amino acid mutations on the thermostability in the nine best Cel7B mutant clones was studied by monitoring the tryptophan fluorescence as a function of temperature. Unfolding temperatures (Tm ) were determined by taking the inflection point of the unfolding curves of the purified Sc Cel7B and the nine mutants (Table 1). Each of the nine mutants had improved thermostability (by 1–3 ◦ C) when compared to the Sc Cel7B (Tm = 65 ◦ C). In addition, the structural consequences of the amino acid changes found in the nine mutant enzymes (Table 1) were estimated by inspecting the three-dimensional structure of the Ma Cel7B. The respective amino acid side-chains were replaced, and the changes in packing of atoms and intermolecular interactions such as hydrogen bonds were analyzed (see Table 1 and also Section 4).

Table 1 The measured unfolding temperatures of the nine thermostable Cel7B mutant enzymes from screen 3 Tm (Trp)a ◦ C

Tm (CD)b ◦ C

65c 68 68 nd

64.5c 68 64.5 66

Loop Loop Loop Loop

66

nd

Surface Surface Inside

Loop Loop Loop

66 66 66

nd nd nd

A3D P114T

Surface Surface

Loop Loop

66

nd

Y100N R166Q

Surface Surface

Loop Loop

66

nd

Clone

Mutation

Location of the mutation in the 3D structure

Location of the mutation in the structural element

Suggested structural consequence of the mutation

Sc Cel7B 50-G9 65-B3 56-B11

– S290T G184D A30T

– Inside Surface Surface

␤-Strand Loop ␤-Strand

Improved packing Possible salt bridge to K186 Improved packing

81-C1

T56S Y100N G386V D411G

Surface Surface Surface Surface

51-D6 73-B11 49-H5

G346D G75D A163T

90-F8 65-E4

Weakened packing

nd = not determined. a The unfolding temperature (T ) measured using tryptophan fluorescence. m b The unfolding temperature (T ) measured using CD. m c The T values for Sc Cel7B and the mutant S290T were measured as duplicates and the error is ±1 ◦ C. m

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Fig. 2. Temperature induced unfolding measured with CD spectroscopy, and the calculated unfolding temperatures (Tm ) for the Sc Cel7B and three mutant enzymes measured at pH 6.0. The curves of Sc Cel7B (), S290T (), G184D () and A30T (䊉) are shown. The unfolding curves for the Sc Cel7B and mutant S290T were measured as duplicates and the curves have been averaged (error ±1 ◦ C); curves for the other mutants were measured once.

Based on the measured thermostability data and the structural analysis, three most interesting mutant enzymes, each containing a single amino acid mutation (S290T, G184D or A30T), were chosen for a more detailed characterisation. 3.5. Unfolding temperature measurements with CD spectroscopy To further investigate the thermodynamic stability of the Sc Cel7B and the selected three mutant enzymes, temperature induced unfolding at pH 6.0 was measured by CD spectroscopy. The CD spectra at 25 ◦ C of the Sc Cel7B and mutant enzymes were measured from 240 to 190 nm at pH 6.0, and were identical. After this, the enzyme samples were heated from 40 to 80 ◦ C and the unfolding temperatures (Tm ) were determined by taking the point of inflection of the unfolding curves. The unfolding temperature for the Sc Cel7B was 64.5 ◦ C. The mutants S290T and A30T showed 3.5 and 1.5 ◦ C higher unfolding temperature, respectively, than the Sc Cel7B (Fig. 2). The mutant G184D had similar unfolding temperatures as the Sc Cel7B enzyme, contrary to the tryptophan fluorescence data (Tm improved by 3 ◦ C as compared to the Sc Cel7B, see Table 1). 3.6. Characterisation of the activity on a soluble substrate and crystalline cellulose

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Fig. 3. The temperature optimum curves of the Sc Cel7B and the three mutant enzymes. The specific activities of the Sc Cel7B (, dashed line), and the mutants A30T (䊉), S290T () and G184D () were determined with MULac at different temperatures in 50 mM sodium phosphate buffer pH 6.0. At each temperature, rates (determined by taking samples at four time points, stopping the reactions with 0.5 M Na2 CO3 , measuring the fluorescence of the samples and using the standard curve as described in Section 2) were linear within experimental error for at least 10 min, and are assumed to be initial rates.

temperature, S290T mutant seemed to have slightly lower specific activity, whereas the G184D mutant had somewhat higher specific activity than the Sc Cel7B enzyme (Fig. 3). The activity of the Sc Cel7B and the three mutant enzymes was also measured on microcrystalline cellulose (Avicel), in order to see how these enzyme variants hydrolyse polymeric substrates at elevated temperatures. Our results demonstrated that the S290T mutant could hydrolyse crystalline cellulose at 70 ◦ C almost two times more effectively than the Sc Cel7B enzyme, when calculated as the time to reach the same level of hydrolysis (Fig. 4A). Mutant G184D had a lower activity against Avicel at 70 ◦ C (Fig. 4A). A striking difference was observed with the mutant A30T which, although having even a slightly better temperature activity profile on MULac than the S290T mutant (Fig. 3), it had lost most of its ability to degrade crystalline cellulose at 70 ◦ C (Fig. 4A). The hydrolysis of Avicel by the Sc Cel7B and S290T mutant enzyme was also measured in the temperature range 55–75 ◦ C (Fig. 4B). The temperature optimum on Avicel was about 68 ◦ C for the S290T mutant and about 65 ◦ C for Sc Cel7B. Thus, the temperature optima of both enzymes were higher during reaction on microcrystalline cellulose than on the small, soluble substrate, MULac, by 10–15 ◦ C (compare Figs. 3 and 4B). 4. Discussion

Specific activity of the three purified Cel7B mutant proteins against MULac was determined as a function of temperature (45–75 ◦ C) at pH 6.0, measuring the apparent initial rates (over 10 min) at each temperature (Fig. 3). Under these conditions, the temperature optimum for the Sc Cel7B enzyme was around 55 ◦ C (rate = 19 ± 3 min−1 ). The temperature optimum of the S290T or the G184D mutant had not changed as compared to the Sc Cel7B enzyme, whereas the temperature optimum of the A30T mutant had shifted to a slightly higher temperature (Topt ∼ 60 ◦ C; rate = 23 ± 1 min−1 ). Below the optimum

Enzyme thermostability is a desired property in industrial biocatalysis, when combined with a high enzymatic activity at these elevated temperatures [24]. Most commercially available cellulases originate from fungi. Thermophily in fungi is, however, not as extreme as in various bacterial species, and the reported temperature optima for various fungal hydrolases are usually lower than 80 ◦ C [7]. To engineer fungal cellulases, a heterologous expression system in bacteria or yeast is preferred, as these hosts allow relatively fast generation of large enough

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Fig. 4. Degradation of microcrystalline cellulose (Avicel) by the Sc Cel7B and three mutants at elevated temperatures, pH 6.0. (A) The time-course of Avicel hydrolysis was followed at 70 ◦ C for 17 h for the Sc Cel7B () and mutants S290T (), G184D () and A30T (䊉). (B) The temperature optimum curve of the Avicel hydrolysis measured after 6 h at 55, 60, 65, 70 and 75 ◦ C for the Sc Cel7B () and the mutant S290T (). The soluble reducing sugars from the Avicel hydrolysis were measured with the PAHBAH reagent using cellobiose (Glc2 ) as a standard. All the measurements were done as duplicates. See Section 2 for details.

mutant libraries, and expression in a cellulase-free background. We therefore expressed the Cel7B gene from M. albomyces under a strong, constitutive promoter in S. cerevisiae to create a system suitable for random mutagenesis. Heterologous expression of fungal cellulases, especially those belonging to the GH family 7, in bacterial or yeast hosts has been a challenge because of the low expression levels and, in yeast, hyper-glycosylation usually obtained [25–27]. Our heterologous expression system was successful, leading to expression levels of 3–5 mg/l in shake flask cultivations, and one major Cel7B protein band with the expected molecular size. This system facilitated random mutagenesis of the Sc Cel7B protein, and convenient purification of mutated enzymes for kinetic and structural characterisation. Our goal was to obtain and identify mutant forms of Cel7B with improved thermostability. Mutants with industrially useful properties can be transferred to a suitable production strain, such as the fungus T. reesei. Although thermostability is one of the most studied and engineered protein properties, no generally applicable rules

have been established so far. Increased thermostability is thought to result from many small cumulative changes in the hydrophobic interactions in the core of a protein structure, in electrostatic interactions and in hydrogen bonds. Directed evolution to improve thermostability has been applied to various enzymes [28]. The prerequisite is that an effective and reliable screening method can be developed [29–31]. After a robotic high-throughput screening work, manual steps are usually needed to assay the properties of the positive hits (clones) more thoroughly. When considering real industrial applications, the robotic screening conditions should mimic the application conditions as closely as possible. In reality, however, compromises have to be made. To select enzymes for high activity at elevated temperatures i.e. for thermoactivity, measuring activities at desired elevated temperatures would be the most optimal screening method. However, in most cases there are technical obstacles, as many high-throughput robotic systems have incubator and microtiter plate readers that can only reach temperatures up to 50 ◦ C. In addition, high temperatures can cause evaporation, pH changes and/or substrate instability. Therefore, a more convenient experimental set-up is often a residual activity measurement at a lower temperature after incubation at elevated temperature, i.e. a thermostability screen. This was also the rationale for our thermostability screen. Concerning cellulases, there are surprisingly few publications on thermostability, or other protein property improvements [32–35]. To our knowledge, there are no published articles on the thermostability engineering of the GH family 7 cellobiohydrolases, which are nevertheless considered important enzymes in total hydrolysis of cellulose. Family 7 cellobiohydrolases are particularly active on crystalline cellulose and they hydrolyse cellulose in a processive manner from the reducing end, releasing mainly cellobiose and using a retaining acid hydrolysis mechanism [36–38]. The three-dimensional crystal structure of GH family 7 cellobiohydrolase catalytic modules from Trichoderma reesei (mesophilic fungus), Phanerochaete chrysosporium (mesophilic fungus) and Talaromyces emersonii (thermophilic fungus) have been solved [39–41]. The structure of the M. albomyces Cel7B has also been solved (T. Parkkinen, A. Koivula, J. Vehmaanper¨a and J. Rouvinen, unpublished work). The amino acid sequences of the three published cellobiohydrolases are 50, 49 and 53% identical with that of the Ma Cel7B sequence. Ma Cel7B has a typical family 7 cellobiohydrolase fold, where four surface loops create an ˚ long active site tunnel extending through the entire about 50 A molecule and providing nine subsites (−7 → +2) for binding of the cellulose chain (Fig. 5) [37,39]. The crystal structures of the cellobiohydrolases from mesophilic organisms T. reesei and P. chrysosporium were compared to those of the thermostable cellobiohydrolases from M. albomyces and T. emersonii. As the structural comparison did not show clear reasons for different thermal stabilities, we decided to randomize the whole gene sequence of Ma Cel7B by error-prone PCR, and screen the mutant clones by a robotic high-throughput thermostability screen. We found 49 positive clones among 14,600 random clones, and picked the nine best ones for purification and thermostability characterization with tryptophan fluorescence

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Fig. 5. The three-dimensional structure of the M. albomyces Cel7B (T. Parkkinen, A. Koivula, J. Vehmaanper¨a and J. Rouvinen, unpublished work) showing all the mutated amino acid side-chains (in yellow) in the nine thermostable mutant clones listed in Table 1. The active site tunnel containing the catalytic amino acids and the nine subsites for the cellulose binding is located along the longitudinal axel of the catalytic molecule and facing upwards in the figure. The three amino acid residues in the three mutant proteins (A30T, S290T and G184D) characterised in more details, are labelled in red. The picture was created with the program PyMol (DeLano Scientific, San Carlos, CA, USA).

(Table 1). As can be seen from Table 1, all nine purified mutant enzymes showed improved thermostability (Tm = 1–3 ◦ C) when compared to that of the Sc Cel7B cellulase (Tm = 65 ◦ C). According to the structural analysis of the amino acid alterations included in the nine Cel7B variants, there were only three amino acid changes, for which we could rationalize their potential role in improving the thermostability of Ma Cel7B. Mutations S290T and A30T, both located in the ␤-strands, could improve packing, and furthermore, there is a possibility for the formation of a new salt bridge between the residues G184D and K186 (Table 1). All three amino acid residues (A30, S290 and G184) are also located further away from the active site tunnel of the Cel7B enzyme, and not likely to directly interact with the bound cellulose chain. Based on these considerations, the more detailed characterisation was performed with three Cel7B mutants S290T, A30T and G184D, each containing a single amino acid change. CD spectroscopy instead of intrinsic tryptophan fluorescence was used in order to get a better picture of the unfolding of the secondary structure of the Cel7B variants upon heating. With the tryptophan fluorescence monitoring, changes in the local environment around the tryptophan residues upon unfolding are detected, and the results may therefore deviate from those obtained with CD spectroscopy (see also below) [42]. CD measurements showed an increase in the unfolding temperature for the S290T mutant by 3.5 ◦ C and for the A30T mutant by 1.5 ◦ C as compared to the Sc Cel7B protein (Fig. 2), consistent with the tryptophan fluorescence measurements (Table 1). However, the Tm value for the Cel7B G184D measured by CD did not show any improvement (Fig. 2), contrary to the tryptophan fluorescence measurements (Table 1), which may therefore be detecting a local stabilisation not detectable by CD spectroscopy. Single beneficial amino acid changes usually increase the Tm by 0.5–3 ◦ C [11,12], as we found here.

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We were also interested in testing the Cel7B mutants on polymeric substrate, although the Ma Cel7B cellobiohydrolase lacks a cellulose-binding module (CBM), and has limited action on crystalline substrates. The hydrolysis of insoluble polymeric substrates does not, in general, follow Michaelis-Menten type of kinetics, as the substrate changes during the hydrolysis and as the intermediately formed soluble oligomers are much better substrates than the insoluble polymer. The GH family 7 cellobiohydrolases have a long active site tunnel, which is expected to restrict the binding and activity of an isolated catalytic module at loose polysaccharide chains and at cellulose chain ends on the crystalline surfaces [43]. Under our assay conditions, 0.2–2% of the Avicel was solubilised by the different Cel7B variants after 17 h of hydrolysis (Fig. 4A). The thermostable mutant A30T (Fig. 2), which had the highest activity towards the soluble substrate (Fig. 3), had the lowest activity towards Avicel hydrolysis (0.2% of the Avicel solubilised after 17 h at 70 ◦ C). On the other hand, S290T mutant, which had the highest thermostability, but similar, or even lower specific activity and temperature optimum on MULac as the Sc Cel7B, was clearly the most active enzyme on Avicel (2% of the Avicel solubilised after 17 h at 70 ◦ C as compared to 1.2% by the Sc Cel7B) (Fig. 4A and B). The poor activity of the A30T mutant on crystalline cellulose might be explainable by the larger threonine residue, located on the surface of the Sc Cel7B variant, interfering with the binding and/or activity of the enzyme on the polymeric substrate. In the S290T mutant protein the residue S290 is buried between two ␤-sheets, and we speculate that in this case the threonine mutation improves the packing of the hydrophobic core, thus increasing the thermostability of the enzyme. S290T is also a consensus mutation, as the corresponding amino acid position contains frequently a threonine residue in many other cellobiohydrolases in the GH family 7. It has been shown for various other enzymes, that consensus mutations often lead to stabilised protein variants [44–46]. The increase in Avicel hydrolysis by the S290T mutation is apparently solely due to the increased thermostability of the mutant enzyme, as the specific activity on MULac seemed actually slightly lowered as compared to the Sc Cel7B (Fig. 3). It is also worth noting that the polymeric substrate stabilised both the Sc Cel7B and S290T protein substantially. The temperature optimum of the Sc Cel7B and S290T enzyme for the MULac hydrolysis was around 55 ◦ C, but for Avicel hydrolysis around 65 ◦ C for the Sc Cel7B, and close to 70 ◦ C for the S290T mutant (Figs. 3 and 4B). We have reported earlier, that the presence of a substrate, bound presumably to the active site tunnel of the enzyme, improved the thermostability of another fungal GH family 7 cellobiohydrolase [42]. In conclusion, we have established a functional heterologous expression system for a fungal GH family 7 cellobiohydrolase and demonstrated that it can be used to improve the thermostability of a cellobiohydrolase by random mutagenesis and high throughput screening. Increases in thermostability of 1–3 ◦ C were obtained, and apparently led to increased temperature optima for hydrolysis of small substrates and crystalline microcellulose (Avicel). One mutant, S290T, exhibited a two-fold increase in the rate of hydrolysis of Avicel at 70 ◦ C. Thus, this expression, mutagenesis and screening system can be used to

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