Variants of exoglucanases having improved activity and uses thereof

Abstract

The present invention relates to the expression and optimization of enzymes involved in the breakdown of lignocellulosic biomass. Disclosed are variants of the exoglucanase 1 of Trichoderma reesei, as well as the use of said variants with improved efficiency in methods for breaking down cellulose and for producing biofuel.

Claims

1. An isolated or purified polypeptide having exoglucanase activity which is improved by at least 10% at a temperature of 35° C. compared with the exoglucanase activity of the exoglucanase (CBH1) reference protein of SEQ ID NO: 2, said polypeptide comprising an amino acid sequence selected from the group consisting of: an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 12, the amino acid sequence of SEQ ID NO: 16, an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 18, and an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

2. A purified or isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of claim 1.

3. The purified or isolated nucleic acid of claim wherein the nucleotide sequence is selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17 and SEQ ID NO: 19.

4. A vector comprising the nucleic acid of claim 2.

5. An isolated host cell comprising the polypeptide of claim 1 or the nucleic acid of claim 2.

6. The isolated host cell of claim 5, wherein the host cell is selected from the group consisting of a Trichoderma cell, an Aspergillus cell, a Neurospora cell, a Humicola cell, a Penicillium cell, a Fusarium cell, a Thermomonospora cell, a Myceliophthora cell, a Chrysosporium cell, a Bacillus cell, a Pseudomonas cell, an Escherichia cell, a Clostridium cell, a Cellulomonas cell, a Streptomyces cell, a Yarrowia cell, a Pichia cell, and a Saccharomyces cell.

7. The isolated host cell of claim 5, wherein the host cell is selected from the group consisting of Trichoderma reesei, Trichoderma viridae, Trichoderma koningii, Aspergillus niger, Aspergillus nidulans, Aspergillus wentii, Aspergillus oryzae, Aspergillus phoenicis, Neurospora crassa, Humicola grisae, Myceliophthora thermopila, Chrysosporium lucknowense, Penicillium pinophilum, Penicillium oxalicum, Escherichia coli, Clostridium acetobutylicum, Clostridium saccharolyticum, Clostridium benjerinckii, Clostridium butylicum, Pichia pastoris, Yarrowia lipolityca and Saccharomyces cerevisiae.

8. An enzymatic composition capable of hydrolyzing lignocellulosic biomass, said enzymatic composition comprising the polypeptide of claim 1.

9. A process for producing an alcohol from lignocellulosic biomass, comprising the following successive steps: suspending the lignocellulosic biomass in an aqueous phase; contacting the suspended lignocellulosic biomass with the enzymatic composition of claim 8 to hydrolyze the lignocellulosic biomass and produce a hydrolysate containing glucose; fermenting the glucose of the hydrolysate with a fermentative microorganism to produce a fermentation must comprising the alcohol; and separating the alcohol from the fermentation must, wherein the alcohol is selected from the group consisting of ethanol, butanol, isopropanol, 1,2-propanediol, 1,3-propanediol, 1,4-propanediol, and 2,3-butanediol.

10. A process for producing an alcohol from lignocellulosic biomass, comprising the following successive steps: suspending the lignocellulosic biomass in an aqueous phase; contacting the suspended lignocellulosic biomass with the enzymatic composition of claim 8 and a fermentative microorganism to simultaneously hydrolyze the lignocellulosic biomass to produce glucose and ferment the glucose to produce a fermentation must comprising the alcohol; and separating the alcohol from the fermentation must, wherein the alcohol is selected from the group consisting of ethanol, butanol, isopropanol, 1,2-propanediol, 1,3-propanediol, 1,4-propanediol, and 2,3-butanediol.

11. The process of claim 9, wherein the fermentative microorganism comprises at least one isolated or purified polypeptide, or at least one nucleic acid comprising a nucleotide sequence encoding the at least one isolated or purified polypeptide, wherein the polypeptide has exoglucanase activity which is improved by at least 10% at a temperature of 35° C. compared with the exoglucanase activity of the exoglucanase (CBH1) reference protein of SEQ ID NO: 2, said polypeptide comprising an amino acid sequence selected from the group consisting of: an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 12, the amino acid sequence of SEQ ID NO: 16, an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 18, and an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

12. The process of claim 10, wherein the fermentative microorganism comprises at least one isolated or purified polypeptide, or at least one nucleic acid comprising a nucleotide sequence encoding the at least one isolated or purified polypeptide, wherein the polypeptide has exoglucanase activity which is improved by at least 10% at a temperature of 35° C. compared with the exoglucanase activity of the exoglucanase (CBH1) reference protein of SEQ ID NO: 2, said polypeptide comprising an amino acid sequence selected from the group consisting of: an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 12, the amino acid sequence of SEQ ID NO: 16, an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 18, and an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

13. The process of claim 10, wherein the fermentative microorganism comprises at least one vector comprising at least one nucleic acid comprising a nucleotide sequence encoding an isolated or purified polypeptide, wherein the polypeptide has exoglucanase activity which is improved by at least 10% at a temperature of 35° C. compared with the exoglucanase activity of the exoglucanase (CBH1) reference protein of SEQ ID NO: 2, said polypeptide comprising an amino acid sequence selected from the group consisting of: an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 12, the amino acid sequence of SEQ ID NO: 16, an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 18, and an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

14. An isolated host cell comprising the vector of claim 4.

15. The process of claim 9, wherein the fermentative microorganism comprises at least one vector comprising at least one nucleic acid comprising a nucleotide sequence encoding an isolated or purified polypeptide, wherein the polypeptide has exoglucanase activity which is improved by at least 10% at a temperature of 35° C. compared with the exoglucanase activity of the exoglucanase (CBH1) reference protein of SEQ ID NO: 2, said polypeptide comprising an amino acid sequence selected from the group consisting of: an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 12, the amino acid sequence of SEQ ID NO: 16, an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 18, and an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

16. The process of claim 9, wherein the enzymatic composition is produced by the fermentative microorganism.

17. The process of claim 10, wherein the enzymatic composition is produced by the fermentative microorganism.

18. The isolated or purified polypeptide of claim 1, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 12, the amino acid sequence of SEQ ID NO: 16, an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 18, and an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

19. The isolated or purified polypeptide of claim 1, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 12, the amino acid sequence of SEQ ID NO: 16, the amino acid sequence of SEQ ID NO: 18, and the amino acid sequence of SEQ ID NO: 20.

Description

(1) Brief Description of the Figures

(2) FIG. 1 is a MALDI-TOF mass spectrum representing the DP3 to DP11 cellodextrins used for the screening.

(3) FIG. 2 is a photo showing the hydrolysis halos generated by the enzymes secreted by the pure clones isolated during the screening of T. reesei in Walseth Petri dishes.

(4) FIG. 3 is a two-dimensional electrophoresis gel comparing the secretomes of the T. reesei clones; CL847 reference strain; CL847ΔCBH1 reference strain; isolated pure clones No. 6 and No. 20 of the 130G9 variant (SEQ ID NO: 8).

(5) FIG. 4 is a two-dimensional electrophoresis gel comparing the secretomes of the T. reesei clones: CL847 reference strain; CL847ΔCBH1 reference strain and isolated pure clone No. 24 of the 453E8 variant (SEQ ID NO: 16).

(6) FIG. 5 is a graph presenting the results of SHF for the 453E8-24 cocktail, derived from strain No. 24 expressing the 453E8 variant (SEQ ID NO: 16) and the CL847 reference cocktail supplemented with β-glucosidase.

(7) FIG. 6 is a graph presenting the results of SHF for the 130G9-6 and 130G9-20 cocktails derived from strains No. 6 and No. 20 expressing the 130G9 variant (SEQ ID NO: 8) and the CL847 reference cocktail supplemented with β-glucosidase.

(8) FIG. 7 is a graph presenting the results of SSF for the 453E8-24 cocktail, derived from strain No. 24 expressing the 453E8 variant (SEQ ID NO: 16) and the CL847 reference cocktail supplemented with β-glucosidase.

(9) FIG. 8 is a graph presenting the results of SSF for the two cocktails 130G9-6 and 130G9-20 derived from strains No. 6 and No. 20 expressing the 130G9 variant (SEQ ID NO: 8) and the CL847 reference cocktail supplemented with β-glucosidase.

EXAMPLES

Example 1

Preparation of DP 3-11 Reduced Cellodextrins

(10) 1—Cellulose Hydrolysis

(11) Adapted from Y-H. Percival Zhang, L. R. Lynd Analytical Biochemistry 322 (2003), 225-232.

(12) ##STR00001##

(13) 20 g of cellulose (Avicel, CAS Number 9004-34-6, Sigma-Aldrich Saint-Quentin Fallavier) are added portionwise and with vigorous stirring to 160 ml of a hydrochloric acid solution cooled to 0° C. Precooled sulfuric acid is added to the solution in several steps (4×10 ml). The reaction is kept stirring for four hours at 24° C. before being poured into 1.8 l of acetone cooled to −20° C. After two hours of stirring, the precipitate is filtered off, taken up in 400 ml of cooled acetone and then again filtered. The solid is then taken up in 600 ml of water, and then stirred overnight in order to dissolve the cellodextrins (CDs). After the solid has been filtered off, the soluble fraction containing the cellodextrins is neutralized with 300 g of Amberlite IRA 400 OH.sup.− resin and then lyophilized. The lyophilisate is then resuspended in 500 ml of methanol in the presence of ultrasound for 30 minutes in order to dissolve the low-molecular-weight sugars, before being filtered and then lyophilized again so as to give 6.8 g of DP 3-11 cellodextrins. For the screening, it was chosen to work with substrates of the highest possible molecular weight in order to mimic as closely as possible the structure of cellulose. However, high-molecular-weight cellodextrins are not soluble, which prevents good reproducibility of the tests.

(14) A range of cellodextrins of DP 5-7 was therefore chosen, which represents a good compromise between the high molecular weight required and the solubility of the cellodextrins.

(15) FIG. 1 presents a MALDI-TOF mass spectrum typically obtained according to the process described above.

(16) FIG. 1 shows that the isolated oligosaccharides are predominantly of DP 5-7.

(17) 2—Cellodextrin Reduction

(18) 400 mg of sodium borohydride are added to 2 g of DP 3-11 cellodextrins diluted in 120 ml of water. After three hours with stirring at ambient temperature, the solution is neutralized by adding Amberlite H.sup.+ IR 120 resin, filtered, and then lyophilized, so as to give 2 g of quantitatively reduced cellodextrins (C. Schou, G. Rasmussen, M-B. Kaltoft, B. Henrissat, M. Schulein Eur. J. Biochem. 217, 947-953 (1993)).

(19) Assaying of the isolated cellodextrins with BCA (bicinchoninic acid) makes it possible to verify the total reduction of the ends (Y.-H. Percival Zhang, L. R. Lynd Biomacromolecules 2005, 6, 1510-1515).

Example 2

Evolution by L-Shuffling

(20) The sequence of the cellobiohydrolase 1 gene (cbh1, SEQ ID NO: 1) from Trichoderma reesei was subjected to a round of L-shuffling according to the patented process described in patent EP 1 104 457 with the genes of a cellobiohydrolase from Talaromyces stipitatus ATCC 10500 (TS, SEQ ID NO: 23) and of a cellobiohydrolase from Neosartorya fischeri NRRL 181 (NF, SEQ ID NO: 21) having respectively 62% and 61% homology with the cbh1 parental gene.

(21) 1—High-Throughput Screening

(22) A high-throughput screening test was developed in order to select the best clones resulting from the L-shuffling, i.e. those exhibiting at least 20% improvement in the cellobiohydrolase activity compared with the cbh1 reference enzyme (SEQ ID NO: 2).

(23) The high-throughput screening test was carried out according to the following steps: isolation on agar of the clones of Y. lipolytica expressing the L-shuffling variants of the enzyme according to the invention and preculturing in YNB casa medium (yeast nitrogen base 1.7 g/l, NH.sub.4Cl 10 g/l, glucose 10 g/l, casamino acids 2 g/l, pH 7) of said colonies for 36 hours at 28° C.; inoculation of a YTD medium (yeast extract 10 g/l, tryptone 20 g/l, glucose 2.5 g/l, pH 6.8) supplemented with tetracycline at 12.5 μg/ml at 5% with the preculture and then incubation for 20 hours at 28° C.; inoculation of the expression medium containing the inducer (oleic acid) in an amount of 20 g/l at 10% with the previous culture and then incubation for 96 hours at 28° C.; centrifugation for five minutes at 1500 rpm; removal of 100 μl of supernatant; addition of 100 μl of reduced CDs at 1 g/l in 0.1 M citrate phosphate buffer at pH 6; incubation for 17 hours at 50° C.; centrifugation for five minutes at 2500 rpm; removal of 80 μl of supernatant; addition of 80 μl of DNS reagent; incubation for 12 minutes at 105° C. and then five minutes on ice; reading of the optical density (OD) at 540 nm on 120 μl.

(24) Under these screening conditions, an improvement in the cellobiohydrolase activity (increase in the OD at 540 nm) compared with the cbh1 reference enzyme (SEQ ID NO: 2) was found in several clones, including in particular the 32F9, 64C2, 130G9, 224C11, 225B11 and 453E8 clones (respectively SEQ ID NO: 4, 6, 8, 10, 12 and 16).

(25) 2—Determination of the Improvement in the Cellobiohydrolase Activity

(26) 2-1/On the Reduced-Cellodextrin Substrate

(27) In order to estimate the relative kcat of the variants selected in the first round of L-shuffling with respect to the cbh1 reference enzyme (SEQ ID NO: 2), the following process is carried out: preparation of a stock culture of Y. lipolytica expressing a recombinant enzyme according to the invention, overnight at 28° C.; inoculation of an expression medium with a volume of stock culture making it possible to have an OD at 600 nm equal to 0.2 at the beginning of the culture; culture of said cells at 28° C. for 96 hours; centrifugation at 8000 rpm for five minutes; incubation of 100 μl of supernatant with 100 μl of 0.1 M citrate phosphate buffer, pH 6, containing 1% of reduced CDs, for 24 hours at 35° C. and 50° C.; removal of 100 μl of reaction; addition of 100 μl of DNS reagent; incubation for five minutes at 100° C.; incubation for three minutes on ice; centrifugation for 10 minutes at 3000 rpm; reading of the OD at 540 nm on 150 μl.

(28) According to the invention, the calculation of the kcats is carried out in the following way: plotting the curve of the ODs at 540 nm as a function of the amount of protein of interest (in nM); subtracting the value of the negative control; dividing by the direction coefficient of the glucose standard rate (various amounts of glucose are revealed with the DNS); dividing by the reaction time (1440 minutes).

(29) Table 2 gives the value of the kcats and also the improvement factor obtained for the 32F9, 64C2, 130G9, 224C11, 225B11 and 453E8 clones (respectively SEQ ID NOs: 4, 6, 8, 10, 12 and 16) compared with the cbh1 reference protein (SEQ ID NO: 2) under these experimental conditions.

(30) TABLE-US-00002 TABLE 2 improvement in the cellobiohydrolase activity on reduced CDs 35° C. 50° C. Kcat Kcat Improvement Clone (min.sup.−1) (min.sup.−1) factor First- 32F9 0.0024 0.0116 1.5 round 64C2 0.0165 0.019 2.4 clones 130G9 0.0031 0.0071 0.9 224C11 0.0032 0.0112 1.4 225B11 0.0022 0.0092 1.2 453E8 0.0043 0.019 2.4 Reference cbh1 0 0.008 1 protein

(31) At 35° C., the improvement factor compared with the cbh1 reference enzyme (SEQ ID NO: 2) could not be calculated because, under these experimental conditions, the activity of cbh1 is not measurable. The enzymatic activity of the 32F9, 64C2, 224C11, 225B11 and 453E8 clones is improved at 35° C. and 50° C. compared with the enzymatic activity of the cbh1 reference enzyme (SEQ ID NO: 2). The enzymatic activity of the 130G9 enzyme (SEQ ID NO: 8) is improved at 35° C. compared with the enzymatic activity of the cbh1 reference enzyme (SEQ ID NO: 2).

(32) 2-2/On the Avicel Substrate

(33) The improvement in activity of the 32F9, 64C2, 130G9, 224C11, 225B11 and 453E8 clones (respectively SEQ ID NOs: 4, 6, 8, 10, 12 and 16) was then measured with a second substrate: Avicel.

(34) The activity of these clones was determined by measuring the end-point OD at 540 nm according to the protocol described above. The reduced-cellodextrin substrate is replaced with the Avicel substrate at the same concentration. The activity test is carried out with 100 μl of culture supernatant containing the protein of interest, for 48 hours.

(35) Table 3 presents the value of the ODs at 540 nm after subtraction of the OD value obtained with the negative control and also the improvement factor of the 32F9, 64C2, 130G9, 224C11, 225B11 and 453E8 clones (respectively SEQ ID NOs: 4, 6, 8, 10, 12 and 16) compared with the cbh1 reference enzyme (SEQ ID NO: 2) under these experimental conditions.

(36) TABLE-US-00003 TABLE 3 improvement in the cellobiohydrolase activity on Avicel 35° C. 50° C. Delta Delta OD Improvement OD Improvement Clone 540 nm factor 540 nm factor First- 32F9 0.023 1.6 0.033 1.5 round 64C2 0.007 0.5 0.0176 0.8 clones 130G9 0.076 5.4 0.065 3.0 224C11 0.014 1.0 0.046 1.0 225B11 0.008 0.6 0.009 0.4 453E8 0.029 2.1 0.05 2.3 Reference cbh1 0.014 1 0.022 1 protein

(37) These results show an improvement in the enzymatic activity, compared with the cbh1 reference enzyme (SEQ ID NO: 2) for the 32F9, 130G9 and 453E8 clones (respectively SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 16) at 35° C. and 50° C.

Example 3

Evolution by Recombination

(38) The 32F9, 130G9 and 453E8 genes (respectively SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 15) were chosen because the enzymes that they encode are improved on reduced CDs and Avicel. The 242D11 gene (SEQ ID NO: 13) was selected because its sequence differs from that of the 32F9, 130G9 and 453E8 clones and thus makes it possible to improve the sequence diversity. The 32F9, 130G9, 453E8 and 242D11 genes were recombined to generate new mutants. The activity of the mutants obtained was first of all evaluated with the reduced-CD substrate according to the protocol described in section 2-1 of example 2.

(39) 1—Determination of the Improvement in the Cellobiohydrolase Activity

(40) 1-1/On the Reduced-Cellodextrin Substrate

(41) Mutant B (SEQ ID NO: 18) has an improved cellobiohydrolase activity (increase in the OD at 540 nm) compared with the 453E8 variant (SEQ ID NO: 16). The 453E8 variant is the best variant resulting from the evolution by L-shuffling.

(42) Table 4 presents the value of the kcats and also the improvement factor obtained for clone B compared with the 453E8 protein (SEQ ID NO: 16) under these experimental conditions. The kcats are calculated according to the protocol described in section 2-1 of example 2.

(43) TABLE-US-00004 TABLE 4 improvement in the cellobiohydrolase activity on reduced cellodextrins 35° C. 50° C. Kcat Improvement Kcat Improvement Clone (min.sup.−1) factor (min.sup.−1) factor B 0.0054 2.2 0.0136 0.9 Reference 453E8 0.0025 1 0.015 1 protein

(44) The result show an improvement in the enzymatic activity compared with the reference enzyme (SEQ ID NO: 16) for clone B (SEQ ID NO: 18) at 35° C.

(45) 1-2/On the Avicel Substrate

(46) The improvement in activity of clone B was then confirmed with a second substrate: Avicel.

(47) The activity of these clones was determined by measuring the end-point OD at 540 nm according to the protocol described in section 2-2 of example 2.

(48) Table 5 presents the value of the kcats and also the improvement factor obtained for clone B compared with the 453E8 reference protein (SEQ ID NO: 16) under these experimental conditions.

(49) TABLE-US-00005 TABLE 5 improvement in the cellobiohydrolase activity on Avicel 35° C. 50° C. Delta OD Improvement Delta OD Improvement Clone 540 nm factor 540 nm factor B 0.041 2.15 0.008 0.2 Reference 453E8 0.019 1 0.039 1 protein

(50) These results show an improvement in the enzymatic activity compared with the 453E8 enzyme (SEQ ID NO: 16) for clone B (SEQ ID NO: 18) at 35° C.

Example 4

Evolution by Evosight

(51) In order to improve the cellobiohydrolase activity, the Evosight strategy (patent application WO 2006/003298) was applied to the 453E8 mutant (SEQ ID NO: 15), the best variant resulting from the L-shuffling.

(52) 1—High-Throughput Screening

(53) The high-throughput screening test used to select the best clones, i.e. those exhibiting at least 20% improvement in cellobiohydrolase activity compared with the 453E8 enzyme (SEQ ID NO: 16), is the same as that described in section 1 of example 2. The variants generated by Evosight are compared with the 453E8 clone (SEQ ID NO: 16) because it is the best clone resulting from the L-shuffling.

(54) Under these screening conditions, an improvement in the cellobiohydrolase activity (increase in the OD at 540 nm) compared with the 453E8 enzyme (SEQ ID NO: 16) was found in several clones, in particular the 91D9 clone (SEQ ID NO: 20).

(55) 2—Determination of the Improvement in the Cellobiohydrolase Activity

(56) 2-1/On the Reduced-Cellodextrin Substrate

(57) The protocol used to determine the relative kcat of the 91D9 clone (SEQ ID NO: 20) compared with the 453E8 enzyme (SEQ ID NO: 16) is identical to that described in section 2-1 of example 1.

(58) Table 6 presents the value of the kcats and also the improvement factor obtained for the 91D9 clone compared with the 453E8 enzyme (SEQ ID NO: 16) under these experimental conditions.

(59) TABLE-US-00006 TABLE 6 improvement in the cellobiohydrolase activity on reduced cellodextrins 35° C. 50° C. Kcat Improvement Kcat Improvement Clone (min.sup.−1) factor (min.sup.−1) factor 91D9 0.0072 2.88 0.0174 1.2 Reference 453E8 0.0025 1 0.015 1 protein

(60) These results show an improvement in the enzymatic activity compared with the 453E8 enzyme (SEQ ID NO: 16) for the 91D9 clone (SEQ ID NO: 20) at 35° C. and 50° C.

(61) 2-2/On the Avicel Substrate

(62) The improvement in activity of the 91D9 clone was then confirmed with a second substrate: Avicel.

(63) The activity of this clone was determined by measuring the end-point OD at 540 nm according to the protocol described in section 2-2 of example 2.

(64) Table 7 presents the value of the kcat and also the improvement factor obtained for the 91D9 clone compared with the 453E8 protein (SEQ ID NO: 16) under these experimental conditions.

(65) TABLE-US-00007 TABLE 7 improvement in the cellobiohydrolase activity on Avicel 35° C. 50° C. Delta OD Improvement Delta OD Improvement Clone 540 nm factor 540 nm factor 91D9 0.05 2.63 0.004 0.2 Reference 453E8 0.019 1 0.039 1 protein

(66) These results show an improvement in the enzymatic activity compared with the 453E8 enzyme (SEQ ID NO: 16) for the 91D9 enzyme (SEQ ID NO: 20) at 35° C.

Example 5

Cloning of the Exoglucanase 1 Variants 130G9 and 453E8 in the T. reesei CL847 ΔCBH1 Strain

(67) The 130G9 and 453E8 variants are clones resulting from the L-shuffling. Each variant was cloned into a T. reesei CL847 ΔCBH1 strain.

(68) The coding sequences of the 130G9 and 453E8 variants were amplified by PCR using the following oligonucleotides:

(69) TABLE-US-00008 For: (SEQ ID NO: 25) TCCATCctcgagatgtatcggaagttggccgtc Rev: (SEQ ID NO: 26) TCCATCctcgagttacaggcactgagagtagtaag

(70) The fragments obtained were digested with XhoI and then cloned into an expression vector between the cbh1 promoter and terminator, according to methods known to those skilled in the art (Wang et al., 2012, Microb Cell Fact. 2012 Jun. 18; 11:84. doi: 10.1186/1475-2859-11-84). The selectable marker of the vector is phleomycin (Calmels et al., 2011, Curr Genet. 1991 September; 20(4):309-14).

(71) The strain used for the construction is a CL847 strain (Durand et al., 1988, Enz. Microb Technol, 10, 341-346), the CBH1 gene of which has been removed beforehand according to a method known to those skilled in the art (Suominen et al., MGG, 1993, 241; 523-530) to give the CL847ΔCBH1 strain. Protoplasts of the T. reesei CL847ΔCBH1 strain were transformed according to a conventional method known to those skilled in the art, by calcium and PEG shock, with 5 μg of the DNA fragment containing the sequences encoding the 130G9 or 453E8 variant. The clones thus obtained were selected on PDA/sucrose selective medium containing 50 g/ml of phleomycin. The number of clones obtained after purification and isolation is presented in table 8.

(72) TABLE-US-00009 TABLE 8 Selection of the clones having integrated the variant of interest Number of clones subcultured after Number of pure Variant name transformation clones isolated 130G9 231 19 453E8 189 11

(73) The activity of the isolated pure clones is screened on cellulose dishes coupled with analysis of the secretome on a 2D gel.

(74) The screening medium, termed “Walseth Cellulose” medium, is prepared in the following way: 250 ml/l of “4N” medium (KOH 3.32 g/l, 85% H.sub.3PO.sub.4 5 ml/l, (NH.sub.4).sub.2SO.sub.4 5.6 g/l, MgSO.sub.4.7H.sub.2O 1.2 g/l, CaCl.sub.2.2H.sub.2O 1.2 g/l, Na.sub.2HPO.sub.4.12H.sub.2O: 0.23 g/l, pH adjusted to 1.5 with H.sub.2SO.sub.4); 1 ml/l of a solution of trace elements (FeSO.sub.4.7H.sub.2O 30 g/l, Co(NO.sub.3).sub.2.6H.sub.2O 9 g/l, MnSO.sub.4.1H.sub.2O 6.4 g/l, ZnSo.sub.4.7H.sub.2O 8.4 g/l, boric acid 0.4 g/l, sodium molybdate 1.04 g/l, pH adjusted to 1.5 with H.sub.3PO.sub.4); 2 g/l of peptone; 2 g/l of agar; 50 g/l of 8% cellulose prepared according to the Walseth method (Walseth, 1952, Tappi, 225; 228-232).

(75) The whole mixture is homogenized using a homogenizer (Ultra Turrax, Ika, Germany) for five minutes. The pH is adjusted to 6.0 with a 3 M KOH solution. The medium obtained is autoclaved at 110° C. for 30 minutes. When the temperature of the medium is 50° C., the phleomycin is added in an amount of 50 μg/ml. The medium is then transferred into the Petri dishes in an amount of 20 ml/dish. The solidification is monitored until complete setting of the agar, on which a disk of perforated Plexiglass is then placed; 24 wells per dish are thus created.

(76) The screening step is carried out by depositing extracts of agar carrying isolated clones resulting from the transformation in the wells of the Walseth Petri dishes (one isolated pure clone/well). This system makes it possible to obtain enzymatic hydrolysis halos since the mycelium remains confined in the well, whereas the cellulolytic enzymes secreted diffuse into the agar. The dishes are incubated at 30° C. for seven days, at the end of which a visual evaluation of the halos is carried out by difference in color between the opaque agar and the transparent hydrolyzed zones.

(77) FIG. 2 illustrates this technique and its discriminating capacity by showing clones of interest identified by comparisons with the two control strains: the CL847ΔCBH1 strain (denoted ΔC1 on the dish) and the CL847 strain from which the cbh1 reference gene has not been deleted (SEQ ID NO: 1).

(78) Thus, any isolated clone of which the halo is smaller than that of CL847ΔCBH1 is discarded, whereas those of which the halo is at least larger than that of CL847ΔCBH1 are retained.

(79) By following this procedure for all of the clones obtained, clones No. 6 and No. 20 resulting from the transformation of the CL847ΔCBH1 strain with the sequence encoding the 130G9 gene (SEQ ID NO: 7) and isolated clone No. 24 resulting from the transformation of the CL847ΔCBH1 strain with the sequence encoding the 453E8 gene (SEQ ID NO: 15) were thus selected and retained.

(80) In order to confirm this choice, the three isolated clones selected were cultured for seven days at 30° C. with shaking at 150 rpm in liquid medium having the following composition:

(81) 3.4 g K.sub.2HPO.sub.4, 1.68 g (NH.sub.4).sub.2SO.sub.4, 0.12 g MgSO.sub.4, 0.6 g cornsteep, 1 ml of trace element solution (30 g/l FeSO.sub.4.7H.sub.2O, 9 g/l Co(NO.sub.3).sub.2.6H.sub.2O, 6.4 g/l MnSO.sub.4.1H.sub.2O, 8.4 g/l ZnSO.sub.4.7H.sub.2O, 0.4 g/l boric acid, 1.04 g/l sodium molybdate, pH adjusted to 1.5 with H.sub.3PO.sub.4), 4.64 g maleic acid, 4 g lactose, 4 g Solka Floc cellulose (Nutrafiber, USA) for 1 l of medium. The whole mixture is homogenized using an Ultra Turrax for five minutes. The pH is adjusted to 6.0 with a 3 M KOH solution. The medium obtained is autoclaved at 110° C. for 30 minutes. The phleomycin is added in an amount of 50 μg/ml when the medium is at ambient temperature.

(82) An assay of protein concentration of the extracellular medium is carried out using a DC Protein Assay colorimetric kit (BioRad, California, United States) on the basis of a bovine serum albumin (BSA) standard range. The supernatants are then subjected to two-dimensional electrophoresis as described by Herpoël-Gimbert et al. (Biotechnol Biofuels. 2008 Dec. 23; 1(1):18. doi: 10.1186/1754-6834-1-18), using 7 cm strips, pH 4.0-7.0.

(83) The protein profiles obtained for clones No. 6 and No. 20 of the 130G9 variant (SEQ ID NO: 8) and for clone No. 24 of the 453E8 variant (SEQ ID NO: 16) are compared with those of the CLN847 and CL847ΔCBH1 reference strains (FIG. 3 and FIG. 4).

(84) The results presented in FIG. 3 and in FIG. 4 show that the intensity of the spots, which correspond to the proteins of the secretome, is similar in each strain. This makes it possible to verify that the expression of these proteins is preserved regardless of the strain. The band indicated by the arrows makes it possible to confirm the presence of CBH1 in these strains, in comparison with the strain having been used for the CL847ΔCBH1 transformations.

(85) The clones selected at the end of these screening steps are referred to as “strains” in the rest of the examples.

Example 6

Production of Enzyme Cocktails

(86) Strains No. 6 and No. 20 having integrated the 130G9 variant (SEQ ID NO: 8) and strain No. 24 having integrated the 453E8 variant (SEQ ID NO: 16), constructed in example 5, were the subject of enzyme productions according to the miniaturized protocol described in patent application FR 2 989 385 and Jourdier et al. (Microb Cell Fact.2012 May 30; 11:70. doi: 10.1186/1475-2859-11-70). All of the proteins secreted by a given strain constitute its cocktail.

(87) The protein production by the T. reesei strains is carried out in two phases: a first batch phase for biomass production and a second fed-batch phase for protein production.

(88) The production is carried out according to the following protocol: In 250 ml flasks, 55 ml of F45 medium (10 g/l of dipotassium phthalate buffer, pH 6, 4.2 g/l (NH.sub.4).sub.2SO.sub.4, 300 mg/l MgSO.sub.4.7H.sub.2O, 150 mg/l CaCl.sub.2.2H.sub.2O, 1.5 g/l cornsteep, 0.07% of ortho-phosphoric acid, 5 mg/l FeSO.sub.4, 1.4 mg/l MnSO.sub.4, 1.4 mg/l ZnSO.sub.4, 3.7 mg/l CoCl.sub.2 and 12.5 g/l glucose) were inoculated with spores of the respective strains and shaken at 150 rpm and 30° C. Samples were taken every 24 hours in order to determine the pH and the glucose concentration.

(89) As soon as the glucose concentration is below 3 g/l, the fed-batch phase is launched by adding a solution of 50 g/l lactose and 0.3% NH.sub.3 at a flow rate of 40 mg of sugar/g of biomass per hour. Daily samples were taken in order to determine the pH, the dry weight and the protein concentration in the supernatant. After five days of fed-batch culture, the culture is filtered on a 0.45 μm filter and the supernatant is frozen after measuring the protein concentration. Said concentration was measured by the Lowry method using BSA to produce the standard range.

(90) The protein concentrations of the supernatants obtained for the 453E8-24, 130G9-6 and 130G9-20 strains and also the CL847 reference strain are given in table 9.

(91) TABLE-US-00010 TABLE 9 Protein concentration of the culture supernatants Protein concentration Strain (g/l) 453E8-24 5.3 130G9-6 7.4 130G9-20 5.8 CL847 5.2

Example 7

Efficiency of the Enzymes Resulting From the L-Shuffling in Lignocellulosic Biomass Hydrolysis According to an SHF Process

(92) The reference substrate used is a wheat straw having undergone a vapor-explosion pretreatment (19 bar-3 minutes). The biomass undergoes the explosion after acid impregnation at 0.01% H.sub.2SO.sub.4 for 10 hours. It is then washed, adjusted to pH 5, pressed and dried. The characteristics of the straw are given in table 10.

(93) TABLE-US-00011 TABLE 10 Composition of the straw used for the hydrolysis tests Composition % w/w WIS 97.52 Ash content 5 Cellulose 51.7 Corrected xylans 3.57 Hemicellulose 4.14 Klason lignin 36.49 (overestimated) Acetyl 0.6

(94) The hydrolyses were carried out at 10% of solids w/w, i.e. an equivalent of 5.4% of cellulose w/w. The WIS (Water Insoluble Solids) content is systematically determined before each series of microhydrolyses. The reference WIS value is 93.7%. The lignocellulosic solids content in the tests was set at 10%, i.e. ˜5.4% of cellulose.

(95) The protein content is set at 10 mg/g solids, i.e. approximately 19 mg/g cellulose. The enzymatic cocktails were supplied with β-glucosidase activity in an amount of 120±2 IU/g cellulose, by adding SP188 β-glucosidase (Novozymes, Denmark). This addition of β-glucosidase makes it possible to limit the cellobiohydrolase inhibition by cellobiose.

(96) The tests are carried out in Eppendorf tubes with a 2 ml working volume (1 g reactional) containing: 0.11±0.001 g of washed straw substrate; 0.9±0.02 ml of hydrolysis reaction medium composed of 50 mM acetate buffer—pH 4.8 and chloramphenicol (0.05 g/l); between 0.1 and 0.2±0.02 g of enzymatic cocktail as a function of their protein content.

(97) The enzymatic hydrolyses are carried out at 45±2° C. with vortexing at 900 revolutions per minute in an

(98) Eppendorf Thermomixer Comfort.

(99) All the tests are carried out in duplicate with sampling times set at 24, 48 and 96 hours with, for some, samplings at 72 hours.

(100) At each sampling time, the hydrolysates are warmed for five minutes in sacrificed Eppendorf tubes. These tubes are then cooled and centrifuged. The glucose is assayed by HPLC. In parallel, the solid residues of each Eppendorf tube are washed and centrifuged three times before being dried at 105° C. for 24 hours so as to evaluate the WIS. The hydrolysis yield is calculated by taking into account the amount of WIS in the straw used in the hydrolysis tests.

(101) The three cocktails resulting from the 130G9-6, 130G9-20 and 453E8-24 recombinant strains of example 6 were evaluated. A control test is carried out with the CL847 reference cocktail comprising the native CBH1 enzyme also supplemented with β-glucosidase for comparison.

(102) FIG. 5 gives the hydrolysis results for the 453E8-24 cocktail comprising the 453E8 enzyme (SEQ ID NO: 16).

(103) The results given in FIG. 5 show that the initial rate of hydrolysis of the 453E8-24 cocktail is close to that of the CL847 reference cocktail. The final hydrolysis yield of the 453E8-24 cocktail is greater than that of the CL847 reference cocktail.

(104) FIG. 6 gives the hydrolysis results for the two cocktails 130G9-6 and 130G9-20 resulting from the strains expressing the 130G9 enzyme (SEQ ID NO: 8).

(105) The results given in FIG. 6 show, for the two cocktails 130G9-6 and 130G9-20, that the initial rate of hydrolysis is greater than the rate of hydrolysis of the CL847 reference cocktail for the first 20 hours of reaction. The final hydrolysis yield of the two cocktails 130G9-6 and 130G9-20 is also greater than that of the CL847 reference cocktail.

Example 8

Efficiency of the Enzymes in Lignocellulosic Biomass Hydrolysis According to an SSF Process

(106) The substrate used is the same as that described in table 10 of example 7.

(107) The SSFs are carried out in triplicate in laboratory reactors. These reactors consist of the following elements: a glass flask with a 30 ml working volume; a polyether ether ketone (PEEK) safety stopper; a DV-118 one-way valve (Vaplock, United States) fitted through the stopper. The valve is configured so as to open in the outlet direction when the relative pressure in the flask is greater than 70 mbar; a first hollow polypropylene tube, the lower end of which is equipped with a septum. This tube is fitted through a second tube which passes through the safety stopper; a flat seal placed between the neck of the flask and the safety stopper.

(108) The principle for using the bioreactors is the following: the CO.sub.2 produced during the ethanolic fermentation accumulates in the top located above the reaction medium, leading by accumulation to an increase in the pressure in the bioreactor (P.sub.G). When P.sub.G becomes higher than the one-way valve opening pressure (P.sub.S), the valve opens to allow an amount of gas to escape, which amount is for example determined by weighing.

(109) When P.sub.G<P.sub.S, the valve closes again until P.sub.G is higher than P.sub.S. Thus, the bioreactor when operating is always pressurized so as to ensure a stable anaerobic environment for the fermentation. The amount of ethanol produced is evaluated by the CO.sub.2 production estimated by loss of weight on the basis of the following stoichiometric equation for fermentation of glucose to ethanol:
C.sub.6H.sub.12O.sub.6 (glucose).fwdarw.2 CO.sub.2+2CH.sub.3CH.sub.2OH (ethanol)+energy

(110) The culture medium used for the SSF is an aqueous medium which comprises: a 50 mM acetate buffer for pH 5; chloramphenicol at 0.1 g/l; nutritive medium containing 3 g/l of KH.sub.2PO.sub.4, 2 g/l of (NH.sub.4).sub.2SO.sub.4, 0.4 g/l of MgSO.sub.4.7H.sub.2O and 1 g/l of yeast extract.

(111) The SSFs were carried out at 10±0.01% w/w of solids, i.e. an equivalent of 5.4% cellulose w/w for a total reaction weight of 15±0.003 g. The protein content is set at 10±0.01 mg of cellulases per gram of solids, i.e. approximately 19 mg/g of cellulose. The enzymatic cocktails were supplemented with β-glucosidase activity in an amount of 120±2 IU/g cellulose, by adding SP188 β-glucosidase (Novozymes, Denmark).

(112) The yeast for fermentation of the sugars (Saccharomyces cerevisiae, Ethanol Red strain, Fermentis, France) is added to the medium so as to obtain a content of 2±0.1 g/kg.

(113) The enzymes and the yeast are added to the bioreactors after one hour of conditioning of the wheat straw pretreated at 35° C. with the culture medium.

(114) The SSF reaction is carried out at a temperature of approximately 35° C., by placing the laboratory bio reactor in an Infors Multitron HT Standard incubator with an orbital rotation speed of 150 revolutions per minute.

(115) Over time, the loss of weight was monitored by weighing the bioreactors. At the end of the reaction, the fermentation must is heated at 100° C. for 5 minutes, cooled and centrifuged to separate the non-hydrolyzed solids from the fermentation liquor. The fermentation liquor is then analyzed by gas chromatography in order to determine its ethanol concentration.

(116) The three cocktails resulting from the 130G9-6, 130G9-20 and 453E8-24 recombinant strains of example 6 were evaluated. An SSF is carried out with the reference cocktail comprising the native CBH1 enzyme also supplemented with β-glucosidase for comparison.

(117) FIG. 7 gives the results of progression of the SSF for the 453E8-24 cocktail resulting from the strains expressing the 453E8 exoglucanase.

(118) The results given in FIG. 7 show that the ethanol concentration after 100 hours of SSF is equivalent in the fermentation liquor of the 453E8-24 cocktail and in that of the CL847 reference strain.

(119) FIG. 8 gives the results of progression of the SSF for the 130G9-6 and 130G9-20 cocktails resulting from the strains expressing the enzyme of the 130G9 clone (SEQ ID NO: 8).

(120) The results given in FIG. 8 show, for the two cocktails 130G9-6 and 130G9-20, that the initial rate of fermentation is greater than that of the CL847 reference cocktail for the first 20 hours of reaction. The final yield of the two cocktails 130G9-6 and 130G9-20 is also greater than that of the CL847 reference cocktail.