EXPRESSION OF RECOMBINANT BETA-XYLOSIDASE ENZYMES

Abstract

The present invention relates to a Myceliophthora thermophila cell, which expresses a nucleotide sequence that codifies a recombinant beta-xylosidase enzyme comprising an amino-acid sequence having at least 70% identity with SEQ ID NO: 1, an enzymatic composition comprising said cell and/or the recombinant enzyme with beta-xylosidase activity expressed by said cell, the use of this host cell, the recombinant enzyme with beta-xylosidase activity expressed by said cell or the composition for the degradation of biomass, and a method of producing biological products, preferably bioethanol, comprising the use of said host cell, the recombinant enzyme with the beta-xylosidase activity expressed by said cell or said composition.

Claims

1. A Myceliophthora thermophila cell comprising a nucleotide sequence encoding a recombinant beta-xylosidase enzyme comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1.

2. The cell according to claim 1, wherein the recombinant beta-xylosidase enzyme comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

3. The cell according to claim 2, wherein the recombinant beta-xylosidase enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1.

4. The cell according to claim 3, wherein the recombinant beta-xylosidase enzyme comprises the amino acid sequence SEQ ID NO: 1.

5. The cell according to any of claims 1 to 4, wherein the recombinant beta-xylosidase enzyme comprises a signal peptide.

6. The cell according to claim 5, wherein the signal peptide is the glucoamylase A signal peptide, preferably, the glucoamylase A signal peptide from Aspergillus awamori.

7. The cell according to claim 6, wherein the glucoamylase A signal peptide from Aspergillus awamori comprises the amino acid sequence SEQ ID NO: 2.

8. The cell according to claim 7, wherein the recombinant beta-xylosidase enzyme comprises the amino acid sequence SEQ ID NO: 3.

9. The cell according to any of claims 1 to 8, wherein the nucleotide sequence comprises the sequence SEQ ID NO: 4.

10. The cell according to claim 9, wherein the nucleotide sequence is bound to a nucleotide sequence encoding a signal peptide, preferably, the nucleotide sequence encoding the signal peptide of the glucoamylase A, preferably, the nucleotide sequence is the glucoamylase A signal peptide from Aspergillus awamori, more preferably, the nucleotide sequence is the glucoamylase A signal peptide from Aspergillus awamori of SEQ ID NO: 5.

11. The cell according to claim 10, wherein the nucleotide sequence comprises the sequence SEQ ID NO: 6.

12. A recombinant beta-xylosidase enzyme expressed by the host cell of any of claims 1 to 11.

13. A composition comprising the host cell according to any of claims 1 to 11 and/or the recombinant beta-xylosidase enzyme according to claim 12.

14. A composition which is an enzymatic mixture obtained by the cell of any of claims 1 to 11.

15. The composition according to claim 14, further comprising other cellulolytic enzymes derived from the host cell according to any of claims 1 to 3.

16. Use of the host cell according to any of claims 1 to 11, the recombinant beta-xylosidase enzyme according to claim 12, or the composition according to any of claims 13 to 15, for the degradation of biomass.

17. Use according to claim 16 for the degradation of biomass in a bioproduct production process.

18. Use according to claim 17 wherein the bioproduct is biofuel.

19. Use according to claim 18, wherein the biofuel is bioethanol.

20. A method of producing fermentable sugars comprising: a. Incubating biomass with the recombinant beta-xylosidase enzyme according to claim 12 or the composition according to any of claims 13 to 15, and b. Recovering the fermentable sugars obtained after the incubation in step (a).

21. A method of producing a bioproduct from biomass comprising: a. Incubating biomass with the recombinant beta-xylosidase enzyme according to claim 12 or the composition according to any of claims 13 to 15, b. Fermenting the fermentable sugars obtained after the incubation of step (a) with at least one fermenting microorganism, and c. Recovering the bioproduct obtained after the fermentation in step (b).

22. The method according to claim 21, wherein the bioproduct is biofuel.

23. The method according to claim 22, wherein the biofuel is bioethanol.

Description

DESCRIPTION OF THE DRAWINGS

[0121] FIG. 1. Expression vector pBase-5K-4. Expression vector with Pcbh1 as promoter, Tcbh1 as terminator sequences and pyr4 as selection marker.

[0122] FIG. 2. Expresion plasmid pABC656. This plasmid allows the expression of native Anbxl cDNA from A. nidulans with glucoamylase A signal peptide (SPGA) from A. awamori.

[0123] FIG. 3. Xylose production profiles during the enzymatic hydrolysis of biomass by the enzymatic mixtures produced by M. thermophila C1 and transformants expressing SPGA-AnBxl. Xylose yield is calculated as the percentage of xylose released compared to the maximum (%), according to the analysis of pre-treated material. Data represent the average of three independent samples, and bars indicate the standard deviation. Control cocktail: cocktail produced by M. thermophila C1 parent strain; Cocktails 1-5: cocktails produced by M. thermophila C1 strain successfully expressing SPGA-AnBxl.

[0124] FIG. 4. Xylobiose consumption profiles during the enzymatic hydrolysis of biomass by the enzymatic mixtures produced by M. thermophila C1 and transformants expressing SPGA-AnBxl. Xylobiose consumption is calculated as the percentage of xylobiose hydrolysated compared to the value of control enzyme cocktail (expressed as 100%). Data represent the average of three independent samples, and bars indicate the standard deviation. Control cocktail: cocktail produced by M. thermophila C1 parent strain; Cocktails 1-5: cocktails produced by M. thermophila C1 strain successfully expressing SPGA-AnBxl.

[0125] FIG. 5. Comparative xylose release percentage by the host cell of the invention expressing different betaxylosidases.

[0126] SPGA-AnBxl: M. thermophila C1 cell expressing the mature beta-xylosidase enzyme from A. nidulans (SEQ ID NO: 1) with the signal peptide of glucoamylase A from A. awamori (SEQ ID NO: 2). SPGA-HiBxl: M. thermophila C1 cell expressing the mature beta-xylosidase enzyme from Humicola insolens (SEQ ID NO: 8) with the signal peptide of glucoamylase A from A. awamori (SEQ ID NO: 2), resulting in SEQ ID NO: 10.

EXAMPLES

Example 1

Expression of Beta-Xylosidase AnBxl from A. nidulans with Signal Peptide of Glucoamylase A from A. awamori, in M. thermophila C1. Construction of the Expression Vector and Beta-Xylosidase Activity Analysis in M. thermophila Transformants

[0127] M. thermophila C1 is a good host for expressing and secreting heterologous proteins and polypeptides. The beta-xylosidase gene anbxl (AN8401.2, accession number: Q5ATH9) from A. nidulans was used to express the enzyme and test its enzymatic performance in the present invention.

[0128] The anbxl cDNA sequence was synthesized in vitro after optimization, leading to remove the recognition sites for the most common restriction enzymes without altering the amino acid sequence. The cDNA nucleotide sequence of anbxl and the deduced amino acid sequence are shown in SEQ ID NO: 4 and SEQ ID NO: 1 respectively. The coding sequence is 2289 in length by including the stop codon. The encoded predicted protein is 763 amino acids long with a predicted molecular mass of 82.2 KDa and an isoelectric point of 4.52 Using the Signal IP program (Petersen et al., 2011, Signal IP 4.0, Nature Methods, 8:785-786), a signal peptide of 23 residues was predicted. The predicted mature protein of SEQ ID NO: 3 contains 740 amino acids with a predicted molecular mass of 79.6 KDa and an isoelectric point of 4.50 (data obtained using ProtParam Tool (Gasteiger E., et al, 2005))

[0129] Before synthesis in vitro the signal peptide from AnBxl native protein was replaced to increase secretion of AnBxl mature protein in M. thermophila. Native signal peptide from AnBxl was substituted by the signal peptide of glucoamylase A from Aspergillus awamori shown in SEQ ID NO: 2.

[0130] The gene anbxl was synthesized in vitro in the plasmid pBase-5K-4, which contains the promoter sequence of cellobiohydrolase 1 gene (Pcbh1), corresponding to an upstream region of 1796 bp of the cellobiohydrolase 1 gene (cbh1, NCBI Accession number XP_003660789.1) of M. thermophila C1. The expression vector pBase-5K-4 also contained the terminator sequence of the cellobiohydrolase 1 gene from M. thermophila C1 (Tcbh1, corresponding to a downstream region of 1014 bp of cbh1). The pBase 5K-4 plasmid also contains pyr4 gene (NCBI Accession number XP_003660657.1) from the same strain as selection marker. The pyr4 gene encodes for a functional orotidine-5-monophosphate decarboxylase and its expression allows complementation of the uridine auxotrophy in the corresponding auxotrophic M. thermophila C1 host strain (pyr4). The map of the expression vector pBase-5K-4 is shown in FIG. 1.

[0131] The expression vector pBase-5K-4 with synthetized SPGA-anbxl gene was transformed in XL1Blue MRF E. coli electro-competent cells following the protocol provided by the manufacturer (Stratagene). The recombinant plasmid obtained was named pABC656 and is shown in FIG. 2.

[0132] The plasmid pABC656 containing anbxl from A. nidulans under Pcbh1 promoter sequence and pyr4 as selection marker, was transformed in the M. thermophila C1 pyr4 derivative (Verdoes et al., 2007, Ind. Biotechnol. 3 (1)), auxotrophic host strain previously used in other high-throughput screening in M. thermophila. The DNA was introduced in the host strain using protoplast transformation (U.S. Pat. No. 7,399,627B2). The transformants were plated out in agar plates without uridine supplementation. After 5 days of incubation at 35 C., resulting prototrophic transformants (expressing pyr4 gene) were analysed.

Example 2

Beta-Xylosidase Activity Determination on Enzymatic Mixtures Produced by M. thermophila C1 and Transformants Expressing the SPGA-AnBxl

[0133] The SPGA-AnBxl transformants were inoculated in 96-well microtitter plates (MTPs) cultures to identify beta-xylosidase activity in transformants expressing anbxl. Hydrolytic activity on pNXP (Sigma N2132) as substrate was measured.

[0134] Most of the transformants tested showed an increase of beta-xylosidase activity compared to untransformed M. thermophila C1 control. All the transformants with beta-xylosidase activity were confirmed in a second round test as defined in U.S. Pat. No. 7,794,962B2. Some of the positive transformants were confirmed at flask scale (Verdoes et al., 2007, Ind. Biotechnol. 3 (1); Visser et al., 2011, Ind. Biotechnol. 7 (3)) and beta-xylosidase activity was measured from culture supernatants. Six different enzymatic cocktails were produced (see Table 1): a control from parent strain (control cocktail) and five transformants expressing SPGA-AnBxl (Enzyme cocktails 1-5). The control cocktail consisted of the mixture of extracellular enzymes produced by M. thermophila C1 strain under the production conditions described in the references given above. The SPGA-AnBxl enzyme cocktails (cocktails 1-5) consisted of the mixtures of enzymes produced by this C1 strain successfully expressing pABC656 (described in example 1) under identical production conditions.

TABLE-US-00007 TABLE 1 Specific activity of enzymatic mixtures produced by M. thermophila C1 and transformants expressing SPGA-AnBxl. Analyses were carried out using pNXP as substrate. Errors are indicated as the standard deviation (SD) of three independent measurements. Enzyme cocktail BXL activity (U mg prot..sup.1) SD Control cocktail 15.31 0.01 1 320.57 24.29 2 447.82 25.82 3 195.89 11.18 4 313.35 10.43 5 171.98 7.24

[0135] Beta-Xylosidase Activity Determination

[0136] Beta-xylosidases (EC 3.2.1.27) are hydrolytic enzymes that catalyze the cleaving off the terminal xylose units from the non-reducing end of the short xylose oligomers arising from the endoxylanase (EC 3.2.1.8) activity towards xylan.

[0137] Beta-xylosidase activity was determined using p-nitrophenyl-beta-D-xylopyranoside (pNXP, Sigma N2132) as substrate. For this pNXP assay, the enzymatic reaction mixtures (1 mL final volume) containing 100 mol sodium acetate buffer (pH 5.0), 100 g pNXP (0.33 mol) and proper amount of respective enzyme cocktail were incubated at 50 C. for 10 minutes. The amount of p-nitrophenol released was measured at A.sub.410 (410=15.2 mM.sup.1 cm.sup.1) after addition of 100 g sodium carbonate to stop the reaction mixtures. One unit of pNXP hydrolysing activity was defined as the amount of enzyme needed to release 1 mol p-nitrophenol per minute. The specific activities obtained are shown in Table 1.

[0138] The control cocktail exhibits significantly less pNXP activity than the cocktails produced by M. thermophila C1 successfully expressing pABC656 under identical production conditions.

[0139] Total protein of the enzymatic mixtures was determined by the BCA method (Applichem, A7787 0500).

Example 3

Effect of Transformants Expressing SPGA-AnBxl Cocktails on the Release of Xylose During the Enzymatic Hydrolysis of Xylan-Containing Biomass

[0140] Enzymatic Hydrolysis Experiments

[0141] Unwashed pretreated corn stover (PCS) was used as substrate for enzymatic hydrolysis. Pre-treatment of the biomass was performed by a modification of the steam explosion system described by Nguyen et al., 1998, Appl. Biochem. Biotechnol. 70-72, in which no acid treatment was applied so that xylan hydrolysis was impaired. Incomplete release of xylose from pre-treated material was necessary for the evaluation of the effect of the SPGA-AnBxl activity.

[0142] The compositional analysis of this material was performed accordingly to the Standard Biomass Analytical Procedures from National Renewable Energy Laboratory (NREL) (http://www.nrel.gov/biomass/analytical_procedures.html).

[0143] Hydrolysis of the pre-treated biomass (20 grams of total reaction mass) was performed in 100 mL ISO bottles. Water was added to adjust the solid loading to 20 total solids (based on pre-treated substrateafter the pH was adjusted to 5.5 by addition of 25% ammonia solution (NH.sub.4OH). The enzyme loading was 12 mg protein per g glucan content of each enzymatic cocktail produced by M. thermophila C1 strain and AnBxl transformants. The hydrolysis was performed by incubating at 50 C. inside 25 mm orbit diameter shakers at 150 rpm for 72 hours.

[0144] Xylose release and xylobiose hydrolysis profiles obtained during the enzymatic hydrolysis are shown in FIG. 3 and FIG. 4, wherein it can be seen that the use of cocktails obtained by transformants expressing SPGA-AnBxl (SEQ ID NO: 4) leads to a high xylose production and xylobiose consumption as compared with the control M. thermophila C1 parent strain.

Example 4

Performance of SPGA-AnBxl Compared to Beta-Xylosidases from Other Organisms Expressed in M. thermophila C1

[0145] Following the conditions described at Example 3, a similar experiment was carried out in order to compare a transformant expressing SPGA-AnBxl with a transformant expressing a beta-xylosidase from H. insolens (Hi) (SPGA-HiBxl). The gene that codifies for this protein was synthetized in vitro and it was cloned in the same vector than anbxl. This enzyme did display a clear signal peptide, so glucoamylase A signal peptide from A. awamori was added as described for anbxl in Example 1.

[0146] The mature beta-xylosidase enzyme from H. insolens (SEQ ID NO: 8) with the signal peptide of glucoamylase A from A. awamori (SEQ ID NO: 2), resulting in the construct SPGA-HiBxl (SEQ ID NO: 10). Nucleotide sequences encoding mature protein of SEQ ID NO: 8 and protein SEQ ID NO: 10 are shown as SEQ ID NO: 9 and SEQ ID NO: 11.

TABLE-US-00008 SEQIDNO:8: MAPLITNIYTADPSAHVFNGKLYIYPSHDRETDIQFNDNGDQYDMADYHV FSLDSLDPPSEVTDHGVVLKVEDIPWVSKQLWAPDAATKDGKYYLYFPAR DKEGIFRIGVAVSDKPEGPFTPDPEPIKGSYSIDPAVFVDDDGSAYMYFG GLWGGQLQCYQKGNNIFDAEWSGPKEPSGSGAKALGPRVAKLTDDMRQFA EEVREIVILAPETGEPLAADDHDRRFFEAAWMHKYNGKYYFSYSTGDTHY LVYAVGDSPYGPFTYGGRILEPVLGWTTHHSIVEFQGRWWLFHHDCELSK GVDHLRSVKVKEIWYDKDGKIVTEKPE SEQIDNO:9: gcccccctcatcaccaacatctacaccgccgacccctccgcccacgtctt caacggcaagctctacatctacccctcccacgaccgcgagaccgacatcc agttcaacgacaacggcgaccagtacgacatggccgactaccacgtcttc tccctcgactccctcgaccccccctccgaggtcaccgaccacggcgtcgt cctcaaggtcgaggacatcccctgggtctccaagcagctctgggcccccg acgccgccaccaaggacggcaagtactacctctacttccccgcccgcgac aaggagggcatcttccgcatcggcgtcgccgtctccgacaagcccgaggg ccccttcacccccgaccccgagcccatcaagggctcctactccatcgacc ccgccgtcttcgtcgacgacgacggctccgcctacatgtacttcggcggc ctctggggcggccagctccagtgctaccagaagggcaacaacatcttcga cgccgagtggtccggccccaaggagccctccggctccggcgccaaggccc tcggcccccgcgtcgccaagctcaccgacgacatgcgccagttcgccgag gaggtccgcgagatcgtcatcctcgcccccgagaccggcgagcccctcgc cgccgacgaccacgaccgccgcttcttcgaggccgcctggatgcacaagt acaacggcaagtactacttctcctactccaccggcgacacccactacctc gtctacgccgtcggcgactccccctacggccccttcacctacggcggccg catcctcgagcccgtcctcggctggaccacccaccactccatcgtcgagt tccagggccgctggtggctcttccaccacgactgcgagctctccaagggc gtcgaccacctccgctccgtcaaggtcaaggagatctggtacgacaagga cggcaagatcgtcaccgagaagcccgagtaa SEQIDNO:10: MSFRSLLALSGLVCSGLAAPLITNIYTADPSAHVFNGKLYIYPSHDRETD IQFNDNGDQYDMADYHVFSLDSLDPPSEVTDHGVVLKVEDIPWVSKQLWA PDAATKDGKYYLYFPARDKEGIFRIGVAVSDKPEGPFTPDPEPIKGSYSI DPAVFVDDDGSAYMYFGGLWGGQLQCYQKGNNIFDAEWSGPKEPSGSGAK ALGPRVAKLTDDMRQFAEEVREIVILAPETGEPLAADDHDRRFFEAAWMH KYNGKYYFSYSTGDTHYLVYAVGDSPYGPFTYGGRILEPVLGWTTHHSIV EFQGRWWLFHHDCELSKGVDHLRSVKVKEIWYDKDGKIVTEKPE SEQIDNO:11: ATGTCGTTCCGATCTCTTCTCGCCCTGAGCGGCCTTGTCTGCTCGGGGTT GGCAGCCCCCCTCATCACCAACATCTACACCGCCGACCCCTCCGCCCACG TCTTCAACGGCAAGCTCTACATCTACCCCTCCCACGACCGCGAGACCGAC ATCCAGTTCAACGACAACGGCGACCAGTACGACATGGCCGACTACCACGT CTTCTCCCTCGACTCCCTCGACCCCCCCTCCGAGGTCACCGACCACGGCG TCGTCCTCAAGGTCGAGGACATCCCCTGGGTCTCCAAGCAGCTCTGGGCC CCCGACGCCGCCACCAAGGACGGCAAGTACTACCTCTACTTCCCCGCCCG CGACAAGGAGGGCATCTTCCGCATCGGCGTCGCCGTCTCCGACAAGCCCG AGGGCCCCTTCACCCCCGACCCCGAGCCCATCAAGGGCTCCTACTCCATC GACCCCGCCGTCTTCGTGGACGACGACGGCTCCGCCTACATGTACTTCGG CGGCCTCTGGGGCGGCCAGCTCCAGTGCTACCAGAAGGGCAACAACATCT TCGACGCCGAGTGGTCCGGCCCCAAGGAGCCCTCCGGCTCCGGCGCCAAG GCCCTCGGCCCCCGCGTCGCCAAGCTCACCGACGACATGCGCCAGTTCGC CGAGGAGGTCCGCGAGATCGTCATCCTCGCCCCCGAGACCGGCGAGCCCC TCGCCGCCGACGACCACGACCGCCGCTTCTTCGAGGCCGCCTGGATGCAC AAGTACAACGGCAAGTACTACTTCTCCTACTCCACCGGCGACACCCACTA CCTCGTCTACGCCGTCGGCGACTCCCCCTACGGCCCCTTCACCTACGGCG GGCGCATCCTGGAGCCCGTCCTCGGCTGGACCACCCACCACTCCATCGTC GAGTTCCAGGGCCGCTGGTGGCTCTTCCACCACGACTGCGAGCTGTCCAA GGGCGTGGACCACCTCCGCTCCGTCAAGGTCAAGGAGATCTGGTACGACA AGGACGGCAAGATCGTCACCGAGAAGCCCGAGTAA

[0147] As shown in FIG. 5, xylose release is higher by transformant expressing SPGA-AnBxl than the one expressing SPGA-HiBxl.