Expression of recombinant beta-xylosidase enzymes

Abstract

The present invention relates to a Myceliophthora thermophila host cell which expresses a recombinant enzymes from Fusarium oxysporum with beta-xylosidase activity. The invention also refers to an enzymatic composition comprising the host cell of the invention and/or the recombinant enzyme with beta-xylosidase activity expressed by the host cell of the invention. The invention further relates to the use of the host cell of the invention, the recombinant enzyme with beta-xylosidase activity expressed by the host cell of the invention or the composition of the invention for the degradation of biomass and to a method of producing bioproducts, preferably bioethanol, which comprises the use of the host cell of the invention, the recombinant enzyme with beta-xylosidase activity expressed by the host cell of the invention or the composition of the invention.

Claims

1. A Myceliophthora thermophila host cell which expresses the recombinant beta-xylosidase enzyme which consists of the amino acid sequence SEQ ID NO: 4.

2. A composition comprising the host cell according to claim 1.

3. The composition according to claim 2, further comprising other cellulolytic enzymes expressed by a Myceliophthora thermophila host cell which expresses the recombinant beta-xylosidase enzyme which consists of the amino acid sequence SEQ ID NO: 4, wherein the cellulolytic enzymes are selected from the list consisting of: endoglucanases, beta-glucosidases, cellobiohydrolases, endoxylanases or any combination thereof.

4. A method of producing fermentable sugars comprising: a. Incubating biomass with the composition according to claim 2, and b. Recovering the fermentable sugars obtained after the incubation in step (a).

5. A method of producing a bioproduct from biomass comprising: a. Incubating biomass with the composition according to claim 2, 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).

6. The method according to claim 5, wherein the bioproduct is biofuel.

7. The method according to claim 6, wherein the biofuel is bioethanol.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Shows the vector named pBASE1. Expression vector with Tcbh1 as terminator sequence and pyr5 as selection marker. XbaI and BamHI were the restriction sites chosen for the cloning of Pcbh1-fobxl cassette.

(2) FIG. 2. Shows the vector named pABC341. Expression plasmid of native fobxl cDNA from F. oxysporum.

(3) FIG. 3. Shows the beta-xylosidase activity (U/L) of some fobxl transformants analysed using pNXP as substrate.

(4) FIG. 4. Shows the vector named pBASE5. Expression vector with Pcbh1 as promoter sequence, Tcbh1 as terminator sequence and pyr5 as selection marker. NdeI and BamHI were the restriction sites chosen for the cloning of genetic fusion SPGA-fobxl.

(5) FIG. 5. Shows the vector named pABC397. Expression plasmid containing the genetic fusion SPGA-fobxl.

(6) FIG. 6. Shows the beta-xylosidase activity (U/L) of some SPGA-fobxl transformants analysed using pNXP as substrate.

(7) FIG. 7. Shows the xylose production profiles during the enzymatic hydrolysis of biomass by the enzymatic mixtures produced by M. thermophila C1 and transformants expressing the FoBxl or SPGA-FoBxl. Xylose yield is calculated as the percentage of xylose released compared to the maximum (%), according to the analysis of pre-treated material. Shown 72 h of process correspond to the phase 2 of enzymatic hydrolysis described in examples below. Data represent the average of three independent samples, and bars indicate the standard deviation.

(8) FIG. 8. Shows the xylobiose consumption profiles during the enzymatic hydrolysis of biomass by the enzymatic mixtures produced by M. thermophila C1 and transformants expressing the FoBxl or SPGA-FoBxl. Xylose consumption is calculated as the percentage of xylobiose hydrolysated from the initial value at the beginning of the enzymatic hydrolysis. Shown 72 h of process correspond to the phase 2 of enzymatic hydrolysis described in examples below. Data represent the average of three independent samples, and bars indicate the standard deviation.

EXAMPLES

Example 1. Expression of Beta-Xylosidase FoBxl from Fusarium oxysporum (Strain Fo5176) in M. thermophila C1. Construction of the Expression Vector and Beta-Xylosidase Activity Analysis in M. thermophila Transformants

(9) M. thermophila C1 has been described as a good quality transformation system for expressing and secreting heterologous proteins and polypeptides. The beta-xylosidase gene fobxl (FOXB_13892 Accession number: EGU75604) from F. oxysporum (Fo5176) was the target to express the enzyme and test its enzymatic quality in the present invention.

(10) The fobxl 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 fobxl and the deduced amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively. The coding sequence is 1047 in length by including the stop codon. The encoded predicted protein is 348 amino acids long with a predicted molecular mass of 40 KDa and an isoelectric point of 9.02. Using the Signal IP program (Petersen et al., 2011, Signal IP 4.0, Nature Methods, 8:785-786), a signal peptide of 20 residues was predicted. The predicted mature protein (SEQ ID NO: 3) contains 328 amino acids with a predicted molecular mass of 37 KDa and an isoelectric point of 8.81.

(11) The gene fobxl was in vitro synthesized together with the promoter sequence of cellobiohydrolase 1 gene (Pcbh1), corresponding with an upstream region of 1796 bp of the cellobiohydrolase 1 gene (cbh1, NCBI Accession number XP_003660789.1) of M. thermophila C1. This cassette (Pcbh1-fobxl) was synthesized in vitro including the sequence of the restriction enzymes XbaI and BamHI at the ends (5′ and 3′ ends, respectively) in order to be cloned into an expression vector named pBASE1. The expression vector pBASE1 also contained the terminator sequence of the cellobiohydrolase 1 gene from Myceliophthora thermophila C1 (Tcbh1, corresponding with a downstream region of 1014 bp of cbh1) and pyr5 gene (NCBI Accession number XP_003660657.1) from the same strain as selection marker. The pyr5 gene encodes for a functional orotate-phosphoribosyl transferase and its expression allows complementation of the uridine auxotrophy in the corresponding auxotrophic M. thermophila C1 host strain (pyr5). The expression vector pBASE1 is shown in FIG. 1.

(12) The cassette Pcbh1-fobxl was digested with the restriction enzymes XbaI and BamHI and cloned in the pBASE1 previously digested with the same restriction enzymes. The expression vector pBASE1 and the cassette Pcbh1-fobxl were ligated and the ligation product was transformed in XL1 Blue MRF Escherichia coli electro-competent cells following the protocol provided by the manufacturer (Stratagene). The recombinant plasmid obtained was named pABC341 and is shown in FIG. 2.

(13) The pABC341 plasmid containing fobxl from F. oxysporum under Pcbh1 promoter sequence and pyr5 as selection marker, was transformed in the M. thermophila pyr5 (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 a protoplast transformation method (U.S. Pat. No. 7,399,627B2). The transformants were plated out in agar plates with no uridine supplementation. After 5 days of incubation at 35° C., resulting prototrophic transformants (expressing pyr5 gene) were analysed.

(14) The transformants obtained were inoculated in 96-well microtitter plates (MTPs) cultures to carry out a high throughput screening (U.S. Pat. No. 7,794,962B2). The aim of the screening was to identify beta-xylosidase activity in transformants expressing fobxl. Hydrolytic activity on p-nitrophenyl-beta-D-xylopyranoside (pNXP, Sigma N2132) as substrate was measured. Percentage of beta-xylosidase activity was measured by the release of p-nitrophenol (and consequent increase of A.sub.410) in units per litter of culture (U/L). One unit of pNXP hydrolysing activity was defined as the amount of enzyme needed to release 1 μmol p-nitrophenol per minute. Beta-xylosidase activity of 50 μl of the culture supernatants of each transformant was assayed with 200 mg/L of pNXP for 10 minutes at 50° C. in a final volume of 100 μL. The reaction was stopped by adding 100 μL of carbonate 1M to the reaction mixtures. The hydrolytic capacity was measured by the release of p-nitrophenol (and consequent increase of A.sub.410).

(15) Among the transformants tested, most of them showed an increase of beta-xylosidase activity using M. thermophila C1 as negative control. The results of beta-xylosidase activity are shown in FIG. 3. 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 with grown at flask scale production (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.

Example 2. Genetic Fusion of Glucoamylase Signal Peptide from A. niger with Beta-Xylosidase FoBxl Mature Protein Sequence from F. oxysporum. Construction of an Expression Vector and Beta-Xylosidase Activity Analysis in M. thermophila Transformants

(16) The signal peptide from Fobxl native protein was exchanged to increase secretion of Fobxl mature protein in M. thermophila. Native signal peptide from Fobxl was substituted by the signal peptide of glucoamylase from Aspergillus niger (glaA, accession number An03g06550). Glucoamylase is a naturally highly secreted enzyme and its signal peptide was used to reach a highly secretion of the recombinant protein in the filamentous fungi.

(17) For the native signal peptide substitution, the fragment of the fobxl gene encoding the mature protein (excluding sequence coding native signal peptide) was amplified by PCR using oligonucleotide 1 and 2. The oligonucleotide 1 (SEQ ID NO: 7) includes NdeI restriction site and the sequence coding glucoamylase signal peptide (SPGA). The oligonucleotide 2 (SEQ ID NO: 8) includes SmaI and BamHI restriction sites and includes the stop codon. The amplification from oligonucleotide 1 allows the genetic fusion of glucoamylase signal peptide and mature protein of Fobxl (SPGA-Fobxl).

(18) Oligonucleotide 1 (SEQ ID NO: 7): NdeI restriction site is underlined. SPGA is framed. 5′ end sequence of FoBxl mature protein is shadow texted.

(19) TABLE-US-00001

(20) Oligonucleotide 2 (SEQ ID NO: 8): SmaI and BamHI restriction sites are underlined. Stop codon is framed.

(21) TABLE-US-00002 GTTCTTGTC-3′

(22) Amplification of genetic fusion SPGA-fobxl was performed using the oligonucleotides 1 and 2 using plasmid DNA pABC341 (previously described in Example 1) as target with iProof High-Fidelity DNA Polymerase (BioRad) and programmed for one cycle at 95° C. for 2 minutes and 30 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 1 minute and one cycle of 72° C. for 10 minutes. The fragment of DNA amplified was digested with NdeI and BamHI restriction enzymes and cloned into pBASE5 previously digested with the same restriction enzymes (shown in FIG. 4). pBASE5 comes from pBASE1 (described in Example 1) where the promoter sequence Pcbh1 was cloned including NdeI restriction site. pBASE5 also contains Tchb1 as terminator sequence and pyr5 as selection marker (described in Example 1). The plasmid with SPGA-fobxl cloned under Pcbh1 was named pABC397 and is shown in FIG. 5.

(23) The pABC397 plasmid containing the genetic fusion SPGA-fobxl under Pcbh1 promoter sequence and pyr5 as selection marker, was transformed in the M. thermophila pyr5- (Verdoes et al., 2007, Ind. Biotechnol. 3 (1)). The DNA was introduced in the host strain using a protoplast transformation method (U.S. Pat. No. 7,399,627B2). The transformants were plated out in agar plates with no uridine supplementation. After 5 days of incubation at 35° C., resulting prototrophic transformants (expressing pyr5 gene) were analysed.

(24) High throughput screening of the transformants obtained was carried out as described in Example 1. The aim of the screening was to identify the beta-xylosidase activity in transformants expressing fobxl (as described in Example 1).

(25) Among the transformants tested, most of them showed higher beta-xylosidase activity than the observed with the transformants expressing fobxl with native signal peptide. M. thermophila C1 was used as negative control. The results of beta-xylosidase activity are shown in FIG. 6. All the transformants with higher beta-xylosidase activity were confirmed in a second round test in MTPs and flask fermentation was performed as described in Example 1. Higher beta-xylosidase activity was confirmed in all of them.

Example 3. Beta-Xylosidase Activity Determination on Enzymatic Mixtures Produced by M. thermophila C1 and Transformants Expressing the FoBxl or SPGA-FoBxl

(26) Production of Enzymatic Cocktails

(27) Production of the enzyme cocktails was performed as described in Verdoes et al. (2007) and Visser et al., 2011, Ind. Biotechnol. 7 (3), using the industrial platform for the expression of industrial enzymes based on M. thermophila C1 developed by Dyadic Netherlands.

(28) Three different enzymatic cocktails were produced: a control cocktail, the FoBxl cocktail and the SPGA-FoBxl cocktail. The control cocktail consisted of the mixture of extracellular enzymes produced by Myceliophthora thermophila C1 strain under the production conditions described in the references given above. The FoBxl and SPGA-FoBxl enzyme cocktails consisted of the mixtures of enzymes produced by this C1 strain successfully expressing respective constructions (described in examples 1 and 2) under identical production conditions.

(29) Beta-Xylosidase Activity Determination

(30) 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.

(31) 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 μmol sodium acetate buffer (pH 5.0), 100 μg pNXP (0.33 μmol) and proper amount of respective enzyme cocktail were incubated at 50° C. for 10 min. The amount of p-nitrophenol released was measured at A.sub.410 (ε410=15.2 mM.sup.−1 cm.sup.−1) after addition of 100 μg sodium carbonate to the reaction mixtures. One unit of pNXP hydrolysing activity was defined as the amount of enzyme needed to release 1 μmol p-nitrophenol per minute. Obtained specific activities are shown in Table 1.

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

(33) TABLE-US-00003 TABLE 1 Specific activity of enzymatic mixtures produced by M. thermophila C1 and transformants expressing the FoBxl or SPGA-FoBxl. Errors are indicated as the standard deviation (SD) of three independent measurements. BXL activity Enzyme cocktail (U mg prot..sup.−1) SD Control cocktail 11.47 0.04 FoBxl cocktail 36.05 0.19 SPGA-FoBxl 154.06 0.77 cocktail

Example 4. Effect of FoBxl and SPGA-FoBxl Cocktail Supplementation on the Production of Xylose During the Enzymatic Hydrolysis of Xylan-Containing Biomass

(34) Enzymatic Hydrolysis Experiments

(35) 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 FoBxl and SPGA-FoBxl activities.

(36) The compositional analysis of this material was performed accordingly to the Standard Biomass Analytical Procedures (http://www.nrel.gov/biomass/analytical_procedures.html), and revealed to contain a 4.06% and 11.11% (w/w, D.M.) of xylan and xylose concentration, respectively, and a 12.24% and 3.61% (w/w, DM) of glucan and glucose, respectively.

(37) Hydrolysis reactions were performed in two phases. An initial phase was carried out by the control enzymatic cocktail during 24 hours at 25% dry matter (DM) concentration. This initial reaction mixture contained, in a total mass of 200 g: pretreated corn stover corresponding to 50 g DM; NaOH. 1.6 g; and control enzymatic cocktail with a content of 3 g of total protein (measured as previously described).

(38) This initial hydrolysis phase was performed in 2 L ISO flasks to ensure liquefaction of the PCS; afterwards, resulting slurry was aliquoted into 10 mL tubes (4 g per tube), in which a second hydrolysis phase was performed.

(39) Effect of FoBxl and SPGA-FoBxl was indeed studied during the second hydrolysis phase, in which 4 g of slurry were mixed with either 1 g of water (experimental control), or 1 g aqueous dilutions of corresponding cocktail. Therefore, DM of slurry was adjusted to 20% during this second phase of enzymatic hydrolysis, which was performed for 72 h. Enzymatic cocktail dosage was adjusted to 0.1% (w/w, protein/DM), the equivalent to 8 mg prot. g glucan.sup.−1. Both phases of enzymatic hydrolysis were performed at 50° C. inside 25 mm orbit diameter shakers at 150 rpm.

(40) Xylose production and xylobiose hydrolysis profiles obtained during this second phase of the enzymatic hydrolysis are shown in FIG. 7 and FIG. 8, wherein it can be seen that the use of cocktails obtained by transformants expressing FoBxl (SEQ ID NO: 3) or SPGA-FoBxl (SEQ ID NO: 4) leads to a great xylose and xylobiose production as compared with the control cocktail produced by the control (wild type) Myceliophthora thermophila C1 strain.