Patent Application
20180230446
The invention relates to the field of bioproducts, preferably biofuels, and more particularly, to the expression of a recombinant enzyme with beta-xylosidase activity in host cells and its use in the production of bioproducts, preferably biofuels, from biomass.
Nowadays, many efforts are being made in order to obtain less expensive and renewable sources of fuel. Biofuels offer an attractive alternative to petroleum based fuels and can be obtained through the fermentation of monomeric sugars derived from starch or cellulose. However, current economics do not support the widespread use of biofuels due to the high cost of generating them.
Plant biomass provides a plentiful source of potential energy in form of carbohydrates that can be utilized for numerous industrial and agricultural processes, and is therefore a significant renewable resource for generating fermentable sugars. Fermentation of these sugars can commercially produce valuable end-products, such as biofuel and biochemicals. However, the enormous potential energy of these carbohydrates is currently under-utilized because the sugars are locked in complex polymers and, hence, are not readily accessible for fermentation (WO2012018691A2).
Wood, agricultural residues, herbaceous crops, and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and/or lignin. Once the cellulose is converted to glucose by means of an enzymatic hydrolytic process, the glucose is easily fermented by yeast into ethanol. Thus, the more amounts of complex sugars remaining at the end of the hydrolytic process the lower the yield of ethanol production at the end of the fermentation process. Therefore, one area of research aimed at decreasing costs and enhancing the yield of biofuel production processes is focus on the enhancement of the technical efficacy of the hydrolytic enzymes that can be used to generate fermentable sugars from biomass.
Due to the complexity of biomass, its conversion to monomer sugars involves the action of several different enzyme classes, which digest cellulose and hemicellulose, major polysaccharides comprised in cellulosic materials. After cellulose, hemicellulose is the second most abundant fraction available in nature. It is a storage polymer in seeds and it forms the structural component in cell walls of woody plants. The classification of these hemicellulose fractions depends on the types of sugar moieties present. The principal monomers present in most of the hemicelluloses are D-xylose, D-mannose, D-galactose and L-arabinose. Thus, hemicellulose includes xylan, mannan, galactan and arabinan as the main heteropolymers. Specifically, xylan contains 85 to 93% of D-xylose, a small amount of arabinose and traces of glucuronic acid residues. The main chain of xylan is composed of beta-(1-4) linked beta-xylopyranose residues, and several side chains have been described to be present. Among them, most usually found are xylopiranose, glucuronic acid and arabinofuranose linkages, as well as acetyl groups (Bastawde, 1992, World Journal of Microbiology and Biotechnology (8) 353-368).
The presence of lignin in biomass leads to a protective barrier that prevents proper enzymatic hydrolysis of glucan and xylan. Thus, a pretreatment process of the biomass is required for increasing the access of the enzymes to their substrates and consequent efficient hydrolysis. Pretreatment uses various techniques, including ammonia fiber explosion, chemical treatment and steam explosion at high temperatures to alter the structure of cellulosic biomass and make cellulose more accessible. Hemicellulose can be readily hydrolysed under moderate conditions, but much more extreme conditions are needed for cellulose hydrolysis. Therefore, the pretreated material (substrate for the enzymatic hydrolysis) usually contains a high concentration of xylose, whereas glucose content is rather low (Kumar et al, 2009. Ind. Eng. Chem. Res., 48 (8), 3713-3729).
Single component enzymes have been shown to only partially digest cellulose and hemicellulose and thus the concerted action of different classes of enzymes is required to complete their conversion to monomeric sugars. Many more enzymes are required to digest hemicellulose to sugar monomers including xylanase, xylosidase, arabinofuranosidase, mannanase, galactosidase and glucuronidase. Non-glycosyl hydrolases such as acetyl xylan esterase and ferulic acid esterase may also be involved.
A large number of naturally-occurring organisms have been found to produce enzymatic hydrolysis of cellulosic materials to produce fermentable sugars. Organisms capable of carry out a complete cellulose and hemicellulose degradation, that subsequently allows an efficient fermentation, would greatly enhance the cost effectiveness of bioethanol production.
The hydrolytic efficiency of a multi-enzyme complex in the process of cellulosic saccharification (or hydrolysis) depend both on properties of the individual enzymes and the ratio of each enzyme within the complex. It is therefore desirable to generate cellulolytic enzymes expressing-microorganisms which improve the yield of cellulosic material degradation process, increasing the amount of released fermentable sugars and thus improving the yield of final biofuel production.
Thus, some efforts carried out in order to generate improved cellulolytic enzymes expressing-microorganisms have involved inserting a gene encoding the specific hydrolytic enzyme to be expressed under the control of strong expression signals, which leads to an increased stability of the transcribed mRNA or an increased number of copies of the gene in the produced organism (US20080194005A1).
A number of host cells used for heterologous gene expression, such as bacteria Escherichia coli, and methods of transformation have been disclosed in the prior art. In this context, also a number of fungal expression systems have been developed, for instance Aspergillus niger, Aspergillus awamori, Aspergillus nidulans, Trichoderma reesei. However, for various reasons many of these recombinant microorganisms have not found wide-spread acceptance or use. In general terms, the ideal host cell must fulfill a large number of criteria, such as, uses the medium efficiently, produces the polypeptide or protein of interest in high yield, should be capable of efficient secretion of the protein or polypeptide, allows a wide range of expression regulatory elements to be used thus ensuring ease of application and versatility, allows the use of easily selectable markers that are cheap to use, and produce stable transformants.
The present invention relates to the recombinant expression of a beta-xylosidase enzyme which comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 1, preferably the beta-xylosidase enzyme of SEQ ID NO: 1, in a host cell, more preferably in Myceliophthora thermophila, even more preferably in Myceliophthora thermophila strain C1. Said recombinant expression leads to a cell with improved efficiency of hydrolysis of biomass into fermentable sugars, more particularly the degradation of xylan oligomers to xylose (see
The present invention represents a solution to the need to provide a microorganism that expresses a mixture of cellulolytic enzymes which improves the yield of biomass hydrolytic process or saccharification, increasing the amount of released fermentable sugars and thus improving the yield of bioproducts, preferably biofuel, obtained after the fermentative process.
An important percentage of xylose of constituent biomass polysaccharides is not released in the process of enzymatic hydrolysis of biomass. The host cell of the invention expresses a recombinant beta-xylosidase enzyme which is capable of degrading xylan oligomers to xylose in a more efficient way than other beta-xylosidase enzymes. Thus, this host cell and the enzymatic cocktail produced by it are useful for the optimization of the hydrolysis step of biomass into fermentable sugars.
The inventors have demonstrated that the incorporation, and successful later expression, of a recombinant beta-xylosidase enzyme which comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, preferably the mature beta-xylosidase AnBxl from the fungus Aspergillus nidulans which comprises the amino acid sequence SEQ ID NO: 1, in a Myceliophthora thermophila host cell, enhances the concentration of released xylose from biomass when the transformed cell or the enzymatic cocktail produced by said cell is used in a process of hydrolysis of biomass. This represents an increase in the final concentration of fermentable sugars, and hence of the overall yield of bioproducts, preferably biofuel, production.
Therefore, a first aspect of the present invention is related to a Myceliophthora thermophila host cell which expresses 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, from now on referred to as “host cell of the invention”.
The “host cell”, as used herein, includes any cell type which is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a nucleotide sequence or polynucleotide encoding the recombinant beta-xylosidase enzyme referred to above. The choice of a host cell will to a large extend depend upon the nucleotide sequence encoding the polypeptide and its source. The host cell may be eukaryote, such as mammalian, insect, plant or fungal cell. In a preferred embodiment, the host cell is a filamentous fungal cell. Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. In a more preferred embodiment, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. In a most preferred embodiment, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred embodiment, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium pseudograminearum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In another most preferred embodiment, the filamentous fungal host cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Gibberella zeae, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell. In an even more preferred embodiment, the host cell of the invention is any strain of the species Myceliophthora thermophila. In a preferred embodiment, the host cell of the invention is Myceliophthora thermophila strain C1.
It will be understood that for the aforementioned species the invention encompasses both perfect and imperfect states, and other taxonomic equivalents, e. g. anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. For instance, Myceliophthora thermophila is equivalent to Chrysosporium lucknowense.
The host cell of the invention expresses 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.
The term “beta-xylosidase” refers to a protein that hydrolyses short 1,4-beta-D-xylooligomers into xylose. The “recombinant beta-xylosidase” of the invention is a beta-xylosidase enzyme which is naturally expressed in a microorganism other than the host cell of the invention, i.e. a heterologous beta-xylosidase, the amino acid sequence of which has not been modified or has been modified preferably by means of one or more deletions, insertions, substitutions, etc. In the context of the present invention, the terms “foreign” and “heterologous” are equivalent and mean derived from a different cell type or a different species from the recipient.
In a preferred embodiment, the recombinant beta-xylosidase is a naturally occurring beta-xylosidase coming from or isolated from a microorganism other than the host cell of the invention, more preferably from an Aspergillus cell, even more preferably from an Aspergillus nidulans cell.
The recombinant beta-xylosidase referred to in the present invention comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1.
In the present invention, the terms “sequence identity”, “identity” and “similarity” are considered equivalent and can be used interchangeably. The term “sequence identity” (or its grammatical equivalents) means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified reference sequence using an alignment algorithm. The degree of identity can be determined by, for example, the Clustal method, the Wilbur-Lipman method, the GAG program, including GAP, BLAST or BLASTN, EMBOSS Needle, FASTA, etc. Furthermore, the Smith Waterman algorithm can be used in order to determine the degree of identity between two sequences.
An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
Recombinant beta-xylosidases comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 may be obtained from a filamentous fungus, such as, Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Gibberella, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma. In a more preferred embodiment, the recombinant beta-xylosidase is an Aspergillus aculeatus, A. awamori, A. foetidus, A. fumigatus, A. japonicus, A. nidulans, A. niger, A. oryzae, F. bactridioides, F. cerealis, F. crookwellense, F. culmorum, F. graminearum, F. graminum, F. heterosporum, F. negundi, F. oxysporum, F. pseudograminearum, F. reticulatum, F. roseum, F. sambucinum, F. sarcochroum, F. sporotrichioides, F. sulphureum, F. torulosum, F. trichothecioides, F. venenatum, G. zeae, H. insolens, H. lanuginosa, M. miehei, N. crassa, P. purpurogenum, T. harzianum, T. koningii, T. longibrachiatum, T. reesei, or T. viride beta-xylosidase.
In a more preferred embodiment, the recombinant beta-xylosidase enzyme comprises an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.
In a more preferred embodiment, the recombinant beta-xylosidase enzyme comprises, or consists of, the amino acid sequence SEQ ID NO: 1. The sequence SEQ ID NO: 1 corresponds to the mature beta-xylosidase enzyme from the fungus A. nidulans named AnBxl. An example of a beta-xylosidase enzyme comprising the amino acid sequence SEQ ID NO: 1 is the polypeptide of SEQ ID NO: 7 corresponding to the preprotein of the beta-xylosidase enzyme from A. nidulans, wherein the amino acids 1 to 23 refer to signal peptide.
The recombinant beta-xylosidase enzyme can be bound/linked to a signal peptide which directs the enzyme into the cell's secretory pathway. Thus, in another preferred embodiment, the beta-xylosidase enzyme comprises a signal peptide or signal sequence. Said signal peptide may be heterologous to the enzyme, i.e. the signal peptide and the enzyme comes from different microorganisms, or be the native signal sequence of the beta-xylosidase enzyme, i.e. the signal peptide of the beta-xylosidase enzyme from A. nidulans.
When the recombinant beta-xylosidase enzyme comprises, or consist of, the amino acid sequence SEQ ID NO: 7, the host cell is not an Aspergillus nidulans cell.
In a preferred embodiment, the signal peptide is heterologous to the beta-xylosidase enzyme.
Effective signal peptide sequences for bacterial host cells are the signal peptide obtained from Bacillus stearothermophilus maltogenic amylase, Bacillus licheniformis subtilisin, B. licheniformis beta-lactamase, B. stearothermophilus alpha-amylase, B. stearothermophilus neutral proteases (nprT, nprS, nprM), Bacillus subtilis prsA, etc. Effective signal peptide sequences for filamentous fungal host cells are the signal peptide obtained from Aspergillus niger neutral amylase, A. niger glucoamylase A (SEQ ID NO: 12), Aspergillus shirousami glucoamylase A, Aspergillus kawachii glucoamylase I, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, H. insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
In a preferred embodiment, the signal peptide is the glucoamylase A signal peptide, preferably, the glucoamylase A signal peptide from Aspergillus awamori, more preferably, the signal peptide comprises the sequence SEQ ID NO: 2.
The resulting polypeptide of binding the signal peptide to an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 is a fusion polypeptide or fusion protein, wherein the signal peptide sequence is foreign to, or heterologous to, said an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1. The meaning of the terms “foreign” and “heterologous” has been explained above.
Thus, the beta-xylosidase enzyme may be a fusion polypeptide or cleavable fusion polypeptide in which a signal peptide sequence is fused at the N-terminus of the amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).
A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
In a particular embodiment, the beta-xylosidase enzyme is a fusion protein having the amino acid sequence SEQ ID NO: 3, comprising the glucoamylase A signal peptide from A. awamori of sequence SEQ ID NO: 2 bound to the an amino acid sequence of SEQ ID NO: 1.
As it will be shown in examples below, the highest yield of released xylose and hydrolyzed xylobiose during the hydrolytic process of biomass was obtained when the host cell of the invention expressed this SEQ ID NO: 3 (see
As explained above, the host cell of the invention expresses a nucleotide sequence encoding the recombinant beta-xylosidase enzyme disclosed herein. Nucleotide sequences encoding these recombinant beta-xylosidases can encode the mature polypeptide (such as, SEQ ID NO: 1) or a preprotein thereof (such as SEQ ID NO: 3) comprising a signal peptide (such as SEQ ID NO: 2) linked to the amino acid sequence of said mature enzyme (such as SEQ ID NO: 1). As the skilled person in the art understand, the preprotein of sequence SEQ ID NO: 3 will have to be subsequently processed by the cell giving rise to the mature polypeptide SEQ ID NO: 1.
Thus in another preferred embodiment, the nucleotide sequence encoding the beta-xylosidase enzyme disclosed herein comprises the sequence SEQ ID NO: 4 which preferably is bound to a nucleotide sequence encoding a signal peptide. In a more preferred embodiment, the nucleotide sequence encoding the signal peptide comprises the sequence SEQ ID NO: 5. In a most preferred embodiment, the nucleotide sequence encoding the beta-xylosidase enzyme comprises the sequence SEQ ID NO: 6.
Nucleic acid sequences encoding beta-xylosidases can be included in a genetic construct, preferably in an expression vector. Said genetic construct may further comprise one or more regulatory sequences of gene expression, such as promoters, terminators, etc.
In accordance with the present invention, “nucleic acid sequence” or “polynucleotide” or “nucleotide sequence” is a nucleic acid molecule (polynucleotide) that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA, including cDNA. The nucleotide sequence of the present invention can be or not chemically or biochemically modified and can be artificially performed by means of cloning and selection methods or by sequencing.
The term “nucleic acid construct” as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for the expression of a coding sequence of the recombinant beta-xylosidase.
The term “control sequences” is defined herein to include all components which are necessary or advantageous for the expression of a polynucleotide encoding a recombinant beta-xylosidase of the present invention. Each control sequence may be native or heterologous to the nucleotide sequence encoding the recombinant beta-xylosidase. Such control sequences include, but are not limited to, a leader sequence, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
The control sequence may be a promoter,i.e. a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), B. licheniformis alpha-amylase gene (amyL), B. licheniformis penicillinase gene (penP), B. stearothermophilus maltogenic amylase gene (amyM), B. subtilis levansucrase gene (sacB), B. subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), Escherichia coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.
Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for A. nidulans acetamidase, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger or A. awamori glucoamylase (glaA), A. oryzae TAKA amylase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, F. oxysporum trypsin-like protease (WO 96/00787), F. venenatum amyloglucosidase (WO 00/56900), F. venenatum Dania (WO 00/56900), F. venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, R. miehei aspartic proteinase, T. reesei beta-glucosidase, T. reesei cellobiohydrolase I, T. reesei cellobiohydrolase II, T. reesei endoglucanase I, T. reesei endoglucanase II, T. reesei endoglucanase III, T. reesei endoglucanase V, T. reesei xylanase I, T. reesei xylanase II, T. reesei xylanase III, T. reesei beta-xylosidase, and T. reesei translation elongation factor, as well as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader sequence has been replaced by an untranslated leader sequence from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an A. niger neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an A. nidulans or A. oryzae triose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof. Other promoters are described in U.S. Pat. No. 6,011,147. In a preferred embodiment, the promoter is the promoter from A. nidulans beta-xylosidase.
In a yeast host cell, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), S. cerevisiae galactokinase (GAL1), S. cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), S. cerevisiae triose phosphate isomerase (TPI), S. cerevisiae metallothionein (CUP1), and S. cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3′-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), B. licheniformis alpha-amylase (amyL), and E. coli ribosomal RNA (rrnB).
Preferred terminators for filamentous fungal host cells are obtained from the genes for A. nidulans acetamidase, A. nidulans anthranilate synthase, A. niger glucoamylase, A. niger alpha-glucosidase, A. oryzae TAKA amylase, F. oxysporum trypsin-like protease, T. reesei beta-glucosidase, T. reesei cellobiohydrolase I, T. reesei cellobiohydrolase II, T. reesei endoglucanase I, T. reesei endoglucanase II, T. reesei endoglucanase III, T. reesei endoglucanase V, T. reesei xylanase I, T. reesei xylanase II, T. reesei xylanase III, T. reesei beta-xylosidase, and T. reesei translation elongation factor.
Preferred terminators for yeast host cells are obtained from the genes for S. cerevisiae enolase, S. cerevisiae cytochrome C(CYC1), and S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, cited ad supra.
The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
Examples of suitable mRNA stabilizer regions are obtained from a B. thuringiensis cryIIIA gene (WO 94/25612) and a B. subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).
The control sequence may also be a leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5′-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.
Preferred leader sequences for filamentous fungal host cells are obtained from the genes for A. oryzae TAKA amylase and A. nidulans triose phosphate isomerase.
Suitable leader sequences for yeast host cells are obtained from the genes for S. cerevisiae enolase (ENO-1), S. cerevisiae 3-phosphoglycerate kinase, S. cerevisiae alpha-factor, and S. cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP.
The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for A. nidulans anthranilate synthase, A. niger glucoamylase, A. niger alpha-glucosidase A. oryzae TAKA amylase, and F. oxysporum trypsin-like protease.
Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.
At a minimum, the control sequences comprise a signal peptide sequence as explained previously, and preferably a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding the recombinant beta-xylosidase. The term “operably linked” denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of the recombinant beta-xylosidase.
The nucleotide sequence encoding the beta-xylosidase described herein may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression.
The term “expression vector” is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding the recombinant beta-xylosidase as disclosed herein, and which is operably linked to additional nucleotides that provide for its expression. Said vector comprising a polynucleotide encoding the recombinant beta-xylosidase is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector.
In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression. The expression vectors referred to in the present invention comprise a polynucleotide encoding the beta-xylosidase described herein, a promoter, and transcriptional and translational stop signals. The various nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the enzyme at such sites.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
The vectors used in the present invention preferably contain one or more selectable markers which permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), pyr5, cysC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.
The vectors used in the present invention preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the beta-xylosidase enzyme or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s).
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication which functions in a cell. The term “origin of replication” or “plasmid replicator” is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo. Examples of origins of replication useful in a filamentous fungal cell are AMAI and ANSI.
More than one copy of a nucleotide sequence encoding the beta-xylosidase of the present invention may be inserted into the host cell to increase the production of the gene product. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected by cultivating the cells in the presence of the appropriate selectable agent. The procedures used to ligate the elements described above to construct the recombinant expression vectors referred to in the present invention are well known to one skilled in the art.
The term “expression” includes any step involved in the production of the recombinant beta-xylosidase including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
As explained above, the host cell of the invention expresses a nucleotide sequence encoding a functional recombinant beta-xylosidase enzyme and it is capable of secreting it to the extracellular medium. The term “functional” means that the expressed enzyme retains its capacity to hydrolyse xylan oligomers to xylose. This activity can be measured by means of any suitable method known in the state of the art to assess the beta-xylosidase activity, preferably by means of the method described below in examples of the present invention. The most usual detection method involves the use of the chromogenic substrate para-nitrophenyl beta-D-xylopyranoside (hereinafter pNXP). A preferred method involves the measurement of excision of xylobiose to xylose by HPLC.
The expression of the beta-xylosidase in the host cell of the invention may be performed by means of any method known in the art, such as transformation of a suitable host cell with a nucleotide sequence encoding the recombinant beta-xylosidase, or a genetic construction comprising said nucleotide sequence, and cultivation of the transformed host cell under conditions which induce the expression of said nucleotide sequence in order to obtained the secreted enzyme.
The host cell can be cultivated in a nutrient medium suitable for production of the recombinant beta-xylosidase using methods well known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial bioreactor performed in a suitable medium and under conditions allowing the beta-xylosidase to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the beta-xylosidase is secreted into the nutrient medium, the beta-xylosidase can be recovered directly from the medium.
The recombinant beta-xylosidase expressed may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate.
The resulting beta-xylosidase may be recovered using methods known in the art. For example, the beta-xylosidase may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
The beta-xylosidases produced in the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction, in order to obtain substantially pure beta-xylosidase that can be included in an enzymatic composition together with other cellulolytic enzymes.
Thus, a second aspect of the invention refers to a recombinant beta-xylosidase enzyme expressed by the host cell of the invention. Preferably, said recombinant beta-xylosidase enzyme comprise, or consists of, the amino acid sequence SEQ ID NO: 7 (preprotein beta-xylosidase from A. nidulans), SEQ ID NO: 1 (mature beta-xylosidase from A. nidulans) or SEQ ID NO: 3 (SPCG+mature beta-xylosidase from A. nidulans), more preferably SEQ ID NO: 3.
A third aspect of the invention refers to a composition comprising the recombinant beta-xylosidase enzyme produced by means of the host cell of the invention, preferably the enzyme comprising, or consisting of, the sequence SEQ ID NO: 3 (hereinafter “composition of the invention”), and/or the host cell of the invention. This composition of the invention may further comprise one or more of other enzymatic activities, such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, such as arabinofuranosidase, xyloglucanase, polysaccharide mono-oxygenases (previously known as glycosyl-hydrolases family 61 or GH61), endoglucanases, beta-glucosidases and/or cellobiohydrolases; chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase, or any combination thereof. The additional enzyme(s) may be produced, for example, by a microorganism belonging to the genus Aspergillus, such as A. aculeatus, A. awamori, A. fumigatus, A. foetidus, A. japonicus, A. nidulans, A. niger, or A. oryzae; Fusarium, such as F. bactridioides, F. cerealis, F. crookwellense, F. culmorum, F. graminearum, F. graminum, F. heterosporum, F. negundi, F. oxysporum, F. pseudograminearum, F. reticulatum, F. roseum, F. sambucinum, F. sarcochroum, F. sulphureum, F. toruloseum, F. trichothecioides, or F. venenatum; Gibberella, such as G. zeae; Humicola, such as H. insolens or H. lanuginosa; Trichoderma, such as T. harzianum, T. koningii, T. longibrachiatum, T. reesei, or T. viride; or Myceliophthora, such as M. heterotalica or M. thermophila.
In a preferred embodiment, the composition of the invention further comprises one or more of other cellulolytic enzymes. The term “cellulolytic enzymes” also known as “cellulases”, refers to a category of enzymes capable of hydrolysing cellulose (beta-1,4-glucan or beta-D-glucosidic linkages) or hemicellulose to shorter oligosaccharides, cellobiose and/or glucose. Examples of cellulolytic enzymes are, but not limited to, arabinofuranosidase, xyloglucanase, polysaccharide mono-oxygenases, endoglucanases, beta-glucosidases, cellobiohydrolases or endoxylanases. Thus, in a more preferred embodiment, these cellulolytic enzymes are selected from the list consisting of: arabinofuranosidase, xyloglucanase, polysaccharide mono-oxygenases, endoglucanases, beta-glucosidases, cellobiohydrolases, endoxylanases and any combination thereof. These cellulolytic enzymes can derive from the host cell of the invention or other cellulolytic enzymes producers-microorganisms different from the host cell of the invention. Likewise, they can be naturally or recombinantly produced.
Preferably, the composition of the invention comprises the recombinant beta-xylosidase enzyme produced by means of the host cell of the invention, preferably the enzyme comprising, or consisting of, the SEQ ID NO: 1, and other cellulolytic enzymes derived from the host cell of the invention. In a more preferred embodiment, the composition of the invention is an enzymatic mixture obtained by the host cell of the invention. In an even more preferred embodiment, the composition of the invention is an enzymatic mixture obtained by the M. thermophila host cell of the invention, preferably M. thermophila C1, wherein said cell comprises a nucleotide sequence encoding the recombinant beta-xylosidase enzyme which comprises, or consists of, the amino acid sequence SEQ ID NO: 3.
The term “endoglucanase” or “EG” refers to a group of cellulase enzymes classified as E.C. 3.2.1.4. These enzymes hydrolyse internal beta-1,4 glucosidic bonds of cellulose. Endoglucanase activity may be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481).
The term “cellobiohydrolase” refers to a protein that catalyzes the hydrolysis of cellulose to cellobiose via an exoglucanase activity, sequentially releasing molecules of cellobiose from the reducing or non-reducing ends of cellulose or cellooligosaccharides. Cellobiohydrolase activity may be determined according to the procedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288; and Tomme et al. , 1988, Eur. J. Biochem. 170: 575-581.
The term “beta-glucosidase” as used herein refers to an enzyme which catalyses the hydrolysis of a sugar dimer, including but not limited to cellobiose, with the release of a corresponding sugar monomer, used, but not limited, for the synthesis of ethanol. Beta-glucosidase enzyme acts upon beta1→4 bonds linking two glucose or glucose-substituted molecules (i.e., the disaccharide cellobiose). It is an exocellulase with specificity for a variety of beta-D-glycoside substrates. It catalyzes the hydrolysis of terminal non-reducing residues in beta-D-glucosides with release of glucose. Beta-glucosidase activity may be determined using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66.
The term “endoxylanase” refers to an enzyme which catalyzes the endohydrolysis of 1,4-beta-D-xylosidic linkages in xylanes.
The term “arabinofuranosidase” or “alpha-L-arabinofuranosidase” means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)- and/or (1,5)-linkages, arabinoxylans, and arabinogalactans. Alpha-L-arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of the present invention, alpha-L-arabinofuranosidase activity is determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40° C. followed by arabinose analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).
The term “xyloglucanase” refers to any enzyme which has an activity towards the substrate xyloglucan. Examples of useful xyloglucanases are family 12 xyloglucan hydrolyzing endoglucanases, in particular family 12 xyloglucan hydrolyzing endoglucanases, obtained from e.g. A. aculeatus as described in WO 94/14953. Another useful example is a xyloglucanase produced by Trichoderma, especially EGIII. The xyloglucanase may also be an endoglucanase with xyloglucanase activity and low activity towards insoluble cellulose and high activity towards soluble cellulose, e.g., family 7 endoglucanases obtained from, e.g., H. insolens. The xyloglucanase activity may be measured by means of measuring the residual sugar: A purified source of xyloglucan (e.g. from Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) is dissolved in a suitable buffer (250 μg xyloglucan in 100 μL buffer) and incubated with 30-400 ng of enzyme for 1 or 20 hours. The increase in reducing sugar is determined according to the procedure of Somogyi using glucose for calibration.
The term “polysaccharide mono-oxygenases”, formerly classified as GH61 and CBM33 enzymes in the CAZy database, refers to enzymes having cellulolytic enhancing activity and means a polypeptide that catalyzes the enhancement of the hydrolysis of a cellulosic material. For purposes of the present invention, cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of total protein/g of cellulose in pretreated corn stover (PCS), wherein total protein is comprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of a GH61 polypeptide having cellulolytic enhancing activity for 1-7 days at a suitable temperature, such as 40° C.-80° C., e.g., 50° C., 55° C., 60° C., 65° C., or 70° C., and a suitable pH, such as 4-9, e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5, compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g of cellulose in PCS).
The composition of the invention may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the composition may be in the form of a granulate or a microgranulate. The enzymes to be included in the composition may be stabilized in accordance with methods known in the art.
As stated above, the host cell of the invention expresses a recombinant beta-xylosidase enzyme comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, preferably the beta-xylosidase enzyme from A. nidulans, more preferably, the beta-xylosidase enzyme from A. nidulans of SEQ ID NO: 1, which is capable of degrading xylan oligomers to xylose when secreted to the extracellular medium. This host cell is capable of secrete this enzyme to the medium together with other native or recombinantly produced cellulolytic enzymes, being thus useful for the optimization of the hydrolysis step of biomass into fermentable sugars.
Therefore, a fourth aspect the invention relates to the use of the host cell of the invention, the use of the recombinant beta-xylosidase enzyme produced by means of the host cell of the invention or the use of the composition of the invention for the degradation of biomass.
The term “biomass” means the biodegradable fraction of products, waste and residues from biological origin from agriculture (including vegetal, such as crop residues, and animal substances), forestry (such as wood resources) and related industries including fisheries and aquaculture, as well as biodegradable fraction of industrial and municipal waste, such as municipal solid waste or wastepaper. In a preferred embodiment, the biomass is straw or organic fraction of municipal solid wastes. In a more preferred embodiment, the biomass is plant biomass, more preferably selected from the list consisting of: fermentable sugar-rich biomass, such as sugarcane, starchy biomass, for example, wheat grain, or corn straw.
The recombinant beta-xylosidase enzyme produced by means of the host cell of the invention, as well as the host cell or the composition of the present invention may be used in the production of monosaccharides, disaccharides, and polysaccharides as chemical or fermentation feedstocks from biomass for the production of ethanol, plastics, or other products or intermediates.
The host cell of the present invention may be used as a source of the polypeptide having beta-xylosidase activity, and other cellulolytic enzymes, in a fermentation process with the biomass.
The predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemi-cellulose, and the third is pectin. The secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened through polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses usually bind through hydrogen bonds to cellulose, as well as to other hemicelluloses, which helps stabilize the cell wall matrix. The recombinant beta-xylosidase enzymes produced by the host cell of the invention may be used in conjunction with the other cellulolytic enzymes to further degrade the cellulose component of the biomass substrate.
The degradation or hydrolysis of biomass into fermentable sugars, process also known as “saccharification”, by means of the recombinant beta-xylosidase enzyme expressed by the host cell of the invention, the host cell of the invention or the composition of the invention can be followed by a fermentation process in which the obtained fermentable sugars are used in order to finally obtain a bioproduct such as bioethanol.
Thus, another preferred embodiment of this aspect of the invention refers to the use of the recombinant beta-xylosidase enzyme produced by means of the host cell of the invention, the use of the host cell of the invention or the use of the composition of the invention for the degradation of biomass in a bioproduct production process.
The term “bioproduct” or “bio-based products” refers to those materials, chemicals and energy derived from renewable biological resources. Examples of these bioproducts are, but not limited to, hydrocarbon compounds in their different forms, such as aliphatic (saturated, insaturated, cyclic) or aromatic, as alkanes, alkenes, alkines, cyclic forms of these compounds or aromatic hydrocarbons; oxygenated substances as alcohols, ethers, aldehydes, ketones or carboxylic acids; nitrogenated substances as amines, amides, nitrocompounds or nitriles; halogenated substances as halures. The term “bioproducts” includes also any combination of the compounds described above, compounds further derived from the compounds described above by any kind of physical, chemical or biological treatment, polymers from the compounds described above, compounds described above substituted by any functional group or element in one or more of their bounds and branched forms of the compounds described above.
Furthermore, the term “bioproduct” or “bio-based products” also refers to high-value-added products which can be obtained by chemical transformation of sugars or by biological fermentation of said sugars. Examples of microorganisms which can carry out the fermentation of sugars are, without limiting to, Bacillus thermoglucosidaisus, Clostridium butyricum, Clostridium acetobutylicum, Clostridium beijerinckii, Corynebacterium glutamicum, Enterobacter aerogenes, E. coli, Geobacillus themoglucosidasius, Klebsiella oxytoca, Lactobacillus sp. Leunoscoc mesenteroides, Thermoanaerobacter BG1L1, Thermoanaerobacter ethanolicus, Thermoanaerobacter mathranii, Thermoanaerobacter thermosaccharolyticum, Zymobacter palmae, Zymomonas mobilis Candida arabinofermentans, Candida boidinii, Candida diddensis, Candida fermentans, C. lucknowense, Candida pastoris, Candida shehatae, Candida sonorensis, Candida tropicalis, Hansenula anómala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces marxianus, Pichia pastoris, Pichia stipitis, S. cerevisiae, Saccharomyces bulderi, Saccharomyces barnetti, Saccharomyces exiguus, Saccharomyces diastaticus, Saccharomyces uvarum, Schizosaccharomyces pombe, Pseudomonas sp., such as, P. aeruginosa, P. putida, or combinations thereof.
Non-limiting examples of bioproducts obtained as consequence of fermenting sugars are acids, alcohols, aromatics compounds, aldehydes, ketones, triglycerides, gases, fatty acids (such as palmitic acid, lauric acid, fatty acids-Δ2, etc.), biopolymers, proteins, peptides, amino acids, vitamins (such as B12, riboflavin, beta-carotene, etc.), antibiotics (such as penicillin, cephalosporins, tetracycline, etc.), pharmaceuticals, industrial enzymes, and combinations thereof.
Non-limiting examples of acids include acetic acid, lactic acid, propionic acid, 3-hydroxypropionic, butyric acid, gluconic acid, itaconic acid, citric acid, succinic acid, levulinic acid, glutamic acid, succinic acid, beta-cetoacid, beta-cetoalcohol, beta-hydroxiacid, and combinations thereof.
Non-limiting examples of alcohols include methanol, ethanol, propanol, hexanol, octanol, dodecanol, 1,3-butanodiol(1-3 diol), 1-alcohol, isopropanol, butanol, ethylene glycol, propanediol, butanediol, glycerol, erythritol, xylitol, sorbitol, and combinations thereof.
Non-limiting examples of amino acids include glutamic acid, aspartic acid, methionine, lysine, glycine, arginine, threonine, phenylalanine, tyrosine, and combinations thereof.
Other non-limiting examples of fermentation products include gases, such as methane, butane, ethylene, acetone, hydrogen, carbon dioxide, etc.; hydrocarbon, such as alkanes, alkenes, alkines, aromatic hydrocarbons, etc.; nitrogen compounds such as amines, amides, nitrocompounds or nitriles; halides; etc.
Ethanol can be produced by enzymatic degradation of biomass and conversion of the released saccharides to ethanol. This kind of ethanol is often referred to as bioethanol. It can be used as a fuel additive or extender in blends of from less than 1% and up to 100% (a fuel substitute).
In a more preferred embodiment the bioproduct is biofuel. The term “biofuel” as used herein refers to a hydrocarbon, or a mixture thereof, which can be used as fuel and is obtained using fermentable biomass as starting material. Examples of biofuels include, but are not limit to, ethanol or bioethanol, butanol and biodiesel. In a more preferred embodiment, the biofuel is bioethanol.
The term “bioethanol” refers to an alcohol made by fermentation, mostly from fermentable biomass, such as carbohydrates produced in sugar or starch crops such as corn or sugarcane.
Non-limiting examples of fermentation organisms and associated product include the following. Fermentation of carbohydrates to acetone, butanol and ethanol by: (i) solventogenic Clostridia as described by Jones and Woods (1986) Microbiol. Rev. 50:484-524; (ii) a mutant strain of Clostridium acetobutylicum as described in U.S. Pat. No. 5,192,673; and (iii) a mutant strain of Clostridium beijerinckii as described in U.S. Pat. No. 6,358,717 is known. Fermentation of carbohydrates to ethanol by modified strains of E. coli has been described by Underwood et al., (2002) Appl. Environ. Microbiol.68:6263-6272 and by a genetically modified strain of Zymomonas mobilis is described in US 2003/0162271 A1. Preparation of lactic acid by recombinant strains of E. coli (Zhou et al., (2003) Appl. Environ. Microbiol. 69:399-407), natural strains of Bacillus (US20050250192), and Rhizopus oryzae (Tay and Yang (2002) Biotechnol. Bioeng. 80:1-12) is known. Recombinant strains of E. coli have been used as biocatalysts in fermentation to produce 1,3 propanediol (U.S. Pat. Nos. 6,013,494 and 6,514,733) and adipic acid (Niu et al., (2002) Biotechnol. Prog. 18:201-211). Acetic acid has been produced using recombinant Clostridia (Cheryan et al., (1997) Adv. Appl. Microbiol. 43:1-33) and newly identified yeast strains (Freer (2002) World J. Microbiol. Biotechnol. 18:271-275). Production of succinic acid by recombinant E. coli and other bacteria is disclosed in U.S. Pat. No. 6,159,738 and by mutant recombinant E. coli in Lin et al., (2005) Metab. Eng. 7:116-127). Pyruvic acid has been produced by mutant Torulopsis glabrata yeast (Li et al., (2001) Appl. Microbiol. Technol. 55:680-685) and by mutant E. coli (Yokota et al., (1994) Biosci. Biotech. Biochem. 58:2164-2167). Recombinant strains of E. coli have been used for production of para-hydroxycinnamic acid (US20030170834) and quinic acid (US20060003429).
In a fifth aspect, the present invention refers to a method of producing fermentable sugars, hereinafter “first method of the invention”, comprising:
A pretreatment process of the biomass is often required for increasing the access of the enzymes to their substrates and consequent efficient hydrolysis. Pretreatment uses various techniques, including but not limited to ammonia fiber explosion, chemical treatment and steam explosion at high temperatures to alter the structure of cellulosic biomass and make cellulose more accessible. The use of the host cell of the invention, the recombinant beta-xylosidase enzyme produced by means of the host cell of the invention or the composition of the invention in the methods of the present invention is advantageous since high temperatures are not required in the pretreatment process of the biomass.
The term “fermentable sugar”, as used herein, refers to simple sugars, such as glucose, xylose, arabinose, galactose, manose, rhanmose, sucrose or fructose, among others.
A sixth aspect of the present invention refers to a method of producing a bioproduct from biomass, hereinafter “second method of the invention”, comprising:
The term “fermenting or fermentation” as used herein, refers to a biological transformation process caused by the activity of some microorganisms in which sugars such as glucose, fructose, and sucrose are converted into ethanol. The microorganisms used therefore, are fermenting microorganisms, which have a fermentation capacity, such as yeasts, preferably S. cerevisiae.
The term “recovery” as used herein, refers to the collection of fermentable sugars obtained after the incubation in step (a) of the first method of the invention or bioproduct obtained after fermentation of step (b) of the second method of the invention. The recovery may occur by any method known in the art, including mechanical or manual ones.
In a preferred embodiment of the second method of the invention, the bioproduct is biofuel, more preferably bioethanol.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word “comprise” and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples, drawings and sequence listing are provided by way of illustration and are not intended to be limiting of the present invention.
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.
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.
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))
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.
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
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
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.
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.
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.
M. thermophila C1 and transformants expressing SPGA-AnBxl.
Beta-Xylosidase Activity Determination
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.
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 minutes. The amount of p-nitrophenol released was measured at A410 (ε410=15.2 mM−1 cm−1) after addition of 100 μg sodium carbonate to stop 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. The specific activities obtained are shown in Table 1.
The control cocktail exhibits significantly less pNXP activity than the cocktails produced by M. thermophila C1 successfully expressing pABC656 under identical production conditions.
Total protein of the enzymatic mixtures was determined by the BCA method (Applichem, A7787 0500).
Enzymatic Hydrolysis Experiments
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.
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).
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 (NH4OH). 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.
Xylose release and xylobiose hydrolysis profiles obtained during the enzymatic hydrolysis are shown in
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.
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.
As shown in
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/ES2015/070589 | 7/29/2015 | WO | 00 |
