TWI444477B - Polycistronic expression cassettes for producing cellulosomes and applications thereof - Google Patents

Description

Polycistronic expression of cellulase hydrolase complex and its application

The present invention relates to a polycistronic expression cassette for the manufacture of a cellulolytic enzyme complex and uses thereof.

Lignocellulose is abundant in the natural world, mainly in the plant cell wall, and the sugar produced after decomposition can be converted into ethanol, which is an important raw material for biomass energy. However, in plant cell walls, lignocellulose forms complex and compact structures with other components such as hemicellulose and lignin, which require pre-treatment to improve conversion efficiency.

The pre-treatments currently used include physical and chemical methods such as pulverization, crushing, high temperature, high pressure, and acid-base treatment; however, these methods consume energy, produce toxic waste liquid, are harmful to the environment, and are prone to other by-products. The biological rule is to add enzymes or special strains to avoid the above shortcomings, but only need many types of enzymes to achieve effective decomposition of cellulose.

Clostridium thermocellum is a heat-resistant anaerobic, highly capable cellulolytic agent . Studies have shown that Clostridium thermocellum forms a high-molecular-weight and complex multi-enzyme complex called cellulosome, which is efficiently decomposed by the synergistic action of various decomposing enzymes. Cellulose. In addition to Clostridium thermocellum, there are many other microorganisms in the natural world with similar cellulolytic enzyme complexes, for example, C. cellulovorans , C. cellulolyticum , and paper-coated C. papyrosolvens , Trichoderma. longibrachiatum , Baceroides cellulosolvens , Acetivibrio cellulolyticusas , Neocallimastix frontalis , Ruminal fungus P ( Promyces spp ), Bacillus megaterium , Bacillus licheniformis, Bacillus cellulosolvens , Ruminococcus flavefaciens , and Vibrio anguillarum Acetivibrio cellulolyticus ).

Studies have shown that the cellulolytic enzyme complex of Clostridium thermocellum mainly contains three subunits: scaffold protein, decomposing enzyme and cell surface anchoring protein; Type I cohesin domains, which bind to the type I dockerin domains of the degrading enzyme; the scaffold protein has a type II dockerin domain, Binding to the type II cohesin domain of the cell surface anchoring protein allows the cellulolytic enzyme complex to be immobilized on the surface of the cell; the scaffold protein has a carbohydrate-binding domain (carbohydrate-binding) Domain, CBM), which binds to cellulose carbohydrates. Figure 1 is a schematic view showing the structure of a cellulolytic enzyme complex of Clostridium thermocellum.

In the cellulolytic enzyme complex of Clostridium thermocellum, the scaffold protein (CipA) has nine first-type binding domains, which can bind nine decomposing enzymes; cell surface anchoring proteins (OlpB, SdbA and Orf2p) can each be linked One or more scaffold proteins; and the degrading enzymes include at least exoglucanases (exoglucanases CelS, CblA, and CelK), endoglucanases (such as CelR, CelA, CelF, CelN, and CelB), poly-wood Carbohydrases (such as XynC, XynY and XynZ) and hemicellulases (such as LicB and ChiA). Studies have shown that Clostridium thermocellum adjusts the correct proportion of various degrading enzymes due to different environments (for example, facing different carbon sources) to achieve synergistic decomposition efficiency. For example, when the carbon source is derived from microcrystalline cellulose (avicel), the highest amount of the degrading enzyme of Clostridium thermocellum is CelK, followed by Celsi, CelR, CelA, XynC and XynZ, etc.; Decomposing enzymes When the carbon source is derived from cellobiose, Clostridium thermocellum adjusts its highest performing decomposition enzyme to XynZ, followed by XynC, CelA, CelK, CelR and CelS. Studies have shown that the proportion of degrading enzymes is related to the efficiency of the decomposition of a particular carbon source. However, the molecular mechanism by which Clostridium thermocellum regulates the correct ratio of various degrading enzymes is not well understood.

For related literature, see Gold et al, J. Bacteriol. 189(19): 6787-6795, 2007; Bayer et al, J. Structural Biol . 124:221-234, 1998; Demain et al, Microbiol. Mol. Biol. Rev. 69:124-154,2005; and Wu, ACS Symp. Ser. 516:251-264, 1993 Bayer et al., Curr Opin Biotechnol. 18(3): 237-45, 2007; Cha et al., J Microbiol Biotechnol 17(11): 1782-8, 2007; Blouzard et al, JOURNAL OF BACTERIOLOGY 189(6): 2300-9, 2007.

The artificially designed "designer cellulosome" is considered to be an important tool for studying the hydrolysis mechanism of cellulolytic enzyme complexes, and can also be applied to the industrial process of biomass energy. However, due to the complexity of the cellulolytic enzyme complex itself and the technical bottleneck of multi-gene transfer, it is still impossible to construct a complete or nearly complete cellulose hydrolase complex by artificial techniques.

In this case, the EngB-degrading enzyme and the fragment scaffold protein (mini-CbpA), which each exhibit E. coli, were used, and the interaction between the EngB-degrading enzyme and the fragment scaffold protein was observed in vitro. Fierobe et al. propose a technique for fused cellulonic enzyme complexes. Fierobe et al. used recombinant technology to produce chimeric scaffoldins, which contain two binding domains from different bacterial sources, each of which has strong specificity for the anchoring domain corresponding to the degrading enzyme of the species. Without affecting each other, the degrading enzyme containing the corresponding anchor domain can be precisely bound to the corresponding binding domain of the fusion scaffold protein, thereby combining the expected cellulolytic enzyme complex in vitro (J Biol. Chem. 276(24): 21257-21261). However, this technique purifies each recombinant protein and combines the cellulolytic enzyme complex in vitro. There are many limitations. For example, the pairing between the selected binding domain and the anchoring domain must be specific and must not interfere with other Pairing between anchoring domains and anchoring domains to avoid affecting the formation of cellulolytic enzyme complexes; currently there are only a limited number of combinations available for this technology, no more than three; and even if new pairs of species appear in the future, Each time, a new fusion scaffold protein must be reconstructed. It is confirmed that the new pairing type will not interfere with the original pairing type before it can be used. The involved project is very cumbersome, very inconvenient, and it is not possible to adjust various decomposition enzymes. The ratio is to achieve synergistic decomposition efficiency for a specific quality.

On the other hand, Cho et al. have constructed a expression vector carrying the gene of Clostridium clostridium fragment (mini-CbpA) and EngB degrading enzyme, and transferred it into Bacillus subtilis, confirming that these genes can be Bacillus subtilis expresses and constitutes a tiny cellulolytic enzyme complex (Cho et al. 2004. Production of Minicellulosome form Clostridium cellulovorans in bacillus subtilis WB800. Applied and Environmental Microbiology 70(9): 5071-5707). Arai et al. constructed a fragment carrier protein, an EngB degrading enzyme and a separate vector of XynB, and confirmed the co-cultivation of Bacillus subtilis containing the gene of the fragment scaffold protein and the EngB degrading enzyme, or co-cultivation of a gene containing the fragment scaffold protein and the XynB degrading enzyme. Bacillus subtilis can produce tiny cellulolytic enzyme complexes (minicellulosomes), one consisting of fragment scaffold proteins and EngB degrading enzymes, and the other consisting of fragment scaffold proteins and XynB degrading enzymes (Arai et al. 2007. Synthesis Of Clostridium cellulovorans minicellulosomes by intercellular complementation. Proc Natl Acad Sci US A. 104(5): 1456-60). However, the scaffold protein used in the above technique has only a small fraction of the fragment, and the number of the binding domains contained is only for testing the binding to the degrading enzyme, and only a maximum of one decomposing enzyme gene is transferred in one host. There is a considerable gap in constructing a complete cellulolytic enzyme complex, and there is no way to adjust the ratio of various decomposing enzymes to achieve synergistic decomposition efficiency for a specific substrate.

The present invention provides, for the first time, a novel technical solution for successfully producing a cellulolytic enzyme complex containing a plurality of decomposing enzymes in a host cell, and wherein the ratio between the decomposing enzymes can be arbitrarily adjusted as needed to mimic the microorganism to achieve a specific Synergistic decomposition efficiency. Compared with the prior art (for example, the fusion type cellulolytic enzyme complex), the genetic recombination step used in the present invention is simple, and is not limited to the pairing type between the binding domain and the anchoring domain, and can be simply and conveniently hosted. A cellulolytic enzyme complex in which the ratio between the plurality of decomposing enzymes contained in the cells can be arbitrarily adjusted is produced in the cells. The invention breaks through the bottlenecks and difficulties of the prior art, and greatly contributes to the subsequent research of the cellulose hydrolase complex and the development of the biomass energy.

In one aspect, the present invention provides a polycistronic expression cassette for producing a mimicking a natural microbial cellulolytic enzyme complex in a host cell, the cellulolytic enzyme complex comprising a scaffold protein a unit (scaffoldin subunits) and a plurality of enzymatic subunits, wherein the plurality of decomposing enzyme subunits have a natural ranking order according to the performance of the environmental growth period, wherein the polycistronic expression cassette include:

(a) a promoter; and

(b) a polycistronic nucleotide sequence operably linked to the promoter, the polycistronic nucleotide sequence comprising a scaffold protein nucleotide sequence encoding the scaffold protein subunit and encoding the plural a plurality of degrading enzyme nucleotide sequences of the decomposing enzyme subunit, wherein the plurality of decomposing enzyme nucleotide sequences are sequentially arranged in the natural ranking order in the polycistronic nucleus relative to the position of the promoter In the nucleotide sequence, the ranking order of the performance of the plurality of decomposing enzyme subunits under the control of the promoter is consistent with the above natural ranking order.

In another aspect, the invention provides a vector comprising the polycistronic expression cassette described above.

In still another aspect, the present invention provides a host cell comprising the aforementioned vector.

Further, the present invention also provides a method for producing a cellulolytic enzyme complex in vivo, which comprises culturing the aforementioned host cell in a condition exhibiting the cellulolytic enzyme complex to obtain the cellulolytic enzyme complex.

Further, the present invention provides a method of decomposing a lignocellulosic biomass comprising contacting the lignocellulosic biomass with the aforementioned host cell.

In another aspect, the present invention provides a method for formulating a plurality of degrading enzyme subunits by preparing the aforementioned vector and introducing it into a host cell and culturing in a suitable environment to prepare a plurality of decomposing enzyme subunits. The amount of performance is achieved by blending the ratio of the content of the plurality of decomposing enzyme subunits on the cellulolytic enzyme complex.

Detailed descriptions of various specific examples of the invention are given below. Other features of the present invention will be apparent from the following detailed description and claims.

Without further elaboration, it is believed that those of ordinary skill in the art of Therefore, the following description is to be construed as illustrative rather than limiting

All technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art to which the invention pertains, unless otherwise indicated.

The term "a" as used herein, unless otherwise specified, refers to the quantity of at least one (one or more).

As used herein, the term "cellulolytic enzyme complex" is a multi-enzyme complex composed of a plurality of protein subunits and capable of efficiently decomposing cellulose substrates. A cellulolytic enzyme complex comprises scaffoldin subunits and a plurality of enzymatic subunits. The scaffold protein subunit serves as a backbone of the native cellulolytic enzyme complex, typically comprising one or more type I cohesive domains, which are used in combination with the first type of each decomposing enzyme subunit. Type II dockerin domains are combined with each other. A plurality of decomposing enzyme subunits have two or more decomposing enzyme subunits, including but not limited to cellulase, exoglucanases, enoglucanases, Xylanases, hemicellulases, cellobiohydrolase, β-glucosidase, exo-β-1,4-glucanase ( BGLU, EC3.2.1.21), lichenase (β-1,3-1,4-endoglucanase), mannanase, chitinase, chitinase Endopolygalacturonase, laccases, pectinase, carbohydrate esterases, proteases, which are different due to microbes The growth environment (for example, different carbon sources) adjusts the ratio of the performance of each decomposition enzyme subunit to achieve synergistic decomposition efficiency. The cellulolytic enzyme complex further includes a cell surface anchoring protein subunit that immobilizes the multi-enzyme complex on the cell surface of the microorganism. Typically, the cell surface anchoring protein subunit comprises one or more second type of binding domains that bind to the second type of anchoring domain of the scaffold protein.

As used herein, the "scaffold protein nucleotide sequence", "plurality of decomposing enzyme nucleotide sequences" and "cell surface anchoring protein nucleotide sequence" are respectively referred to as scaffold protein subunits and plural decomposing enzyme subunits, respectively. And a nucleotide sequence of a cell surface anchoring protein subunit, which encodes a full length amino acid sequence of the corresponding native protein subunit, or a fragment thereof or equivalent thereof, including genetically engineered variants, preferably There is at least one corresponding bonding domain and/or anchoring domain.

In the natural world, microorganisms capable of producing a cellulolytic enzyme complex include, but are not limited to, C. thermocellum , C. cellulovorans , C. cellulolyticum , and lysate. C. papyrosolvens , Trichoderma. longibrachiatum , Baceroides cellulosolvens , Acetivibrio cellulolyticusas , rumen fungus N-type bacteria ( Neocallimastix frontalis) , rumen fungi P ( Promyces spp ), Bacillus megaterium , Bacillus licheniformis, Bacillus cellulosolvens , Ruminococcus flavefaciens , and fiber vinegar arc Acetivibrio cellulolyticus . Table 1 lists information about the cellulolytic enzyme complexes of various microorganisms.

Table 1:

As used herein, the term "performance card" refers to a nucleotide sequence produced by recombinant techniques or synthesis that enables gene expression in a host cell. Performance cards can be embedded in plastids or chromosomes. Typically, the expression cassette portion of a performance vector comprises a nucleotide sequence to be transcribed, a promoter and a polyadenylation instruction. As used herein, the term "polycistronic expression cassette" refers to a performance cassette containing a nucleotide sequence to be transcribed that encodes two or more gene products. As used herein, the term "polycistronic nucleotide sequence" refers to a nucleotide sequence to be transcribed in a polycistronic expression cassette encoding two or more gene products.

As used herein, the term "encoding" refers to the intrinsic property of a nucleotide sequence or gene sequence that can be used as a template in biological action to synthesize a corresponding nucleotide sequence (ie, rRNA, tRNA, and A polymer or macromolecule of an mRNA) or amino acid sequence. It will be apparent to those skilled in the art that due to the degeneracy of the genetic code, different gene sequences can encode the same polypeptide. Thus, unless otherwise indicated, "nucleotide sequence encoding a protein" or the like includes all nucleotide sequences which are degenerate versions of each other and which encode the same amino acid sequence.

As used herein, the term "expression" refers to the amount of a gene product (such as a transcript or protein) produced by a nucleotide sequence in a cell.

The term "natural ranking order" as used herein is used to describe the order in which a plurality of degrading enzyme subunits are ranked in terms of their performance or enzyme activity in an environment. Taking Clostridium thermocellum as an example, when the culture environment contains microcrystalline cellulose (avicel) as a carbon source, the order of the number of decomposition enzyme subunits of the cellulolytic enzyme complex in terms of the amount of expression or the activity of the enzyme is CelK, CelS, CelR, CelA, XynC, and XynZ; when the culture environment contains cellobiose as a carbon source, the plurality of decomposing enzyme subunits of these cellulolytic enzyme complexes are ranked according to the amount of expression or the activity of the enzyme. For XynZ, XynC, CelA, CelK, CelR and CelS.

As used herein, the term "lignocellulosic biomass" refers to a lignocellulosic material that can be converted into an energy source by decomposition of a cellulolytic enzyme complex. In particular, the lignocellulosic biomass comprises one or more ingredients selected from any one or a combination of cellulose, hemicellulose, and lignin.

In one aspect, the invention provides a polycistronic expression cassette for use in a host cell to mimic a natural microbial cellulolytic enzyme complex comprising a scaffold protein subunit and a plurality of degrading enzymes The subunit, the plurality of decomposing enzyme subunits have a natural ranking order according to their performance in an environment growth, wherein the polycistronic performance card includes:

(a) a promoter; and

(b) a polycistronic nucleotide sequence operably linked to the promoter, the polycistronic nucleotide sequence comprising a scaffold protein nucleotide sequence encoding the scaffold protein subunit and encoding the plural a plurality of degrading enzyme nucleotide sequences of the decomposing enzyme subunit, wherein the plurality of decomposing enzyme nucleotide sequences are sequentially arranged in the natural ranking order in the polycistronic nucleus relative to the position of the promoter In the nucleotide sequence, the ranking order of the performance of the plurality of decomposing enzyme subunits under the control of the promoter is consistent with the above natural ranking order.

The polycistronic expression cassette of the present invention has at least the following advantages: First, the nucleotide sequence encoding a plurality of cellulolytic enzyme complexes can be simultaneously introduced into a suitable host cell, thereby avoiding the consumption of individual nucleotide sequences. Time and technically arduous multiple selection process; secondly, because the position of the introduced multiple nucleotide sequences in the expression cassette is related to its expression, the nucleotide positions can be adjusted according to the design to adjust the nucleotide sequence. The relative amount of the corresponding product, in turn, obtains a cellulolytic enzyme complex which simulates the optimum ratio of hydrolase subunits contained in the cellulolytic enzyme complex of the natural microorganism to achieve optimal synergistic decomposition. Efficiency (also known as "biomimic").

In one embodiment of the invention, the scaffold protein subunit of the cellulolytic enzyme complex comprises one or more type I cohesive domains, and the plurality of decomposing enzyme subunits comprise a first type anchor A type I dockerin domain, wherein the first type of binding domain and the first type of anchoring domain can be combined with each other, and thus, the scaffold protein subunit can be complexed with a plurality of decomposing enzyme subunits. In one example, the scaffold protein subunit comprises nine first type of binding domains, ie, nine decomposing enzyme subunit binding sites (eg, CipA of Clostridium thermocellum) are provided.

In another embodiment of the invention, the cellulolytic enzyme complex may further comprise cell surface anchoring protein subunits, which may be immobilized on the cell surface. Typically, the cell surface anchoring protein subunit comprises one or more type II cohesive domains, and the scaffold protein further comprises a type II dockerin domain, wherein the second The type of binding domain and the second type of anchoring domain can be combined with each other. Therefore, the scaffold protein subunit and the plurality of decomposing enzyme subunits can be complexed with the cell surface anchoring protein subunit and immobilized on the cell surface. In one example, the cell surface anchoring protein subunit comprises one, two or seven second type of binding domains, ie, one, two or seven scaffold protein binding sites, respectively (eg, Clostridium thermocellum) SdbA, Orf2p and OlpB).

Thus, the polycistronic expression cassette of the present invention constructs a high order protein complex. For example, in a particular example, when the selected scaffold protein subunit comprises nine first type of binding domains (a binding site providing nine degrading enzyme subunits, such as CipA) and a cell surface anchoring protein subunit comprising seven A second type of binding domain (which provides a binding site for seven scaffold proteins, such as OlpB) provides a total of up to 63 binding enzyme subunit binding sites.

According to the present invention, a plurality of decomposing enzyme subunits have a natural ranking order according to their performance in an environmental growth, and in the polycistronic performance cassette of the present invention, a plurality of the plurality of decomposing enzyme subunits are encoded. The degrading enzyme nucleotide sequences are arranged in order according to the position of the promoter in the natural ranking order, such that the ranking order of the performance of the plurality of decomposing enzyme subunits under the control of the promoter is the natural ranking The order matches. More specifically, the plurality of degrading enzyme nucleotide sequences form an arrangement sequence according to the distance from the promoter, and the closer to the promoter gene sequence, the higher the expression amount, and the further the gene sequence from the promoter The lower the amount of performance, therefore, the order of the arrangement may be arbitrarily adjusted as needed, so that the ranking order of the corresponding plurality of decomposing enzyme subunits under the control of the promoter is in accordance with the natural ranking order, and the plurality of decomposing enzymes generated are generated. The secondary unit binds itself to the binding site provided by the scaffold protein subunit in a competitive manner to form a biomimetic cellulose hydrolase complex.

According to the present invention, we can design a cellulolytic enzyme complex in a host cell according to the cellulolytic enzyme complex of any natural microorganism (especially according to the natural ranking order of the amount of the decomposing enzyme subunit). The body is more cistern and positive. The cellulolytic enzyme complex according to the present invention may be derived from various microorganisms including, but not limited to, C. thermocellum , C. cellulovorans , C. cellulolyticum . , papyrus solution C. (C. papyrosolvens), Trichoderma longibrachiatum (Trichoderma. longibrachiatum), dissolved cellulose Bacteroides (Baceroides cellulosolvens), Vibrio cellulose acetate solution (Acetivibrio cellulolyticusas), N-type rumen bacteria fungi ( Neocallimastix frontalis ), rumen fungus Promyces spp , Bacillus megaterium , Bacillus licheniformis, Bacillus cellulosolvens , Ruminococcus flavefaciens , and defibrin Acetivibrio cellulolyticus .

In a preferred embodiment, the polycistronic expression cassette of the present invention is designed according to a cellulolytic enzyme complex derived from a thermophilic microorganism, and the various hydrolyzing enzyme complexes of the microorganism contain various degrading enzymes. The secondary unit has thermal stability. Therefore, the polycistronic expression of the cellulose hydrolase produced by the calf exhibits high enzyme activity at high temperatures, which is beneficial to the process of industrial biomass energy.

In a preferred embodiment, the thermophilic microorganism is Clostridium thermocellum. In a specific embodiment, the scaffold protein subunit is CipA.

In a specific embodiment of the present invention, the scaffold protein subunit CipA and the plurality of degrading enzyme subunits include exo-glucanase CelS and CelK, endoglucanase CelA, and polyxylase XynC and XynZ. In a specific example, the cleavage enzyme nucleotide sequence of the polycistronic nucleotide sequence encodes CelS, CelK, CelA, XynC, and Xynz in sequence. In yet another particular example, the polycistronic nucleotide sequence encodes CipA, CelS, CelK, CelA, XynC, and Xynz in sequence.

In another preferred embodiment, the polycistronic expression cassette of the present invention is designed according to a cellulolytic enzyme complex derived from Clostridium thermocellum, and the natural cellulolytic enzyme complex further comprises cell surface anchoring. Protein subunit. In a specific embodiment, the scaffold protein subunit is CipA. In yet another embodiment, the cell surface anchoring protein subunit is selected from the group consisting of OlpB, SdbA, and Orf2p. In another embodiment, the plurality of degrading enzyme subunits include exoglucanases CelS and CelK, endoglucanase CelR and CelA, and polyxylases XynC and XynZ.

In a specific example, the cleavage enzyme nucleotide sequence of the polycistronic nucleotide sequence encodes CelK, CelS, CelR, CelA, XynC, and XynZ in sequence; wherein, preferably, the cell surface anchor protein subunit Preferably, the SdbA, more preferably the polycistronic nucleotide sequence encodes CipA, CelK, CelS, CelR, SdbA, CelA, XynC and XynZ.

In another specific example, the degrading enzyme nucleotide sequence of the polycistronic nucleotide sequence encodes XynZ, XynC, CelA, CelK, CelR, and CelS in sequence; wherein, preferably, the cell surface anchor protein is The unit line SdbA, more preferably the polycistronic nucleotide sequence, encodes CipA, XynZ, XynC, CelA, SdbA, CelK, CelR, and CelS in sequence.

In yet another embodiment of the invention, the scaffold protein subunit is CipA, and the cell surface anchoring protein subunit is OlpB.

A promoter is a nucleotide sequence that contains elements that initiate transcription of an operably linked nucleic acid sequence. At the very least, the promoter contains an RNA polymerase binding site. It may further comprise one or more enhancer elements which by definition may enhance transcription or comprise one or more regulatory elements that control the on/off state of the promoter. The appropriate promoter selected for construction of this performance cassette is determined by the type of host cell into which the expression cassette is introduced. When using E. coli as a host cell, suitable promoters include, but are not limited to, beta-endoprostanase and lactose promoter systems (see Chang et al, Nature 275:615-624, 1978); SP6, T3 and T7 RNA polymerase promoter (Studier et al, Meth. Enzymol . 185: 60-89, 1990); lambda promoter (Elvin et al, Gene 87: 123-126, 1990); trp promoter (Nichols and Yanofsky) , Meth. in Enzymology 101: 155-164, 1983); tac and trc promoters (Russell et al, Gene 20: 231-243, 1982); and pCold (see U.S. Patent No. 6,479, 260). When Bacillus subtilis is selected as the host cell, exemplary promoters include the Pr promoter, the Spol promoter, the Tac promoter, and the LacI promoter. These promoters may also be used for other bacterial hosts, e.g., E. coli (Escherichia. Coli), Clostridium (Clostridium), mycoplasma (Mycoplasma), Lactococcus (Lactococcus), lactic acid bacteria (Lactobacillus), Vibrio ( Vibrio ) and Cyanobacteria . Promoters for yeast (eg, Saccharomyces cerevisiae) or other fungi (eg, Kluyveromyces, Pichia, Aspergillus, Trichoderma, and Candida) include the Lac4 promoter, the Adh4 promoter, the GapDH promoter, Adh1 promoter, Pgk promoter, Aac promoter, Pho5 promoter and Gal7 promoter.

In a preferred embodiment, the expression cassette is constructed using an inducible promoter. Such promoters are only activated under certain conditions, such as the presence of a particular compound (eg, IPTG or tetracycline) or at a particular temperature (eg, 40 ° C or above).

It is possible to confirm that the target gene and its position are contained in the expression cassette by a conventional method such as gene-specific PCR or restriction enzyme sequencing. This performance cassette is then introduced into a suitable host cell for the production of a protein complex. If desired, two or more of the performance cassettes described herein can be introduced into a host cell for expression of multiple proteins. If the target gene encodes a thermophilic enzyme, the host cell is preferably mesophilic. Positive selection strains can be identified by, for example, antibiotic resistance selection methods, and by determining the extent of expected enzyme activity. It can then be cultured under appropriate conditions to express the protein encoded by the gene and to combine the desired recombinant protein complex.

In a preferred embodiment, the expression cassette is constructed using an inducible promoter. Such promoters are only activated under certain conditions, such as the presence of a particular compound (eg, IPTG or tetracycline) or at a particular temperature (eg, 40 ° C or above).

The polycistronic expression cassette of the present invention can be incorporated into a vector and introduced into a suitable host cell for the production of a protein complex to produce a cellulolytic enzyme complex.

Thus, in another aspect, the invention provides a vector comprising the polycistronic expression cassette described above.

In still another aspect, the present invention provides a host cell comprising the aforementioned vector.

It is possible to confirm that the target gene and its position are contained in the expression cassette by a conventional method such as gene-specific PCR or restriction enzyme sequencing. This performance cassette is then introduced into a suitable host cell for the production of a protein complex. If the target gene encodes a thermophilic enzyme, the host cell is preferably mesophilic (eg, Bacillus subtilis). Positive selection strains can be identified by, for example, antibiotic resistance selection methods, and by determining the extent of expected enzyme activity. It can then be cultured under appropriate conditions to express the protein encoded by the gene and to combine the protein complex. Examples of host cells include, but are not limited to, Escherichia coli, Bacillus subtilis, Clostridium, Mycoplasma, Lactococcus, Lactobacillus, Vibrio, Blue-green algae, Yeast (eg, Saccharomyces cerevisiae) or other fungi (eg, gram Rubella, Pichia, Aspergillus, Trichoderma and Candida).

Accordingly, the present invention also provides a method of producing a cellulolytic enzyme complex in vivo comprising culturing said host cell in a condition exhibiting said cellulolytic enzyme complex to obtain said cellulolytic enzyme complex.

In one embodiment, the method of the invention further comprises the step of purifying the cellulolytic enzyme complex. Typically, since the scaffold protein has a carbohydrate-binding domain (CBM) that binds to the cellulose carbohydrate substrate, the purification step can be carried out using a cellulose adsorption method. On the other hand, since the cell surface anchoring protein can bind to the cells, a simple separation method such as centrifugation of the cell culture can be performed. In a specific example, the host cell culture can be mixed with cellulose and the cellulose pellets collected by centrifugation for purification purposes. The following examples illustrate implementation details.

Further, the present invention provides a method of decomposing a lignocellulosic biomass comprising contacting the cellulosic biomass with the aforementioned host cell. Typically, the lignocellulosic biomass comprises one or more components of cellulose, hemicellulose, and/or lignin.

In still another aspect, the present invention provides a plurality of degrading enzyme subunits by preparing the aforementioned vector and introducing it into a host cell and culturing in a suitable environment to formulate the performance between the plurality of decomposing enzyme subunits A method for blending the ratio of the content of the plurality of decomposing enzyme subunits on the cellulolytic enzyme complex. In particular, the method of the invention comprises:

(1) preparing a vector comprising a polycistronic expression cassette, the polycistronic expression cassette comprising:

(a) a promoter; and

(b) a polycistronic nucleotide sequence operably linked to the promoter, the polycistronic nucleotide sequence comprising a scaffold protein nucleotide sequence encoding a scaffold protein subunit and encoding the plurality of Decomposing a plurality of degrading enzyme nucleotide sequences of the enzyme subunit, wherein the plurality of decomposing enzyme nucleotide sequences are sequentially arranged in the polycistronic nucleotide sequence in a position relative to the position of the promoter, Having the ranks of the plurality of decomposing enzyme subunits under the control of the promoter are ranked in accordance with the order of the plurality of decomposing enzyme nucleotide sequences relative to the promoter; and

(2) introducing the vector into a host cell and culturing it in an appropriate environment to express the plurality of decomposing enzyme subunits, wherein the content ratio between the plurality of decomposing enzyme subunits is thus adjusted.

Detailed descriptions of various specific examples of the invention are given below. The technical features of the present invention will be more clearly apparent from the following detailed description of the specific embodiments and claims.

Example 1: Construction of the polycistronic expression card of the present invention

The KOD-Plus kit of Japan TOYOBO Co., Ltd. was used to separately amplify the scaffold protein CipA, exo-glucanase CelS and CelK, endoglucanase CelA, and polyxylase encoding Clostridium thermocellum by PCR. DNA fragments of cell body proteins such as XynC and XynZ; wherein CipA contains a cellulose binding domain (CBM), a surface layer homology module (SLH), and nine type I binding domains. The resulting PCR products were separately cloned into plastid pCR-XL-TOPO and introduced into E. coli host cells. DNA plastids were prepared from positive transformants using Qiagen plastid Midi kit (Qiagen, Calif.) and subjected to restriction enzyme digestion. After separation and purification by agarose gel electrophoresis, DNA fragments encoding the above proteins were obtained.

The above DNA fragment was ligated into the shuttle vector PGEST 118 of Escherichia coli and Bacillus subtilis using the "Ordered gene assembly in bacillus subtilis (OGAB)" proposed by Tsuge and Itaya et al. a heat-inducible Pr promoter and an increase in the number of copies of Bacillus subtilis induced by isopropylthio-β-D-galactoside (IPTG) ( Nucleic Acids Res. 31:e133 (2003)), The sequence of the gene designed is cipA-celS-celK-celA-xynC-xynZ. The sequence of these decomposing enzyme genes and the degrading enzyme produced by Clostridium thermocellum from the carbon source derived from crystallized cellulose (Avicel) The order of expression is consistent; the higher the expression of the gene closer to the promoter, and the decreasing amount of the gene away from the promoter, the sequence of the gene sequence designed here can simulate the thermofibromus The amount of performance of the crystallized cellulose (Avicel) derived from the produced enzyme is high.

The experimental method was to mix the DNA fragment (equal molar number) with the carrier, and then use the Takara binding kit Ver. 1, in 2 times concentrated buffer (132 mM Tris ± HCl (pH 7.6), 13.2 mM MgCl ₂ , 20 mM dithiothreitol, 0.2 mM ATP, 300 mM NaCl, 20% (w/v) polyethylene glycol 6000; Japan Wako Purification Co., Ltd., a bonding reaction was carried out at 16 ° C for 30 minutes, thereby producing Recombinant DNA molecule, wherein the expression cassette includes a promoter and a polycistronic nucleotide sequence encoding cipA-celS-celK-celA-xynC-xynZ, the sequence of which is shown in Figure 8 (SEQ ID NO: 1) .

Example 2: Screening of a transgenic strain containing the polycistronic expression cassette of the present invention

The polycistronic DNA fragment obtained in Example 1 was introduced into the B. subtilis mutant strains RM125 and BUSY9166. Briefly, a competent two-stage culture method was used to prepare competent Bacillus subtilis cells ( J. Bacteriol. 1961 81(5): 741-6 (1961)), and then an appropriate amount of polycistronic DNA fragments and 100 ml of competent subtilis The bacillus cells were mixed and incubated at 37 ° C for 30 minutes. 300 ml of LB medium was added to the mixture of the DNA and the cells, and then the cells were cultured at 37 ° C for 1 hour. The cultured cells were plated on LB plates containing tetracycline (10 mg/ml), and positive transformants were selected.

The anti-tetracycline-selected strain was cultured in a medium containing 1 mM isopropylthio-β-D-galactoside (IPTG) at 30 ° C for 5 hours and then at 42 ° C for 3 hours. The supernatant was then collected and concentrated by filtration using an Amicon filter (30 kDa threshold). The degree of fluorescence generated by UV irradiation in the filtrate was measured to determine the glucanase activity of the selected strain.

This test screened the selected strains 1 and 13, which exhibited glucanase activity. The results of gene-specific PCR analysis showed that the DNA from these plants could amplify the above six cell body-related genes, and the digestion analysis of restriction enzymes showed that the six cell-related genes were arranged in the order of design, ie cipA-celS-celK-celA-xynC-xynZ.

Example 3: Analysis of enzyme activity

The selected strains 1 and 13 obtained in Example 2, and the Bacillus subtilis containing the empty vector (control selection) were grown in LB medium supplemented with 12.5 μg/ml tetracycline at 30 ° C for 6 hours. The cells were then cultured at 42 ° C for 5 hours to induce the expression of cell body proteins. The cell culture was then centrifuged at 5,000 g for 10 minutes. The supernatant was collected and subjected to Viva Flow 50 (10 kDa threshold) (Sartorius, Goettingen, Germany) in the case of anti-displacement buffer (50 mM Tris, 10 mM CaCl ₂ and 5 mM DTT pH 6.8) at 4 Concentrate under °C. On the other hand, cell pellets were also collected, resuspended in PBS, and solubilized by ultrasonic oscillation (pulse: 3 seconds; stop: 2 seconds every 12 minutes), and then centrifuged at 13,200 rpm for 40 minutes to shift A sample containing intracellular proteins ("intracellular sample") is formed in addition to the particles. Alternatively, the cell pellet is collected and then resuspended in PBS to produce a sample containing intact cells. The protein content of the supernatant and the intracellular sample was measured by the Bradford method, and then the enzyme activities of the samples were analyzed by the following methods.

3.1 endoglucanase activity

Endoglucanase activity was determined using azurite cross-linking (AZCL)-β-glucan (dye CMC) (purchased from Megazyme) as a substrate. Briefly, 1% (v/w) dye CMC with 50 mM sodium acetate was incubated with the above supernatant sample and intracellular sample at 60 ° C for 3 hours, respectively, and then the absorbance at 590 nm of each sample was measured (OD _590). Value), which is related to the activity intensity of the glucanase. Figures 2(a) and (b) show the activity and specific activity of the endoglucanase of the sample, respectively.

The results showed that the supernatant samples of the selected strain 1 and the selected strain 13 had more than 2 times the endoglucanase activity compared to the supernatant sample of the control colony; Intracellular samples of the colonies, and intracellular samples of the selected strains 1 and 13 were also measured for significantly enhanced endoglucanase activity, see Figure 2(a). In addition, the intracellular protein content of the selected strains 1 and 13 was significantly higher than that of the control colonies, indicating that a certain amount of exogenous protein was retained in the cells, which was compared with the specific activity shown in Fig. 2(b). The results of the enzyme activity normalizing the total protein content were consistent.

3.2 Total glucanase activity

To test the activity of total glucanase, the supernatant and intracellular samples were incubated at 60 ° C in 50 mM sodium acetate buffer (pH 5.0) at a final concentration of 1 mg/ml with 4-methylumbelliferone-β. -d-cellulose diglucoside (MUC) was mixed for 3 hours. The enzyme activity is measured by fluorescence assay at 365 nm UV irradiation in 1% NaCO _3. Figures 2(c) and (d) show the activity and specific activity of the total glucanase of the sample, respectively.

The results showed that the glucanase activity observed from the supernatants of the selected strains 1 and 13 was much higher than that observed from the supernatant from the control colonies, see Figure 2(c); A certain amount of exogenous protein was found in the cells, see Figure 2(d).

Example 4: Detection of the formation of cellulose hydrolase complex

The formation of the cellulolytic enzyme complex in the selected strains 1 and 13 was examined by SDS-PAGE. First, an extracellular protein sample (not boiled) from the two selected strains was subjected to electrophoresis analysis on a 5-15% (w/v) polyacrylamide gel containing 0.1% SDS. The polyacrylamide gel was then placed on an agar gel containing xylan or CMC and the bubbles between the two gels were removed. The two gels were wrapped with a plastic film and incubated at 40 ° C for 3 hours (if the agar gel contained CMC) or overnight at 60 ° C (if the agar gel contained xylan). Thereafter, the agar gel was separated from the polyacrylamide gel, fixed, and subjected to protein fluorescent dye (sypro ruby) protein staining. The agar gel was soaked in 1 mg/ml Congo red for 30-60 minutes and then soaked in 1 M NaCl for 10-60 minutes. The position where the CMC or xylan matrix is decomposed on the agar gel is yellow, and the position where no decomposition occurs is dark red. Polyacrylamide gel was also incubated in a solution (pH 5.0) containing 0.2 mg/ml of MUC and 50 mM NaOAc, and incubated at 60 ° C for 30 minutes to determine glucanase activity. The degree of decomposition of the MUC was detected by examining the luminescence at 365 nm on the gel.

The results obtained from the above experiments showed that the selected strains 1 and 13 exhibited the activity of the extracellular endoglucanase, xylan and total glucanase.

The protein on the above polyacrylamide gel was as described in Matsudaira, J. Biol. Chem . 262: 10035~10038 (1987) or Salinovich and Montelaro, Anal. Biochem . 156:341-347 (1986). Transfer to polyvinylidene chloride film (GE). The membrane was blocked with PBS containing 5% skim milk, washed, and then cultured with an anti-rCipA antibody (1:5000-fold dilution) at 4 ° C for 16 hours. After several washes, the film was co-cultured with HRP-conjugated goat anti-rabbit IgG (1:5000 dilution). After washing with PBS (pH 7.4), the film was incubated with a solution containing NBT/BCIP to form a signal. The results showed that it was observed that the position of the CipA protein in the polyacrylamide gel overlaps with the above-described positions exhibiting the activities of various decomposing enzymes.

The protein located at a position exhibiting the activity of the degrading enzyme was extracted from the gel, and then analyzed by a 5-15% (w/v) polypropylene guanamine gel and two-dimensional DIGE gel electrophoresis. In this analysis, it was found that each peptide was highly similar to the fragments of CelA, CelK, CelS, CipA and XynZ proteins, indicating that these proteins form a protein complex and exhibit the desired degrading enzyme activity.

Example 5: Determination of Enzyme Thermal Stability

According to the above method, CMC and MUC were used as the test substance for the control strain and the strain 1 for extracellular decomposition enzyme activity at different temperatures. Figure 3 shows the results.

The results showed that the selected strain 1 showed higher decomposing enzyme activity above 50 °C and greater difference at higher temperature than the control colony, see Fig. 3(a). In addition, the selected strains had similar extracellular protein content at each test temperature, see Fig. 3(b), indicating that the selected strain 1 has elevated degrading enzyme activity at high temperature due to the degrading enzyme expressed by the selected strain 1. It has thermal stability.

Example 6: Other examples 6.1 Construction of other polycistronic expression cassettes and selection of transgenic plants

According to the foregoing bionic strategy, the order of the performance of the decomposition enzymes of the cellulolytic enzyme complex is predicted by Clostridium thermocellum in response to different carbon sources, and other examples of the polycistronic expression of the present invention are designed, wherein the mode I The polycistronic expression card is based on microcrystalline cellulose (avicel) as the carbon source, and its genetic sequence is cipA-celK-celS-celR-sdbA-celA-xynC-xynZ; The sub-expression cassette is designed based on cellobiose as a carbon source, and its genetic sequence is cipA-xynZ-xynC-celA-sdbA-celK-celR-celS.

The experimental method is the same as described above. First, DNA fragments encoding cellulosic proteins such as CipA, CelS, CelK, CelA, CelR, XynC, XynZ and sdbA were cloned into the Topo vector system and subjected to restriction enzyme map analysis, sequence verification and colloidal extraction. purification. Then, the aforementioned DNA fragment encoding each cell body protein was ligated to the shuttle vector pGETS 188 in the designed sequence using the OGAB method. The ligation product was introduced into Bacillus subtilis mutant BUSY9797 (strain of CI inhibitor mutation), and a positive selection plant having tetracycline resistance was selected under the culture condition of 37 °C. By restricting the digestion analysis of the enzyme, it was confirmed that the recombinant vector produced by the positive selection plant had the desired polycistronic expression cassette, and the polycistronic expression of the pattern I showed that the gene sequence of the cassette was cipA-celK-celS-celR- sdbA-celA-xynC-xynZ (Fig. 4(a)), the sequence of which is shown in Figure 9 (SEQ ID NO: 2), and the polycistronic expression of the pattern II is cipA-xynZ-xynC - celA-sdbA-celK-celR-celS (Fig. 4(b)), the sequence of which is shown in Figure 10 (SEQ ID NO: 3).

6.2 Gene transcription analysis

Total RNA was isolated from the bacilli after 20 hours of culture using the RNeasy Protect Bacteria kit (High Purity RNA Isolation Kit, Roche). Use the reverse transcription kit (iScript cDNA synthesis kit, BioRad) and the real-time quantitative SYBR green IRT-PCR kit (Roche 480 SYBR green I Master, Roche) and the gene-specific primer set (the size of the amplified product is 113 to 137 bp; see below RT-PCR was performed on a LightCycler (LightCycler 480, Roche) instrument with absolute quantitative analysis of RNA.

The results showed that among the B. subtilis strains of Mode I and Mode II, the largest transcript of the eight genes was from the gene located immediately downstream of the promoter, and the number of copies of the remaining transcripts was between the promoter and the promoter. The distance is increased proportionally; it indicates that the eight genes can be successfully transcribed by a single Pr promoter, and the order of the expression of each gene has the same tendency as the position order of these genes in the recombinant plasmid.

6.3 Formation analysis of cellulose hydrolase complex

To determine whether the gene of the cellulolytic enzyme complex of Clostridium thermocellum can be expressed in a heterologous host (Bacillus subtilis) and combined into a cellulolytic enzyme complex, the test is carried out from a Bacillus subtilis culture by cellulose adsorption. The protein was purified and subjected to electrophoretic analysis. Briefly, Bacillus subtilis strains of culture mode I and mode II were obtained to obtain bacterial cultures, which were centrifuged at 3500 g for 20 minutes at 4 °C. A 2 ml supernatant sample was mixed with 4 mg of crystalline cellulose and formulated into a final volume of 4 ml of 50 mM phosphate buffer (pH 7). After incubating for 1 hour at 4 ° C, the cellulose was collected by centrifugation and washed twice with phosphate buffer. The protein adsorbed on the cellulose pellet was dissolved in 100 ml of sodium dodecylsulfate-polyacrylamide colloidal electrophoresis (SDS-PAGE) loading buffer, boiled for 10 minutes, and subjected to SDS-PAGE analysis. . Figure 5 shows the results.

The results showed that the B. subtilis strains of model I and model II exhibited molecular bands of molecular weights of 100.6, 92.2, 83.5, 82, 69.5, and 52.5 kD, corresponding to CelK, XynZ, CelS, CelR, XynC and CelA, respectively. The B. subtilis strains of I and II were induced to exhibit various hydrolases and combined with the scaffold protein CipA having a cellulose-binding region (CBM), so that they could be purified by cellulose adsorption.

6.4 Analysis of zymogram electrophoresis of cellulose hydrolase complex

Add cultures of Mode I and Mode II Bacillus subtilis strains to buffer containing CaCl ₂ and low DTT concentration, without boiling and without adding reducing agent, then perform 5-15% gradient SDS colloidal electrophoresis Analysis, in which each protein molecule can be separated by the force of electrophoresis due to its molecular weight and inherent charge. Figure 6(d) shows the staining results of Commassie Blue Staining Solution.

After completion of electrophoresis, a zymogram assay with poly-xylose and CMC as a substrate was performed, followed by staining with Congo red, and the region degraded by polyxylase and endoglucanase was dark-stained. A transparent ribbon appears in the background; Figures 6(b) and 6(c) show the results. For glucanase activity analysis, the colloid was immersed in a buffer containing MUC, followed by fluorescence measurement by a 365 nm UV light source; Figure 6 (a) shows the results.

The results showed that the secreted proteins of the model I and model II Bacillus subtilis strains exhibited significant effects on MUC, polyxylose and CMC compared to the strains containing only pGETS (control group). Decompose enzyme activity. In addition, the position of the transparent band of the colloidal electrophoresis colloid of MUC, polyxylose and CMC has a smear area to present various glucanases to MUC, polyxylose and CMC. active. These results are one of the evidences showing that each hydrolyzing enzyme-producing protein produced by the B. subtilis strains of Mode I and Mode II can interact with the scaffold protein CipA to form a complex.

6.5 Analysis of Decomposition Enzyme Activity of Cellulose Hydrolase Complex

The Bacillus subtilis strains of the model I and the model II were cultured and induced at 42 ° C for 20 hours. The supernatant and intracellular material of the culture of the Bacillus subtilis strain were collected using a buffer containing CaCl ₂ and a low DTT concentration, and then treated with MUC, crystalline cellulose, dye CMC, and polyxylose. Various decomposition enzyme activity analyses were performed. Figure 7 shows the results.

The results showed that the supernatant of the culture of the B. subtilis strains of the model I and the model II exhibited a considerably higher decomposing enzyme activity than the control group with only the pGETS-plastid. In particular, when the substrate is crystalline cellulose, the activity of exoglucanase in the pattern I and mode II of the B. subtilis strain is significantly improved, and the activity of the type I strain is more than the type II selection. The activity of the colonies was higher (Fig. 8b); and when the substrate was polyxylose, the supernatant of the culture of the model B subtilis strain showed the highest degrading enzyme activity. The two selected strains exhibit different rankings of the decomposition enzyme activities, and the order of the nucleotide sequences of the two selected strains is different from each other.

In summary, the present invention is the first to successfully construct a polycistronic expression cassette which can mimic the natural microbial cellulolytic enzyme complex in a host cell, which can arbitrarily select a protein subunit type of a desired cellulolytic enzyme complex. And by adjusting the order of the corresponding nucleotide sequences, the relative expression amount of each protein subunit is controlled to achieve the purpose of simulating natural microorganisms, and is applicable to the development of biomass energy. In addition, according to the present invention, the cellulolytic enzyme complex can be immobilized on the cell surface through a cell surface anchoring protein subunit, and the whole cell can be regarded as an immobilized enzyme cell, which is advantageous for purification and subsequent application. In addition, when a thermostable decomposition enzyme is selected to design a polycistronic expression cassette, a thermally stable cellulose hydrolase complex can be produced, which is more advantageous for industrial applications of biomass energy. The platform technology of the present invention breaks through the bottleneck of the prior art, and has made considerable contributions to the research of cellulose hydrolase complex and the development of biomass energy.

Without further elaboration, it is believed that those of ordinary skill in the art of Therefore, it is to be understood that the following description is for illustrative purposes only and is not intended to limit the disclosure.

<110> Academia Sinica

<120> Multi-cistronic expression of cellulose hydrolase complex and its application

<130> ACA0050TW

<150> Republic of China invention patent application No. 098143083

<151> 2009-12-16

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 16446

<212> DNA

<213> Artificial sequence

<220>

<223> Recombination sequence

<400> 1

<210> 2

<211> 20869

<212> DNA

<213> Artificial sequence

<220>

<223> Recombination sequence

<400> 2

<210> 3

<211> 20869

<212> DNA

<213> Artificial sequence

<220>

<223> Recombination sequence

<400> 3

Figure 1 is a schematic view showing the structure of a cellulolytic enzyme complex of Clostridium thermocellum.

Figure 2 is a graph showing the extracellular and intracellular cellulolytic activity of a cellulolytic enzyme complex containing Clostridium thermocellum CipA, CelS, CelK, CelA, XynC and XynZ proteins in Bacillus subtilis host cells, wherein (a ) shows the endoglucanase activity measured using CMC as a substrate; (b) shows the endoglucanase specific activity, by normalizing the protein content by the endoglucanase activity in the sample Determination; (c) shows total glucanase activity measured using MUC as a substrate; and (d) shows total glucanase specific activity, by total endoglucanase activity in the sample for its protein content Determined by normalization.

Figure 3 shows the thermal stability of a glucanase producing a cellulolytic enzyme complex containing Clostridium thermocellum CipA, CelS, CelK, CelA, XynC and XynZ proteins in Bacillus subtilis, wherein (a) shows a control colony And the glucanase activity of the selected strain 1 at different temperatures; and (b) the glucanase activity and the protein content of the control colony and the selected strain 1 at different temperatures.

Figure 4 shows the nucleotide sequence of the polycistronic expression cassette of Example 6.1, wherein (a) shows mode I, and (b) shows mode II.

Figure 5 shows the results of electrophoretic analysis of Example 6.3, where the first lane is the control sample, the second lane is the model I sample, and the second lane is the model II sample.

Figure 6 shows the results of zymography analysis of the cellulolytic enzyme complex of Example 6.4, wherein (a) shows the position of glucanase degradation; (b) shows the position of endoglucanase degradation; (c) shows The location of polyxylase degradation; and (d) the staining results of Coomassie Brilliant Blue; the first lane is the control sample, the second lane is the model I sample, and the second lane is the model II sample.

Figure 7 shows the results of the decomposition enzyme activity analysis of Example 6.5, wherein (a) the MUC specific activity result; (b) the exo-glucanase specific activity result; (c) the endoglucanase specific activity result; (d) shows the results of the specific activity of the xylase.

Figures 8-1 to 8-5 show the nucleotide sequence (SEQ ID NO: 1) of cipA-celS-celK-celA-xynC-xynZ contained in the polycistronic expression cassette of Example 1, wherein the italic portion Representing the restriction enzyme region, the bottom line portion representing each protein coding region, and the shaded portion representing the ribosome binding site.

Figures 9-1 to 9-7 show the nucleotide sequence of cipA-celK-celS-celR-sdbA-celA-xynC-xynZ contained in the polycistronic expression cassette of the mode I described in Example 6.1 (SEQ ID NO: 2), wherein the italic portion represents the restriction enzyme region, the bottom line portion represents each protein coding region, and the shaded portion represents the ribosome binding site.

Figures 10-1 to 10-7 show the nucleotide sequence of cipA-xynZ-xynC-celA-sdbA-celK-celR-celS contained in the polycistronic expression cassette of the model II described in Example 6.1 (SEQ ID NO: 3), wherein the italic portion represents the restriction enzyme region, the bottom line portion represents each protein coding region, and the shaded portion represents the ribosome binding site.

Claims (33)

A polycistronic expression cassette for producing a native microbial cellulolytic enzyme complex in a host cell, the cellulolytic enzyme complex comprising scaffoldin subunits and two Or two or more plurality of enzymatic subunits, wherein the plurality of decomposing enzyme subunits have a natural ranking order according to their performance in an environmental growth, wherein the polycistronic performance card includes (a) a single promoter; and (b) a polycistronic nucleotide sequence operably linked to the single promoter, the polycistronic nucleotide sequence comprising a scaffold encoding the scaffold protein subunit a protein nucleotide sequence and a plurality of degrading enzyme nucleotide sequences encoding the plurality of decomposing enzyme subunits, wherein the plurality of degrading enzyme nucleotide sequences are in a position relative to the promoter in the natural ranking order Arranging sequentially in the polycistronic nucleotide sequence such that the order of the performance of the plurality of decomposing enzyme subunits under the control of the promoter is the same as the above natural ranking The order matches. The polycistronic expression cassette of claim 1, wherein the scaffold protein subunit comprises one or more type I cohesive domains, and the plurality of decomposing enzyme subunits include the first Type I dockerin domain, wherein the first type of bonding domain and the first type of anchoring domain can be combined with each other. The polycistronic expression complex of claim 1, wherein the natural cellulolytic enzyme complex further comprises cell surface anchoring protein subunits, and the polycistronic nucleoside The acid sequence includes a cell surface anchoring protein nucleotide sequence encoding the cell surface anchoring protein subunit. For example, the multi-cistronic performance card of the third paragraph of the patent application scope, The cell surface anchoring protein subunit comprises one or more type II cohesive domains, and the scaffold protein further comprises a type II dockerin domain, wherein the second type of binding domain The second type of anchoring domains can be combined with each other. For example, the polycistronic expression cassette of claim 1 is selected from the group consisting of Clostridium thermocellum , C. cellulovorans , and C. cellulolyticum . , C. papyrosolvens , Trichoderma . longibrachiatum, Baceroides cellulosolvens , Acetivibrio cellulolyticusas , rumen fungus N-type bacteria Neocallimastix frontalis ), rumen fungus Promyces spp , Bacillus megaterium , Bacillus licheniformis, Bacillus cellulosolvens , Ruminococcus flavefaciens , and defibrin One of the groups consisting of Acetivibrio cellulolyticus . For example, the polycistronic performance card of the fifth application of the patent scope is the microorganism, which is Clostridium thermocellum. For example, the multi-cistronic expression of the sixth paragraph of the patent application range, wherein the scaffold protein subunit is CipA. For example, the multi-cistronic expression cassette of claim 7 includes the exoglucanases CelS and CelK, and the enoglucanases CelA. And xylase (xylanases) XynC and XynZ. The polycistronic expression cassette of claim 8 wherein the degrading enzyme nucleotide sequence encodes CelS, CelK, CelA, XynC and Xynz in sequence. The polycistronic nucleotide sequence of claim 9 is wherein the polycistronic nucleotide sequence encodes CipA, CelS, CelK, CelA, XynC and Xynz in sequence. For example, the multi-cistronic performance card of the third application patent scope, the micro The organism is Clostridium thermocellum. For example, the multi-cistronic expression of the 11th article of the patent application range, wherein the scaffold protein subunit is CipA. The polycistronic expression cassette of claim 12, wherein the cell surface anchoring protein subunit is selected from the group consisting of OlpB, SdbA and Orf2p. For example, the polycistronic expression cassette of claim 13 wherein the plurality of degrading enzyme subunits include exo-glucanase CelS and CelK, endoglucanase CelR and CelA, and polyxylase XynC and XynZ. For example, the polycistronic expression cassette of claim 14 wherein the degrading enzyme nucleotide sequence encodes CelK, CelS, CelR, CelA, XynC and XynZ in sequence. For example, the polycistronic expression of the 15th article of the patent application range, wherein the cell surface anchor protein subunit is SdbA. The polycistronic nucleotide sequence of claim 16 of the patent application, wherein the polycistronic nucleotide sequence encodes CipA, CelK, CelS, CelR, SdbA, CelA, XynC and XynZ in sequence. For example, the polycistronic expression cassette of claim 14 wherein the degrading enzyme nucleotide sequence encodes XynZ, XynC, CelA, CelK, CelR and CelS in sequence. For example, the multi-cistronic expression of the 18th item of the patent application range, wherein the cell surface anchor protein subunit is SdbA. The polycistronic nucleotide sequence of claim 19, wherein the polycistronic nucleotide sequence encodes CipA, XynZ, XynC, CelA, SdbA, CelK, CelR, and CelS in sequence. For example, the polycistronic expression cassette of claim 13 of the patent scope, wherein the cell surface anchor protein subunit is OlpB. The polycistronic expression cassette of claim 6 is wherein the promoter is a heat-inducible promoter. A vector comprising the polycistronic expression cassette of any one of claims 1 to 22. A host cell comprising the vector of claim 23 of the patent application. The host cell of claim 24 of the patent application is mesophilic. A host cell as claimed in claim 24, which is B. subtilis . A method for producing a cellulolytic enzyme complex in vivo, which comprises culturing a host cell according to claim 24 of the patent application under conditions capable of expressing the cellulolytic enzyme complex to obtain the cellulolytic enzyme complex body. The method of claim 27, wherein the host cell is B. subtilis . The method of claim 27, further comprising the step of purifying the cellulolytic enzyme complex. The method of claim 29, wherein the step is achieved using a cellulose adsorption method. The method of claim 29, wherein the step is achieved using a separation method. A method of decomposing a lignocellulosic biomass, comprising contacting a lignocellulosic biomass with a host cell as in claim 24 of the patent application. A method for formulating a content ratio between two or more plural decomposition enzyme subunits of a cellulose hydrolase complex, comprising: (1) preparing a carrier comprising a polycistronic expression card The polycistronic expression cassette comprises: (a) a single promoter; and (b) a polycistronic nucleotide sequence operably linked to the single promoter, the polycistronic nucleoside The acid sequence includes a scaffold protein nucleotide sequence encoding a scaffold protein subunit and a plurality of degrading enzyme nucleotide sequences encoding the plurality of decomposing enzyme subunits, wherein the plurality of decomposing enzyme nucleotide sequences are relative Positioning the promoter in the order of the polycistronic nucleotide sequence, such that the plurality of decomposition enzyme subunits are ranked under the control of the promoter and the plurality of the foregoing The degrading enzyme nucleotide sequence is in accordance with the positional order of the promoter; and (2) introducing the vector into a host cell and culturing in an appropriate environment to express the plurality of decomposing enzyme subunits, wherein the complex number is formulated The amount of performance between the subunits of the decomposing enzymes is such that the content ratio between the plurality of decomposing enzyme subunits on the cellulolytic enzyme complex is thus adjusted.