CN109694861B - Artificial cellulose body, method for decomposing cellulose and producing alcohol by using artificial cellulose body - Google Patents

Abstract

The present invention provides a method for decomposing cellulose, comprising: creating a recombinant phage by modifying the genome of the phage such that the surface of the phage expresses: a binding peptide having an affinity for a lytic enzyme, and an anchor peptide having an affinity for an anchor site on the surface of a cell; contacting the recombinant phage with a lytic enzyme; and contacting the cellulose with the lytic enzyme.

Description

Artificial cellulose body, method for decomposing cellulose and producing alcohol by using artificial cellulose body

Technical Field

The present invention relates to a method for decomposing cellulose, and more particularly, to a method for decomposing cellulose using a cellulosome having a phage scaffold and a method for producing alcohol using the same.

Background

In the conventional biomass alcohol process, a Combined Bioconversion Process (CBP) is the most excellent process at present, and by integrating all reactions in the production process in the same strain (for example, Saccharomyces cerevisiae), the production cost and demand can be greatly reduced. Among them, the cellulosome found in anaerobic clostridium thermocellum (c. thermocellum) is considered as one of the key technologies in the development of CBP program.

The cellulosome (cellulosome) mainly takes a protein scaffold as a main body, and combines a plurality of cellulolytic enzymes with the scaffold so as to integrally form a complex with a plurality of decomposition functions, thereby not only overcoming the difficulty that the expression of the cellulolytic enzymes on the cell surface is limited through the prior transgenic technology, but also being proved that the complex has higher enzyme activity compared with the same amount of the cellulolytic enzymes. However, the structure of the cellulosome is very complex, and the existence of the cellulosome is only found in anaerobic bacteria with insufficient fermentation productivity in nature, so that the artificial synthesis of the cellulosome and the expression of the cellulosome on the surface of saccharomyces cerevisiae with high fermentation productivity through a cell surface expression technology are the most important development trend at present.

The single cellulosome found in nature can link about 96 cellulolytic enzymes, while the synthetic cellulosome described in the prior art can link about 63 cellulolytic enzymes, and there is still a difference in productivity compared to the natural cellulosome.

Disclosure of Invention

In view of the above disadvantages, the present invention provides a cellulosome using recombinant phage as protein scaffold, which can link hundreds to thousands of cellulolytic enzymes on the surface of a single phage by self-assembly and anchor them on the surface of saccharomyces cerevisiae, not only overcoming the problem of the present decomposition productivity, but also obtaining an artificial cellulosome capable of high-speed production by the extremely short replication cycle of phage itself, and providing a highly efficient cellulolytic platform.

For the above reasons, the present invention provides a method for decomposing cellulose, comprising: creating a recombinant bacteriophage, wherein the recombinant bacteriophage is obtained by modifying the genome of the bacteriophage to express on the surface of the bacteriophage: a linker peptide having an affinity for a lytic enzyme, and an anchor peptide having an affinity for an anchor site on a cell surface; contacting the recombinant phage with a lytic enzyme; and contacting the cellulose with the lytic enzyme.

Preferably, the adaptor peptide is linked to 1 to 3000 enzymes.

Preferably, the lytic enzyme is an endo-cellulolytic enzyme, an exo-cellulolytic enzyme, a cellobiohydrolase, or a combination thereof.

Preferably, the lytic enzyme expresses SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or a combination thereof.

Preferably, the anchor position is expressed as SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or a combination thereof and is not repeated with the sequence expressed by the aforementioned lytic enzyme.

Preferably, the bacteriophage is selected from lambda phage and filamentous phage.

Preferably, the cells are selected from yeasts.

The present invention also provides a method for producing biomass alcohol using the cellulose decomposition method provided above, comprising: creating a recombinant bacteriophage, wherein the bacteriophage comprises a binding peptide and an anchor peptide; conjugating a lytic enzyme to the conjugating peptide; anchoring the anchoring peptide to an anchoring position on the surface of the yeast; decomposing cellulose with a decomposing enzyme to obtain a saccharide; and fermenting the sugars with yeast to obtain biomass alcohol.

The present invention further provides an artificial cellulosome used in the method for decomposing cellulose and the method for producing biomass alcohol, comprising: a recombinant phage expressing on its surface a conjugate peptide having affinity for a lytic enzyme; and an anchor peptide having affinity with an anchor site on the surface of the yeast; and a peptide-conjugated cellulolytic enzyme conjugated to the surface of the recombinant phage.

In summary, the present invention provides artificial cellulosomes having a phage scaffold and an enzyme, which have higher cellulolytic activity compared to the unassociated enzyme. Therefore, the present invention also provides a method for efficiently decomposing cellulose using the artificial cellulosome, and use of the method for efficiently producing biomass alcohol.

Drawings

FIG. 1 is a flow chart of an embodiment of the present invention.

FIG. 2 is a schematic structural diagram of a recombinant bacteriophage according to an embodiment of the present invention.

FIG. 3 is a schematic diagram of the anchoring of recombinant phage to the surface of yeast according to one embodiment of the present invention.

FIG. 4 is a schematic view of a biological reaction system according to an embodiment of the present invention.

FIGS. 5 and 6 are fluorescence micrographs of an embodiment of the invention.

FIGS. 7A to 7C are graphs comparing the activity of enzymes according to one embodiment of the present invention.

[ notation ] to show

S11, S12, S13, S20, S30: step (ii) of

RP-BGL-SH 3: cellobiase conjugated to recombinant phage surface and having SH3 domain

BGL-SH 3: cellobiase having SH3 domain

RP-celA-SH 3: endonuclear cellulolytic enzyme having SH3 domain and attached to recombinant phage surface

celA-SH 3: endonuclear cellulolytic enzyme having SH3 domain

RP-EC-SH 3: exo-cellulolytic enzyme having SH3 domain and attached to recombinant phage surface

EC-SH 3: exo-cellulolytic enzymes having SH3 domain

Detailed Description

In the prior art, the artificial cellulosome is usually prepared by directly bonding the lytic enzyme to the protein scaffold to mimic the structure of natural cellulosome, however, the amount of the bondable enzyme cannot reach the amount equivalent to that of the natural cellulosome, resulting in the less than expected decomposition capability. In the invention, the protein scaffold is jointed with the peptide with smaller molecular weight, and then the peptide is connected with the lytic enzyme, thereby overcoming the steric hindrance and other adverse factors and leading a single protein scaffold to be jointed with a large amount of lytic enzyme. In addition, the inventor selects phage as the stem to be the protein scaffold of the artificial fibrosome, and expresses a large amount of peptide on the surface of the phage by the gene recombination technology, thereby increasing the bonding amount of the degrading enzyme.

In the present invention, the artificial fibrosomes can be established using recombinant yeast and recombinant phage. The establishment mode comprises that the recombinant yeast expresses the anchoring structure domain; and expressing the surface of the protein outer membrane of the recombinant phage with a conjugating peptide with affinity with the cellulase, and expressing the anchoring peptide corresponding to the anchoring structure domain expressed by the recombinant yeast on the surface of the tail end of the recombinant phage; and a large amount of cellulase is produced by E.coli. The recombinant phage, the recombinant yeast and the cellulase are co-cultured, so that the cellulase is combined with the conjugated peptide expressed on the surface of the recombinant phage to form an artificial cellulosome, and meanwhile, the recombinant phage can be fixed on the surface of the recombinant yeast through the affinity of the anchoring peptide and the anchoring structure domain of the recombinant yeast. In this way, when the cellulose in the biomass raw material contacts the artificial cellulosome, the artificial cellulosome can be efficiently decomposed by the high-density cellulase, and the hexose generated by decomposition can be directly used for fermentation reaction by the anchored recombinant yeast to produce the biomass alcohol.

In order to make the above objects, technical features and gains obvious and understandable after actual implementation, preferred embodiments will be described in more detail with reference to the accompanying drawings.

In one embodiment of the present invention, the process is shown in FIG. 1.

In step S11, recombinant phages are established and produced rapidly and in large quantities by e. Recombinant phage RP are created by modifying the genome of a phage such that the surface of the phage expresses a adaptor peptide with affinity for a particular enzyme and an anchor peptide with affinity for an anchor site on the surface of a particular cell. In particular, the bacteriophage may be lambda and filamentous bacteriophage, preferably filamentous bacteriophage, such as M13, f1, fd phage or other species suitable for use in known phage display (phagedisplay) techniques, the conjugating peptide may be any ligand having affinity for the selected cellulolytic enzyme, and the anchoring peptide may be any ligand having affinity for the anchor site expressed by the recombinant yeast.

In step S12, recombinant yeast is established and produced rapidly and in large quantities by E.coli. Recombinant yeast is created by modifying the genome of the yeast to anchor the surface expression of the yeast. In particular, the yeast may be any yeast commonly used in alcoholic fermentation, for example: yeasts of the genera Schizosaccharomyces, Saccharomyces, Hanseniaspora, and the like; the anchoring site may be any anchoring domain having affinity corresponding to the anchoring peptide of the recombinant bacteriophage of the present invention. In addition, the recombinant yeast can express the five-carbon sugar degrading enzyme according to the requirement, so that the recombinant yeast has the capability of degrading the five-carbon sugar.

In step S13, recombinant escherichia coli is established, and a large amount of the enzyme to be replicated is secreted and expressed by escherichia coli, for example: a cellulolytic enzyme. In the case of replicating cellulolytic enzymes, recombinant E.coli is constructed by modifying the genome of E.coli so that it synthesizes and secretes cellulolytic enzymes extracellularly, and expressing the corresponding domain of synthesized cellulolytic enzymes with affinity for the recombinant phage-expressed adaptor peptides so that it has affinity for the adaptor peptides. Coli itself has a characteristic of rapid division, and thus, it is possible to produce a large amount of cellulolytic enzymes in a short time. In the method for producing biomass alcohol according to the present invention, the cellulolytic enzyme may be an endo-cellulolytic enzyme, an exo-cellulolytic enzyme, or a cellobiohydrolase, and preferably may be an endo-cellulolytic enzyme (celA), a cellobiohydrolase (BGL), an exo-cellulolytic Enzyme (EC), or other enzymes having similar functions. In addition, a single group of recombinant escherichia coli may be established to secrete and express the endo-cellulolytic enzyme, the exo-cellulolytic enzyme or the cellobiohydrolase simultaneously, or three groups of recombinant escherichia coli may be established to express the endo-cellulolytic enzyme, the exo-cellulolytic enzyme or the cellobiohydrolase respectively. Preferably, the cellulolytic enzyme has affinity to the recombinant phage-expressed adaptor peptide of the invention.

In step S20, the recombinant bacteriophage, the recombinant yeast, and the recombinant escherichia coli are co-cultured to form a biological reaction system. The biological reaction system can be any environment which can ensure that the recombinant phage, the recombinant yeast and the recombinant escherichia coli coexist and continuously grow, and biomass raw materials are added into the biological reaction system to produce and extract the biomass alcohol.

In step S30, a biomass feedstock is added to the bioreactor system. In particular, the biomass feedstock may be any feedstock having cellulose, such as: grain raw materials such as barley, wheat, oat, rice, beet, sweet sorghum, cassava, and sweet potato; or non-food feedstocks such as: wheat straw, rice straw, corn straw, and the like; or agricultural, municipal and construction waste, such as kitchen waste, newspaper, wood chips, waste wood, and the like; or fast-growing cellulosic crops such as miscanthus, pennisetum, switchgrass; or easily harvested raw materials such as seaweeds and the like.

The present invention will be described below with reference to the above steps S11-S13, S20 and S30 in an exemplary embodiment.

Corresponding to step S11, recombinant M13 phage was created that surface-expressed the adaptor and anchor peptides. For convenience of description, the following steps are described by taking the engaging peptide as SH3 ligand and the anchoring peptide as PDZ ligand, but the actual implementation is not limited thereto. Among the genes of M13 phage, the gene of SH3 ligand (SEQ ID NO:2) was inserted in the gene sequence of PVIII protein, and the gene of PDZ ligand (SEQ ID NO:1) was inserted in the gene sequence of PIII protein. In designing recombinant plasmids, the PIII protein must retain the intact structure while leaving the M13 phage infectious, allowing rapid replication by infection with E.coli. The final structure of the recombinant phage is shown in FIG. 2, the PIII protein with PDZ ligand is located at the tail end of the recombinant phage, while the PVIII protein with SH3 ligand is located in the long rod-like part. In addition, the recombinant phage produces corresponding amounts of SH3 ligand and PDZ ligand according to the amount of PIII and PVIII proteins per se, and a normal M13 phage may have about 3-5 PIII proteins and about 2700 PVIII proteins, so preferably the recombinant phage may have about 3-5 SH3 ligands and about 2700 PVIII ligands. The recombinant phage is amplified for subsequent use. The choice of the above mentioned linker and anchor peptides may be interchanged or other different ligands or combinations thereof may be used, for example: GBD ligand (SEQ ID NO:3), SH2 ligand (SEQ ID NO:4), etc., but NO repetitive ligands are used for peptide conjugation and ecological anchoring.

Corresponding to step S12, recombinant yeast with surface expression anchor position is established. For example, following the example of step S11, the yeast is Saccharomyces cerevisiae belonging to the genus Saccharomyces, and in the case of PDZ ligand as the anchor peptide, PDZ domain having affinity for PDZ ligand expressed by PIII domain of recombinant phage is used as the anchor site. The recombinant yeast is obtained by inserting a gene (SEQ ID NO:5) of the PDZ domain into a gene of S.cerevisiae yeast to express the PDZ domain on the surface of the S.cerevisiae yeast. The phage are recombined and amplified by culture for subsequent use. It will be appreciated that other anchor peptides may be used with different domain anchor positions, for example: GBD ligand collocation GBD domain (SEQ ID NO:7), SH2 ligand collocation SH2 domain (SEQ ID NO:8) and the like.

Corresponding to step S13, recombinant Escherichia coli having the ability to secrete and express cellulolytic enzymes was established. The recombinant Escherichia coli is obtained by inserting the gene (SEQ ID NO:9) of endo-cellulolytic enzyme, the gene (SEQ ID NO:10) of exo-cellulolytic enzyme and the gene (SEQ ID NO:11) of cellobiohydrolase and the gene (SEQ ID NO:6) of SH3 structural domain into the gene of Escherichia coli, so that the Escherichia coli has the capability of secreting and expressing the endo-cellulolytic enzyme, the exo-cellulolytic enzyme and the cellobiohydrolase which have the affinity with SH3 ligand, namely SH3 structural domain. The recombinant E.coli was amplified by culture for subsequent use. As mentioned above, other anchoring peptides may be used in combination with different domain anchoring sites.

Corresponding to step S20, the recombinant phage, the recombinant yeast and the recombinant escherichia coli are mixed in the same culture system to be used as a biological reaction system for producing biomass alcohol. During the culture process, the recombinant escherichia coli can secrete a large amount of endo-cellulolytic enzyme, exo-cellulolytic enzyme and cellobiohydrolase to be dispersed in a biological reaction system. The recombinant phage binds the cellulolytic enzyme to the surface of the recombinant phage when they come into contact with each other by virtue of the affinity of the SH3 ligand expressed on the surface of the recombinant phage for the cellulolytic enzyme. Theoretically, a recombinant phage has about 2700 SH3 ligands and should have about 2700 cellulolytic enzymes accessible on its surface. However, the amount of the cellulolytic enzyme to be ligated may vary depending on the difference in the amount of SH3 ligand actually expressed by each recombinant phage, the distribution of the position, angle and steric space of actual ligation, and the relationship between the amount and concentration of both in the biological reaction system. The number of recombinant phage-conjugated cellulolytic enzymes may be 1 to 3000, preferably 100 to 2000. On the other hand, the recombinant phage also anchors the recombinant phage to the surface of the recombinant yeast when in contact by virtue of the affinity of its surface-expressed PDZ ligand to the surface-expressed PDZ domain of the recombinant yeast. Theoretically, each recombinant yeast surface can anchor recombinant phage corresponding to the number of PDZ domains it expresses on its surface. Similarly, however, the number of recombinant phage anchored may vary depending on the actual anchoring location, angle and spatial distribution, and the number and concentration of both in the bioreaction system. The above-mentioned joining and anchoring processes are all processes spontaneously occurring in the culture process in the biological reaction system, and finally, the cellulolytic enzymes produced by the recombinant phage, the recombinant yeast and the recombinant escherichia coli can be self-assembled into a whole, as shown in fig. 3, the structure of the recombinant yeast can be regarded as that the surface of the recombinant yeast has an artificial cellulosome which is formed by using the recombinant phage as a protein scaffold and combining with the cellulolytic enzymes, and is similar to anaerobic thermophilic bacteria t. However, the artificial cellulosome of the present invention has a greater amount of cellulolytic enzymes conjugated thereto, and thus has a more efficient cellulolytic ability, compared to native t.

Corresponding to step S30, biomass feedstock is added to the bioreactor system to produce biomass alcohol. The material conversion and supply flow in the biological reaction system is shown in FIG. 4, and the addition of avidine

Microcrystalline cellulose: (

PH) and Carboxymethyl cellulose (Carboxymethyl cellulose)lose) to a biological reaction system as a biomass raw material, so that the cellulose of the biomass raw material is decomposed by cellulolytic enzyme jointed on the surface of the recombinant phage to obtain hexose, and the hexose can be applied to a fermentation reaction by recombinant yeast anchored by the recombinant phage to convert the hexose into alcohol. In addition, the hexose can also be used as an energy source of recombinant escherichia coli and recombinant yeast in a biological reaction system, so that the hexose can be used for continuously producing the cellulolytic enzyme and producing the alcohol respectively. In the case where the recombinant phage is a non-lysogenic recombinant phage, such as the recombinant M13 phage of the exemplary embodiment of the present invention, replication amplification using recombinant E.coli can be continued.

In general, the above-described bioreactor system has both cellulose decomposing ability and hexose fermenting ability, and can be modified to have both five-carbon sugar decomposing ability according to the need. In addition, the recombinant Escherichia coli in the biological reaction system can produce cellulolytic enzyme by itself, and the required energy can be supplied by the hexose produced after the cellulose is decomposed, so that the whole biological reaction system can be operated continuously only by continuously supplying sufficient biomass raw material without providing additional energy.

Hereinafter, the activity data of the recombinant phage surface-bound cellulolytic enzyme in the above examples will be tested, and it is proved that the cellulolytic method of the present invention can achieve sufficient cellulolytic effect and achieve the purpose of efficiently producing biomass alcohol.

As shown in FIG. 5, when the PDZ domain sequence was inserted into the recombinant yeast gene, it was labeled with AlexaFluor 488 fluorescent dye. FIG. 5A is a photograph showing partially the non-recombinant yeast in a bright field of a microscope; and the C part shows a photograph of the recombinant yeast in a bright field of a microscope. FIG. 5B shows the results of wavelength observation of the excited Alexa Fluor 488 fluorochrome in the same field as part A, without any fluorescence signal visible, indicating the absence of PDZ domain; however, in FIG. 5D, which shows the observation that the Alexa Fluor 488 fluorescent dye was excited in the same field as in section C, green fluorescence was clearly observed, indicating that the PDZ domain was indeed expressed on the surface of the recombinant yeast.

As shown in FIG. 6, recombinant phages were labeled with a Syto 9 fluorescent dye. FIG. 6A shows, in part, a photograph under a microscope brightfield after mixing of recombinant phage RP with non-recombinant yeast and removal of unanchored recombinant phage; and part C shows a photograph under a microscope bright field after mixing the recombinant phage with the recombinant yeast and removing the unanchored recombinant phage. FIG. 6B shows the wavelength observation of the excitation of the Syto 9 fluorochrome in the same field as part A, without a green fluorescence signal, indicating that the recombinant phage is not anchored to the non-recombinant yeast; while part D shows the observation that the Syto 9 fluorochrome was excited in the same field as part of the field, and a green fluorescence was clearly observed, indicating that the recombinant phage was indeed anchored to the recombinant yeast.

Table 1 shows the comparison of the activities of cellobiase having SH3 domain (BGL-SH3) and cellobiase having SH3 domain (RP-BGL-SH3) which were attached to the surface of recombinant phages. Et in Table 1 is enzyme concentration; vmax is the maximum reaction rate; kcat is the reaction constant; KM is the Michaelis constant. The activities of RP-BGL-SH3 and BGL-SH3 are obtained by comparing kcat/KM data, namely catalytic efficiency. From the results of table 1 and fig. 7A, it was found that the catalytic efficiency of RP-BGL-SH3 was about 2.33 times as high as that of BGL-SH3 by observing the change in concentration of 4-nitrobenzene β -D-glucopyranoside (pNPG) at the initial rate pair, showing that the difference in catalytic efficiency with or without bonding to the phage surface, which could cause cellulose decomposition, reached about 2.33 times even though BGL-SH3 was present at the same concentration.

TABLE 1

Table 2 shows the comparison of the activities of endo-cellulolytic enzyme having SH3 domain (celA-SH3) and endo-cellulolytic enzyme having SH3 domain (RP-celA-SH3) bound to the surface of recombinant phage, in which the reference numerals of the respective parameters are the same as those in Table 1. From the results of the graphs in tables 2 and 7B, it was found that the catalytic efficiency of RP-celA-SH3 was about 1.81 times that of celA-SH3 by observing the change in the initial rate with respect to the concentration of Carboxymethyl cellulose (CMC), and it was revealed that the difference in the catalytic efficiency of cellulose decomposition caused by the presence or absence of the binding to the phage surface was about 1.81 times even though the same concentration of celA-SH3 was present.

TABLE 2

Table 3 shows the comparison of the activities of the exo-cellulolytic enzyme having SH3 domain (EC-SH3) and the exo-cellulolytic enzyme having SH3 domain (RP-EC-SH3) bound to the surface of recombinant phage, in which the measurement methods and the parameter numbers are the same as in Table 1. From the results of Table 3 and 7C, it was found that the catalytic efficiency of RP-EC-SH3 was about 1.57 times that of CelA-SH3 by observing the change in the concentration of Phosphoric Acid Swollen Cellulose (PASC) at the initial rate, and it was revealed that the difference in the catalytic efficiency of cellulose decomposition caused by the presence or absence of binding to the phage surface was about 1.57 times even though EC-SH3 was present at the same concentration.

TABLE 3

In conclusion, the results obtained all confirm that the artificial cellulosome with phage scaffold of the present invention facilitates the greatly improved cellulose decomposition efficiency under the same enzyme concentration. Moreover, the artificial cellulosome of the invention is used for decomposing cellulose and producing biomass alcohol, and the decomposition efficiency of the cellulose is greatly improved under the same enzyme concentration. Moreover, the artificial cellulosome of the invention forms a complete combined biotransformation process by anchoring to the surface of the recombinant yeast and co-culturing with the recombinant escherichia coli, so that the invention can really break through the capacity limit of the existing combined biotransformation process.

Although the present invention has been described specifically with reference to the above-described embodiments, the cellulose decomposition method, the biomass alcohol production method and the cellulosome used therefor, it will be understood by those skilled in the art that modifications and variations can be made in the embodiments without departing from the technical principles and spirit of the present invention. Therefore, the protection scope of the present invention should be defined by the claims to be described later.

Sequence listing

<110> Zhu Yi Min

CHANG CHUN PLASTICS Co.,Ltd.

Chang Chun Petrochemical Co.,Ltd.

<120> Artificial cellulosome, METHOD for DECOMPOSING CELLULOSE AND producing ALCOHOL USING THE SAME (ARTIFICIAL CELLULOSOME HAVING BACTERIOPHAGE SCAFFOLD, METHOD OF DECOMPOSING CELLULOSE AND fermenting ALCOHOL USING THE SAME)

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aagattgcaa cagtagcaac aagttcaaca actccaatta actggcagtt ggttaatagt 660

actggagcag ctgttttaac aggtaaatca actgttaaag gtgccgaccg tgcatcaggt 720

gataatgtcc atatcattga tttctctagt tacacaacac ctggtaccga ctataagata 780

gtaacagatg tatcagtaac aaaagccgga gacaatgaaa gtatgaagtt caatattgga 840

gatgaccttt ttactcaaat gaaatacgat tcaatgaagt atttctatca caacagaagt 900

gctattccaa tacaaatgcc atactgtgat caatcacaat gggcacgtcc tgcaggacac 960

acaactgata tacttgctcc agatccaaca aaggattaca aggctaacta cacacttgac 1020

gttacaggtg gttggtatga tgccggtgac catggtaagt atgttgttaa tggtggtatt 1080

gcaacctgga ccgtaatgaa tgcatatgag cgtgcactac acatgggtgg agacacttca 1140

gttgctccat ttaaagacgg ttctttagca ataccagaag cggaagtcta tcctgacata 1200

ctggacgaag ctcgttacca gctcattaac atgaaaacat tattaaatag tcaggttcca 1260

gcaggaaagt atgcgggtat ggctcaccac aaagctcatg acgaacgttg gacagctctt 1320

gctgtacgtc ccgaccagga tacaatgaaa cgttggttgc agcctccaag tacagcagct 1380

acattaaatc tggctgctat tgctgcacaa agttcacgtc tttggaaaca gtttgattct 1440

gctttcgcaa ctaagtgttt aactgcagca gaaactgcta gggatgcagc tgtagctcat 1500

ccagaaatat atgcaactat ggaacagggt gccggtggtg gagcatacgg agacaactat 1560

gttcttgatg atttctactg ggcagcatgt gaattgtatg caactacagg cagtgacaag 1620

tatttgaact acataaagag ctcaaagcat tatctcgaaa tgcctacaga attaacaggc 1680

ggtgagaata ctggaattac aggggctttt gactggggtt gtacagcagg tatgggaaca 1740

ataacacttg cacttgtacc tacaaagctt ccggcagcag atgttgctac agctaaagct 1800

aatattcaag ctgcagctga taagttcata tcaatttcaa aagcacaagg ctatggtgta 1860

ccactagaag aaaaagtaat ttcatctcct tttgatgcat ctgttgttaa aggtttccaa 1920

tggggatcaa actcattcgt tattaatgaa gcaatagtta tgtcatatgc ttatgaattc 1980

agcgatgtta atggcacaaa gaataataaa tatattaatg gtgctttaac agcaatggat 2040

tacctcctcg gacgtaaccc aaatattcaa agctatataa ctggttatgg tgacaaccca 2100

cttgaaaatc ctcatcaccg tttctgggca taccaggcag acaacacatt cccaaaacca 2160

cctccgggat gtctgtcagg aggacctaac tccggcttgc aggatccttg ggttaagggt 2220

tcaggctggc agccaggtga aagacctgct gaaaaatgct tcatggacaa tattgaatct 2280

tggtcaacaa acgaaataac catcaactgg aatgctcctc ttgtatggat atcagcttac 2340

cttgatgaaa aggggccaga gattggtggg tcagtgactc ctccaactaa tttaggagat 2400

gttaacggcg atggaaacaa ggatgcattg gacttcgctg cattgaagaa agccttgtta 2460

agccaggata cttctactat aaatgttgct aatgctgata taaacaaaga tggttctatt 2520

gatgcagttg actttgcatt actcaaatca ttcttgttag gaaaaatcac acag 2574

<210> 11

<211> 1341

<212> DNA

<213> Artificial sequence

<220>

<223> cellobiohydrolase (BGL)

<400> 11

tcaaagataa ctttcccaaa agatttcata tggggttctg caacagcagc atatcagatt 60

gaaggtgcat acaacgaaga cggcaaaggt gaatctatat gggaccgttt ttcccacacg 120

ccaggaaata tagcagacgg acataccggc gatgttgcat gcgaccacta tcatcgttat 180

gaagaagata tcaaaataat gaaagaaatc ggtattaaat catacaggtt ttccatctca 240

tggcccagaa tctttcctga aggaacaggt aaattaaatc aaaagggact ggatttttac 300

aaaaggctca caaatctgct tctggaaaac ggaattatgc ctgcaatcac tctttatcac 360

tgggaccttc cccaaaagct tcaggataaa ggcggatgga aaaaccggga caccaccgat 420

tattttacag aatactctga agtaatattt aaaaatctcg gagatatcgt tccaatatgg 480

tttactcaca atgaacccgg tgttgtttct ttgcttggcc actttttagg aattcatgcc 540

cctgggataa aagacctccg cacttcattg gaagtctcgc acaatcttct tttgtcccac 600

ggcaaggccg tgaaactgtt tagagaaatg aatattgacg cccaaattgg aatagctctc 660

aatttatctt accattatcc cgcatccgaa aaagctgagg atattgaagc agcggaattg 720

tcattttctc tggcgggaag gtggtatctg gatcctgtgc taaaaggccg gtatcctgaa 780

aacgcattga aactttataa aaagaagggt attgagcttt ctttccctga agatgacctg 840

aaacttatca gtcagccaat agacttcata gcattcaaca attattcttc ggaatttata 900

aaatatgatc cgtccagtga gtcaggtttt tcacctgcaa actccatatt agaaaagttc 960

gaaaaaacag atatgggctg gatcatatat cctgaaggct tgtatgatct gcttatgctc 1020

cttgacaggg attatggaaa gccaaacatt gttatcagcg aaaacggagc cgccttcaaa 1080

gatgaaatag gtagcaacgg aaagatagaa gacacaaaga gaatccaata tcttaaagat 1140

tatctgaccc aggctcacag ggcaattcag gacggtgtaa acttaaaagc atactacttg 1200

tggtcgcttt tggacaactt tgaatgggct tacgggtaca acaagagatt cggaatcgtt 1260

cacgtaaatt ttgatacgtt ggaaagaaaa ataaaggata gcggctactg gtacaaagaa 1320

gtaatcaaaa acaacggttt t 1341

Claims (9)

1. A method for decomposing cellulose, comprising:

creating a recombinant bacteriophage, wherein the recombinant bacteriophage is produced by modifying the genome of the bacteriophage to express on the surface of the bacteriophage:

at least one conjugated peptide having affinity to at least one lytic enzyme; and

at least one anchor peptide having an affinity to an anchor site on the cell surface;

contacting the at least one lytic enzyme with the recombinant bacteriophage; and

the cellulose is contacted with the lytic enzyme.

2. The lysis method of claim 1, wherein said at least one adaptor peptide is linked to between 1 and 3000 of said at least one lytic enzyme.

3. The method of claim 1, wherein the at least one lytic enzyme comprises an endo-cellulolytic enzyme, an exo-cellulolytic enzyme, a cellobiolytic enzyme, or a combination thereof.

4. The method of claim 1, wherein the at least one lytic enzyme expresses SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, or combinations thereof.

5. The degradation method of claim 4, wherein the anchor position expresses SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or a combination thereof and is not repeated with the sequence expressed by the at least one degradation enzyme.

6. A decomposition process according to any of claims 1 to 5, wherein the bacteriophage comprises a lambda bacteriophage or a filamentous bacteriophage.

7. A decomposition method according to any one of claims 1 to 5, wherein the cells comprise yeast.

8. A method for producing biomass alcohol, comprising:

creating a recombinant bacteriophage, wherein the recombinant bacteriophage comprises at least one adaptor peptide and at least one anchor peptide;

conjugating at least one lytic enzyme to the at least one conjugating peptide;

anchoring the at least one anchoring peptide to an anchoring position on the surface of the yeast;

allowing the at least one lytic enzyme to break down cellulose to obtain sugars; and

fermenting the saccharide with the yeast to obtain biomass alcohol.

9. An artificial fibrosome comprising:

a recombinant bacteriophage which expresses on its surface:

at least one conjugated peptide having affinity to at least one lytic enzyme; and

at least one anchor peptide having an affinity to an anchor site on the cell surface; and

at least one cellulolytic enzyme that binds to the at least one binding peptide expressed on the surface of the recombinant bacteriophage.

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