Method for constructing saccharomyces cerevisiae high-yield cellulase and fermentation application

Abstract

The invention provides a method for constructing a high-yield cellulase saccharomyces cerevisiae strain and fermentation application of the high-yield cellulase saccharomyces cerevisiae strain. The construction method comprises the following steps: 1) connecting a gene for coding cellulase to a yeast expression vector; 2) connecting the yeast expression regulation gene to a yeast expression vector; and (3) respectively transferring the yeast expression vectors obtained in the step (1) and the step (2) into the same saccharomyces cerevisiae strain to obtain the saccharomyces cerevisiae strain with high cellulase yield. Through gene modification, three high-activity cellulases are subjected to efficient co-expression in the yeast, protein secretion key genes are overexpressed, the secretion of the cellulases is greatly improved, the yeast protein can be produced by fermenting agricultural wastes such as corn straws and vinegar residues, and waste is turned into wealth.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for constructing a high-yield cellulase from brewer's yeast and its fermentation application. Background Art

[0002] Cellulose is the main component of plant cell walls, accounting for about 30-50% of the dry weight of plants, and is the largest renewable resource in nature. my country has abundant fiber resources, and the utilization and conversion of high-content lignocellulose biomass such as straw, distiller's grains and vinegar grains is of great significance for solving the current problems of recycling agricultural waste.

[0003] At present, cellulose is mainly degraded by acid or enzyme. Acid degradation requires a certain pressure and temperature, and has high requirements for equipment. A large amount of waste will be generated during the production process, which is difficult to meet environmental protection requirements. Enzyme degradation reaction conditions are mild and are the development goal of cellulose utilization and conversion. Three types of cellulases can degrade cellulose into glucose by synergistic action. The three types of cellulases are endoglucanase (EG), exoglucanase (CBH) and β -Glucosidase ( β -glucosidase, BGL). Cellulose is first hydrolyzed into cellobiose by endoglucanases (EGs) and exoglucanases (CBHs), which are then β -Glucosidases (BGLs) hydrolyze it into glucose.

[0004] Cellulase has a wide range of sources and is widely present in organisms in nature. Bacteria and fungi can produce cellulase. Studies have shown that the amount of cellulase produced by bacteria is relatively low, mainly endoglucanases, and these enzymes are mainly intracellular enzymes and rarely secreted outside the cell, so they are rarely used in industry. Actinomycetes produce almost no cellulase or the production is extremely low, making them difficult to use in industry. Cellulase used for industrial production generally comes from molds, and the more typical ones are Trichoderma, Aspergillus and Penicillium. Trichoderma reesei and its closely related strains are more typical and are currently recognized as better cellulase producers. Trichoderma strains have the advantages of large enzyme production and a relatively complete enzyme system, but their cellulase specific activity and β -Glucosidase activity is not very high and production is slow.

[0005] Using cellulase-producing yeast to transform cellulose materials to produce single-cell protein is more advantageous than bacteria and filamentous fungi, thereby effectively reducing the production cost of protein feed. Therefore, in recent years, there have been continuous studies on screening cellulase-producing yeast strains from nature or using biotechnology to obtain some cellulase-producing yeast strains through recombinant genetic engineering bacteria. The efficiency of screening cellulase-producing yeast strains from nature is very low. Wild yeast rarely produces cellulase or does not produce enzymes. Foreign reports have screened yeast strains from tropical plant leaves to produce cellulase, but the enzyme activity is low. Recently, China reported that yeast that can produce cellulase was screened from a yeast strain library from the marine environment ("Screening, identification and fermentation condition optimization of a cellulase-producing marine yeast". Journal of Ocean University of China. Supplement II, December 2007). The enzyme production culture medium was optimized, and the final cellulase activity of glucanase (CMCase) reached 4.51U / mg, and the filter paper enzyme activity reached 4.75U / mg, which is relatively low.

[0006] At present, the use of biotechnology to recombinant genetic engineering bacteria is an important way to obtain cellulase-producing yeast strains. Although cellulase yeast engineering strains have broad application prospects, the current cellulase yeast engineering strains are still limited by low cellulase activity and low secretion levels. For example, the literature reported that a cellulase-producing yeast strain was cloned from the Trichoderma koningii TK-4-775 strain. i The gene was cloned and the secretory activity of cbh1 was successfully expressed in Pichia pastoris, but the enzyme activity was only 24.1U / L (Study on Cloning and Expression of Trichoderma koningii Cellulase CBHI Gene in Pichia pastoris. Brewing Technology. Issue 1, 2011).

[0007] Therefore, it is necessary to construct highly active cellulase yeast strains through genetic engineering and other methods to achieve efficient expression of cellulase. This can ensure that the pretreated lignocellulose is converted into glucose economically and efficiently, and ultimately supply yeast growth and reproduction to produce yeast protein. Its molecular modification ultimately lays a theoretical foundation and strategic direction for the industrial fermentation production of cellulase yeast strains.

[0008] Chinese patent document CN101319196A discloses a transgenic Saccharomyces cerevisiae for degrading cellulose to synthesize ethanol and a method for constructing the same. β -1,4-Glucanase, exo β The genes of three enzymes, i.e., -1,4-glucanase and cellobiase, are co-transferred into Saccharomyces cerevisiae through a Saccharomyces cerevisiae expression vector to obtain the obtained product.

[0009] Chinese patent document CN103382444A discloses a genetically recombinant brewer's yeast capable of degrading crystalline cellulose. The genetically recombinant brewer's yeast is a yeast that has an endonuclease β -1,4-glucanase gene, exon β -1,4-glucanase gene and β -glucosidase gene was simultaneously transferred into Saccharomyces cerevisiae through Saccharomyces cerevisiae expression vector in a ratio of 1:3:2 and correctly secreted and expressed. The recombinant yeast obtained in Example 3 disclosed therein had a CBH enzyme activity of 0.04 IU / mL after 96h of culture, 0.1 IU / mL after 120h of culture, and 0.15 IU / mL after 168h of culture.

[0010] It can be seen that although cellulase yeast engineering strains have been widely used in the prior art, cellulase yeast engineering strains at this stage are still limited by low cellulase activity and low secretion level. Summary of the invention

[0011] In view of the above technical problems, the present invention provides a method for constructing a high-yield cellulase brewer's yeast strain and its fermentation application, which specifically includes the following technical solutions: The present invention provides a method for constructing a high-yield cellulase Saccharomyces cerevisiae strain, the method comprising the following steps: 1) Connect the gene encoding cellulase to the yeast expression vector; 2) Connect the yeast expression regulatory gene to the yeast expression vector; 3) The yeast expression vectors obtained in step 1) and step 2) are respectively transferred into the same Saccharomyces cerevisiae strain to obtain a Saccharomyces cerevisiae strain with high cellulase production.

[0012] Preferably, the gene encoding the cellulase is selected from β - One or more of the genes for glucosidase, exoglucanase and endoglucanase.

[0013] Further preferably, the gene encoding the cellulase includes β -Genes for glucosidase, exoglucanase and endoglucanase.

[0014] Also preferably, the yeast expression regulatory gene is selected from PDI1 , SSA4 , SEC53 , GCN4 and ERO1 One or more of .

[0015] Most preferably, the yeast expression regulatory gene is SEC53 .

[0016] The invention also provides a saccharomyces cerevisiae strain constructed by the method.

[0017] The present invention also provides the use of the saccharomyces cerevisiae strain in fermentation to produce yeast protein, which is used for fermenting agricultural waste to decompose wood fiber to produce yeast protein.

[0018] Preferably, the agricultural waste is selected from corn stover and vinegar grains.

[0019] The present invention has the following beneficial effects: The present invention firstly uses gene recombination technology to clone and connect three cellulases to a Saccharomyces cerevisiae expression vector, and then transfers them into the Saccharomyces cerevisiae strain to construct a cellulase yeast engineering strain CEL. PDI1 , SSA4 , SEC53 , GCN4 and ERO1 The genes were cloned and connected to the expression vector of Saccharomyces cerevisiae, and then transformed into the CEL strain to construct the cellulase yeast engineering strains CEL+PDI1, CEL+SSA4, CEL+SEC53, CEL+GCN4 and CEL+ERO1, which can overexpress more expression regulatory genes. The effect was investigated and the best strain CEL+SEC53 with high cellulase production was obtained through screening and identification. In a 3L fermenter, the strain expressed β -Glucosidase activity is 21.95 U / g DCW (cell dry weight), exoglucanase activity is 4.04 U / g DCW, and endoglucanase activity is 23.19 U / g DCW. The three cellulases are co-expressed at the same time and have extremely high enzyme activities, which lays a good foundation for the large-scale production of cellulases.

[0020] The present invention also provides a fermentation method for efficiently utilizing agricultural wastes such as corn stalks and vinegar lees by using a cellulase yeast engineering strain. The cellulase yeast engineering strain CEL+SEC53 is used to ferment the acid-pretreated corn stalks and vinegar lees, and the cellulose in the corn stalks and vinegar lees is degraded into glucose, thereby supplying yeast growth and reproduction, and finally generating yeast protein. In a 3L fermentation tank, the net value added of crude protein of 10 g corn stalks fermented by the cellulase yeast engineering strain CEL+SEC53 is 910.9 mg / 779.5 mg of true protein, and the net value added of crude protein of 10 g vinegar lees fermented is 777.3 mg / 686 mg of true protein. Therefore, it is estimated that in a ton-level fermentation tank, the net value added of crude protein of one ton of corn stalks fermented is 91.09 kg / 77.95 kg of true protein, and the net value added of crude protein of one ton of vinegar lees fermented is 77.73 kg / 68.6 kg of true protein. It can be seen that the cellulase Saccharomyces cerevisiae engineered strain CEL+SEC53 can efficiently degrade wood fiber into glucose, convert cellulose into an available carbon source, and finally generate yeast protein, thus turning waste into treasure and having high application value. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The Western blot results of three cellulases in the fermentation broth of cellulase Saccharomyces cerevisiae engineering strain CEL are shown (3 d). (a) Western blot of three cellulases in the fermentation broth of cellulase Saccharomyces cerevisiae engineering strain CEL; (b) β -glucosidase (AnBGL1) by Western blot; (c) exoglucanase (TtCBH2) by Western blot in the fermentation broth of cellulase Saccharomyces cerevisiae engineered strain CEL; (d) endoglucanase (TrEG2) by Western blot in the fermentation broth of cellulase Saccharomyces cerevisiae engineered strain CEL.

[0022] Figure 2 The enzymatic activities of three cellulases in a 3 L fermenter (3 days) are shown. (a) β -glucosidase (AnBGL1) activity; (b) exoglucanase (TtCBH2) activity; (c) endoglucanase (TrEG2) activity.

[0023] Figure 3The crude protein amount generated by the conversion of corn stover or vinegar grains at the 3 L fermentation tank level is shown. (a) The crude protein amount generated by the conversion of corn stover after fermentation by cellulase Saccharomyces cerevisiae engineered strain and Saccharomyces cerevisiae; (b) The crude protein amount generated by the conversion of vinegar grains after fermentation by cellulase Saccharomyces cerevisiae engineered strain and Saccharomyces cerevisiae; (c) The true protein amount generated by the conversion of corn stover after fermentation by cellulase Saccharomyces cerevisiae engineered strain and Saccharomyces cerevisiae; (d) The true protein amount generated by the conversion of vinegar grains after fermentation by cellulase Saccharomyces cerevisiae engineered strain and Saccharomyces cerevisiae.

[0024] Figure 4 Electron microscope images of corn stover and vinegar grains fermented by cellulase-producing Saccharomyces cerevisiae and Saccharomyces cerevisiae are shown (9 d). (a) Corn stover fermented by Saccharomyces cerevisiae; (b) Corn stover fermented by cellulase-producing Saccharomyces cerevisiae; (c) Vinegar grains fermented by Saccharomyces cerevisiae; (d) Vinegar grains fermented by cellulase-producing Saccharomyces cerevisiae. DETAILED DESCRIPTION

[0025] The following is further described in detail through specific implementation methods. However, it should be pointed out that the following embodiments of the present invention are only for better illustrating the content of the present invention, but do not mean that the content of the present invention is limited to the examples. Therefore, those skilled in the art can make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still belongs to the protection scope of the present invention and is subject to the protection scope of the attached claims.

[0026] The term "Saccharomyces cerevisiae" as used in the present invention is Saccharomyces cerevisiae , also known as baker's yeast or budding yeast. Saccharomyces cerevisiae is the yeast with the widest relationship with humans. It is used to make foods such as bread and steamed buns and brew wine. Saccharomyces cerevisiae belongs to the Ascomycetes, Saccharomyces, Saccharomyces, Saccharomyces, and Saccharomyces species. Saccharomyces cerevisiae is the first eukaryotic organism to complete genome sequencing, which was completed in 1996. Saccharomyces cerevisiae has many of the same structures as animal and plant cells, which are also eukaryotic organisms, and is easy to culture. Yeast is used as a model organism for studying eukaryotic organisms and is also the most commonly used biological species in fermentation. Saccharomyces cerevisiae has the advantages of short growth cycle, strong fermentation ability, easy large-scale cultivation, and rich nutrients such as a variety of proteins, amino acids, vitamins, and bioactive substances. It has always been the main object of basic and applied research, and is widely used in the fields of food, medicine, etc. It is also used to produce agricultural fertilizers, single-cell protein feeds, and other agricultural products. Although the present invention uses Saccharomyces cerevisiae as the best engineering bacteria, it is only because Saccharomyces cerevisiae is the most widely used, but the present invention can still use other yeast strains as engineering bacteria objects.

[0027] The term "cellulase" used in the present invention is cellulase, which is a kind of protein enzyme that can decompose cellulose into oligosaccharides or monosaccharides, and plays a biological catalytic role in decomposing cellulose. Cellulase is widely present in organisms in nature. Bacteria, fungi, animals, etc. can produce cellulase. The cellulase generally used in production comes from fungi, and the more typical ones are Trichoderma ( Trichoderma spp.), Aspergillus spp. Aspergillus spp.) and Penicillium ( Penicillium spp.) Cellulase. Cellulase can be divided into two categories according to its catalytic function: endoglucanase (1,4- β -D-glucan glucanohydrolase or endo-1,4- β -D-glucanase, EC3.2.1.4, from fungi referred to as EG, from bacteria referred to as Cen; exoglucanase (1,4- β -D-glucancellobilhydrolase or exo-1,4- β -D-glucannase, EC.3.2.1.91), abbreviated as CBH from fungi and Cex from bacteria; β -Glucosidase ( β -1,4-glucosidase, EC.3.2.1.21) is referred to as BGL. Endoglucanases randomly cut the amorphous regions inside the cellulose polysaccharide chain to produce oligosaccharides of different lengths and the ends of new chains; exoglucanases act on the ends of these reducing and non-reducing cellulose polysaccharide chains to release glucose or cellobiose; β -Glucosidase hydrolyzes cellobiose to produce two molecules of glucose. Although the present invention uses these preferred cellulases simply because they have the best performance, the present invention can still use cellulases from other sources.

[0028] Those skilled in the art know that the protein folding secretion pathway is a complex process involving the participation and regulation of multiple genes. Among them, PDI1 is a protein disulfide isomerase, which plays a key role in the protein folding process. It mainly mediates the formation of disulfide bonds between secretory proteins and cell surface proteins, and cleans up abnormal disulfide bonds; in addition, PDI1 can also form a complex with MNL1 to process the sugar groups of unfolded or misfolded glycoproteins and promote the degradation of unfolded proteins. ERO1 is an oxidase in the endoplasmic reticulum. It cooperates with proteins such as PDI1 to promote the formation of disulfide bonds, and forms disulfide bonds by oxidizing the sulfhydryl groups in proteins, thereby promoting the folding and stabilization of proteins. SSA4 is one of the vesicle transport-related genes. It is involved in the transport process of intracellular substances. In the protein folding secretion pathway, SSA4 is involved in transporting folded proteins from the endoplasmic reticulum to target locations such as the Golgi apparatus. SEC53 is one of the key genes for the transport of substances between the endoplasmic reticulum and the Golgi apparatus. It is involved in the formation of vesicles, which are responsible for transporting proteins synthesized in the endoplasmic reticulum to the Golgi apparatus for further processing and secretion. GCN4 is a stress response gene that is activated when cells face stress conditions such as amino acid starvation. The activation of GCN4 can trigger a series of stress responses, including the inhibition of protein synthesis and the upregulation of the amino acid transport system.

[0029] During the construction of the yeast strain of the present invention, SD defective medium (SD / -ura-leu) (1 L) was used for culturing the yeast: 8 g of uracil and tryptophan nutrient-deficient medium of Panjino yeast (YGM003A-35, purchased from Beijing Panjino Technology Co., Ltd.) was dissolved in ddH2O and the pH was adjusted to 6.0, and the volume was made up to 960 mL with ddH2O, sterilized at 121°C for 20 min, cooled to 55°C in a clean bench, and 40 mL of 50% glucose was added to make the final concentration reach 2% (w / v), and the final volume reached 1 L.

[0030] When the present invention uses a 3L fermentation tank to ferment yeast, a fermentation medium (1L) with corn stover or vinegar dregs as the sole carbon source is used: 700 mL 0.1 M PBS, 75 mL 0.1 M MgCl2, 0.885 g yeast extract and 1.77 g peptone, acid-pretreated corn stover (10 g) or vinegar dregs (10 g), and the volume is fixed to 1 L with ddH2O. The acid pretreatment of corn stover is based on the method recorded in the literature (Zhu Zet al. Comparative study of corn stover pretreated by dilute acid and cellulose solvent-based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility. Biotechnol Bioeng, 2009 (4)) (similar to the vinegar dregs treatment), specifically: 250 g corn stover was ground with a wall breaker; the corn stover was sieved to a particle size of >60 mesh and <40 mesh, i.e., 0.25-0.42 mm in diameter; 1 g corn stover powder was mixed with 8 mL of 84% phosphoric acid using a glass rod, and the mixed slurry was incubated in a 50 °C water bath for 30 minutes, and then the reaction was terminated by an ice water bath; 40 mL acetone was then added (to precipitate dissolved cellulose and hemicellulose); the mixture was centrifuged at 3600 rpm for 20 minutes at room temperature; the supernatant was discarded, the precipitate was resuspended with 40 mL acetone, and centrifuged at 3600 rpm for 10 minutes (the first acetone wash); the supernatant was discarded, the precipitate was resuspended with 40 mL acetone, and centrifuged at 3600 rpm for 10 minutes (the second acetone wash); the supernatant was discarded, the precipitate was resuspended with 40 mL acetone, and centrifuged at 3600 rpm for 10 minutes (the third acetone wash); the supernatant was discarded, the precipitate was resuspended with 40 mL sterile water, and centrifuged at 3600 rpm for 10 minutes. rpm for 10 minutes (wash with sterile water for the first time); discard the supernatant, resuspend the precipitate with 40 mL of sterile water, and centrifuge at 3600 rpm for 10 minutes (wash with sterile water for the second time); discard the supernatant, resuspend the precipitate with 5 mL of sterile water, and measure the pH; neutralize the suspension to a pH of about 5 with 1 M sodium hydroxide.

[0031] However, it is clear to those skilled in the art that the above acid pretreatment is only one method for implementing the present invention, and other acid pretreatment methods known in the art may be applicable.

[0032] If the technology, instruments, equipment and materials used in the present invention are not particularly specified, any other technology, instruments, equipment and materials known to those skilled in the art that can achieve the same purpose may be used; even if the technology, instruments, equipment and materials used in the present invention are particularly specified, it does not mean that the present invention can only use these technologies, instruments, equipment and materials, which only represent the preferred embodiments of the present invention, and those skilled in the art can still use any other technology, instruments, equipment and materials known to those skilled in the art that can achieve the same purpose.

[0033] Example 1 Construction of a yeast engineered strain CEL that co-expresses three cellulase genes First, three cellulase genes were synthesized, namely Trichoderma reesei ( Trichoderma reesei ) of the endoglucanase gene ( TrEG2 )、Thermophyton spp. shell( Thermothelomyces thermophilus ATCC42464) exoglucanase gene ( TcBH2 ) and Aspergillus niger ( Aspergillus niger )of β -glucosidase gene ( AnBGL2 ). The nucleotide sequences of the three genes have been removed by bioinformatics methods, and the signal peptide and all introns. The nucleotide sequence of the TrEG2 gene is shown in SEQ ID No. 1. TcBH2 The nucleotide sequence of the gene is shown in SEQ ID No. 2. AnBGL2 The nucleotide sequence of the gene is shown in SEQ ID No.3.

[0034] The above three cellulases were cloned and connected to the Saccharomyces cerevisiae expression vector P414-GPD (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) through the XhoI and BglII restriction sites to obtain the plasmid P414-GPD- AnBGL1 - TcBH2 - TrEG2 , whose nucleotide sequence is shown in SEQ ID No.4, which includes GAP promoter, α-factor signal peptide, AnBGL1 , 6×His protein tag, CYC1 terminator, GAP promoter, α-factor signal peptide, TcBH2 , 3×HA protein tag, CYC1 terminator, GAP promoter, α-factor signal peptide, TrEG2 , 3×FLAG protein tag, CYC1 terminator. The primer pair P414-GPD- AnBGL1-TtCBH2-TrEG2 -F (forward primer, the sequence of which is shown in SEQ ID NO: 5) and P414-GPD- AnBGL1-TtCBH2-TrEG2 -R (reverse primer, whose sequence is shown in SEQ ID NO: 6) to clone the plasmid.

[0035] The resulting plasmid P414-GPD- AnBGL1 - TcBH2 - TrEG2 The cells were electroporated into the Saccharomyces cerevisiae W303a strain (purchased from Beijing Biobo Biotechnology Co., Ltd.), which is a Ura3 / Trp / Leu / His deficient strain, to construct a yeast engineered strain CEL that co-expresses three cellulase genes.

[0036] like Figure 1 The figure shows the Western blot results of three cellulases in the fermentation broth of the cellulase Saccharomyces cerevisiae engineering strain CEL cultured on a medium with corn straw or vinegar lees cellulose as the sole carbon source (3 days). (a) Western blot of three cellulases in the fermentation broth of the cellulase Saccharomyces cerevisiae engineering strain CEL; (b) Western blot of three cellulases in the fermentation broth of the cellulase Saccharomyces cerevisiae engineering strain CEL β (c) Western blot grayscale analysis of exoglucanase (TtCBH2) in the fermentation broth of cellulase Saccharomyces cerevisiae engineering strain CEL; (d) Western blot grayscale analysis of endoglucanase (TrEG2) in the fermentation broth of cellulase Saccharomyces cerevisiae engineering strain CEL. As can be seen from the figure, the W303a strain itself does not produce three cellulases, while the transgenic cellulase Saccharomyces cerevisiae engineering strain CEL produces three cellulases, which can be detected by the corresponding antibody tags.

[0037] Example 2 Construction of Yeast Engineering Strains Co-expressing Yeast Expression Regulatory Genes First, five genes related to the protein folding secretion pathway were isolated and cloned, including the gene PDI1 , ER01 , SSA4 , SEC53 and GCN4 , which are all present in the W303a yeast strain. These genes were obtained by PCR on the gDNA of the W303a strain. The sequences of these genes are also known to those skilled in the art, among which PDI1 The nucleotide sequence of the gene is shown in GeneID: 854462. ER01 The nucleotide sequence of the gene is shown in Gene ID: 854909. SSA4 The nucleotide sequence of the gene is shown in Gene ID: 856840. SEC53 The nucleotide sequence of the gene is shown in Gene ID: 850499. GCN4The nucleotide sequence of the gene is shown in Gene ID: 856709.

[0038] The above five protein folding secretion pathway-related genes were cloned and connected (the vectors were uniformly cut with SacI and KpnI, the target gene fragments were directly carried with homology arms during PCR, and the target gene fragments and the vectors were connected together by homologous recombination) to the Saccharomyces cerevisiae expression vector P415-GPD (purchased from Wuhan Miaoling Biotechnology Co., Ltd.), and the plasmids P415-GPD- PDI1 、P415-GPD- ER01 、P415-GPD- SSA4 、P415-GPD- SEC53 and P415-GPD- GCN4 . The above five plasmids can be cloned using the following primer pairs: P415-GPD-PDI1-F (forward primer, whose sequence is shown in SEQ ID NO:7) and P415-GPD-PDI1-R (reverse primer, whose sequence is shown in SEQ ID NO:8); P415-GPD-ERO1-F (forward primer, whose sequence is shown in SEQ ID NO:9) and P415-GPD-ERO1-R (reverse primer, whose sequence is shown in SEQ ID NO:10); P415-GPD-SSA4-F (forward primer, whose sequence is shown in SEQ ID NO:11) and P415-GPD-SSA4-R (reverse primer, whose sequence is shown in SEQ ID NO:12); P415-GPD-SEC53-F (forward primer, whose sequence is shown in SEQ ID NO:13) and P415-GPD-SEC53-R (reverse primer, whose sequence is shown in SEQ ID NO:14). NO:14); P415-GPD-GCN4-F (forward primer, whose sequence is shown in SEQ ID NO:15) and P415-GPD-GCN4-R (reverse primer, whose sequence is shown in SEQ ID NO:16).

[0039] One of the above five plasmids was respectively transferred into the Saccharomyces cerevisiae engineering strain CEL obtained in Saccharomyces cerevisiae Example 1 by electroporation, and the corresponding fragments were amplified by designing PCR primers. The size of the fragments was verified by sequencing or electrophoresis to construct cellulase yeast engineering strains CEL+PDI1, CEL+ERO1, CEL+SSA4, CEL+SEC53 and CEL+GCN4 that further expressed genes related to the protein folding and secretion pathway.

[0040] Example 3 Fermentation test The CEL+PDI1, CEL+ERO1, CEL+SSA4, CEL+SEC53 and CEL+GCN4 engineered bacteria constructed in Example 2 were activated respectively, and the bacterial solution was aseptically inoculated into a 3L fully automatic fermenter with an inoculation volume of 5%. The initial fermentation conditions were: liquid volume 1000-1500mL, initial stirring speed 500 rpm, ventilation volume 2.5 vvm (ventilation ratio or ventilation rate, indicating the ratio of ventilation volume per minute to the actual liquid volume of the tank, that is, the amount of air required per minute per cubic meter of fermentation liquid in cubic meters), and pH was controlled to 6 with 30% phosphoric acid solution and 25% concentrated ammonia water. The culture temperature during the growth period was 30°C, and the maximum stirring speed was set to 1000 rpm. The original Saccharomyces cerevisiae strain W303a was fermented under the same conditions.

[0041] As the fermentation time progresses, the content of β -Glucosidase activity, exoglucanase activity, endoglucanase activity, crude protein content.

[0042] β -Glucosidase ( β -glucosidase, BGL) enzyme activity assay method: β -Glucosidase activity was determined by p-nitrophenol spectrophotometry. β -Glucosidase catalyzes the β -D-Glucoside (p-nitrophenyl β -D-glucoside, pNPG) produces p-nitrophenol and glucose. Measure the change in absorbance at 405 nm and calculate based on the results of the standard curve β -Glucosidase activity units.

[0043] Exoglucanase (CBH) activity assay: Exoglucanase activity was assayed using the phenol-sulfuric acid assay. Exoglucanase can release glucose and / or cellobiose from the ends of cellulose chains. Avicel (microcrystalline cellulose) was used to estimate the activity of purified exoglucanase. The change in absorbance at 540 nm was measured, and the enzyme activity was calculated based on the linear relationship between total soluble sugar release and enzyme dilution. One unit of exoglucanase activity was defined as the amount of enzyme that releases one micromole of glucose equivalent from Avicel per minute, and the exoglucanase activity unit was calculated.

[0044] Endoglucanase (EG) activity assay method: Endoglucanase activity was assayed using carboxymethyl cellulose / DN spectrophotometry. Endoglucanase randomly cuts accessible intermolecular bonds on the cellulose surface. β-1,4-glucosidic bond. β -glucosidase bonds have very low fractionation effects on cellulase, and water-soluble cellulose derivatives such as CMC are often used for endoglucanase activity determination. 0.5 mg of absolute glucose is released under the reaction conditions. The reducing end concentration is measured by the DNS method. The change in absorbance at 540 nm is measured, and the endoglucanase activity units are calculated based on the results of the standard curve.

[0045] Crude protein determination method: The crude protein in the fermentation product is determined by a rapid nitrogen analyzer. The solid fermentation product after fermentation is washed twice with sterile water, freeze-dried, and the sample is prepared according to the requirements and placed in a rapid nitrogen analyzer. The crude protein content in the fermentation product is calculated based on the protein ratio result output by the rapid nitrogen analyzer.

[0046] Figure 2 The enzymatic activities of three cellulases in each engineered strain are shown at the 3 L fermentation tank level with corn stover or vinegar cellulose as the sole carbon source (3 days). (a) β -glucosidase (AnBGL1) activity; (b) exoglucanase (TtCBH2) activity; (c) endoglucanase (TrEG2) activity. The results showed that the Saccharomyces cerevisiae strain W303a β -Glucosidase ( β -glucosidase, BGL), exoglucanase (CBH) and endoglucanase (EG) were all 0; cellulase expressed by the engineered strain CEL of Saccharomyces cerevisiae β -Glucosidase ( β -glucosidase, BGL) activity was 14.48 U / g DCW (dry cell weight), exoglucanase (CBH) activity was 2.81 U / g DCW, endoglucanase (EG) activity was 13.63 U / g DCW; cellulase was expressed by the engineered strain CEL+PDI of Saccharomyces cerevisiae. β -Glucosidase ( β -glucosidase, BGL) activity was 21.29 U / g DCW (dry cell weight), exoglucanase (CBH) activity was 3.86 U / g DCW, endoglucanase (EG) activity was 20.89 U / g DCW; cellulase was expressed by the engineered strain CEL+SEC53 of Saccharomyces cerevisiae. β -Glucosidase (β -glucosidase, BGL) activity was 8.96U / g DCW (dry cell weight), exoglucanase (CBH) activity was 1.09U / g DCW, endoglucanase (EG) activity was 10.44U / g DCW; cellulase was expressed by the engineered strain CEL+SEC53 of Saccharomyces cerevisiae. β -Glucosidase ( β -glucosidase, BGL) activity was 21.95 U / g DCW (dry cell weight), exoglucanase (CBH) activity was 4.04 U / g DCW, endoglucanase (EG) activity was 23.19 U / g DCW; cellulase was expressed in the engineered strain CEL+ERO1 of Saccharomyces cerevisiae. β -Glucosidase ( β -glucosidase, BGL) activity was 20.23 U / g DCW (dry cell weight), exoglucanase (CBH) activity was 4.14 U / g DCW, endoglucanase (EG) activity was 21.59 U / g DCW; cellulase was expressed by the engineered strain CEL+GCN4 of Saccharomyces cerevisiae. β -Glucosidase ( β -glucosidase, BGL) activity is 18.77 U / g DCW (dry cell weight), exoglucanase (CBH) activity is 3.49 U / g DCW, endoglucanase (EG) activity is 19.46U / g DCW. Among them, the cellulase Saccharomyces cerevisiae engineered strain CEL+SEC53 has the best comprehensive expression of three enzymes, which lays a good foundation for the large-scale production of cellulase.

[0047] Figure 3The crude protein amount generated by corn stover or vinegar grains at the 3 L fermentation tank level with corn stover or vinegar grains cellulose added as the sole carbon source is shown. (a) The crude protein amount generated by corn stover after fermentation with cellulase Saccharomyces cerevisiae engineered strain CEL+SEC53 and Saccharomyces cerevisiae W303a; (b) The crude protein amount generated by vinegar grains after fermentation with cellulase Saccharomyces cerevisiae engineered strain CEL+SEC53 and Saccharomyces cerevisiae W303a; (c) The true protein amount generated by corn stover after fermentation with cellulase Saccharomyces cerevisiae engineered strain CEL+SEC53 and Saccharomyces cerevisiae W303a; (d) The true protein amount generated by vinegar grains after fermentation with cellulase Saccharomyces cerevisiae engineered strain CEL+SEC53 and Saccharomyces cerevisiae W303a. The results showed that in a 3L fermenter, the net value of crude protein fermented by the cellulase Saccharomyces cerevisiae engineering strain CEL+SEC53 was 910.9 mg, and the net value of true protein (according to the BCA protein detection kit operating instructions purchased from Thermo Company, item number: 23235) was 779.5 mg, and the net value of crude protein fermented by 10 g vinegar lees was 777.3 mg, and the net value of true protein was 686 mg. Therefore, it is estimated that in a ton-level fermenter, the net value of crude protein fermented by one ton of corn stalks is 91.09 kg, and the net value of true protein is 77.95 kg, and the net value of crude protein fermented by one ton of vinegar lees is 77.73 kg, and the net value of true protein is 68.6 kg.

[0048] Figure 4 Electron microscope images of corn stover and vinegar lees fermented by cellulase Saccharomyces cerevisiae engineered strain CEL+SEC53 and Saccharomyces cerevisiae W303a are shown (9 d). (a) Corn stover fermented by Saccharomyces cerevisiae W303a; (b) Corn stover fermented by cellulase Saccharomyces cerevisiae engineered strain CEL+SEC53; (c) Vinegar lees fermented by Saccharomyces cerevisiae W303a; (d) Vinegar lees fermented by cellulase Saccharomyces cerevisiae engineered strain CEL+SEC53. As can be seen from the figure, compared with the control group (a), the electron microscope photo in the experimental group (b) shows that the pores of cellulose in corn stover are more and larger, and the cellulose in corn stover is degraded more thoroughly; compared with the control group (c), the electron microscope photo in the experimental group (d) shows that the pores of cellulose in vinegar lees are more and larger, and the cellulose in vinegar lees is degraded more thoroughly.

[0049] In summary, the cellulase Saccharomyces cerevisiae engineered strain CEL+SEC53 of the present invention can efficiently degrade wood fiber into glucose, convert cellulose into an available carbon source, and finally generate yeast protein, and has a high application value.

[0050] The above is only an embodiment of the present invention. The commonly known technical common sense in the scheme is not described in detail here. The ordinary technicians in the relevant field are aware of all the common technical knowledge in the technical field to which the invention belongs before the application date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical commonly known technologies should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, they can also make some adjustments and improvements, which should also be regarded as the scope of protection of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection claimed by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a high-yield cellulase Saccharomyces cerevisiae strain, the method comprising the following steps: 1) Connect the gene encoding cellulase to the yeast expression vector; 2) Connect the yeast expression regulatory gene to the yeast expression vector; 3) The yeast expression vectors obtained in step 1) and step 2) are respectively transferred into the same Saccharomyces cerevisiae strain to obtain a Saccharomyces cerevisiae strain with high cellulase production.

2. The method according to claim 1, characterized in that The gene encoding the cellulase is selected from β - One or more of the genes for glucosidase, exoglucanase and endoglucanase.

3. The method according to claim 2, characterized in that The cellulase encoding gene includes β -Genes for glucosidase, exoglucanase and endoglucanase.

4. The method according to claim 3, characterized in that The three genes encoding cellulases are Trichoderma reesei ( Trichoderma reesei ) endoglucanase gene, Thermomyces thermophilus shell ( Thermothelomyces thermophilus ) and Aspergillus niger ( Aspergillus niger )of β -glucosidase gene.

5. The method according to any one of claims 1 to 4, characterized in that: The yeast expression regulatory gene is selected from PDI1 , SSA4 , SEC53 , GCN4 and ERO1 One or more of .

6. The method according to claim 5, characterized in that The yeast expression regulatory gene is SEC53 .

7. The Saccharomyces cerevisiae strain constructed by the method according to any one of claims 1 to 6.

8. Use of the Saccharomyces cerevisiae strain according to claim 7 in fermentation production of yeast protein, characterized in that: Used to ferment agricultural waste, decompose wood fiber and produce yeast protein.

9. The use according to claim 8, characterized in that The agricultural waste is selected from corn stalks and vinegar grains.

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