CN105861338A - High-yield cellulase trichoderma reesei engineering bacteria and preparing method and application thereof - Google Patents
High-yield cellulase trichoderma reesei engineering bacteria and preparing method and application thereof Download PDFInfo
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Abstract
The invention discloses high-yield cellulase trichoderma reesei engineering bacteria which are obtained by guiding a gene bg11 of trichoderma reesei Rut-C30 into trichoderma reesei Rut-C30 by the adoption of the method of single enzyme digestion homologous recombination. The invention further discloses a construction method and application of the high-yield cellulase trichoderma reesei engineering bacteria. Compared with the prior art, extracellular beta-glucosidase generated by the engineering bacteria is increased by 17.3 times compared with that of the starting strain Rut-C30; meanwhile, endoglucanase, exoglucanase, filter paper enzyme activity and protein content are increased. In addition, the strain shows a high glucose endurance ability and a better saccharification ability.
Description
Technical field
The invention belongs to bioenergy and biological technical field, be specifically related to a kind of High Cellulase Production trichoderma reesei engineering bacteria and preparation method and application.
Background technology
Petroleum-based energy crisis and petroleum-based energy use the most serious environmental pollution brought, and make people constantly pursue the regenerative resource with substitutability.Cellulose is widespread in nature, and is to be connected, by glucose residue, a kind of macromolecular polysaccharide polymer formed by β-Isosorbide-5-Nitrae glycosidic bond.It can be become glucose by mycetogenetic cellulose degraded, and then is utilized production bioenergy molecule, bio-based fine chemical product and medicine etc. by microorganism.Cellulase is the enzymatic mixture that a class is complicated, and wherein participate in cellulose degradation mainly have three kinds of enzymes: endoglucanase, exoglucanase and beta-glucosidase.First, cellulose degradation is become cell-oligosaccharide by endoglucanase, then cell-oligosaccharide degrade under the effect of cellobiohydrolase fibroblast dimension disaccharide, last cellobiose is hydrolyzed to fermentability glucose under the effect of beta-glucosidase.
Trichoderma reesei because of its cellulase activity height, stabilization characteristics of genetics, growth and breeding is rapid, condition of culture is simple and biological safety is high, become preferable cellulase industrial producing strain (Journal of Biotechnology, 1994,37 (3): 193-200).Cellulase produces in endoplasmic reticulum, arrives mycelia top through Golgi body, is secreted in a large number in the outer culture fluid of born of the same parents.But, the most a small amount of beta-glucosidase BGL1 is secreted into extracellular so that the degraded of cellulose only rests on generation cellobiose.But the generation of the accumulation of cellobiose and metabolic end product glucose all can suppress the generation of cellulase, causes the reduction of cellulose degradation efficiency.Additionally, industrial collection cellulase simply collects the cellulase in supernatant, it is impossible to collect the BGL1 of intracellular simultaneously, the content causing BGL1 in gained cellulase solution is the lowest, it is impossible to fully degraded cellulose.This problem can only carry out mixing solving by extracting the cellulase in different fermentations liquid, so considerably increases production cost.How to improve the beta-glucosidase in cellulase and become a major issue in the urgent need to address during commercial cellulose enzyme produces.Therefore, it is thus achieved that a strain has the trichoderma reesei engineering strain of the high-yield beta-glucosidase of glucose tolerance and will assist in reduction cellulase production cost and improve cellulose enzyme activity.
Research worker obtains High Cellulase Production trichoderma reesei engineered strain by methods such as gene mutation, process LAN genes of interest, optimization strong promoter, increase strong promoter copy numbers both at home and abroad, and has been applied in commercial production.Obtaining the high productive mutant QM9414 of trichoderma reesei QM6a as Mandals etc. passes through radiation, its albumen and yield of cellulase are 2 times and 4 times of wild-type strain respectively.But this bacterial strain has stronger carbon metablism inhibitory action.Later, Kov á cs etc. was by UV, N-nitroguanidine, and the three-step approach of UV obtains mutant Rut-C30.Rut-C30 is the beta-glucosidase superior strain that a strain has the suppression of more weak carbon metablism, and its born of the same parents' outer fiber element enzyme aggregate level improves 150 times than wild mushroom simultaneously, but its poor growth, raw spore postpone, raw spore reduces.At present, certain progress is also achieved by allos or homology process LAN beta-glucosidase to obtain High Cellulase Production bacterial strain.The beta-glucosidase gene that penicillium decumbens is originated by Ma et al. is under cbh1 promoter effect, Li's Trichoderma strains Rut-C30 expresses, the activity of beta-glucosidase of gained transformant improves 6 to 8 times, and filter paper activity also improves 30%, and straw saccharification ability is remarkably improved.Zhang etc., by making bgl1 gene carry out process LAN under four copy cbh1 promoteres of transformation control, make beta-glucosidase and filter paper enzyme activity add 3.7 times and 1.3 times respectively.
The present invention, with trichoderma reesei RUT-C30 as starting strain, utilizes homologous recombination to obtain the trichoderma reesei mutant strain of a strain high-yield beta-glucosidase, and its activity of beta-glucosidase is 17.3 times of starting strain Rut-C30.Meanwhile, this strains expressed has gone out extremely strong glucose tolerance, is resistant to 225mM glucose.This bacterial strain saccharification of cellulose ability of 24 hours is the 120% of starting strain.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of High Cellulase Production trichoderma reesei engineering bacteria, to solve the problems such as poor growth that existing cellulase producing strain exists, inefficient and carbon metablism inhibitory action are stronger.
The present invention also to solve the technical problem that the construction method being to provide above-mentioned High Cellulase Production trichoderma reesei engineering bacteria.
The present invention finally to solve the technical problem that the application being to provide above-mentioned High Cellulase Production trichoderma reesei engineering bacteria.
For solving above-mentioned technical problem, the technical solution used in the present invention is as follows:
A kind of High Cellulase Production trichoderma reesei engineering bacteria, it is that process LAN bgl1 gene obtains in trichoderma reesei Rut-C30, and described bgl1 gene order is as shown in SEQ ID No.:1.
The construction method of above-mentioned High Cellulase Production trichoderma reesei engineering bacteria, it comprises the steps:
(1) design bgl1 gene amplification primer with the cDNA of trichoderma reesei Rut-C30 for masterplate, carry out PCR amplification；
(2) use the method for single endonuclease digestion homologous recombination to insert in plasmid products therefrom after PCR amplification in step (1), obtain expression cassette pBGL, and verify that this gene is correctly integrated by gene sequencing；
(3) being imported by the expression cassette pBGL obtained in step (2) in the spore of trichoderma reesei Rut-C30, screening obtains the transformant of stable heredity, i.e. High Cellulase Production trichoderma reesei engineering bacteria, named TRB1.
In step (1), in described bgl1 gene amplification primer, the nucleotides sequence of forward primer is classified as ACCCAATAGTCAATCTAGAATGCGTTACCGAACAGCAGC；The nucleotides sequence of downstream primer is classified as TCGGCATCTACTTCTAGATTAATGATGATGATGATGATGCGCTACCGACAGAGTGC TCG.
In step (1), when carrying out PCR amplification, denaturation condition is 95 DEG C of reaction 15s, and Denaturing is 95 DEG C of reaction 15s, and annealing conditions is 66 DEG C of reaction 30s, and extension condition is 72 DEG C of reaction 3min, and thorough extension condition is 72 DEG C of reaction 5min.
In step (2), described plasmid is pDHt/sk.
In step (3), it is agrobacterium-mediated transformation by the method that expression cassette pBGL imports trichoderma reesei Rut-C30.
In step (3), the method of the transformant that screening obtains stable heredity is: after expression cassette pBGL imports the spore of trichoderma reesei Rut-C30, screening culture medium is cultivated and within 5 days, obtains transformant, transformant is carried out 3 again and takes turns stable Secondary Culture so that bgl1 stably process LAN in transformant, obtain the transformant of stable heredity；
Wherein, the formula of described screening culture medium is: the PDA culture medium containing corresponding selection markers.
The application in terms of cellulose hydrolysis of the above-mentioned High Cellulase Production trichoderma reesei engineering bacteria is also within protection scope of the present invention.
Wherein, described application process is, is seeded in culture medium by the High Cellulase Production trichoderma reesei engineering bacteria described in claim 1, can produce cellulolytic enzyme.
Wherein, by the method that High Cellulase Production trichoderma reesei engineering bacteria is seeded to culture medium it is: after High Cellulase Production trichoderma reesei engineering bacteria is cultivated 5～10 days in PDA solid medium at 25～30 DEG C, take spore inoculating to activate after 24～50 hours in SDB culture medium, inoculate in the TMM+2% microcrystalline Cellulose culture medium containing glucose and cultivate 5～10 days.
Wherein, the method that High Cellulase Production trichoderma reesei engineering bacteria is seeded to culture medium is preferably: after being cultivated 7 days at 28 DEG C in PDA solid medium by High Cellulase Production trichoderma reesei engineering bacteria, take spore inoculating to activate after 30 hours in SDB culture medium, inoculate in the TMM+2% microcrystalline Cellulose culture medium containing glucose and cultivate 7 days.
Wherein,
The preparation method of PDA solid medium is: takes the 1L that adds water after peeled potatoes 200g is cut into small pieces, goes filtrate after boiling 30 points, melts subpackage after adding glucose 20g and agar 15g, and in 115 DEG C of sterilizings 15 minutes.
The formula of SDB culture medium is: glucose 4g, yeast extract 1g, peptone 1g, water 1L.
The formula of the TMM+2% microcrystalline Cellulose culture medium containing glucose is: (NH4)SO4 5g、KH2PO4 15g、 MgS04 0.6g、CaCl2 0.6g、FeS04·7H2O 0.005g、MnSO4 0.0016g、ZnSO4·7H2O 0.0014g、COCl20.002g, glucose 10～50g, 20g microcrystalline Cellulose, water 1L.
Wherein, described cellulolytic enzyme includes beta-glucosidase, endoglucanase, exoglucanase and filter paper enzyme activity.
Wherein,
Live for improving the enzyme of beta-glucosidase, concentration of glucose preferably 10～50g in TMM+2% microcrystalline Cellulose culture medium.
Live for improving the enzyme of endoglucanase, concentration of glucose preferably 10～50g in TMM+2% microcrystalline Cellulose culture medium.
Live for improving the enzyme of exoglucanase, concentration of glucose preferably 10～50g in TMM+2% microcrystalline Cellulose culture medium.
Live for improving the enzyme of Filter paperlyase, concentration of glucose preferably 10～30g in TMM+2% microcrystalline Cellulose culture medium.
Beneficial effect: the present invention utilizes homologous recombination to obtain the genetic engineering bacterium of high-yield beta-glucosidase.The endoglucanase of this engineering bacteria, exoglucanase and filter paper enzyme activity increase the most accordingly, and in addition its cellular morphology and endoplasmic reticulum form the most do not change, and have stronger tolerance to glucose.
Compared with prior art, the present invention has the advantage that
(1) the outer beta-glucosidase of the born of the same parents that TRB1 produces improves 17.3 times relative to starting strain Rut-C30.While improving beta-glucosidase, endoglucanase, exoglucanase and filter paper enzyme activity and protein content have obtained a certain degree of raising and maintenance.
(2) this strains expressed goes out the glucose tolerance of 550mM.
(3) more preferable saccharification capability is shown.
Accompanying drawing explanation
Fig. 1 is pBGL expression cassette plasmid map in embodiment 1；
Fig. 2 is transformant screening result in embodiment 1；
Fig. 3 is that in embodiment 1, transformant TRB1～5 is lived and the comparison diagram of protein content with the Rut-C30 enzyme after 7 days that ferments；
Fig. 4 is the supernatant enzyme comparison diagram alive of TRB1 and Rut-C30 in embodiment 2；
Fig. 5 A is TRB1 and Rut-C30 supernatant activity of beta-glucosidase in different glucose culture medium in embodiment 3；
Fig. 5 B is TRB1 and Rut-C30 supernatant exoglucanase activity in different glucose culture medium in embodiment 3；
Fig. 5 C is TRB1 and Rut-C30 supernatant endoglucanase activity in different glucose culture medium in embodiment 3；
Fig. 5 D is that in embodiment 3, in different glucose culture medium, TRB1 and Rut-C30 supernatant filter paper enzyme activity is active；
Fig. 6 A is in embodiment 4 under A group condition of culture, the crude enzyme liquid hydrocellulose ability detection figure of TRB1 and Rut-C30；
Fig. 6 B is in embodiment 4 under B group condition of culture, the crude enzyme liquid hydrocellulose ability detection figure of TRB1 and Rut-C30；
Fig. 6 C is in embodiment 4 under C group condition of culture, the crude enzyme liquid hydrocellulose ability detection figure of TRB1 and Rut-C30；
Fig. 7 A is the confocal microscope figure of the endoplasmic reticulum of Rut-C30 in embodiment 5；
Fig. 7 B is the confocal microscope figure of the endoplasmic reticulum of TRB1 in embodiment 5；
Fig. 8 A is the confocal microscope figure of the cell membrane of Rut-C30 in embodiment 5；
Fig. 8 B is the confocal microscope figure executing the cell membrane of TRB1 in example 5；
Fig. 9 A is that the mycelium pellet of Rut-C30 in embodiment 6 forms state；
Fig. 9 B is that the mycelium pellet of TRB1 in embodiment 6 forms state.
Detailed description of the invention
According to following embodiment, the present invention be may be better understood.But, as it will be easily appreciated by one skilled in the art that the content described by embodiment is merely to illustrate the present invention, and should be also without limitation on the present invention described in detail in claims.
Embodiment 1
The structure of pBGL expression cassette, comprises the following steps:
(1) design of bgl1 gene amplification primer is shown in Table one.With the cDNA of trichoderma reesei Rut-C30 as masterplate, with this primer amplification bgl1 gene.Amplification condition is denaturation: 95 DEG C, 15s；Degeneration: 95 DEG C, 15s；Annealing: 66 DEG C, 30s；Extend: 72 DEG C, 3min；Thoroughly extend: 72 DEG C, 5min；
(2) purified genes of interest PCR primer uses single endonuclease digestion homologous recombination to be inserted on the XbaI enzyme cutting site of plasmid pDht/sk acquisition expression cassette pBGL, sees Fig. 1；
(3) use agriculture bacillus mediated way that this expression cassette is transferred to concrete grammar in trichoderma reesei spore as follows:
Agrobacterium tumefaciens transformation:
The a.-70 DEG C of Agrobacterium tumefaciems kept is placed in and hatches 10min on ice；
B. toward the plasmid of addition 0.5-1 μ g, ice bath 30min in Agrobacterium tumefaciems；
C. liquid nitrogen 5min, 37 DEG C of 5min, ice bath 2min；
D. after adding liquid LB, 200rpm, the 28 DEG C of cultivation 3h of 1ml, centrifugal collection thalline, then wash once；
E. with the 100 μ l resuspended thalline of liquid LB and be uniformly coated in LB resistant panel (Carbenicillin containing 50 μ g/ml and the kanamycin of 50 μ g/ml), cultivate two days for 28 DEG C.
The conversion of Agrobacterium tumefaciens mediated trichoderma reesei
A. Filamentous fungi spore requires fresh preparation, and the Tween-80 water of 0.02% washes lower spore from flat board, concentration about 1x106/ml；
B. Agrobacterium: in the LB fluid medium of the kanamycin that first mono-for Agrobacterium AGL1 bacterium colony (containing binary vector) is inoculated in Carbenicillin that 3ml contains 50 μ g/ml and 50 μ g/ml, 200rpm, 28 DEG C of incubated overnight are 0.6 to OD660, it is then centrifuged for collecting thalline and with appropriate IM fluid medium (acetosyringones containing 200 μMs) resuspended thalline, and bacterial concentration is transferred to OD660 is 0.15, then 200rpm, 28 DEG C of cultivations are 0.5-0.8 to OD660.After being mixed by the spore of 100 μ l agrobacterium liquids and the trichoderma reesei of 100 μ l, being evenly coated on solid IM (acetosyringones containing 200 μMs) flat board, 26 DEG C of lucifuges cultivate 48h.After the mixture co-cultured two days later is scraped with the Tween-80 water of 0.02%, it is evenly coated in containing 600 μMs of cefotaxime (cefotaxime, to kill agrobatcerium cell), on the PDA plate of 0.1%Triton X-100 and corresponding antibiotic, cultivate one week for 28 DEG C.Obtain transformant, see Fig. 2；
(4) transformant obtained in (3) step is carried out the 3 stable heredity taken turns, after activating 30h in SDB, be inoculated in TMM+2% microcrystalline Cellulose culture medium and carry out the detection that enzyme is lived, see Fig. 3.
Embodiment 2
By each for trichoderma reesei bacterial strain at the cellulose culture media shaking vase cultivation supernatant of 7 days through 4 DEG C, 8000rpm, 15min are centrifuged the mensuration collecting supernatant for cellulase activity.Cellulase activity assay method is as follows:
(1) beta-glucosidase (pNPGase) enzyme activity determination: the enzyme liquid supernatant 20 μ l suitably diluted is added pNPG (the 50mM NaCl of 90 μ l 4mM, pH5.0), 50 DEG C of insulation 10min, draw 100 μ l and add isopyknic 2%NaCO3, it is placed in ELISA Plate and reads OD405 (Biochem-Tokyo 1999,125 (4): 728-736).
(2) exoglucanase (pNPC) enzyme activity determination: pNPC solution (the 0.05M NaAc of 90 μ l 4mM, pH5.0,1mg/ml 1,5-δ-gluconic acid lactone) add the enzyme liquid of the 20 suitable extension rates of μ l, 50 DEG C of insulation 30min, draw 100 μ l and add isopyknic 2%NaCO3, be placed in ELISA Plate reading OD405 (Anal Biochem 1984,138 (2): 481-487)
(3) endoglucanase (CMCase) enzyme activity determination: take the enzyme liquid 30 μ l of certain extension rate, add 30 μ l CMC (PH4.8,50mM NaAc, 2%), 50 DEG C of insulation 30min, it is subsequently adding the DNS of 120 μ l, 95 DEG C of insulation 5min, takes out 36 μ l afterwards and be added to read in the ELISA Plate equipped with 160 μ l ultra-pure waters the numerical value of OD540.One enzyme unit (IU) alive is defined as: the enzyme amount (Anal Biochem 2005,342 (1): 176-178) needed for interior release 1 μm ol reducing sugar per minute.
null(4) filter paper enzyme activity (FPase) measures: take the fermented liquid supernatant of the 20 certain extension rates of μ l，Add 40 μ l sodium-acetate buffer (50mM，pH4.8)，Join after mixing in the hole of the PCR plate being placed with little filter paper，50 DEG C are incubated one hour，It is subsequently adding the DNS of 120 μ l，95 DEG C of insulation 5min，Take out 36 μ l afterwards and be added to ELISA Plate (the Greiner bio-one equipped with 160 μ l ultra-pure waters,Frickenhausen,Germany) inner at microplate reader Varioskan Flash microplate reader (Thermo electron,Finland) numerical value (the Method Enzymol 1988 of OD492 is read,160:87-112).Measurement result is shown in Fig. 4.
Embodiment 3
Each for trichoderma reesei bacterial strain is collected supernatant by centrifugation at the supernatant that the cellulose culture media shaking vase adding different glucose is cultivated 7 days and is used for the mensuration of cellulase activity.Cellulase activity assay method is with reference to embodiment 3, and measurement result is shown in Fig. 5.
Embodiment 4
The culture supernatant obtained under different condition is for hydrocellulose, the cellulose hydrolysis efficiency of detection trichoderma reesei TRB1 institute cellulase-producing and substrate specificity, concrete grammar is as follows: weigh corn straw (EDAPCS) (this hydrocellulose is provided) 75mg/ml of ethylenediamine process by University Of Tianjin teacher Li Bingzhi in centrifuge tube, it is separately added into 100mM sodium acetate buffer (pH5.0) (A group) to each pipe, the Hydrazoic acid,sodium salt (B group) of 0.2mg/ml and the fermented supernatant fluid (C group) of 150ug/ml, in 50 DEG C, react 72 hours under the conditions of 200rpm, within every 24 hours, measure a glucose content.Result is shown in Fig. 6.In Fig. 6, abscissa C30 represents that Rut-C30 cultivates in the TMM culture medium containing microcrystalline Cellulose, abscissa TRB1 represents that TRB1 cultivates in the TMM culture medium containing microcrystalline Cellulose, abscissa C30-1%Glu represents that Rut-C30 cultivates in the TMM culture medium containing microcrystalline Cellulose and 1% glucose, and abscissa TRB1-1%Glu represents that TRB1 cultivates in the TMM culture medium containing microcrystalline Cellulose and 1% glucose.As can be seen from Figure, the engineering bacteria cultivated in the TMM culture medium comprise only microcrystalline Cellulose exceeds 18% at the hydrolysis efficiency of 24h than Rut-C30, and the engineering bacteria cultivated in the TMM culture medium containing microcrystalline Cellulose and 1% glucose exceeds 35% at the hydrolysis efficiency of 24h than Rut-C30.
Embodiment 5
The red ER-tracker of each bacterial strain of 3 days is cultivated at inducing culture, FITC dyes, observe under Laser Scanning Confocal Microscope, concrete grammar is as follows: takes cultured bacterium solution 8000rpm and is centrifuged 5min, add HBSS with Ca2+&Mg2+ (Hanks ' Balanced Salt Solution with Ca2+&Mg2+) buffer to be centrifuged again twice, removal cleaning mixture takes a small amount of ER-Tracker Red and joins in ER-Tracker Red diluent according to the ratio of 1:1000, that prepare and 37 DEG C of precincubation ER-Tracker Red dyeing working solution, 30 minutes are hatched altogether with 37 DEG C of cell.Laser co-focusing observed result is shown in Fig. 7 and Fig. 8.
Embodiment 6
In the TMM culture medium be added with glucose, cultivation trichoderma reesei Rut-C30 and TRB1 (according to 5% inoculum concentration inoculation) is after 3 days, observes its mycelium pellet and forms state, and result is shown in Fig. 9.
Claims (8)
1. a High Cellulase Production trichoderma reesei engineering bacteria, it is characterised in that it is in trichoderma reesei Rut-C30
Process LAN bgl1 gene obtains, and described bgl1 gene order is as shown in SEQ ID No.:1.
2. the construction method of the High Cellulase Production trichoderma reesei engineering bacteria described in claim 1, it is characterised in that it
Comprise the steps:
(1) design bgl1 gene amplification primer with the cDNA of trichoderma reesei Rut-C30 for masterplate, carry out PCR amplification；
(2) method of single endonuclease digestion homologous recombination is used to insert in plasmid products therefrom after PCR amplification in step (1),
Obtain expression cassette pBGL；
(3) the expression cassette pBGL obtained in step (2) being imported trichoderma reesei Rut-C30, screening obtains stable something lost
The transformant passed, i.e. High Cellulase Production trichoderma reesei engineering bacteria.
Construction method the most according to claim 2, it is characterised in that in step (1), described bgl1 gene
In amplimer, the nucleotides sequence of forward primer is classified as ACCCAATAGTCAATCTAGAATGCGTTACCGA
ACAGCAGC；The nucleotides sequence of downstream primer is classified as TCGGCATCTACTTCTAGATTAATGATGATGA
TGATGATGCGCTACCGACAGAGTGCTCG。
Construction method the most according to claim 2, it is characterised in that in step (2), described plasmid is pDHt/sk.
5. the application in terms of cellulose hydrolysis of the High Cellulase Production trichoderma reesei engineering bacteria described in claim 1.
Application the most according to claim 5, it is characterised in that described application process is, by claim 1
Described High Cellulase Production trichoderma reesei engineering bacteria is seeded in culture medium, can produce cellulolytic enzyme.
Application the most according to claim 5, it is characterised in that High Cellulase Production trichoderma reesei engineering bacteria is connect
Kind to the method for culture medium be: by High Cellulase Production trichoderma reesei engineering bacteria in PDA solid medium in 25～30 DEG C
Lower cultivation 5～after 10 days, takes spore inoculating after SDB culture medium activates 24～50 hours, inoculates TMM and cultivate
Base is cultivated 5～10 days.
Application the most according to claim 5, it is characterised in that described cellulolytic enzyme includes β-glucose
Glycosides enzyme, endoglucanase, exoglucanase and Filter paperlyase.
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| CN108949725B (en) * | 2018-08-17 | 2022-01-21 | 杭州园泰生物科技有限公司 | Production and purification process of complex enzyme preparation |
| CN109852554A (en) * | 2018-12-25 | 2019-06-07 | 山东大学 | The trichoderma reesei engineering bacteria and its construction method of one plant of endoglucanase and the double hypersecretions of beta-glucosidase and application |
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| CN113106114A (en) * | 2020-01-13 | 2021-07-13 | 中国科学院分子植物科学卓越创新中心 | Factor for regulating and controlling trichoderma reesei protein expression efficiency, regulating and controlling method and application |
| CN113106114B (en) * | 2020-01-13 | 2024-04-12 | 中国科学院分子植物科学卓越创新中心 | Factor for regulating expression efficiency of trichoderma reesei protein, regulating method and application |
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| CN116334111B (en) * | 2023-03-03 | 2024-02-09 | 上海市农业科学院 | Straw mushroom cellobiose hydrolase and application thereof |
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