CN107012102A - The trichoderma reesei genetic engineering bacterium of one plant of High Cellulase Production in the case where soluble and non-solubility carbon source is induced and construction method and application - Google Patents
The trichoderma reesei genetic engineering bacterium of one plant of High Cellulase Production in the case where soluble and non-solubility carbon source is induced and construction method and application Download PDFInfo
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- CN107012102A CN107012102A CN201710024825.0A CN201710024825A CN107012102A CN 107012102 A CN107012102 A CN 107012102A CN 201710024825 A CN201710024825 A CN 201710024825A CN 107012102 A CN107012102 A CN 107012102A
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- Prior art keywords
- trichoderma reesei
- carbon source
- genetic engineering
- cellulase
- engineering bacterium
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to genetic engineering and field of microbial fermentation, disclose the trichoderma reesei genetic engineering bacterium SEU 7 of one plant of High Cellulase Production in the case where soluble and insolubility carbon source is induced, its Classification And Nomenclature is Trichoderma reesei, bacterial strain SEU 7, it has been preserved in Chinese Typical Representative Organism Depositary, deposit number is CCTCC M 2016492, and preservation date is September in 2016 18.The application of cellulase is produced the invention also discloses the construction method of the genetic engineering bacterium and its under different carbon source induction.Compared with prior art, the present invention constructs the trichoderma reesei genetic engineering bacterium of High Cellulase Production using homologous recombination technique, and it shows the ability of high cellulase-producing under the induction of soluble carbon source and non-solubility carbon source,.Meanwhile, institute's cellulase-producing liquid of the present invention shows the more preferable saccharification capability of cellulase solution than Rut C30.
Description
Technical field
The invention belongs to biological technical field, and in particular to one plant of high yield fibre under the induction of soluble and non-solubility carbon source
The trichoderma reesei genetic engineering bacterium of the plain enzyme of dimension and its construction method and application.
Background technology
Cellulose is the macromolecular polysaccharide polymer being widely present in nature.Cellulose can be dropped using cellulase
Solution can ferment the carbohydrate utilized into bacterium to produce bio-fuel, bio-based fine chemical product and medicine.Cellulase is
The complicated enzymatic mixture of one class, mainly comprising endoglucanase, exoglucanase and beta-glucosidase.Cellulose it is thorough
Bottom degraded needs the synergy of these three enzymes.Endoglucanase acts on the noncrystalline domain of cellulose, by cellulose degradation
Into the cell-oligosaccharide of different length, then cell-oligosaccharide is degraded into the presence of cellobiohydrolase is exoglucanase
Cellobiose, last cellobiose is hydrolyzed into the glucose of fermentability in the presence of beta-glucosidase.
Trichoderma reesei is the microbial bacteria of manual security because that can produce a large amount of (up to 100g/L) extracellular cellulase
Kind, become important cellulase industrial producing strain (Journal of biotechnology, 1994,37 (3):193-
200).However, only a small amount of beta-glucosidase (BGL1) be secreted into it is extracellular, and industrially collect cellulase be receipts
Collect the cellulase in supernatant, it is impossible to while collecting the beta-glucosidase (BGL1) of intracellular, cause gained cellulase solution
In beta-glucosidase (BGL1) content it is very low, it is impossible to abundant degraded cellulose.Trichoderma reesei cellulase belongs to induction
Type enzyme.01 using cellulose as substrate when can promote the generation of cellulase.But cellulose may not be fiber in itself
The real inducer of plain enzyme, because the cellulose matter of this insolubility can not be entered in fungal cell.Cellulose is not
It is soluble so that the production of cellulase becomes complicated with separating, therefore using the cellulose cellulase induction of insolubility
Produce unsatisfactory in industrial production.Thus, soluble inducer is more concerned, as cellobiose, lactose, sophorose etc. all
It is soluble cellulose enzyme induction thing.Among them, lactose is that most important and with economic feasibility cellulase is lured
Thing is led, but inducing effect is not so good as cellulose.This, which promotes people to strive to find, a kind of can improve the gene work of lactose induced efficiency
Journey bacterium.
Li's Trichoderma strains, which are transformed, by metabolic engineering and genetic engineering means obtains High Cellulase Production genetic engineering
Superior strain is focus of concern under bacterium and the soluble inducer induction of searching.Early in the 1970s, scientist
Using the method for random mutation superior strain QM9414 and RUT-C30 are obtained to QM6a.Rut-C30 is one plant with weaker
The beta-glucosidase superior strain that carbon metablism suppresses, while its extracellular cellulase aggregate level improves 150 times than wild mushroom,
But its slow-growing, raw spore delay, raw spore are reduced.In order to obtain High Cellulase Production bacterial strain, scientists are by trichoderma reesei
Cellulose enzyme gene be overexpressed in other microorganisms, such as saccharomycete (Molecular biotechnology, 2013,54
(2):158-169.Metabolic engineering,2007,9(1):87-94), bacterium (Biotechnology
letters,2012,34(1):91-96；Humana press,1996:389-397), and plant (Journal of
biotechnology,2007,131(3):362-369), but yield of cellulase is all relatively low.1987Et al. report
Road in trichoderma reesei heterogenous expression outer source pollution gene to improve yield of cellulase (Yeast, 1987,3:175-
185).At present, also achieved necessarily by heterologous or homologous overexpression beta-glucosidase with obtaining High Cellulase Production bacterial strain
Progress.The beta-glucosidase gene that Ma et al. originates penicillium decumbens is under the effect of CBH1 promoters, in Li's Trichoderma strains
Expressed in Rut-C30, the activity of beta-glucosidase of gained transformant improves 6 to 8 times, and filter paper activity also improves 30%, straw
Stalk saccharification capability is remarkably improved (Enzyme and microbial technology, 2011,49 (4):366-371).
Zhang etc. makes beta-glucosidase by making bgl1 genes be overexpressed under four copy cbh1 promoter controls of transformation
Add 3.7 times and 1.3 times (Bioresource technology, 2010,101 (24) respectively with filter paper enzyme activity:9815-
9818)。
Forefathers' research shows sophorose (Journal of bacteriology, 1979,139 (3):761-769), fiber two
Sugar (Journal of bacteriology, 1960,79 (6):816), galactolipin (Fungal biology reviews,
2007,21(1):42-48), L- sorboses and lactose (Fungal Biology Reviews, 2007,21 (1):42-48) all
Can be with the generation of cellulase induction, but inducing effect is all less desirable.Sophorose had been considered as once induction trichoderma reesei
Optimal inducer (Journal of bacteriology, 1979,139 (3) that cellulase is produced:761-769), but it not
Can be by other fungies such as Phanerochaete chrysosporium and Aspergillus purpurogenum (Springer
berlin heidelberg,1992:1-27) utilize and produced cellulase.In addition, sophorose price is costly, serious limit
Its large-scale application is industrially made.Lactose is a kind of cheap carbon source as a kind of accessory substance of dairy industry, can be by
Multiple-microorganism utilizes such as bacterium containing lactose operon and some species of yeast.Now, lactose turns into industrial induction
Trichoderma reesei produces a kind of conventional soluble inducer of cellulase, but induced efficiency is nothing like cellulose.In order to
Overcome the shortcoming of lactose inducing effect difference, improving fermentation process or transformation bacterial strain makes trichoderma reesei make it under lactose inductive condition
High Cellulase Production is very necessary.
The present invention obtains one plant with insolubility carbon using trichoderma reesei RUT-C30 as starting strain using homologous recombination
Source and the trichoderma reesei genetic engineering bacterium SEU-7 that soluble carbon source is inducer High Cellulase Production.SEU-7, which ferments 7 days, to be produced
Extracellular beta-glucosidase be 37.9 times of starting strain Rut-C30, pNPCase is 8.7 times of starting strain Rut-C30,
CMCase is 2.6 times of starting strain Rut-C30, and FPase is 1.1 times of starting strain Rut-C30.Under lactose induction,
SEU-7 beta-glucosidase is 153 times of starting strain Rut-C30, and pNPCase is 14 times of starting strain Rut-C30,
CMCase is 9 times of starting strain Rut-C30, and FPase is 1.7 times of starting strain Rut-C30.SEU-7 bacterial strains 24h fiber
Plain saccharification capability improves nearly 50% relative to starting strain RUT-C30.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of trichoderma reesei base of the High Cellulase Production under carbon source induction
Because of engineering bacteria, yielded poorly and soluble inducer induction cellulase-producing production with solving the beta-glucosidase of prior art presence
Measure low problem.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of trichoderma reesei genetic engineering bacterium of the High Cellulase Production in the case where soluble and non-solubility carbon source is induced, its point
Class is named as Moniliales (Moniliales) trichoderma (Penicillium) filamentous fungus T. reesei (Trichoderma
Reesei), bacterial strain SEU-7, has been preserved in Chinese Typical Representative Organism Depositary, and address is Wuhan University of Wuhan, China city,
Postcode is 430072, and deposit number is CCTCC M 2016492, and preservation date is September in 2016 18.
Wherein, above-mentioned trichoderma reesei genetic engineering bacterium SEU-7 is to be overexpressed bgl1-his in trichoderma reesei Rut-C30
Gene is obtained, described bgl1-his gene orders such as SEQ ID No.：Shown in 1.
Wherein, above-mentioned trichoderma reesei genetic engineering bacterium SEU-7 construction method comprises the following steps：
(1) extract trichoderma reesei Rut-C30 RNA and carry out reverse transcription.
(2) bgl1-linker-his gene magnification primers are designed by masterplate of trichoderma reesei Rut-C30 cDNA, are carried out
PCR is expanded.
(3) products therefrom after PCR amplifications in step (2) is inserted to the XbaI enzyme cutting position of plasmid using the method for homologous recombination
In point, recombinant plasmid pBGL-his is obtained, verifies that the gene is correctly integrated by gene sequencing afterwards.
(4) the recombinant plasmid pBGL-his obtained in step (3) is imported in trichoderma reesei Rut-C30 spore, screening
The transformant of the High Cellulase Production of 5 plants of stable heredity is obtained, SEU-2, SEU-5, SEU-6, SEU-7 and SEU- is respectively designated as
8。
(5) the hereditary transformant of the stabilization to acquisition carries out 3 wheels stable Secondary Culture pBGL-his is being converted
Stably it is overexpressed in son, carries out the detection of cellulase activity and protein content to transformant afterwards.
(6) genetic engineering bacterium and starting strain in PDA solid mediums in 28 degree culture 7 days after, take spore inoculating in
SDB culture mediums activate 30h, are inoculated into the TMM culture mediums containing different inducers and cultivate 7 days, determine cellulase activity
And protein content.The cellulase highest that wherein SEU-7 is either produced under cellulose, lactose or glucose induction.
In step (1), extract and reverse transcription process can according to Omega Fungal RNA kit specifications and
Takara reverse transcription reagent box is carried out.
Described linker sequence is catcatcatcatccgggcggcagcgtgaaaaaacgc.
In step (2), in described bgl1-linker-his gene magnification primers,
The nucleotides sequence of sense primer is classified as：acccaatagtcaatctagaatgcgttaccgaacagcagc；
The nucleotides sequence of anti-sense primer is classified as:tcggcatctacttctagattagtggtggtggtggtggtggcgtttt
ttcacgctgccgcccggatgatgatgatgcgctaccgacagagtgctcg。
In step (3), described plasmid is pDHt/sk.
In step (4), the method that screening obtains the transformant of stable heredity is：Recombinant plasmid pBGL-his is imported into Richter scale
After trichoderma Rut-C30 spore, culture obtains transformant for 5 days in screening and culturing medium, then transformant is carried out into the stable biography of 3 wheels
It is commissioned to train and supports so that bgl1 is stably overexpressed in transformant, to produce the transformant of stable heredity.
Wherein, the formula of the screening and culturing medium is：PDA culture medium containing corresponding selection markers.
The application of above-mentioned trichoderma reesei genetic engineering bacterium SEU-7 production cellulases under carbon source induction is also the present invention's
Within protection domain.
Wherein, described carbon source includes insolubility carbon source and soluble carbon source；Wherein, described insolubility carbon source is
Cellulose, described soluble carbon source is lactose, glucose, cellobiose or glycerine.
Wherein, described cellulase includes beta-glucosidase, endoglucanase, exoglucanase, Filter paperlyase
To hemicellulase and the enzyme of other related degraded celluloses, lignin and hemicellulose.
Wherein, described application process is, described trichoderma reesei genetic engineering bacterium SEU-7 is seeded to using carbon source to lure
Lead in the culture medium of thing, cellulolytic enzyme can be produced.
Wherein, specific method is：By described trichoderma reesei genetic engineering bacterium SEU-7 in PDA solid mediums in 25
At~30 DEG C cultivate 5~10 days after, take spore inoculating in SDB culture mediums activate 24~50 hours after, inoculate using carbon source as
Cultivated 5~10 days in the TMM culture mediums of inducer.
Wherein,
The formula of PDA solid mediums is：Add water 1L after 200g peeled potatoes are cut into small pieces, and is gone after boiling 30 minutes
Melt packing after filtrate, plus 20g glucose and 15g agar, and sterilized 15 minutes in 115 DEG C.
The formula of SDB culture mediums is：4g glucose, 1g yeast extracts and 1g peptones are dissolved in 100mL water.
TMM culture medium prescriptions are：(NH4)SO4 5g、KH2PO4 15g、MgS04 0.6g、CaCl2 0.6g、FeS04·
7H2O0.005g、MnSO4 0.0016g、ZnSO4·7H2O 0.0014g、COCl20.002g, 10~50g of carbon source, water 1L.
Beneficial effect：
Compared with prior art, the present invention has following advantage：
1st, the present invention constructs the trichoderma reesei genetic engineering bacterium SEU-7 of High Cellulase Production using homologous recombination technique,
The trichoderma reesei genetic engineering bacterium SEU-7 shows high production under the induction of soluble carbon source and non-solubility carbon source,
The ability of cellulase.Specifically, under cellulose induction, the extracellular beta-glucosidase enzyme activity of SEU-7 institutes, CMCase,
PNPCase and Fpase are 37.9 times, 8.7 times, 2.6 times and 1.1 times of Li's Trichoderma Rut-C30 respectively；In lactose induction
Under, SEU-7 extracellular beta-glucosidase, pNPCase, CMCase and FPase be Li's Trichoderma Rut-C30 respectively
153 times, 14 times, 9 times and 1.7 times.
2nd, trichoderma reesei genetic engineering bacterium SEU-7 of the invention shows saccharification capability more more preferable than Rut-C30, is saccharified
Ability improves 63%.
3rd, trichoderma reesei genetic engineering bacterium SEU-7 of the invention can produce cellulase and Rut-C30 can not.
Brief description of the drawings
Fig. 1 is bgl1-his recombinant plasmid collection of illustrative plates in embodiment 1.
Fig. 2 is transformant screening result in embodiment 1.
Fig. 3 is the supernatant cellulase activity comparison diagram after SEU-7 and Rut-C30 are cultivated 7 days in embodiment 1；It is wherein horizontal
Coordinate represents different transformants, and BGL represents beta-glucosidase, and CBH represents exoglucanase, and CMC represents inscribe Portugal and gathered
Carbohydrase, FPA represents filter paper enzyme activity.
Fig. 4 A are supernatant β-grape after being cultivated 7 days as inducer SEU-7 and Rut-C30 using lactose and cellulose respectively
Glucosides enzyme activity comparison diagram.
Fig. 4 B are the circumscribed Portugal of supernatant after being cultivated 7 days as inducer SEU-7 and Rut-C30 using lactose and cellulose respectively
Glycan enzyme activity comparison diagram；
Fig. 4 C are the supernatant inscribe Portugal after being cultivated 7 days as inducer SEU-7 and Rut-C30 using lactose and cellulose respectively
Glycan enzyme activity comparison diagram.
Fig. 4 D are the supernatant Filter paperlyase after being cultivated 7 days as inducer SEU-7 and Rut-C30 using lactose and cellulose respectively
Comparison diagram living.
Fig. 4 E are that the Supernatant protein after being cultivated 7 days as inducer SEU-7 and Rut-C30 using lactose and cellulose respectively contains
Measure comparison diagram.
Fig. 5 is the cellulase activity situation using glucose as inducer SEU-7.
Fig. 6 is the contrast for the reduced sugar that SEU-7 and Rut-C30 changes under different condition of culture between SEU-7 and Rut-C30
Figure.
Embodiment
The structure of the bgl1-his recombinant plasmids of embodiment 1
(1) bgl1-his gene magnifications primer is with reference to the bgl1-linker-his primers in the content of the invention (6).With Richter scale
Trichoderma Rut-C30 cDNA is that masterplate expands bgl1-his genes with the primer.Amplification condition is pre-degeneration：95 DEG C, 15s；Become
Property：95 DEG C, 15s；Annealing：66 DEG C, 30s；Extension：72 DEG C, 3min；Thoroughly extension：72 DEG C, 5min；
(2) purified target gene PCR primer is inserted into plasmid pDht/sk XbaI enzymes using single endonuclease digestion homologous recombination
Bgl1-his recombinant plasmids are obtained in enzyme site, Fig. 1 is seen；
(3) expression cassette is transferred in trichoderma reesei spore using agriculture bacillus mediated method, specific method is as follows：
Agrobacterium tumefaciens transformation：
The Agrobacterium tumefaciems of a.-70 DEG C of holding is placed in is incubated 10min on ice；
B. toward the plasmid of addition 0.5-1 μ g in Agrobacterium tumefaciems, ice bath 30min；
C. liquid nitrogen 5min, 37 DEG C of 5min, ice bath 2min；
D. after LB, 200rpm, the 28 DEG C of culture 3h of liquid for adding 1mL, thalline is collected by centrifugation, then wash once；
E. thalline is resuspended and is uniformly coated in LB resistant panels with 100 μ L liquid LB and (contain 50 μ g/mL carbenicillin
With 50 μ g/mL kanamycins), 28 DEG C are cultivated two days.
The conversion of Agrobacterium tumefaciens mediated trichoderma reesei
A. trichoderma reesei spore requires fresh preparation, and 0.02% Tween-80 water washes lower spore from flat board, and concentration is about
1x106/mL。
B. Agrobacterium:Agrobacterium AGL1 single bacterium colonies (containing binary vector) are inoculated in the carboxylic benzyl that 3mL contains 50 μ g/mL first
In the LB fluid nutrient mediums of penicillin and 50 μ g/mL kanamycins, 200rpm, 28 DEG C of incubated overnights to OD660For 0.6.Centrifugation
Collect thalline and thalline is resuspended with appropriate IM fluid nutrient mediums (acetosyringone for containing 200 μM).Bacterial concentration is transferred to
OD660For 0.15, then 200rpm, 28 DEG C of cultures to OD660For 0.5-0.8.By 100 μ L agrobacterium liquids and 100 μ L Richter scale wood
After mould spore is mixed, it is evenly coated on solid IM (acetosyringone for containing 200 μM) flat board, 26 DEG C of lucifuge culture 48h.Will be altogether
After the mixture of culture two days later is scraped with 0.02% Tween-80 water, it is evenly coated in containing 600 μM of CTXs
On (cefotaxime, to kill agrobatcerium cell), 0.1%Triton X-100 and corresponding antibiotic PDA plate, 28 DEG C
Culture one week.Transformant is obtained, Fig. 2 is seen.
(4) the stable heredity of 3 wheels is carried out to the transformant obtained in (3) step, activates after 30h, is inoculated in SDB
The detection of enzyme activity is carried out in TMM+2% celluloses or 2% lactose medium.As shown in figure 3, five plants of conversion daughter bacterias show height
In starting strain Rut-C30 cellulose enzyme activity.Wherein SEU-7 cellulase activity highest, BGL reaches 42IU/mL, CBH
It is 10IU/mL to reach 0.9IU/mL, CMC, and FPA is 4.8IU/mL.
Embodiment 2
By each bacterial strain of trichoderma reesei the culture 7 days of cellulose culture media shaking vase supernatant through 4 DEG C, 8000rpm, 15min
Supernatant, which is collected by centrifugation, is used for the measure of cellulase activity.Cellulase activity assay method is as follows：
(1) beta-glucosidase (pNPGase) enzyme activity determination：The μ L of enzyme liquid supernatant 20 suitably diluted are added into 90 μ l4mM
(50mM NaCl, pH 5.0), 50 DEG C of insulation 10min draw 100 μ L and add isometric 2%NaCO3, it is placed in ELISA Plate reading
Take OD405(Biochem-Tokyo,1999,125(4):728-736).All enzyme activity are all with every milliliter of hair of an enzyme-activity unit IU
Zymotic fluid is represented.One enzyme-activity unit (IU) is defined as：1 during with p- nitrobenzene-β-D- synthesis (pNPG) for substrate
Enzyme amount in minute needed for 1 μm of ol products p-nitrophenol (PNP) of release.
(2) exoglucanase (pNPCase) enzyme activity determination：90 μ L 4mM pNPC solution (0.05M NaAc, pH5.0,
1mg/mL 1,5- δ-glucolactone) add the 20 appropriate extension rates of μ l enzyme liquid, 50 DEG C insulation 30min, draw 100 μ L
Isometric 2%NaCO3 is added, is placed in ELISA Plate and reads OD405 (Analytical biochemistry1984,138
(2):481-487).All enzyme activity are all represented with every milliliter of zymotic fluid of an enzyme-activity unit IU.One enzyme-activity unit (IU) definition
For：During using p-nitrophenyl-beta-D- cellobiosides as substrate, in 1 μm of ol product p-nitrophenol of interior release per minute
(PNP) enzyme amount needed for.
(3) endoglucanase (CMCase) enzyme activity determination：The μ L of enzyme liquid 30 of certain extension rate are taken, 30 μ L CMC are added
Then (PH4.8,50mM NaAc, 2%), 50 DEG C of insulation 30min add 120 μ L DNS, 95 DEG C of insulation 5min, take out afterwards
36 μ L are added in the ELISA Plate equipped with 160 μ L ultra-pure waters and read OD540Numerical value.One enzyme-activity unit (IU) is defined as：With carboxylic first
In enzyme amount (the Analytical biochemistry needed for 1 μm of ol reduced sugar of interior release per minute when base cellulose is substrate
2005,342(1):176-178)。
(4) filter paper enzyme activity (FPase) is determined：The fermented liquid supernatant of the 20 certain extension rates of μ L is taken, 40 μ L sodium acetates are added
Buffer solution (50mM, pH 4.8), is added to after mixing and is placed with the hole of the PCR plate of small filter paper.50 DEG C of reaction 1h.It is subsequently added
120 μ L DNS, 95 DEG C of insulation 5min.Then 36 μ L are taken out afterwards is added to the ELISA Plate (Greiner equipped with 160 μ L ultra-pure waters
Bio-one, Frickenhausen, Germany) it is inner, in ELIASA Varioskan Flash microplate reader
(Thermo electron, Finland) reads OD492Numerical value (Method enzymol 1988,160:87-112).It is all
Enzyme activity is all represented with every milliliter of zymotic fluid of an enzyme-activity unit IU.One enzyme-activity unit (IU) is defined as：During using filter paper as substrate,
Enzyme amount needed for 1 μm of ol reduced sugar of interior release per minute.Measurement result is shown in Fig. 4 (A-D).After fermentation seven days, lactose and fiber
The beta-glucosidase of SEU-7 under element induction is respectively 60.6IU/mL and 50.3IU/mL, β-grape far above Rut-C30
Glycosidase (0.4 and 1.5IU/mL).PNPC, CMCase, filter paper enzyme activity and protein content difference of the SEU-7 under lactose induction
4th, 18.4,5.3IU/mL and 4.0mg/mL be respectively Rut-C30 8.7,2.6,1.1 and 2 times.SEU-7 is under cellulose induction
PNPC, CMCase, filter paper enzyme activity and protein content difference 1.3,8.6,4.9IU/mL and 5.3mg/mL, be Rut- respectively
C30 14,9,1.7 and 1.8 times.
Embodiment 3
The culture supernatant obtained under different condition is used for hydrocellulose, and detection trichoderma reesei SEU-7 produces cellulose
The cellulose hydrolysis efficiency and substrate specificity of enzyme, specific method are as follows：Weigh the maize straw (EDA-PCS) of ethylenediamine processing
(Biotechnology for biofuels,2015,8:1-15) 75mg/mL is separately added into 100mM in centrifuge tube to each pipe
The crude enzyme liquid of sodium acetate buffer (pH 5.0), 0.2mg/ml sodium azide and 150ug/mL.In 50 DEG C, 200rpm conditions
Lower reaction 72h.A content of reducing sugar is determined per 24h.As a result as shown in figure 5, producing 11mg/mL's after cellulose hydrolysis 72h
Glucose, is 2.6 times of the glucose that Rut-C30 is produced.
SEQUENCE LISTING
<110>Southeast China University
<120>The structure of the lower High Cellulase Production trichoderma reesei genetic engineering bacterium of lactose induction and its application
<130> SG20160809
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 2232
<212> DNA
<213>Bgl1 gene orders
<400> 1
atgcgttacc gaacagcagc tgcgctggca cttgccactg ggccctttgc tagggcagac 60
agtcactcaa catcgggggc ctcggctgag gcagttgtac ctcctgcagg gactccatgg 120
ggaaccgcgt acgacaaggc gaaggccgca ttggcaaagc tcaatctcca agataaggtc 180
ggcatcgtga gcggtgtcgg ctggaacggc ggtccttgcg ttggaaacac atctccggcc 240
tccaagatca gctatccatc gctatgcctt caagacggac ccctcggtgt tcgatactcg 300
acaggcagca cagcctttac gccgggcgtt caagcggcct cgacgtggga tgtcaatttg 360
atccgcgaac gtggacagtt catcggtgag gaggtgaagg cctcggggat tcatgtcata 420
cttggtcctg tggctgggcc gctgggaaag actccgcagg gcggtcgcaa ctgggagggc 480
ttcggtgtcg atccatatct cacgggcatt gccatgggtc aaaccatcaa cggcatccag 540
tcggtaggcg tgcaggcgac agcgaagcac tatatcctca acgagcagga gctcaatcga 600
gaaaccattt cgagcaaccc agatgaccga actctccatg agctgtatac ttggccattt 660
gccgacgcgg ttcaggccaa tgtcgcttct gtcatgtgct cgtacaacaa ggtcaatacc 720
acctgggcct gcgaggatca gtacacgctg cagactgtgc tgaaagacca gctggggttc 780
ccaggctatg tcatgacgga ctggaacgca cagcacacga ctgtccaaag cgcgaattct 840
gggcttgaca tgtcaatgcc tggcacagac ttcaacggta acaatcggct ctggggtcca 900
gctctcacca atgcggtaaa tagcaatcag gtccccacga gcagagtcga cgatatggtg 960
actcgtatcc tcgccgcatg gtacttgaca ggccaggacc aggcaggcta tccgtcgttc 1020
aacatcagca gaaatgttca aggaaaccac aagaccaatg tcagggcaat tgccagggac 1080
ggcatcgttc tgctcaagaa tgacgccaac atcctgccgc tcaagaagcc cgctagcatt 1140
gccgtcgttg gatctgccgc aatcattggt aaccacgcca gaaactcgcc ctcgtgcaac 1200
gacaaaggct gcgacgacgg ggccttgggc atgggttggg gttccggcgc cgtcaactat 1260
ccgtacttcg tcgcgcccta cgatgccatc aataccagag cgtcttcgca gggcacccag 1320
gttaccttga gcaacaccga caacacgtcc tcaggcgcat ctgcagcaag aggaaaggac 1380
gtcgccatcg tcttcatcac cgccgactcg ggtgaaggct acatcaccgt ggagggcaac 1440
gcgggcgatc gcaacaacct ggatccgtgg cacaacggca atgccctggt ccaggcggtg 1500
gccggtgcca acagcaacgt cattgttgtt gtccactccg ttggcgccat cattctggag 1560
cagattcttg ctcttccgca ggtcaaggcc gttgtctggg cgggtcttcc ttctcaggag 1620
agcggcaatg cgctcgtcga cgtgctgtgg ggagatgtca gcccttctgg caagctggtg 1680
tacaccattg cgaagagccc caatgactat aacactcgca tcgtttccgg cggcagtgac 1740
agcttcagcg agggactgtt catcgactat aagcacttcg acgacgccaa tatcacgccg 1800
cggtacgagt tcggctatgg actgtcttac accaagttca actactcacg cctctccgtc 1860
ttgtcgaccg ccaagtctgg tcctgcgact ggggccgttg tgccgggagg cccgagtgat 1920
ctgttccaga atgtcgcgac agtcaccgtt gacatcgcaa actctggcca agtgactggt 1980
gccgaggtag cccagctgta catcacctac ccatcttcag cacccaggac ccctccgaag 2040
cagctgcgag gctttgccaa gctgaacctc acgcctggtc agagcggaac agcaacgttc 2100
aacatccgac gacgagatct cagctactgg gacacggctt cgcagaaatg ggtggtgccg 2160
tcggggtcgt ttggcatcag cgtgggagcg agcagccggg atatcaggct gacgagcact 2220
ctgtcggtag cg 2232
Claims (10)
1. a kind of trichoderma reesei genetic engineering bacterium of the High Cellulase Production in the case where soluble and non-solubility carbon source is induced, it is classified
Trichoderma reesei, bacterial strain SEU-7 are named, Chinese Typical Representative Organism Depositary has been preserved in, deposit number is
CCTCC M 2016492, preservation date is September in 2016 18.
2. trichoderma reesei genetic engineering bacterium according to claim 1, it is characterised in that it is in trichoderma reesei Rut-C30
Middle overexpression bgl1-his genes are obtained, described bgl1-his gene orders such as SEQ ID No.：Shown in 1.
3. the construction method of the trichoderma reesei genetic engineering bacterium described in claim 2, it is characterised in that comprise the following steps：
(1) extract trichoderma reesei Rut-C30 RNA and carry out reverse transcription；
(2) bgl1-linker-his gene magnification primers are designed by masterplate of trichoderma reesei Rut-C30 cDNA, enter performing PCR expansion
Increase；
(3) products therefrom after PCR amplifications in step (2) is inserted in plasmid using the method for homologous recombination, obtains recombinant plasmid
pBGL-his；
(4) the recombinant plasmid pBGL-his obtained in step (3) is imported into trichoderma reesei Rut-C30, screening obtains stable heredity
Transformant, the i.e. lower High Cellulase Production of lactose induction trichoderma reesei genetic engineering bacterium, be named as SEU-7.
4. construction method according to claim 3, it is characterised in that in step (2), described linker sequence is
catcatcatcatccgggcggcagcgtgaaaaaacgc；In described bgl1-linker-his gene magnification primers,
The nucleotides sequence of sense primer is classified as：acccaatagtcaatctagaATGCGTTACCGAACAGCAGC；
The nucleotides sequence of anti-sense primer is classified as：
TCGGCATCTACTTCTAGATTAGTGGTGGTGGTGGTGGTGgcgttttttcacgctgccgcccggatgatgatga
tgCGCTACCGACAGAGTGCTCG。
5. construction method according to claim 3, it is characterised in that in step (3), described plasmid is pDHt/sk.
6. the trichoderma reesei genetic engineering bacterium described in claim 1 produces the application of cellulase under carbon source induction.
7. application according to claim 6, it is characterised in that described carbon source includes insolubility carbon source and soluble carbon
Source；Wherein, described insolubility carbon source is cellulose, and described soluble carbon source is lactose, glucose, cellobiose or sweet
Oil.
8. application according to claim 6, it is characterised in that described cellulase includes beta-glucosidase, inscribe
Dextranase, exoglucanase, Filter paperlyase and hemicellulase.
9. application according to claim 6, it is characterised in that described application process is：By described trichoderma reesei base
In being seeded to because of engineering bacteria SEU-7 using carbon source as the culture medium of inducer, cellulolytic enzyme can be produced.
10. application according to claim 9, it is characterised in that specific method is：By described trichoderma reesei genetic engineering
Bacterium SEU-7 after being cultivated 5~10 days at 25~30 DEG C, takes spore inoculating to activate 24 in SDB culture mediums in PDA solid mediums
After~50 hours, inoculate in the TMM culture mediums using carbon source as inducer and cultivate 5~10 days.
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| CN114107261A (en) * | 2021-10-27 | 2022-03-01 | 山东理工大学 | Method for producing cellulase by using trichoderma reesei through sophora fruit induction culture |
| CN116334111A (en) * | 2023-03-03 | 2023-06-27 | 上海市农业科学院 | Straw mushroom cellobiose hydrolase and application thereof |
| 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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