CN105802854A - Cellulase high-yielding bacterial strain and application thereof - Google Patents
Cellulase high-yielding bacterial strain and application thereof Download PDFInfo
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Abstract
The invention relates to a cellulase high-yielding bacterial strain and an application thereof. The high-yielding cellulase trichoderma reesei strain PPL3-1 is disclosed for the first time, and produced cellulase has significant advantages in both enzyme activity and yield. The cellulase yield of the bacterial strain is improved through further optimization.
Description
Technical field
The present invention relates to microbiological art, more particularly it relates to an cellulase high-yield and application thereof.
Background technology
Cellulose is the polymer that multiple glucose residue is formed by connecting with β-Isosorbide-5-Nitrae-glycosidic bond, is reproducible biomass resource the abundantest on the earth.It is raw material with lignocellulose, generates glucose with cellulase hydrolysis cellulose, and then fermentation becomes the important outlet of the problems such as reply world today energy crisis, environmental pollution for alcohol fuel.
Cellulase refers to change into cellulose the general name of a series of enzymes of glucose, mainly include endoglucanase (endo-β-1,4-glucanase, EC3.2.1.4), exoglucanase (exoglucanase, and beta-glucosidase (β-glucosidase, EC3.2.1.21) EC3.2.1.91).Endoglucanase acts on the inside of cellulose long-chain molecule by long fibre cutting short-forming fiber, exoglucanase acts on one end of cellulosic molecule, carrying out cutting in units of two glucose residues and generate cellobiose, beta-glucosidase cutting fibre disaccharide and some fibre oligosaccharide ultimately generate single glucose molecule.One of important composition as cellulose complex enzyme system, the pivotal role of beta-glucosidase is mainly reflected in two aspects: on the one hand, owing to the activity of cellobiose accumulation trip endoglucanase and exoglucanase on which has significant feedback inhibition, therefore, cellulosic thorough degraded is played vital effect by the effectively hydrolyzing ability of cellobiose by beta-glucosidase.On the other hand, except hydrolysing activity, beta-glucosidase also has transglycosylation, can pass through transglucosidation under certain conditions and two glucose molecules synthesize a sophorose molecule, and have been found that sophorose is the strong inducer that cellulose enzyme gene is expressed.It is generally acknowledged that the abduction mechanism that filamentous fungi cellulase synthesizes is: a small amount of composing type cellulase first degraded cellulose being present in conidium and hyphal surface generates the oligosaccharide such as cellobiose, under the transglucosidation of the glucosidase then combined at plasma membrane, generate the inducers such as sophorose, composing type permease system on cell membrane is entered in people's cell, starts the synthesis of cellulase.As can be seen here, improve the beta-glucosidase catalysis activity in cellulose degradation system, for improving cellulose degradation system transformation efficiency and reducing cellulosic ethanol industry production cost, there is huge commercial value and realistic meaning.
Cellulase is of a great variety, and many microorganisms particularly fungus has the ability producing this compound enzyme, and what wherein enzymatic productivity was stronger has Trichoderma spp., aspergillosis, rhizopus and penicillium sp etc., especially in the majority with trichoderma strain.Trichoderma reesei (Trichodermareesei) is then utilize maximum bacterial strains in cellulase industry.Li's Trichoderma strains Qm6a is the strain of setting out of each High Cellulase Production bacterial strain, in order to obtain the new strains of High Cellulase Production, Qm6a obtains more efficient cellulase-producing mutant strain Qm9414 through mutation and the screening of two-wheeled linear accelerator, but nevertheless suffer from carbon metablism to check, produce enzyme situation to increase along with fermentation time, concentration of glucose raises, and the synthesis of cellulase is suppressed gradually.In order to obtain higher yield of cellulase, Qm6a is re-started breeding, obtain, by three-wheel mutation and screening, the superior strain Rut-C30 that anti-carbon metablism checks: the first round checks screening by ultraviolet mutagenesis and antimetabolic and obtains bacterial strain M7；Second takes turns by N-nitroguanidine mutation, M7 is obtained mutant NG14, compares Qm9414, NG14 extracellular protein yield and filter paper enzyme activity has been respectively increased 1 times and 4 times；Third round obtains more efficient cellulase producing strain Rut-C30 through what ultraviolet mutagenesis and antimetabolic checked screening on the basis of NG14.Additionally, ultraviolet mutagenesis obtains another mutant RL-P37 on the basis of NG14, another industrial wide variety of cellulase high-yield CL-847 is by Qm9414 mutation.Due to the ability of its High Cellulase Production, industrially it is widely used in the fields such as weaving, papermaking, slurrying and bioenergy.The highest fermentation level protein yield up to 100g/L before this Zoopagales, has very strong protein excretion ability, simultaneously to person poultry safety, is developed to the good fungal host expressing homologous protein and heterologous protein.In cellulase commercial Application, the raising further producing enzyme and protein excretion ability of trichoderma reesei contributes to opening further of application of cellulase market.But, the superior strain of trichoderma reesei is monopolized by American-European major company, limits China's cellulase industrial expansion.Therefore, it is necessary to trichoderma reesei is carried out more deep transformation, improve its enzymatic productivity and protein excretion ability, reduce the commercial production cost of cellulase, and then exploitation has the industrial strain of autonomous property right.
In forming due to the enzyme system of trichoderma reesei, cellulose excision enzyme and restriction endonuclease account for 99%, and beta-glucosidase and other cellulase and hemicellulase are then less than 1%；Fungal enzyme system great majority are slant acidity simultaneously, and thermostability is not good yet, and these all become the bottleneck of trichoderma reesei cellulase.Although traditional mutagenic breeding can obtain much excellent industrial strain, but Kubicek etc. think, from 1978 to 1991, the simple mutant being obtained trichoderma reesei by classic mutagenesis method, did not have big change on enzymatic productivity.In recent years, along with molecular biological further development, people are gradually from conventional mutagenic breeding with improve the traditional biological such as fermentation condition and turn to systems biologies such as utilizing comparative genomics, proteomics, transcription group and metabolism group, analyze the function understanding the important mutant gene of some superior strains and correlation molecule mechanism with this, and utilize these mechanism to carry out further genetic modification to obtain the production bacterial strain more meeting industrial requirement.Especially 2008, the gene order-checking of trichoderma reesei Qm6a and Rut-C30 completes and in succession open, the molecular mechanism of Rut-C30 high yield is resolved by the method for Comparative genomic strategy, such as a series of transcription factor, basal metabolism relevant enzyme and the isogenic disappearance of transport protein.These achievements in research are applied in the transformation of industrial strain by many researcheres: the particularly discovery of the cre1 sudden change of most critical in Rut-C30, explain the derepression phenomenon of mutant.Therefore by knocking out repressor cre1 at wild-type strain or introducing cre1 sudden change, recombinant bacterial strain expression of cellulase under glucose and inductive condition can be significantly increased；And Ace1 is the inhibitive factor of another cellulase-producing, in ace1 knock-out bacterial strain, the expression of cellulose enzyme gene main under cellulose or sophorose inductive condition is obtained for rise.
To sum up, this area is necessary research cellulase production bacterial strain further, the bacterial strain of exploitation function admirable.
Summary of the invention
It is an object of the invention to provide a kind of cellulase high-yield and application thereof.
In a first aspect of the present invention, it is provided that the Li's Trichoderma strains of a kind of separation or its spore, mycelium, protoplast, described bacterial strain is CCTCCNO:M2014561 at the preserving number of China typical culture collection center.
In a preference, the enzyme of the cellulase that described preserving number is the Li's Trichoderma strains of CCTCCNO:M2014561 or its spore produces is lived higher than 15IU/mL；It is preferably higher than 20IU/mL.
In another preference, preserving number is in the bacterial strain of CCTCCNO:M2014561 or its spore, mycelium, protoplast, converts and has hygromycin (hygromycin) resistant gene hph.
In another aspect of this invention, it is provided that a kind of Li's Trichoderma strains or its spore, mycelium, protoplast, described bacterial strain is the cultivation offspring of foregoing Li's Trichoderma strains, and its ura5 gene generation deletion mutation.It is preferred that the enzyme of the cellulase of this offspring's bacterial strain generation is lived higher than 25IU/mL.
In another aspect of this invention, a kind of genetic engineering modified Li's Trichoderma strains or its spore, mycelium, protoplast are provided, described bacterial strain is the bacterial strain obtained after the ura5 (it is preferred that it derives from penicillium oxalicum) and cellulase activity factor Xyr1 in the described outside the pale of civilization source of Li's Trichoderma strains transfer.
In another preference, shown in the nucleotide sequence of described ura5 such as SEQIDNO:5 or 6.
In another preference, the nucleotide sequence of described cellulase activity factor Xyr1 is such as shown in SEQIDNO:7.
In another aspect of this invention, thering is provided a kind of Li's Trichoderma strains or its spore, mycelium, protoplast, described bacterial strain is described Li's Trichoderma strains or its spore, mycelium, the protoplast of process LAN (or conversion has) cellulase activity factor Xyr1.Such as, described dimension element enzyme activition factor Xyr1 is the endogenous process LAN of bacterial strain, or foreign transforming enters to make process LAN after described bacterial strain after dimension element enzyme activition factor Xyr1 coded sequence.
In another aspect of this invention, it is provided that the above purposes of arbitrary described Li's Trichoderma strains or its spore, mycelium, protoplast, it is used for producing cellulase (it is preferred that by utilizing agriculture and industry waste to produce cellulase)；Or be used for being hydrolyzed β-1,4-glycosidic bond (acquisition reducing sugar).
In a preference, described cellulase is used for hydrolysis of lignocellulose；It is preferred that described lignocellulose includes, but is not limited to: corn stalk, corn cob, Caulis et Folium Oryzae, rice husk, wheat straw bar, kaoliang stalk, xylose residue, bagasse or its combination.
In another aspect of this invention, it is provided that a kind of method producing cellulase, described method includes: cultivate above arbitrary described Li's Trichoderma strains or its spore, mycelium, protoplast so that it is produce cellulase.
In a preference, described cultural method includes:
(1) by after above arbitrary described Li's Trichoderma strains or the activation of its spore, making concentration is 106～108The spore suspension of individual/mL, then prepare for seed liquor, then seed liquor is inoculated in liquid fermentation medium, initial pH5.0 ± 0.2, in 28 ± 2 DEG C, 200 ± 50rpm shaking table is cultivated 5-7 days；Described fermentation medium is containing according to mass volume ratio 3 ± 1% microcrystalline Cellulose with according to the inorganic salt culture fluid of mass volume ratio 2 ± 0.5% wheat bran；
(2) fermentation liquid centrifugation step (1) obtained, takes supernatant as crude enzyme liquid.
In another aspect of this invention, a kind of method that hydrolysis of lignocellulose is provided, described method includes: (i) utilizes above arbitrary described Li's Trichoderma strains or its spore, mycelium, protoplast production cellulase, and (ii) utilizes the hydrolyzing ligno-cellulose with cellulosic enzyme obtained.
The other side of the present invention, due to this disclosure, is apparent to those skilled in the art.
Accompanying drawing explanation
The transmission electron microscope photo of Fig. 1, trichoderma reesei Rut-C30 and superior strain PPL3-1 spore.Figure 1A is control strain Rut-C30, Figure 1B is the vesicle showed increased that can be seen that PPL3-1 in superior strain PPL3-1, figure, and sporoderm also has the phenomenon that part thickens.This is likely to relevant with the ability of superior strain secreting, expressing albumen.
Filter paper enzyme activity after the fermentation of Fig. 2, trichoderma reesei different strains bottle.Rut-C30 bacterial strain is in the little shake flask fermentation system of 10ml, after wheat bran and cellulose are induced 7 days, filter paper enzyme activity (FPA) is 13U/ml, and PPL3-1 is 20U/ml after fermenting under the same conditions 7 days, the superior strain PPLU4-6 that genetic breeding obtains further is then up to 26U/ml, and the superior strain PPLXIM after further process LAN Xyr1 is more up to 30U/ml.
Detailed description of the invention
The genetic and breeding method screenings such as the domestication of the present inventor room by experiment, physics and chemistry behavior obtain Li's Trichoderma strains Trichodermareesei (anamorph)/Hypocreajecorina (teleomorph) PPL3-1 (hyg of a plant height cellulase-producingr) (hereinafter referred to as PPL3-1), preserving number is CCTCCNO:M2014561.There is obvious advantage in it compared with industrial conventional trichoderma reesei superior strain, and the cellulase that PPL3-1 bacterial strain produces is lived at enzyme and all significantly improves in yield.The present inventor is also by optimizing the yield of cellulase that improve PPL3-1 bacterial strain further.Therefore, the PPL3-1 bacterial strain of the present invention and engineered bacterial strain thereof have good prospects for commercial application.
As used herein, term " the trichoderma reesei superior strain PPL3-1 of the present invention ", " the trichoderma reesei high yield muton PPL3-1 of the present invention ", " the fusant PPL3-1 of the present invention ", " Trichodermareesei (anamorph)/Hypocreajecorina (teleomorph) PPL3-1 (hygr) ", " TrichodermareeseiPPL3-1 ", " HypocreajecorinaPPL3-1 ", " PPL3-1 " be used interchangeably.
Trichoderma reesei is the main production microorganism of commercial fibres element enzyme, but the β-glucosyl enzym ratio in the cellulase system that the common bacterial strain of current industrial application produces is all on the low side with activity, become effectively hydrolyzing cellulose, particularly the restriction factor of industrial pretreated straw.Therefore, current commercial fibers element enzyme, is all add or the composite β-glucosyl enzym produced from other funguses (aspergillus niger of such as high-yield beta-glucosidase) in the enzyme preparation that trichoderma reesei produces again.
The present inventor by carrying out Laboratory Acclimation to trichoderma reesei wild strain on Cellulose Plate, the bacterial strain that yield of cellulase is higher is selected according to the hydrolysis circle size on Cellulose Plate, then genetic breeding is carried out by methods such as physical chemistry, the high productive mutant obtained carries out protoplast fusion again and obtains a series of fusants, these fusants are carried out extensive filter paper enzyme activity screening operation again, final acquisition superior strain PPL3-1.The cellulase systems of this cellulase high-yield is compared with other trichoderma reeseis industrial strain Rut-C30, its optimum pH is consistent with optimum temperature, beta-glucosidase enzyme is lived and then be there is certain advantage, the pNPGase enzyme of crude enzyme liquid is lived and is reached more than 30IU/mL, compensate for common fiber element enzyme industrial producing strain beta-glucosidase this bottleneck not enough, filter paper enzyme activity, then at more than 20IU/mL, compares, than Rut-C30, the advantage of there is also, and is therefore the good industrial fiber element enzyme bacterial strain of a strain.Its cellulase is the same with other trichoderma reesei industrial strains, can be used for the fields such as food, feedstuff, health care, bioenergy, has very much using value.
The present invention obtains superior strain, the activity of beta-glucosidase obtains and is obviously improved, carbon source plus transformant fermentation culture is the agricultural waste gurrys simple and easy to get such as wheat bran, fairly obvious effect is had in improving the hydrolysis efficiency of cellulase preparation, as being applied in the production of food other industry, also will be substantially reduced its production cost, have commercial Application potentiality more widely.
Further, the High Cellulase Production bacterial strain PPL3-1 of the present invention can as starting strain, and the means such as room domestication by experiment, genetic breeding, molecular genetic manipulation improve further and obtain the derivative strain that yield is higher or enzyme system more optimizes.The enzyme bacterial strain more improved alive is obtained by PPL3-1 being carried out genetic breeding screening, and then carry out single spore separation, the superior strain PPLU4-6 obtaining filter paper enzyme activity raising 30% can be screened further, this superior strain can as the object of molecular genetic manipulation, carry out the genetic manipulation of screening marker-free, industrially have huge application prospect.
Further, the derivative strain PPLU4-6 (uracil-deficient type bacterial strain) of High Cellulase Production bacterial strain PPL3-1 of the present invention, object as molecule manipulation, can knock in and knock out the genetic manipulations such as gene, carry out purposive genetic modification, or the cellulose enzyme gene that process LAN trichoderma reesei expression amount is very few, such as process LAN cellulase activity factor Xyr1 etc., or knock out cellulase repressor Cre1 etc..
The bacterial strain of the present invention is active somatic cell, once obtain the spore of bacterial strain of the present invention, mycelia, protoplast and the relevant culture mix also having active somatic cell thereof, it is possible to inoculate go down to posterity, the means such as regeneration obtain the bacterial strain of the present invention in large quantity.This active somatic cell being usually inoculated into the amplification culture carrying out bacterial strain in solid plate culture medium or fluid medium and obtaining the present invention.And the active somatic cell obtained can carry out Laboratory Acclimation, genetic breeding and molecular genetic manipulation etc. further and obtain mutant and transformant.It is possible with the present invention host cell as heterogenous expression.
Method well-known to those having ordinary skill in the art can be used for the active somatic cell of the mutation present invention, and cause the gene code of active somatic cell to change, enzyme lives characteristic and morphologic change.These methods include utilizing the physical methods such as ray, particle, laser, ultraviolet light, utilize the chemomorphosis methods such as alkylating agent, base analogue (baseanalog), azanol (hydroxylamine), acridine pigment.But mutation one of the above method or multiple method is many for mutation, and is not limited to these methods.Based on bacterial strain provided by the invention, the modes such as physical chemistry can be carried out further and carry out breeding, new cellulose enzyme gene and related regulatory genes can also be imported, the mutant obtained and transformant produce enzyme performance can obtain further raising, and described breeding method is that above-mentioned one or more combine.
Method well-known to those having ordinary skill in the art can be used for construction expression construction (carrier) and transform bacterial strain of the present invention further.Such as, for bacterial strain having been found that or the newfound signal pathway relevant to cellulase production, signal path and the albumen that is directed to carry out further improvement (such as increasing the expression of beneficial agents, reduce the expression of injurious factor).
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.Step used is generally well-known in the art.Such as agriculture bacillus mediated fungal transformation method, protoplast transformation method, electric shock transformation method, CRISPR-Cas9 genome Editing Method, particle bombardment etc., and it is not limited to these methods.
Li's Trichoderma strains PPL3-1 disclosed by the invention can utilize the industrial and agricultural production garbage containing lignocellulose, such as cheap raw materials such as wheat bran, Semen Maydis pulp, soybean cake powder, by liquid or producing cellulase through solid-state fermentation, it is advantageous that institute's cellulase-producing vigor is high, component is reasonable, and hydrolysis of lignocellulose ability is strong.Under identical fermentation condition, this bacterial strain is the twice of conventional industrial strain Rut-C30 yield of cellulase.The present invention solves that during cellulose resource utilizes, enzyme activity is not high, the unreasonable production cost caused of enzyme component is high, the problems such as saccharification efficiency is low provide new microorganism resource.
Present invention application in preparing cellulase can be realized by liquid fermentation, as a preferred embodiment mode, fermentation process includes: (1), by trichoderma reesei PPL3-1 strain after the inclined-plane that Rhizoma Solani tuber osi culture medium is made or flat board activate, making concentration is 106～108mL-1Spore suspension, inoculum concentration by 10% accesses in husky Bao Shi culture medium (seed culture medium: 1% yeast extract, 1% peptone, 4% glucose), 28 DEG C, 200rpm shaken cultivation, obtains seed liquor, then accesses in liquid fermentation medium by seed liquor with 10% inoculum concentration, initial pH5.0, liquid amount be 10mL in 50mL triangular flask, in 28 DEG C, in 200rpm shaking table cultivate 5-7 days；(2) fermentation liquid centrifugation step (1) obtained, takes supernatant as crude enzyme liquid；Described fermentation medium is the inorganic salt culture fluid (0.4%KH containing 5% inducer (3% microcrystalline Cellulose and 2% wheat bran)2PO4, 0.28% (NH4)2SO4, 0.06%MgSO4·7H2O, 0.05%CaCl2, 0.06%urea, 0.3%tryptone, 0.1%Tween80,0.5%CaCO3, 0.001%FeSO4·7H2O, 0.00032%MnSO4·H2O, 0.00028%ZnSO4·7H2O, 0.0004%CoCl2)。
The culture medium and the cultural method that are applied to the bacterial strain of the cultivation present invention are not limited to disclosed above those, and other culture medium being conventionally applied to cultivate trichoderma reesei and cultural method can also be applied in the present invention.
Fermentation system described above can carry out multiplication of system and carry out commercial production, varying in size according to system, and those skilled in the art can carry out suitable adjustment growth or production to be conducive to bacterial strain according to the general knowledge grasped.
Liquid fermentation crude enzyme liquid described above, can through ultrafiltration, saltout or the method such as organic solvent deposit obtains purer cellulase or enzyme powder.Live by fermenting and measuring the filter paper enzyme activity (FPA) of institute's cellulase-producing, the work of CMC enzyme and beta-glucosidase enzyme.Should be understood that separate and the method for purifying cellulose enzyme is not only restricted in the present invention to provide those, other method known to those skilled in the art also apply be applicable in the present invention.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally conventionally condition such as J. Pehanorm Brooker etc. are write, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to manufacturer it is proposed that condition.
Embodiment 1, trichoderma reesei superior strain is utilized to carry out cellulase production
In order to ensure the ability of the cellulase-producing of the PPL3-1 (preserving number is CCTCCNO:M2014561) of acquisition, by liquid fermentation the filter paper enzyme activity realization measuring crude enzyme liquid:
(1) be seeded on the flat board that Rhizoma Solani tuber osi culture medium is made by trichoderma reesei PPL3-1 strain to be placed in 28 DEG C cultivate 7 days after, making concentration is 106～108The spore suspension of individual/mL, access in husky Bao Shi culture medium (seed culture medium (v/v): 1% yeast extract, 1% peptone, 4% glucose) by the inoculum concentration of 10% (v/v), 28 DEG C, 200rpm shaken cultivation, obtains seed liquor, then accesses in liquid fermentation medium by seed liquor with 10% (v/v) inoculum concentration, initial pH5.0, liquid amount be 10mL in 50mL triangular flask, in 28 DEG C, in 200rpm shaking table cultivate 5-7 days；
(2) fermentation liquid centrifugation step (1) obtained, takes supernatant as crude enzyme liquid；Described fermentation medium is the inorganic salt culture fluid (0.4%KH containing 5% inducer (3% microcrystalline Cellulose and 2% wheat bran)2PO4, 0.28% (NH4)2SO4, 0.06%MgSO4·7H2O, 0.05%CaCl2, 0.06% carbamide, 0.3% peptone, 0.1%Tween80,0.5%CaCO3, 0.001%FeSO4·7H2O, 0.00032%MnSO4·H2O, 0.00028%ZnSO4·7H2O, 0.0004%CoCl2),
(3) in the Acetic acid-sodium acetate of 0.05mol/LpH5.0 or citric acid-sodium citrate buffer, joining in filter paper by trichoderma reesei PPL3-1 crude enzyme liquid, 50～60 DEG C of hydrolysis 60min, the enzyme measuring the enzyme on filter paper is lived.Result records, and enzyme work is up to more than 20IU/mL.
Embodiment 2, application cellulase carry out the degraded of lignocellulose
In order to verify the trichoderma reesei High Cellulase Production bacterial strain PPL3-1 degradation capability to natural wooden fiber's element.The present inventor adopts the corn straw that 10% dilute sulfuric acid processes, pretreated straw after washing is dried is as material, in the Acetic acid-sodium acetate or citric acid-sodium citrate buffer of 1mL0.05mol/LpH5.0,30 μ l crude enzyme liquids of trichoderma reesei PPL3-1 institute cellulase-producing are joined in 30mg pretreatment corn straw, in earthquake shaking table, shake with the speed of 200rpm, 50 DEG C are hydrolyzed 24 hours, supernatant is taken after centrifugal, measure the reducing sugar in supernatant by the method for DNS, converse the total amount of reducing sugar in system.Research finds to utilize the cellulase that bacterial strain of the present invention produces can produce 12～16mg/mL reducing sugar (simple sugars), and the reducing sugar (simple sugars) of 5-8mg/mL is only produced as the cellulase of Rut-C30 of comparison, there is fairly obvious advantage.
From the above results, the cellulase that bacterial strain of the present invention produces can be hydrolyzed β-Isosorbide-5-Nitrae-glycosidic bond, it is thus achieved that reducing sugar.
Embodiment 3, trichoderma reesei superior strain PPL3-1 characteristic research
Transmission electron microscope observing is carried out, it has been found that the vesicle number of PPL3-1 compares Rut-C30 substantially to be increased, and cell wall also substantially thickens (Fig. 1), and this is likely to relevant with the ability of PPL3-1 High Cellulase Production to after the spore film-making of PPL3-1 bacterial strain.
Embodiment 4, Li's Trichoderma strains PPL3-1 genetic engineering modified
Trichoderma reesei PPL3-1 strain is seeded on the flat board that Rhizoma Solani tuber osi culture medium is made, and cultivates 7 days, is washed down by spore with tween normal saline (containing 0.85%NaCl, 0.02%Tween-80) for 28 DEG C, and making concentration is 106～108mL-1Spore suspension, and be spread evenly across on the flat board that trichoderma reesei minimal medium (adding final concentration of 2% and 3% ball milling cellulose powder and 0.1%tritonX-100) is made, cultivate.After flat board having single bacterium colony grow, single bacterium colony that hydrolysis circle significantly increases is received in 24 orifice plates and cultivates 7 days in 28 DEG C further, repaste and be distributed in above-mentioned flat board, hydrolysis is again selected to enclose single bacterium, the cultivation significantly increased, and then measured the bacterial strain choosing High Cellulase Production by cellulase activity, thus choose the bacterial strain that a plant height produces.Based on described trichoderma reesei minimal medium, inorganic salt culture fluid is (according to w/v, 1%KH2PO4, 0.6% (NH4)2SO4, 0.1%MgSO4·7H2O, 0.3% 3 sodium citrate 2H2O, 0.0005%FeSO4·7H2O, 0.00016%MnSO4·H2O, 0.00014%ZnSO4·7H2O, 0.0002%CaCl2·2H2O, pH5.8), and add the uridnine of final concentration of 0.5% (w/v).
The superior strain of above-mentioned acquisition carries out monospore separation, after being not less than the going down to posterity of 10 generations, chooses cellulase and stablizes the bacterial strain PPLU4-6 of high yield.
By PPLU4-6 inoculation on the flat board that the Rhizoma Solani tuber osi culture medium containing 0.5% (w/v) uridnine is made, cultivate 7 days for 28 DEG C, with tween normal saline (containing 0.85%NaCl, 0.02%Tween-80) spore is washed down, and synchronize inoculation and set out strain PPL3-1, cultivate after 7 days for 28 DEG C, liquid fermentation is carried out by method described in embodiment 1, measure the filter paper enzyme activity of crude enzyme liquid, wherein the filter paper enzyme activity of PPLU4-6 single-ascospore strain reaches 26U/mL, and the filter paper enzyme activity comparing starting strain PPL3-1 improves 30% (Fig. 2).
The inventors discovered that PPLU4-6 cannot be survived in the culture medium lacking uridnine, therefore investigate the defect that whether there is uridnine synthesis related gene ura3, ura5 in PPLU4-6.According to the genome sequence of Trichoderma reesei Rut C 30 in JGI, the amplimer of design ura3, ura5 gene, primer sequence is respectively as follows:
Ura3F:5 '-GCTCTAGAATGGCACCACACCCGACGCT-3 ' (SEQIDNO:1)；
Ura3R:5 '-GCTCTAGACTATCGCAGCAGCCTCTCGG-3 ' (SEQIDNO:2)；
Ura5F:5 '-GCTCTAGAATGGCTACCACCTCCCAGCT-3 ' (SEQIDNO:3)；
ura5R:5’-GCTCTAGATCAGTCAGTCGCCTTGTACT-3’(SEQIDNO:4)。
Utilizing ura3 and the ura5 genetic fragment of KOD high-fidelity enzymatic amplification trichoderma reesei PPL3-1,3 ' ends utilize TA Cloning Kit to be cloned in pMD18-Tsimple carrier after adding A process, and after PCR verifies positive colony, sample sample presentation checks order.Shown in the sequence such as SEQIDNO:5 (711bp) of ura5 gene wild type.Sequencing result for the sequence of the present embodiment amplification acquisition shows, the aminoacid sequence of ura5 gene exists sudden change (L135P, the corresponding site of its coded sequence is sported CCG by CTG).Hence, it can be determined that the uridine auxotrophy of PPL3-1 is caused by ura5 gene mutation.This sudden change can be utilized as selection markers, PPLU4-6 is carried out further genetic engineering modified.
The ura5 gene (SEQIDNO:6) of promoter sequence is included as selection markers from penicillium oxalicum amplification, the primer by PCR:
Forward primer: 5 ' TCTAGAGCCGCATAGTTAAGCC3 ' (SEQIDNO:10), its 5 ' end adds XbaI recognition site: TCTAGA；
Reverse primer: 5 ' ACTAGTCAGGGCTGGTGACGGAA3 ' (SEQIDNO:11), its 5 ' end adds SpeI recognition site ACTAGT,
It is connected into after amplified production XbaI and SpeI double digestion in the pHDt/sk (referring to MicrobialCellFactories, 2012,11:21) through same double digestion, it is thus achieved that pHDt/sk-ura5.
Separating trichoderma reesei cellulase activating transcription factor Xyr1 (SEQIDNO:7) from the genome of bacterial strain PPL3-1 by PCR, the primer is as follows:
Forward primer: 5 ' TCTAGAATGTTGTCCAATCCTCTCCGTCG3 ' (SEQIDNO:8), its 5 ' end adds XbaI recognition site: TCTAGA；
Reverse primer is 5 ' TCTAGATTAGAGGGCCAGACCGGTTCCGT3 ' (SEQIDNO:9), and its 5 ' end adds XbaI recognition site: TCTAGA.
XbaI enzyme cutting will be used after PCR primer purification, application AxygenPCR product post reclaims test kit and reclaims the DNA fragmentation of enzyme action, by this DNA fragmentation with after the carrier pHDt/sk-ura5 dephosphorylation of the recovery of same enzyme action processes, connect overnight with T4DNA ligase at 16 DEG C, obtain recombinant expression carrier pHDt/sk-ura5-Xyr1.The His label (6 × His-Tag) that the N-terminal of expression product provides, it is simple to subsequent purification.The above-mentioned plasmid pHDt/sk-ura5-Xyr1 built is transformed in Agrobacterium tumefaciems AGL1, is transformed under the mediation of Agrobacterium tumefaciems in trichoderma reesei PPLU4-6 bacterial strain.The bacterial strain obtained is the bacterial strain of process LAN activity factor.
The transformant obtained carries out liquid fermentation by method described in embodiment 1, measures the filter paper enzyme activity of crude enzyme liquid, and wherein the filter paper enzyme activity of PPLXIM single-ascospore strain reaches 30U/mL, and enzyme is lived in having had and significantly increased very much (Fig. 2).
Biomaterial preservation
The Li's Trichoderma strains Trichodermareesei (anamorph) of the present invention/Hypocreajecorina (teleomorph) PPL3-1 (hygr) (be called for short PPL3-1) be deposited in China typical culture collection center (CCTCC, Wuhan, China), preserving number CCTCCNO:M2014561；November 11 2014 preservation day.
The all documents mentioned in the present invention are incorporated as reference all in this application, are individually recited as reference such just as each section of document.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, the present invention can be made various changes or modifications by those skilled in the art, these equivalent form of values fall within the application appended claims limited range equally.
Claims (11)
1. the Li's Trichoderma strains separated or its spore, mycelium, protoplast, it is characterised in that described bacterial strain is CCTCCNO:M2014561 at the preserving number of China typical culture collection center.
2. a Li's Trichoderma strains or its spore, mycelium, protoplast, it is characterised in that described bacterial strain is the cultivation offspring of the Li's Trichoderma strains described in claim 1, and its ura5 gene generation deletion mutation.
3. Li's Trichoderma strains one kind genetic engineering modified or its spore, mycelium, protoplast, it is characterized in that, described bacterial strain is the Li's Trichoderma strains described in claim 2 of the bacterial strain obtained after the ura5 and cellulase activity factor Xyr1 converting and having external source.
4. Li's Trichoderma strains as claimed in claim 3 or its spore, mycelium, protoplast, it is characterised in that shown in the nucleotide sequence of described ura5 such as SEQIDNO:5 or 6.
5. Li's Trichoderma strains as claimed in claim 4 or its spore, mycelium, protoplast, it is characterised in that the nucleotide sequence of described cellulase activity factor Xyr1 is such as shown in SEQIDNO:7.
6. a Li's Trichoderma strains or its spore, mycelium, protoplast, it is characterised in that described bacterial strain is the Li's Trichoderma strains described in claim 1 or its spore, mycelium, the protoplast of process LAN cellulase activity factor Xyr1.
7. the purposes of the arbitrary described Li's Trichoderma strains of claim 1-6 or its spore, mycelium, protoplast, it is characterised in that be used for producing cellulase；Or be used for being hydrolyzed β-1,4-glycosidic bond.
8. purposes as claimed in claim 7, it is characterised in that described cellulase is used for hydrolysis of lignocellulose；It is preferred that described lignocellulose includes: corn stalk, corn cob, Caulis et Folium Oryzae, rice husk, wheat straw bar, kaoliang stalk, xylose residue, bagasse or its combination.
9. the method producing cellulase, it is characterised in that described method includes: cultivate the arbitrary described Li's Trichoderma strains of claim 1-6 or its spore, mycelium, protoplast so that it is produce cellulase.
10. method as claimed in claim 9, it is characterised in that cultural method includes:
(1) by after arbitrary for claim 1-6 described Li's Trichoderma strains or the activation of its spore, making concentration is 106～108The spore suspension of individual/mL, then prepare for seed liquor, then seed liquor is inoculated in liquid fermentation medium, initial pH5.0 ± 0.2, in 28 ± 2 DEG C, 200 ± 50rpm shaking table is cultivated 5-7 days；Described fermentation medium is containing according to mass volume ratio 3 ± 1% microcrystalline Cellulose with according to the inorganic salt culture fluid of mass volume ratio 2 ± 0.5% wheat bran；
(2) fermentation liquid centrifugation step (1) obtained, takes supernatant as crude enzyme liquid.
11. the method for a hydrolysis of lignocellulose, it is characterized in that, described method includes: (i) utilizes the arbitrary described Li's Trichoderma strains of claim 1-6 or its spore, mycelium, protoplast production cellulase, and (ii) utilizes the hydrolyzing ligno-cellulose with cellulosic enzyme obtained.
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