CN114854655A - Engineering strain capable of efficiently secreting xylanase, construction method and application - Google Patents
Engineering strain capable of efficiently secreting xylanase, construction method and application Download PDFInfo
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- CN114854655A CN114854655A CN202210428826.2A CN202210428826A CN114854655A CN 114854655 A CN114854655 A CN 114854655A CN 202210428826 A CN202210428826 A CN 202210428826A CN 114854655 A CN114854655 A CN 114854655A
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- xylanase
- clostridium thermocellum
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- 238000010276 construction Methods 0.000 title claims description 13
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- 108010001817 Endo-1,4-beta Xylanases Proteins 0.000 claims abstract description 13
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- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 1
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- 239000001508 potassium citrate Substances 0.000 description 1
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- HNWCOANXZNKMLR-UHFFFAOYSA-N pyridoxamine dihydrochloride Chemical compound Cl.Cl.CC1=NC=C(CO)C(CN)=C1O HNWCOANXZNKMLR-UHFFFAOYSA-N 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 229960000999 sodium citrate dihydrate Drugs 0.000 description 1
- MWEMXEWFLIDTSJ-UHFFFAOYSA-M sodium;3-morpholin-4-ylpropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CCCN1CCOCC1 MWEMXEWFLIDTSJ-UHFFFAOYSA-M 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
The invention relates to the technical field of genetic engineering, and particularly provides a clostridium thermocellum engineering strain capable of efficiently secreting xylanase, wherein the strain is a strain obtained by adding xylanase A in clostridium thermocellumxynAXylanase C xynCAnd xylanase YxynYAnd (3) overexpressing the obtained clostridium thermocellum engineering strain. By increasing on the C.thermocellum genomeAdding xylanase AxynAXylanase CxynCAnd xylanase YxynYThe gene copy number is added with a strong promoter at the same time, so that the clostridium thermocellum efficiently expresses xylanase, thereby enhancing the xylanase enzyme activity of clostridium thermocellum engineering bacteria. The invention also discloses application of the clostridium thermocellum engineering strain in hemicellulose hydrolysis saccharification, which can obviously improve the hydrolysis rate of hemicellulose in straw fibers, and the hemicellulose hydrolysis rate of the straw fibers can be improved by 50% under the same enzymolysis condition.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an engineering strain capable of efficiently secreting xylanase, a construction method and application.
Background
Xylan is the main component of hemicellulose, and is a polysaccharide that is abundant next to cellulose. The xylanase is an enzyme capable of hydrolyzing xylan to produce xylo-oligosaccharide or xylose, and mainly comprises exo-beta-1, 4-xylanase, endo-beta-1, 4-xylanase, beta-xylosidase and the like.
The clostridium thermophilum is one of the microorganisms which are known in nature to degrade cellulose most efficiently at present, and the secreted cellulosome has extremely strong cellulose degradation capability which is about 50 times that of trichoderma. However, in the lignocellulosic hydrolysis application of clostridium thermocellum, the presence of hemicellulose, one of the components in lignocellulose, increases the structural strength and steric resistance of the plant cell wall, resulting in a decrease in the efficiency of lignocellulosic hydrolysis. Therefore, the problem to be solved is to construct the clostridium thermocellum engineering bacteria capable of efficiently expressing xylanase so as to improve the hemicellulose hydrolysis efficiency and further improve the whole lignocellulose hydrolysis efficiency.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides an engineering strain capable of efficiently secreting xylanase, a construction method and application, the xylanase enzyme activity of the clostridium thermocellum engineering strain is enhanced by efficiently expressing the xylanase of clostridium thermocellum, and the engineering strain can be applied to hemicellulose hydrolysis saccharification to obviously improve the hydrolysis rate of hemicellulose in straw fibers.
In order to achieve the purpose, the invention adopts the following technical scheme:
one aspect of the present invention provides a composition capable of efficiently secretingAn engineered strain of xylanase carrying a recombinant plasmid for recombinant expression of the xylanase. The strain is xylanase A in clostridium thermocellumxynAGene, xylanase CxynCGene and xylanase YxynYThe clostridium thermocellum engineering strain is obtained by gene overexpression.
The clostridium thermocellum is wild clostridium thermocellum, preferably clostridium thermocellumClostridium thermocellumPN2102 with preservation date of 2021 year, 07 month and 09 day, and preservation unit of China general microbiological culture Collection center with preservation number of CGMCC No. 22869.
The nucleotide sequence of the expression gene is shown as SEQ ID No. 1.
The invention also provides a construction method of the clostridium thermocellum engineering strain capable of efficiently secreting xylanase, wherein xylanase A for overexpression is introduced into the original clostridium thermocellum xynAGene, xylanase CxynCGene and xylanase YxynYRecombinant plasmids of the genes.
Preferably, by increasing xylanase A on the C.thermocellum genomexynAXylanase CxynCAnd xylanase YxynYThe method of simultaneously adding strong promoter to gene copy number of the xylanase AxynAGene, xylanase CxynCGene and xylanase YxynYA gene.
Preferably, the gapD promoter and xylanase expression gene sequences of C.thermocellum are inserted downstream of the L-lactate dehydrogenase gene on the C.thermocellum genome. The inserted expression gene sequence has a nucleotide sequence shown as SEQ ID NO. 1.
As a further preferable scheme, the construction method of the engineering strain specifically comprises the following steps:
(1) designing and constructing xylanase gene insertion recombinant plasmid PUC-xyn; (2) electrotransformation is carried out to transfer the recombinant plasmid PUC-xyn into clostridium thermocellum, and a target gene sequence is inserted into a genome through homologous recombination and resistance screening; (3) and selecting and verifying positive monoclonals.
The invention also provides application of the clostridium thermocellum engineering strain capable of efficiently secreting xylanase in hemicellulose hydrolysis saccharification, and fermentation liquor obtained by anaerobic fermentation of the clostridium thermocellum engineering strain is compounded with cellulase to hydrolyze straw fibers. The method specifically comprises the following steps:
(1) pretreatment of lignocellulose: common agricultural straws are taken as raw materials, and are pretreated by one or two of physical and chemical methods;
(2) anaerobic fermentation of clostridium thermocellum: culturing and fermenting the clostridium thermocellum engineering bacteria by seed liquid to obtain fermentation liquid;
(3) fiber hydrolysis: and (3) compounding the fermentation liquor obtained in the step (2) with cellulase (0-10 FPU) to hydrolyze the fiber obtained by the treatment in the step (1).
As a further preferable scheme, in the step (1), the pretreatment of the lignocellulose comprises the following specific steps: cleaning and chopping straw raw materials, carrying out hydrothermal reaction under an alkaline condition, fully removing lignin, extracting straw fiber, dehydrating and fully washing off residual alkali liquor; the inorganic alkali is sodium hydroxide and sodium sulfite; the weight of the sodium hydroxide is as follows: the absolute dry weight of the straw raw material is 1 (4-6), sodium sulfite: the weight ratio of the sodium hydroxide is 1 (3-5); the treatment conditions were: the reaction temperature is 150 ℃ and 160 ℃, and the reaction time is 1-3 h.
In a further preferable scheme, in the step (2), the culture medium is an MTC culture medium which is divided into five ABCDE solutions, wherein the solution A is 5 g/L carbon source (microcrystalline cellulose or straw fiber) and 10.00 g/L MOPS; the solution B is 50.00 g/L tripotassium citrate, 31.25 g/L citric acid monohydrate, and 25.00 g/L Na 2 SO 4 、25.00 g/L KH 2 PO 4 、62.50 g/L NaHCO 3 (ii) a The solution C is 250.00 g/L urea, and the solution D is 50.00 g/L MgCl 2 ·6H 2 O、10.00 g/L CaCl 2 ·2H 2 O、5.00 g/L FeCl 2 ·4H 2 O, 50.00 g/L cysteine salt; the solution E is 1.00 g/L pyridoxamine dihydrochloride, 0.20 g/L p-aminobenzoic acid, 0.10 g/L biotin and 0.10 g/L VB 12. After the preparation of the solution A is finished, sterilizing at 121 ℃ for 20 min; B. c, D, E the solution is prepared according to the above concentration, and is filled into an anaerobic bottle, and an aluminum cap is addedRepeatedly vacuumizing and filling high-purity nitrogen for 3 times after sealing, and finally keeping the interior of the anaerobic bottle at positive pressure. The 5 culture solutions are injected into the sterilized anaerobic bottle filled with the solution A in a super clean bench by using a disposable sterile syringe and a sterile filter membrane of 0.22 mu m according to a ratio of 40:2:1:1:1 (v/v), and the total volume of the culture solutions does not exceed 40% of the volume of the anaerobic bottle.
As a further preferred technical scheme, in the step (2), the seed liquid culture and fermentation process comprises: thawing the strain of Clostridium thermocellum engineering bacteria preserved at-80 deg.C in a refrigerator at 4 deg.C, sucking 1mL strain under aseptic condition, injecting into seed culture medium, culturing at 55 deg.C under 200 rpm for 16-24 h; then inoculating into fermentation medium with an inoculum size of 5% -10% (v/v), and culturing at 55 deg.C and 200 rpm for 16-24 h. Wherein the carbon source in the seed culture medium A is 5.00 g/L microcrystalline cellulose (Avicel PH 105), and the carbon source in the fermentation culture medium is 5.00 g/L straw fiber (the preparation method is as described in step (1)).
As a further preferable technical solution, in the step (3), the specific steps of fiber hydrolysis are: pH is 4.5-5.5, the solid-liquid ratio (absolute dry weight of straw fiber) is 1 (10-30), the reaction temperature is 45-55 ℃, 10-20 mL of clostridium thermocellum fermentation liquor and 0-10 FPU of cellulase are added into the straw fiber per unit mass, the hydrolysis time is 12-48 h, and the stirring speed is 200 rpm.
The invention has the beneficial effects that:
(1) the invention provides a clostridium thermocellum engineering bacterium, which is subjected to metabolic modification to enhance xylanase AxynAXylanase C xynCAnd xylanase YxynYThe clostridium thermocellum can efficiently express xylanase by using the promoter, so that the xylanase activity of the clostridium thermocellum is enhanced. Compared with the starting strain of clostridium thermocellum, the hydrolysis efficiency of hemicellulose can be improved by 50 percent under the same enzymolysis condition.
(2) The preparation of the straw fiber and the method for hydrolyzing hemicellulose by clostridium thermocellum provided by the invention have high hydrolysis efficiency, and most of xylan in the straw fiber is hydrolyzed into xylose after 24 hours of hydrolysis. The method can be used for industrial production and has good application prospect.
Drawings
FIG. 1 is a schematic diagram showing the structure of the gene sequence of Clostridium thermocellum insert in example 1.
FIG. 2 is a map of the recombinant plasmid PUC-xyn inserted with xylanase gene of example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following embodiments, and it is apparent that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The engineering strain capable of efficiently secreting xylanase takes wild clostridium thermocellum as an initial strain, and xylanase A in clostridium thermocellum is usedxynAGene, xylanase CxynCGene and xylanase YxynYThe clostridium thermocellum engineering strain is obtained by gene overexpression.
Specifically, the clostridium thermocellum engineering strain is obtained by adding xylanase A on the genome of clostridium thermocellum through genetic modification of wild clostridium thermocellumxynAXylanase CxynCAnd xylanase YxynYThe engineering strain of gene copy number and strong promoter includes the nucleotide sequence shown in SEQ ID No.1 in the sequence list.
The wild clostridium thermocellum is separated from cotton piles, cow dung and the like, is a thermophilic and strictly anaerobic gram-positive bacterium, is rod-shaped, has wet and low-outburst surface bacterial colonies and high growth speed at the growth temperature of 50-65 ℃. Preferred of the present invention is Clostridium thermocellumClostridium thermocellumPN2102 with preservation date of 2021 year, 07 month and 09 day, and preservation unit of China general microbiological culture Collection center with preservation number of CGMCC No. 22869.
The invention also provides a construction method of the clostridium thermocellum engineering strain capable of efficiently secreting xylanase, and xylanase A for recombinant expression is introduced into clostridium thermocellumxynAGene, xylanase CxynCGenes andxylanase YxynYRecombinant plasmids of the genes.
By adding xylanase A to the C.thermocellum genomexynAGene, xylanase CxynCGene and xylanase Y xynYMethod for expressing xylanase A by adding strong promoter to gene copy number of genexynAGene, xylanase CxynCGenes and xylanase YxynYA gene.
Preferably, the strong promoter is the gapD promoter of Clostridium thermocellum.
Preferably, the gapD promoter and xylanase expression gene sequence of Clostridium thermocellum are inserted downstream of the L-lactate dehydrogenase gene on the Clostridium thermocellum genome.
The nucleotide sequence of the expression gene is shown as SEQ ID No. 1.
The invention also provides application of the clostridium thermocellum engineering strain capable of efficiently secreting xylanase in hemicellulose hydrolysis saccharification, fermentation liquor obtained by anaerobic fermentation of the clostridium thermocellum engineering strain can be compounded with cellulase to hydrolyze straw fibers, and 10-20 mL of clostridium thermocellum fermentation liquor and 0-10 FPU of cellulase are added into the straw fibers per unit mass. Comprises the steps of straw fiber preparation, clostridium thermocellum seed liquid anaerobic fermentation and fiber hydrolysis.
In order to better understand the technical solutions, the technical solutions will be described in detail with reference to specific embodiments. The test materials used in the examples were purchased from a conventional biochemical reagent store unless otherwise specified. The experimental procedures in the examples are conventional unless otherwise specified.
The CTFUD culture medium used by the invention comprises the following components in percentage by weight (g/L): 3.00 sodium citrate dihydrate, 1.30 (NH) 4 ) 2 SO 4 、1.50 KH 2 PO 4 、0.13 CaCl 2 •2H 2 O, 0.5L-cysteine hydrochloride, 11.56 MOPS-Na, 2.60 MgCl 2 •6H 2 O、0.001 FeSO 4 •7H 2 O, 5.00 Avicel pH105, 4.50 YE, 10 g/L agar was added if a solid medium was prepared.
Example 1 construction of Clostridium thermocellum xylanase high efficiency expression engineering bacteria
The gene sequence (shown in SEQ ID No: 1) is inserted and expressed on the genome of the Clostridium thermocellum PN2102 by replacing an L-lactate dehydrogenase gene. Using electric transformation to recombine the plasmid (PUC-xyn) Transferred into clostridium thermocellum PN2102, and the target gene (x) is recombined through homologous recombinationyn A 、xyn C、xyn Y) The sequence is inserted into a clostridium thermocellum PN2102 genome, the schematic diagram of the structure of the inserted gene sequence is shown in figure 1, and the inserted gene sequence is a nucleotide sequence shown as SEQ ID NO.1 in a sequence table.
The method comprises the following specific steps:
(1) recombinant plasmid PUC-xynDesign and construction of
Xylanase gene sequence (x)yn A、xyn C、xyn Y) Both the gapD promoter and the gapD promoter are derived from C.thermocellum and are known from the literature (Application of Long Sequence Reads to uplink Genomes for) Clostridium thermocellum AD2, Clostridium thermocellum LQRI, and Pelosinus fermentansR7) to obtain the xylanase gene sequence of Clostridium thermocellum, wherein GenBank number is GenBank: CP016502.1, synthesized by Nanjing Kingsrei Biotech Co. The expression vector for constructing the recombinant plasmid is pPN01 template plasmid, and can be purchased from the market or stored in a laboratory after being synthesized by a gene design company. The template plasmid contains gapDH promoter, thiamphenicol resistance gene,hptGene, puc19 initiation region,tdkGenes, replication proteins and ampicillin resistance genes.
1) PCR Gene amplification
(ii) a xylanase target gene inserted upstream of L-lactate dehydrogenase ldh in the upstream of the promoter (gapD promoter) using pPN01 as a template plasmidxyn A 、xyn C、xyn Y) The downstream gene recombination segment of the L-lactate dehydrogenase ldh is inserted into the downstream gene recombination segment of the L-lactate dehydrogenase at the rear end of the hpt gene to obtain a recombination plasmid PUC-xyn. The upstream, middle and downstream gene recombination segments of the L-lactate dehydrogenase gene for gene insertion plasmid construction are obtained by PCR of the following three pairs of primers and the head-to-tail connecting segment of 20-25 bp template plasmid.
An upstream primer 1: TCTTCCTCTGTCCTGGCT the flow of the air in the air conditioner,
an upstream primer 2: TGCACCAACTACGGTTACTTTT, respectively;
the primer for midstream 1: AAAAAGCCGACGGAGAAG the flow of the air in the air conditioner,
the middle primer 2: ATACCGTTTACACCCACGA, respectively;
a downstream primer 1: AGTCCGGAAACACTCTAAAA the flow of the air in the air conditioner,
a downstream primer 2: GTATAAAGCCCATGCCTG are provided.
The PCR reaction system is as follows: the primer concentration is respectively 0.4 muL for the upstream primer, 0.4 muL for the intermediate primer and the downstream primer, 1 muL for the bacterial liquid or plasmid, 5 muL for the prime star (efficiency is 10 s/kb), and 3.2 muL for the sterile water, wherein the primer concentration is 10 muM.
The PCR reaction program is: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 s, annealing at 55 ℃ for 10 s, extension at 72 ℃ for 30 s to 2 min for 30 s, cycle number 30; further extension at 72 deg.C for 2 min, and heat preservation at 12 deg.C for 20 min.
2) Linearization of template plasmid pPN01 and vector fragment recovery:
the pPN01 template plasmid was linearized using the following primers and PCR conditions:
primer 1: CTTACTCTAGCAGACTTGGCAATG the flow of the air in the air conditioner,
primer 2: TCCATCTGTTTCGTTTGCCCTTTCC are provided.
The PCR reaction system is as follows: the primer 1 dosage is 0.4 muL, the primer 2 dosage is 0.4 muL, the bacterial liquid or plasmid dosage is 1 muL, the prime star dosage is 5 muL (efficiency is 10 s/kb), and the sterile water dosage is 3.2 muL. The primer concentration was 10. mu.M.
The PCR reaction program is: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 s, annealing at 55 ℃ for 10 s, extension at 72 ℃ for 30 s to 2 min for 30 s, cycle number 30; further extension at 72 deg.C for 2 min, and heat preservation at 12 deg.C for 20 min.
Connecting the upper, middle and lower stream gene recombinant fragments, xylanase gene fragments and linearized template plasmid pPN01 by using Gibson Assembly (three sections of recombinant fragments are 30 ng, xylanase gene fragments are 60 ng, linearized template plasmid is pPN 0130 ng, Gibson Assembly 5 mu L, sterile water is supplemented to 10 mu L, heat preservation is carried out for 1 h at 50 ℃), then transferring into escherichia coli competent cells BL21 and coating on a rubber plate containing benzyl ammonia, and after bacteria grow out, carrying out PCR screening confirmation by using the next pair of primers.
Primer 1: AGCGGTAAAAGTGAAGAAC, primer 2: TGGGCCCCTACTAAAATGA are provided.
The PCR reaction system is as follows: the primer 1 is 0.4 muL, the primer 2 is 0.4 muL, the bacterial liquid or plasmid is 1 muL, the prime star is 5 muL (efficiency is 10 s/kb), and the sterile water is 3.2 muL, wherein the concentration of each primer is 10 muM.
The PCR reaction program is: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 s, annealing at 55 ℃ for 10 s, extension at 72 ℃ for 30 s to 2 min for 30 s, cycle number 30; further extension at 72 deg.C for 2 min, and heat preservation at 12 deg.C for 20 min.
And (3) carrying out agarose gel electrophoresis (2% agarose, 110V and 30min) on the reaction solution after PCR amplification, wherein the size of a positive verification gel picture strip is 473 bp, which indicates that the PUC-xyn recombinant plasmid is successfully constructed.
FIG. 2 shows a PUC-xyn map of a recombinant plasmid into which a xylanase gene is inserted, wherein the upstream of ldh is 967 bp, the downstream of ldh is 948 bp, and the midstream of ldh is 727 bp.
(2) Transfer of recombinant plasmid PUC-xyn
The recombinant plasmid PUC-xyn is transferred into clostridium thermocellum PN2102 by utilizing electric transformation, and a target gene sequence is deleted on a genome through homologous recombination and resistance screening. The operation steps are as follows:
1) cell growth
After 50 mL of the bacterial solution was cultured to OD600=0.6-1, it was placed on ice for 20 minutes.
2) Cell collection and washing
The cells were collected by centrifugation at 6500 g for 10 minutes at 4 ℃ and the supernatant was carefully decanted after centrifugation, washing buffer (steam sterilized reverse osmosis purified water) was carefully added to the centrifuge tube or flask without agitation during the addition, and the cell was centrifuged again at 6500 g for 10 minutes at 4 ℃ and the supernatant carefully decanted, and the above procedure was repeated twice.
3) Electric conversion
The collected cells were placed in an anaerobic chamber and gently resuspended in 100. mu.L of anaerobic wash buffer. Subsequently, 20. mu.L of the cell suspension and 1. mu.g of DNA were added to a standard 1 mm cuvette and mixed well. The electrotransformation conditions are set to be 1500V and the duration time is 1.5 ms for electrotransformation.
4) Incubating the electrically transformed cells
The electrotransferred Clostridium thermocellum PN2102 competent cells were remixed with 1mL of CTFUD medium and incubated at 51 ℃ for 16 hours.
(3) Positive monoclonal selection and validation
1) Selection of transformed cells
The CTFUD solid medium was thawed and cooled to 55 ℃, 1mL of thiamphenicol (6 mg/mL) and 50. mu.L of the incubated electro-transformed cells were added to 20 mL of the CTFUD solid medium and poured into a plate, the medium in the plate was allowed to solidify for 30 minutes at room temperature, and then the plate was left to incubate at 55 ℃ for 3-5 days.
2) A monoclonal colony is selected, and the following pair of primers is used for PCR verification to determine whether the plasmid PUC-xyn has been transferred into clostridium thermocellum PN2102 competent cells.
Primer 1: AGCGGTAAAAGTGAAGAAC, respectively; primer 2: TGGGCCCCTACTAAAATGA are provided.
The PCR reaction system is as follows: the primer 1 is 0.4 muL, the primer 2 is 0.4 muL, the bacterial liquid or plasmid is 1 muL, the prime star is 5 muL (efficiency is 10 s/kb), and the sterile water is 3.2 muL, wherein the concentration of each primer is 10 muM.
The PCR reaction program is: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 s, annealing at 55 ℃ for 10 s, extension at 72 ℃ for 30 s to 2 min for 30 s, cycle number 30; further extension at 72 deg.C for 2 min, and heat preservation at 12 deg.C for 20 min.
And (3) carrying out gel running verification (2% agarose gel electrophoresis under the condition of 110v for 30min) on the PCR amplification product, wherein the size of a positive verification gel picture strip is 473 bp, and the fact that the recombinant plasmid PUC-xyn is transferred into the clostridium thermocellum competent cells is proved.
The single clone colony after verification is inoculated into CTFUD liquid medium containing thiamphenicol (6 mg/mL) and cultured for 1-2 days at 55 ℃.
3) mu.L to 1mL of the culture solution was added to 20 mL of CTFUD solid medium containing thiamphenicol (6 mg/L) and FUDR (10 mg/L), poured onto a plate, the medium in the plate was allowed to solidify at room temperature for 30 minutes, and then the plate was left to stand at 55 ℃ for culture for 2 to 5 days.
4) A single colony was picked and PCR verified using the following pair of primers to verify that the upstream and downstream ldhs in the plasmid PUC-xyn had recombined onto the C.thermocellum PN2102 genome.
Primer 1: TCTTCCTCTGTCCTGGCT, respectively; primer 2: GTATAAAGCCCATGCCTG are provided.
The PCR reaction system is as follows: the primer 1 is 0.4 muL, the primer 2 is 0.4 muL, the bacterial liquid or plasmid is 1 muL, the prime star is 5 muL (efficiency is 10 s/kb), and the sterile water is 3.2 muL, wherein the concentration of each primer is 10 muM.
The PCR reaction program is: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 s, annealing at 55 ℃ for 10 s, extension at 72 ℃ for 30 s to 2 min for 30 s, cycle number 30; further extension at 72 deg.C for 2 min, and heat preservation at 12 deg.C for 20 min.
The PCR amplification products were subjected to 2% agarose gel electrophoresis (110V, 30 min). The size of the positive validation gel strip is 13262 bp. Indicating that ldh upstream and ldh downstream have recombined onto the C.thermocellum PN2102 genome.
The confirmed monoclonal cell colonies were added to 20 mL of CTFUD solid medium containing 8AZH (500 mg/L) and poured into a plate, the medium in the plate was allowed to solidify at room temperature for 30 minutes, and the plate was then incubated at 55 ℃ for 2-5 days.
5) A single colony was picked and PCR verified using the following pair of primers to verify whether the two ldh downstream sequences on the recombinant C.thermocellum genome had recombined.
Primer 1: TCTTCCTCTGTCCTGGCT, respectively; primer 2: GTATAAAGCCCATGCCTG is added.
The PCR reaction system is as follows: the primer 1 is 0.4 muL, the primer 2 is 0.4 muL, the bacterial liquid or plasmid is 1 muL, the prime star is 5 muL (efficiency is 10 s/kb), and the sterile water is 3.2 muL, wherein the concentration of each primer is 10 muM.
The PCR reaction program is: performing pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 s, annealing at 55 ℃ for 10 s, extension at 72 ℃ for 30 s to 2 min for 30 s, cycle number 30; further extending for 2 min at 72 ℃, and keeping the temperature for 20 min at 12 ℃.
The PCR amplification product was run on a gel (2% agarose gel electrophoresis, 110V, 30min) and the band size of the positive validation gel was 9684 bp. Indicating that two ldh genes downstream have recombined onto the C.thermocellum genome.
And adding the verified monoclonal cell colony into 5 mL of culture medium, culturing for 1-2 days, and storing to obtain the recombinant clostridium thermocellum engineering bacterium.
Example 2 fermentation culture of Clostridium thermocellum engineering bacteria
(1) Preparation of culture medium and deoxidization
The culture medium was MTC and prepared as shown in Table 1. After the preparation of the solution A is finished, sterilizing at 121 ℃ for 20 min; B. c, D, E the liquid is prepared according to the table, and is filled in an anaerobic bottle, the bottle is sealed by a rubber plug and an aluminum cover, and then the bottle is repeatedly vacuumized and filled with high-purity nitrogen for 3 times, and finally the interior of the anaerobic bottle is kept at positive pressure. The 5 culture solutions were injected into the sterilized anaerobic bottles containing solution A in an ultra clean bench using disposable sterile syringes and 0.22 μm sterile filter membranes according to a ratio of 40:2:1:1:1 (v/v), and the total volume was not more than 40% of the volume of the anaerobic bottles. Wherein, the carbon source (microcrystalline cellulose or straw fiber) in the solution A is microcrystalline cellulose Avicel PH105 in the seed culture medium, and the concentration is 5 g/L; straw fiber is used in the fermentation process for producing enzyme, and the concentration is 5 g/L.
TABLE 1 culture Medium formulation
(2) Preparing a seed solution: thawing the engineering bacteria of the clostridium thermocellum preserved at the temperature of-80 ℃ in a refrigerator at the temperature of 4 ℃, sucking 1mL of the engineering bacteria of the clostridium thermocellum under the aseptic condition, injecting the engineering bacteria of the clostridium thermocellum into a seed culture medium, and culturing for 24 hours at the temperature of 55 ℃ and at the speed of 200 rpm; the formulation of the seed medium is shown in Table 1, and the carbon source in solution A is microcrystalline cellulose (Avicel PH 105) at 5.00 g/L.
(3) Fermentation: the seed solution was inoculated into a fermentation medium at an inoculum size of 10% (v/v), and cultured at 55 ℃ and 200 rpm for 16 hours. The formulation of the fermentation medium is shown in Table 1, the carbon source is 5.00 g/L straw fiber, which can be extracted by pre-treating agricultural straw, see step (1) of example 3.
Example 3 application of Clostridium thermocellum engineering bacteria in hemicellulose hydrolysis saccharification
(1) Preparing wheat straw fiber:
cleaning and chopping wheat straw raw materials, and treating by using sodium hydroxide and sodium sulfite, wherein the weight of the sodium hydroxide is as follows: the absolute dry weight of the wheat straw raw material is 1:6, and the weight of sodium sulfite: the weight ratio of the sodium hydroxide is 1:3, and the treatment conditions are as follows: the reaction temperature is 150 ℃ and the reaction time is 2 h. After the lignin is fully removed, extracting the wheat straw fiber, dehydrating and fully washing off residual alkali liquor; the straw fiber obtained by the method contains about 70% of cellulose, 15% of hemicellulose and 15% of ash and lignin.
(2) Hydrolysis of wheat straw fiber:
soaking the straw fiber in acetic acid-sodium acetate (or similar buffer pair with buffer effect), and adding clostridium thermocellum fermentation liquor to hydrolyze the straw fiber. The hydrolysis conditions were as follows: the pH value is 5, the solid-to-liquid ratio is 1:10, the addition amount of the clostridium thermocellum fermentation liquor of unit mass straw fibers (per gram of straw fibers) is 20 mL, the cellulase is 10 FPU, the reaction temperature is 50 ℃, the reaction time is 24 hours, and the oscillation rate is 200 r/min.
At the end of the hydrolysis, the xylose concentration in the hydrolysate was about 12.50 g/L and the glucose concentration was 65.00 g/L. Under the same condition, when fermentation liquor of a clostridium thermocellum wild strain PN2102 (namely the original strain modified by the strain of the invention) is used for hydrolysis, the hydrolysis time is 24 hours, the xylose concentration is about 8.10 g/L, and the glucose concentration is 56.00 g/L.
The present invention has been described in detail with reference to the examples, but the present invention is only preferred examples of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
SEQUENCE LISTING
<110> Suzhou Polyvier Biotech limited
<120> engineering strain capable of efficiently secreting xylanase, construction method and application
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 7769
<212> DNA
<213> Clostridium thermocellum engineering strain
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ctggaacaac acccaagttc ttccgtccgc cgaacctcga aacaagccca acattattca 2340
acaatgttga cttggtgttt gtcggcggct taacggcaaa tgactggatt ccatccacaa 2400
ccgccgaaca gagggctgcc gcagttataa acggtgtcag agacggtaca ataattcttt 2460
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cacttaagag ccggggctat gaatttgtga ccttgactga gttgttcacg ttaaagggtg 2580
tgccaattga cccatcagtc aaaagaatgt ataactctgt accgtaattt taaattttgg 2640
gaggtagatc tatgctgaag aaaaaactgt tgaccctttt gacagtcttt gctctgctga 2700
ctgtcggtat ctgcggaagt tttttgccgt tacccaaagc atccgcagca gctctgattt 2760
acgatgattt tgaaacaggt ctgaacggat ggggaccaag aggaccggaa accgtcgaac 2820
ttaccaccga ggaagcttac tcgggaagat acagtttgaa ggtcagcgga cgtaccagca 2880
catggaacgg gcccatggtt gacaaaaccg atgtgttgac tttgggcgaa agctataagt 2940
tgggcgtata tgtaaaattc gtgggtgatt cctattcaaa tgagcaaaga ttcagtttgc 3000
agcttcaata taacgacgga gcaggagatg tataccaaaa tataaaaacc gccacggttt 3060
acaagggaac atggactttg ctggaaggac agcttacagt tcccagccat gcaaaggacg 3120
taaaaatata tgtggaaacc gaatttaaaa attctccgag tccgcaggac ttgatggatt 3180
tctatattga cgatttcaca gcaacacctg caaatttgcc tgaaattgag aaagatattc 3240
caagcttgaa agatgtcttt gccggttatt tcaaagtggg tggtgccgca actgtggcgg 3300
aactggcgcc gaagcctgca aaagagcttt tcctcaagca ttataacagc ttgacttttg 3360
gtaatgagtt aaaaccggaa agtgtacttg actatgatgc tacaattgct tatatggagg 3420
caaacggagg cgaccaggtt aatccgcaga taaccttgag agcggcaaga cccctgttgg 3480
agtttgcgaa agaacacaac atacctgtaa gaggacatac ccttgtatgg cacagccaga 3540
caccggactg gttcttcaga gaaaattact ctcaggacga aaatgctccc tgggcatcca 3600
aggaagtaat gctgcaaagg ttggaaaact acataaagaa tttaatggaa gctttggcga 3660
ccgaatatcc gacggttaag ttctatgcat gggacgttgt gaatgaggct gttgatccta 3720
atacttcaga cggtatgaga actccgggtt cgaataacaa aaatcccgga agctccctgt 3780
ggatgcaaac cgttggaaga gattttattg ttaaagcttt tgaatatgca agaaaatatg 3840
ctcctgcgga ttgtaaactc ttctacaatg actataatga atatgaagac agaaaatgtg 3900
attttattat tgaaattctt accgaactta aagccaaagg cctggttgac ggtatgggta 3960
tgcaatccca ctgggttatg gattatccaa gcataagcat gtttgaaaaa tccatcagaa 4020
gatatgcagc attgggattg gaaattcagc ttaccgagct ggatataaga aatcctgaca 4080
acagccagtg ggctttggaa cgtcaggcta atcgttataa ggagcttgta acaaaattgg 4140
tcgatttgaa aaaagaaggc ataaacatta cggcattggt attctgggga ataaccgacg 4200
cgacaagctg gcttggagga tatccgctcc tgtttgacgc ggaatacaag gcaaaacctg 4260
cattttatgc tatagttaac agcgttccgc cgcttccgac agaaccgccg gttcaggtta 4320
tacccggtga cgtaaacggt gacggtcgtg taaattcatc cgacttgact cttatgaaaa 4380
gatacctttt aaaatccata agcgacttcc cgacaccgga aggaaaaatt gcggcggatt 4440
taaacgaaga cggcaaggta aactcgacag atttgttagc gctgaaaaaa ctcgttctga 4500
gagaactttg attaaaattt aaaaggaggt tgcttatgaa aaacaagaga gttttggcaa 4560
aaataacggc tcttgtggta ttgctgggag tgttttttgt attaccgtca aacataagtc 4620
agctatatgc tgattatgaa gtggttcatg acacttttga agttaacttt gacggatggt 4680
gtaacttggg agtcgacaca tatttaacgg cagttgaaaa tgaaggaaac aacggtacaa 4740
gaggtatgat ggtaataaat cgctccagtg cgagtgacgg tgcgtattcg gaaaaaggtt 4800
tctatctcga cggtggtgta gaatacaagt acagtgtttt tgtaaaacac aacgggaccg 4860
gcaccgaaac tttcaaactt tctgtgtcct atttggattc ggaaacagaa gaagaaaata 4920
aggaagtaat tgcaacaaag gatgttgtgg ccggagaatg gactgagatt tcggcaaaat 4980
acaaagcacc caaaactgca gtgaatatta ctttgtcaat tacaaccgac agcactgtag 5040
atttcatttt tgacgatgta accataaccc gtaaaggaat ggctgaggca aacacagtat 5100
atgcagcaaa cgctgtgctg aaagatatgt atgcaaacta tttcagagtt ggttcggtac 5160
ttaactccgg aacggtaaac aattcatcaa taaaggcctt gattttaaga gagtttaaca 5220
gtattacctg tgaaaatgaa atgaagcctg atgccacact ggttcaatca ggatcaacca 5280
atacaaatat cagggtttct cttaatcgtg cagcaagtat tttaaacttc tgtgcacaaa 5340
ataatatagc cgtcagaggt catacactgg tttggcacag ccagacacct caatggtttt 5400
tcaaagacaa tttccaggac aacggaaact gggtttccca atcagttatg gaccagcgtt 5460
tggaaagcta cataaaaaat atgtttgctg aaatccaaag acagtatccg tctttgaatc 5520
tttatgccta tgacgttgta aatgaggcag taagtgatga tgcaaacagg accagatatt 5580
atggcggggc gagggaacct ggatacggaa atggtagatc tccatgggtt cagatctacg 5640
gagacaacaa atttattgag aaagcattta catatgcaag aaaatatgct ccggcaaatt 5700
gtaagcttta ctacaacgat tacaacgaat attgggatca taagagagac tgtattgcct 5760
caatttgtgc aaacttgtac aacaagggct tgcttgacgg tgtgggaatg cagtcccata 5820
ttaatgcgga tatgaatgga ttctcaggta tacaaaatta taaagcagct ttgcagaaat 5880
atataaatat cggttgtgat gtccaaatta ccgagcttga tattagtaca gaaaacggca 5940
aatttagctt acagcagcag gctgataaat ataaagctgt tttccaggca gctgttgata 6000
taaacagaac ctccagcaaa ggaaaggtta cggctgtctg tgtatgggga cctaatgacg 6060
ccaatacttg gctcggttca caaaatgcac ctcttttgtt taacgcaaac aatcaaccga 6120
aaccggcata caatgcggtt gcatccatta ttcctcagtc cgaatggggc gacggtaaca 6180
atccggccgg cggcggagga ggaggcaaac cggaagagcc ggatgcaaac ggatattatt 6240
atcatgacac ttttgaagga agcgtaggac agtggacagc cagaggacct gcggaagttc 6300
tgcttagcgg aagaacggct tacaaaggtt cagaatcact cttggtaagg aaccgtacgg 6360
cagcatggaa cggagcacaa cgggcgctga atcccagaac gtttgttccc ggaaacacat 6420
attgtttcag cgtagtggca tcgtttattg aaggtgcgtc ttccacaaca ttctgcatga 6480
agctgcaata cgtagacgga agcggcactc aacggtatga taccatagat atgaaaactg 6540
tgggtccaaa tcagtgggtt cacctgtaca atccgcaata cagaattcct tccgatgcaa 6600
cagatatgta tgtttatgtg gaaacagcgg atgacaccat taacttctac atagatgagg 6660
caatcggagc ggttgccgga actgtaatcg aaggacctgc tccacagcct acacagcctc 6720
cggtactgct tggcgatgta aacggtgatg gaaccattaa ctcaactgac ttgacaatgt 6780
taaagagaag cgtgttgagg gcaatcaccc ttaccgacga tgcaaaggct agagcagacg 6840
ttgacaagaa tggatcgata aacagcactg atgttttact tctttcacgc taccttttaa 6900
gagtaatcga caaatttcct gtagcagaaa atccttcttc ttcttttaaa tatgagtcgg 6960
ccgtgcaata tcggccggct cctgattctt atttaaaccc ttgtccgcag gcgggaagaa 7020
ttgtcaagga aacatataca ggaataaacg gaactaagag tcttaatgta tatcttccat 7080
acggttatga tccgaacaaa aaatataaca ttttctacct tatgcatggc ggcggtgaaa 7140
atgagaatac gattttcagc aacgatgtta aattgcaaaa tatccttgac cacgcgatta 7200
tgaacggtga acttgagcct ttgattgtag taacacccac tttcaacggc ggaaactgca 7260
cggcccaaaa cttttatcag gaattcaggc aaaatgtcat tccttttgtg gaaagcaagt 7320
actctactta tgcagaatca acaaccccac agggaatagc cgcttcaaga atgcacagag 7380
gtttcggcgg attctcaatg ggaggattga caacatggta tgtaatggtt aactgccttg 7440
attacgttgc atattttatg cctttaagcg gtgactactg gtatggaaac agtccgcagg 7500
ataaggctaa ttcaattgct gaagcaatta acagatccgg actttcaaag agggagtatt 7560
tcgtatttgc ggccaccggt tccgaggata ttgcatatgc taatatgaat cctcaaattg 7620
aagctatgaa ggctttgccg cattttgatt atacttcgga tttttccaaa ggtaattttt 7680
actttcttgt agctccgggc gccactcact ggtggggata cgtaagacat tatatttatg 7740
atgcacttcc atatttcttc catgaatga 7769
Claims (8)
1. An engineering strain capable of efficiently secreting xylanase, which is characterized by comprising the following components in percentage by weight: the strain is xylanase A in clostridium thermocellumxynAGene, xylanase CxynCGene and xylanase YxynYThe clostridium thermocellum engineering strain is obtained by gene overexpression.
2. An engineered strain of clostridium thermocellum according to claim 1, characterized in that: the Clostridium thermocellum is Clostridium thermocellumClostridium thermocellumPN2102 with preservation date of 2021 year, 07 month and 09 day, and preservation unit of China general microbiological culture Collection center with preservation number of CGMCC No. 22869.
3. An engineered strain of clostridium thermocellum according to claim 1, characterized in that: the nucleotide sequence of the overexpression gene is shown as SEQ ID No. 1.
4. The method for constructing engineered strain of Clostridium thermocellum according to any one of claims 1-3, wherein: introduction of xylanase A for overexpression in Clostridium thermocellumxynAGene, xylanase C xynCGene and xylanase YxynYRecombinant plasmids of the genes.
5. The construction method according to claim 4, wherein: the gapD promoter and xylanase expression gene sequence of Clostridium thermocellum were inserted downstream of the L-lactate dehydrogenase gene on the Clostridium thermocellum genome.
6. The construction method according to claim 5, wherein: the inserted expression gene sequence has a nucleotide sequence shown as SEQ ID NO. 1.
7. The application of the clostridium thermocellum engineering strain capable of efficiently secreting xylanase in hemicellulose hydrolysis saccharification according to claim 1.
8. Use according to claim 7, characterized in that: and (3) compounding fermentation liquor obtained by anaerobic fermentation of the clostridium thermocellum engineering strain with cellulase to hydrolyze the straw fiber.
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| US20060105442A1 (en) * | 2004-11-10 | 2006-05-18 | Wu J H D | Promoters and proteins from Clostridium thermocellum and uses thereof |
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| CN108977421A (en) * | 2018-08-17 | 2018-12-11 | 中国科学院青岛生物能源与过程研究所 | For being catalyzed the full bacterial enzyme preparation of lignocellulosic saccharification |
| CN111386341A (en) * | 2017-06-07 | 2020-07-07 | Ptt全球化学公共有限公司 | Mutant strain clostridium thermocellum for producing cellulase and xylanase and preparation method thereof |
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| US20060105442A1 (en) * | 2004-11-10 | 2006-05-18 | Wu J H D | Promoters and proteins from Clostridium thermocellum and uses thereof |
| CN103429751A (en) * | 2010-12-22 | 2013-12-04 | 马斯科马公司 | Genetically modified clostridium thermocellum engineered to ferment xylose |
| CN111386341A (en) * | 2017-06-07 | 2020-07-07 | Ptt全球化学公共有限公司 | Mutant strain clostridium thermocellum for producing cellulase and xylanase and preparation method thereof |
| CN108977421A (en) * | 2018-08-17 | 2018-12-11 | 中国科学院青岛生物能源与过程研究所 | For being catalyzed the full bacterial enzyme preparation of lignocellulosic saccharification |
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