CN114717174B

CN114717174B - Engineering strain for producing high-quality reducing sugar, construction method and application thereof

Info

Publication number: CN114717174B
Application number: CN202210444632.1A
Authority: CN
Inventors: 张天元; 邵雄俊; 薛怡芸
Original assignee: Suzhou Juwei Yuanchuang Biotechnology Co ltd
Current assignee: Suzhou Juwei Yuanchuang Biotechnology Co ltd
Priority date: 2022-04-26
Filing date: 2022-04-26
Publication date: 2023-10-20
Anticipated expiration: 2042-04-26
Also published as: CN114717174A

Abstract

The application belongs to the technical field of microorganism application, and particularly relates to an engineering strain for producing high-quality reducing sugar, a construction method and application thereof, wherein the engineering strain is a clostridium thermocellum engineering strain for knocking out genes of lactate dehydrogenase, phosphoacetyl transferase and acetate kinase. The clostridium thermocellum original strain isClostridium thermocellumPN2102, the preservation date is 2021, 07 and 09, the preservation unit is China general microbiological culture Collection center, and the preservation number is CGMCC No.22869. The beneficial effects of the application are as follows: through metabolic transformation, lactic dehydrogenase, phosphoacetyl transferase and acetate kinase genes of clostridium thermocellum PN2102 are knocked out, so that clostridium thermocellum engineering bacteria are obtained, lactic acid and acetic acid are not generated in the fermentation and hydrolysis processes, difficult-to-remove fermentation inhibitors are avoided, and the quality of hydrolysate products is improved.

Description

Engineering strain for producing high-quality reducing sugar, construction method and application thereof

Technical Field

The application belongs to the technical field of microorganism application, and particularly relates to an engineering strain for producing high-quality reducing sugar, a construction method and application thereof.

Background

Clostridium thermocellum is one of the most efficient microorganisms known in nature to degrade cellulose, and secreted fiber corpuscles have extremely strong cellulose degradation ability. The ability of the fiber corpuscles to degrade cellulose is about 50 times greater than that of trichoderma, and this efficiency depends on the specific structure of the fiber corpuscles. Clostridium thermocellum is a strictly anaerobic thermophilic microorganism, and the special growth conditions lead the secreted lignocellulose hydrolase to have better heat resistance and minimal risk of mixed bacteria pollution.

In the application of lignocellulose hydrolysis of clostridium thermocellum, due to the complex metabolic pathway of clostridium thermocellum, the secretion of fiber corpuscles is accompanied by metabolic byproducts such as acetic acid, lactic acid and the like, and the byproducts have obvious inhibition effect on the growth of microorganisms.

In the prior art, the use of lignocellulose by clostridium thermocellum mainly involves the following aspects: 1) Clostridium thermocellum directly utilizes lignocellulose to produce hydrogen, ethanol and the like, as in application patent CN111394394a; 2) Clostridium thermocellum and other anaerobic strains co-produce biological products through lignocellulose saccharification technology, such as application patent CN108893501A, CN111349565a and the like. The application patent CN108866025A discloses a cellulase preparation and application thereof, and the yield of the obtained reducing sugar is 90-500 mM, and the cellulase preparation is a high-quality carbon source for microorganism culture; however, in addition to fermentable sugars, the saccharification liquid contains up to 4 g/L acetic acid and 5 g/L ethanol, and too high acetic acid and ethanol are unfavorable for the growth of microorganisms, such as chlorella, rhodozyma and the like. Currently, no studies on metabolic byproducts of clostridium thermocellum and related metabolic pathway modification are found in the existing studies.

Disclosure of Invention

In order to overcome the defects in the prior art, the application provides an engineering strain for producing high-quality reducing sugar, a construction method and application thereof, and provides a genetically modified clostridium thermocellum for secretion of metabolic byproducts during anaerobic fermentation of clostridium thermocellum in the prior art, which can effectively reduce the production of the metabolic byproducts and greatly improve the quality of hydrolysate products.

In order to achieve the above purpose, the present application adopts the following technical scheme:

an engineering strain for producing high-quality reducing sugar, which is characterized in that: the engineering strain is clostridium thermocellum, which is subjected to gene modification and knockout of L-lactate dehydrogenase geneldh) Phosphoacetyl transferase/acetate kinase gene ]pta-ack) Engineering strain.

The clostridium thermocellum is wild clostridium thermocellum, preferably clostridium thermocellum @, and the clostridium thermocellum is a wild clostridium thermocellumClostridium thermocellum) PN2102, the preservation date is 2021, 07 and 09, and the preservation unit is China general microbiological culture Collection center (address: the preservation number is CGMCC No.22869.

Preferably, the knocked-out L-lactate dehydrogenase geneldhThe nucleotide sequence of the gene is shown as SEQ ID No.2, and the knocked-out phosphoacetyl transferase/acetate kinase gene is shown as the expression of [ (]pta-ack) The nucleotide sequence of (2) is shown as SEQ ID No. 3.

The application also provides a construction method of the engineering strain of the high-quality reducing sugar, which knocks the L-lactate dehydrogenase gene on the clostridium thermocellum genome through homologous recombinationldh) And phosphoacetyl transferase/acetate kinase gene ]pta-ack) Obtaining engineering strain.

The L-lactate dehydrogenase geneldhThe nucleotide sequence of the phosphoacetyl transferase/acetate kinase gene is shown as SEQ ID No.2pta-ack) The nucleotide sequence of (2) is shown as SEQ ID No. 3.

The construction method specifically comprises the following steps: (1) Construction of wild type Clostridium thermocellum as starting strainldhGene knockout recombinant plasmid PUC-ldhThe method comprises the steps of carrying out a first treatment on the surface of the (2) Recombinant plasmid PUC-ldhTransfer into clostridium thermocellum to construct L-lactate dehydrogenase geneldhThe deleted clostridium thermocellum engineering bacteria; (3) Constructionpta-ackGene knockout recombinant plasmid PUC-pta-ackThe method comprises the steps of carrying out a first treatment on the surface of the (4) Recombinant plasmid PUC-pta-ackTransfer-inldhConstruction of L-lactate dehydrogenase Gene from deleted Clostridium thermocellum engineering bacterialdh、Phosphoacetyl transferase/acetate kinase gene%pta-ack) Lack of supplyThe clostridium thermocellum engineering bacteria are lost.

When clostridium thermocellum PN2102 is used as an original strain, the construction method specifically comprises the following steps: (1) Construction of Clostridium thermocellum PN2102 as initial strainldhGene knockout recombinant plasmid PUC-ldhThe method comprises the steps of carrying out a first treatment on the surface of the (2) Construction of Clostridium thermocellum L-lactate dehydrogenase GeneldhDeletion engineering bacterium PN 2102-ΔldhThe method comprises the steps of carrying out a first treatment on the surface of the (3) ConstructionptaackGene knockout recombinant plasmid PUC-pta-ackThe method comprises the steps of carrying out a first treatment on the surface of the (4) ConstructionldhGene, phosphoacetyl transferase/acetate kinase genepta-ack) Deleted clostridium thermocellum engineering bacteria PN 2102-ΔldhΔptaack。

Preferably, the recombinant plasmid PUC of step (1)ldhThe construction process of (1) is as follows: 1) Inserting upstream and downstream gene recombination fragments of L-lactate dehydrogenase in the front of the promoter P-gapD by using pPN01 as template plasmidhptThe rear end of the gene is inserted into a gene recombination fragment of the L-lactate dehydrogenase midcourse; 2) Linearization of template plasmid pPN; 3) Connecting the upper, middle and downstream gene recombination fragments with a linearized template plasmid pPN by Gibson Assembly, transferring into BL21, and performing PCR screening and confirmation; 4) Recovering the DNA carrier fragment.

Further preferably, the upper, middle and downstream gene recombination fragments of the L-lactate dehydrogenase gene used for gene deletion plasmid construction are obtained by adding 20-25 bp template plasmid head-to-tail connection fragments to the following three pairs of primers through PCR:

upstream primer 1: TCTTCCTCTGTCCTGGCT;

upstream primer 2: TGCACCAACTACGGTTACTTTT;

midstream primer 1: AAAAAGCCGACGGAGAAG;

midstream primer 2: ATACCGTTTACACCCACGA;

downstream primer 1: AGTCCGGAAACACTCTAAAA;

downstream primer 2: GTATAAAGCCCATGCCTG.

Preferably, in step (2), the plasmid PUC-is transformed by electrotransformationldhTransferring into Clostridium thermocellum PN2102, deleting target gene sequence on genome via homologous recombination and resistance screening, and selecting and verifying positive monoclonal to obtain Clostridium thermocellum L-lactic dehydrogenase baseBecause ofldhDeletion engineering bacterium PN 2102-Δldh。

Further preferred, the phosphoacetyltransferase/acetate kinase gene for gene deletion plasmid constructionpta-ackThe upper, middle and downstream gene recombination fragments of (2) are obtained by adding 20-25 bp template plasmid head-tail connection fragments to the following three pairs of primers through PCR:

upstream primer 1: GGCAGACCTTCGGTTAAAA;

upstream primer 2: CGTCTGATTTCGCCCTTT;

midstream primer 1: AAGCCGCATCCATGATAG;

midstream primer 2: TTCAAAAACTCACTCCCGTC;

downstream primer 1: CCCGATGCGAAAGTAAAG;

downstream primer 2: CGTCACCATACAACAAACC.

Preferably, the recombinant plasmid PUC of step (3)pta-ackThe construction process of (1) is as follows: 1) pPN01 is used as a template plasmid and inserted into the front section of the promoter P-gapDpta-ackRecombinant fragments of upstream and downstream genes, inhptGene back end insertionpta-ackA midstream gene recombination fragment; 2) Linearization of template plasmid pPN; 3) Connecting the upper, middle and downstream gene recombination fragments with a linearized plasmid template pPN by Gibson Assembly, transferring into BL21, and performing PCR screening confirmation; 4) Recovering the DNA carrier fragment.

Preferably, in step (4), the plasmid PUC-is transformed by electrotransformationpta-ackTransfer into Clostridium thermocellum PN 2102-ΔldhIn engineering bacteria, target genes are subjected to homologous recombination and resistance screeningpta-ackDeleting the sequence on the genome, selecting and verifying positive monoclonal to obtain clostridium thermocellum engineering bacterium PN 2102-ΔldhΔptaack。

The application also provides application of the engineering strain for producing high-quality reducing sugar in efficient hydrolysis of fibers, wherein the fermentation liquor of clostridium thermocellum engineering bacteria is compounded with cellulase to hydrolyze straw fibers, 10-40-mL of fermentation liquor is added into each gram of straw fibers, 3-6-mg of xylanase is added into each gram of straw fibers, and the obtained hydrolysate is separated and concentrated to obtain a high-quality reducing sugar product.

The application of the engineering strain for producing high-quality reducing sugar in efficient fiber hydrolysis comprises the following steps:

(1) Pretreatment of lignocellulose: the method comprises the steps of taking common agricultural straws as raw materials, carrying out pretreatment by a physical or chemical method, removing lignin and extracting straw fibers; (2) anaerobic fermentation of clostridium thermocellum engineering bacteria: sequentially carrying out seed culture and anaerobic fermentation on clostridium thermocellum engineering bacteria to obtain fermentation liquor; (3) fiber hydrolysis: hydrolyzing the straw fiber obtained in the step (1) by compounding the fermentation liquor obtained in the step (2) with cellulase to obtain a hydrolysate; (4) separating and concentrating the fiber hydrolysate: and (3) carrying out solid-liquid separation on the hydrolysate obtained in the step (3), and concentrating the collected hydrolysate to obtain a reducing sugar product.

Preferably, in step (1), the pretreatment of lignocellulose comprises the steps of: carrying out hydrothermal reaction on the washed and chopped rice straw raw materials and alkaline substances (inorganic alkali), fully removing lignin, extracting straw fibers, dehydrating, and fully washing away residual alkali liquor to obtain straw fibers; wherein the alkaline substance comprises sodium hydroxide and sodium sulfite, and the ratio of the weight of the sodium hydroxide to the absolute dry weight of the rice straw raw material is 1: (4-6), wherein the weight ratio of the sodium sulfite to the sodium hydroxide is 1: (3-5), wherein the temperature of the hydrothermal reaction is 150-160 ℃, and the time of the hydrothermal reaction is 1-3 h.

Preferably, in the step (2), the seed culture and anaerobic fermentation process of the clostridium thermocellum engineering bacteria comprises the following steps: placing the clostridium thermocellum strain preserved at-80deg.C in a refrigerator at 4deg.C for thawing, sucking and injecting into seed culture medium under aseptic condition, and culturing at 50-65deg.C and 150-200 rpm for 16-24 h to obtain clostridium thermocellum strain seed solution; inoculating clostridium thermocellum seed liquid into fermentation medium in the inoculation amount of 5-10% (v/v), and culturing at 50-65 deg.c and 150-200 rpm for 16-24 h to obtain fermentation liquid.

Wherein the seed culture medium or the fermentation culture medium comprises the components with the volume ratio of 40:2:1:1: liquid A, liquid B, liquid C, liquid D and liquid E of 1; the A liquid comprises 5.00-10.00 g/L microcrystalline cellulose or straw fiber, 10.00 g/L3-morpholineA propane sulfonic acid; the solution B comprises 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 The method comprises the steps of carrying out a first treatment on the surface of the The solution C comprises 250.00 g/L urea; the D solution comprises 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 E solution comprises 1.00 g/L pyridoxamine dihydrochloride, 0.20 g/L para-aminobenzoic acid, 0.10 g/L biotin, 0.10 g/L VB ₁₂ The method comprises the steps of carrying out a first treatment on the surface of the Preferably, the straw fiber is the straw fiber obtained in the step (1).

Preferably, in the step (3), the specific steps of fiber hydrolysis are as follows: soaking the straw fiber in the step (1) in buffer solution with the pH value of 4.5-5.5, and adding the fermentation liquor and xylanase in the step (2) for hydrolysis; wherein: the solid-to-liquid ratio is 1: (10-30), the temperature is 45-55 ℃, the hydrolysis time is 12-48 and h, the oscillation rate is 150-250 r/min, the clostridium thermocellum fermentation liquid added per unit mass of straw fiber (i.e. per gram of straw fiber) is 10-40 mL, and the xylanase added per unit mass of straw fiber (i.e. per gram of straw fiber) is 3-6 mg.

The beneficial effects of the application are as follows: the clostridium thermocellum engineering bacteria provided by the application is subjected to metabolic modification on clostridium thermocellum PN2102, and lactic dehydrogenase, phosphoacetyl transferase gene and acetate kinase gene are knocked out, so that clostridium thermocellum engineering bacteria are obtained, the engineering bacteria cannot generate lactic acid and acetic acid in the fermentation and hydrolysis processes, the engineering bacteria can be effectively applied to hydrolysis of lignocellulose, fermentation inhibitors which are difficult to remove are avoided, the quality of hydrolysate products is improved, and the concentration of glucose and xylose in hydrolysate after concentration is 6.0 times and 6.1 times that before concentration.

Drawings

FIG. 1 is a recombinant plasmid PUC for knocking out the L-lactate dehydrogenase geneldhA map;

FIG. 2 is a recombinant plasmid PUC for knocking out genes of phosphoacetyltransferase and acetate kinasepta-ackA map;

FIG. 3 is a diagram of a DNA deletion proof gel for lactate dehydrogenase gene knockout in example 1, wherein wild-type strain PCR gave a DNA fragment of 2777 bp, and lactic dehydrogenase knockout strain PCR gave a DNA fragment of 1915 bp;

FIG. 4 is a diagram of a DNA deletion verification gel for knocking out genes of phosphoacetyltransferase and acetate kinase in example 2, wherein a wild-type strain PCR gives a DNA fragment of 4017 bp, and a strain PCR after knocking out the phosphoacetyltransferase and acetate kinase gives a DNA fragment of 1834 bp;

FIG. 5 is a pPN01 template plasmid map.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail below with reference to the accompanying drawings and examples, it being apparent that the described examples are only some, but not all, examples of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

The present application uses conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as those described in MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed (Sambrook, 2001) and CURRENTPROTOCOLSIN MOLECULAR BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, it is not intended that the application be limited to any particular method, protocol, or reagents described, as they may vary.

The application provides an engineering strain for producing high-quality reducing sugar, which is clostridium thermocellum and is subjected to genetic modification to knock out an L-lactate dehydrogenase geneldh) Phosphoacetyl transferase/acetate kinase gene ]pta-ack) Is a strain of the engineering strain of (a).

The clostridium thermocellum is wild clostridium thermocellum, which is separated from cow dung, is thermophilic and strictly anaerobic gram-positive bacterium, and has a growth temperature of 50-65 ℃ and a rapid growth speed. Preferably clostridium thermocellum @ isClostridium thermocellum) PN2102, the preservation date is 2021, 07 and 09,the preservation unit is China general microbiological culture Collection center (address: north Chen West Lu No.1, 3 of the Chaoyang district of Beijing city), and the preservation number is CGMCC No.22869. Clostridium thermocellum @Clostridium thermocellum) The nucleotide sequence of PN2102 is shown as SEQ ID No. 1.

Preferably, the knocked-out L-lactate dehydrogenase geneldhThe nucleotide sequence of the knocked-out phosphoacetyl transferase/acetate kinase is shown as SEQ ID No.2pta-ackThe nucleotide sequence of (2) is shown as SEQ ID No. 3.

The construction method takes clostridium thermocellum PN2102 as an original strain, and specifically comprises the following steps: (1) ConstructionldhGene knockout recombinant plasmid PUC-ldhThe method comprises the steps of carrying out a first treatment on the surface of the (2) Construction of Clostridium thermocellum L-lactate dehydrogenase GeneldhDeletion engineering bacterium PN 2102-Δ ldhThe method comprises the steps of carrying out a first treatment on the surface of the (3) Constructionpta-ackGene knockout recombinant plasmid PUC-pta-ackThe method comprises the steps of carrying out a first treatment on the surface of the (4) Construction of Clostridium thermocellum phosphoacetyltransferase/acetate kinase Gene (pta-ack) deletion engineering bacterium PN 2102-ΔldhΔpta-ack。

The application also provides application of the engineering strain for producing high-quality reducing sugar in efficient hydrolysis of fibers, wherein the fermentation broth of clostridium thermocellum engineering bacteria is used for compounding cellulase to hydrolyze straw fibers, and the obtained hydrolysate is separated and concentrated to obtain high-quality reducing sugar products (xylose and glucose). The method comprises the following steps: (1) pretreatment of lignocellulose; (2) anaerobic fermentation of clostridium thermocellum engineering bacteria; (3) Hydrolyzing the straw fiber obtained in the step (1) by compounding the fermentation liquor obtained in the step (2) with cellulase; (4) Separating and concentrating the fiber hydrolysate to obtain a reducing sugar product.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this application belongs.

The culture medium used in the application is as follows: CTFUD medium (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, 0.5. 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, if solid medium is formulated, 10 g/L agar is added. The test materials used in the examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The experimental methods in the examples are all conventional methods unless otherwise specified.

The clostridium thermocellum of the application is clostridium thermocellum @Clostridium thermocellum) PN2102, the preservation date is 2021, 07 and 09, and the preservation unit is China general microbiological culture Collection center (address: the preservation number is CGMCC No.22869. Has a 16s rDNA sequence shown as SEQ ID No. 1.

L-lactate dehydrogenase Gene [ (]ldh) And phosphoacetyl transferase/acetate kinase gene ]pta-ack) The sequence is derived from clostridium thermocellum, and L-lactate dehydrogenase gene of clostridium thermocellum is obtained from the literature (Application of Long Sequence Reads to Upgrade Genomes for Clostridium thermocellum AD2, clostridium thermocellum LQRI, and Pelosinus fermentans R)ldh) And phosphoacetyl transferase/acetate kinase gene ]pta-ack) The sequence, genBank number GenBank: CP016502.1, was synthesized by Nanjing Jinsri Biotechnology Co.

Example 1 thermal fiber shuttleBacterial L-lactate dehydrogenase geneldhDeletion engineering bacterium PN 2102-ΔldhConstruction of (3)

Construction of L-lactate dehydrogenase Gene knockout recombinant plasmid PUC-ldhThe recombinant plasmid was transferred into clostridium thermocellum PN2102 by electrotransformation. Knocked out L-lactate dehydrogenase geneldhThe nucleotide sequence of (2) is shown as SEQ ID No. 2.

The expression vector for constructing the recombinant plasmid is pPN01 template plasmid (as shown in FIG. 5), and can be stored in a laboratory after being purchased from the market or synthesized by a gene design company. The template plasmid contained gapDH promoter, thiamphenicol resistance gene, hpt gene, puc19 initiation region, tdk gene, replication protein and ampicillin resistance gene.

(1) L-lactate dehydrogenase gene knockout recombinant plasmid PUC-ldhDesign and construction of (a)

PCR amplification of target Gene

Inserting upstream and downstream gene recombination fragments of L-lactate dehydrogenase in the front of the promoter P-gapD by using pPN01 as template plasmidhptThe gene rear end is inserted into the gene recombination fragment of L-lactic dehydrogenase. The upper, middle and downstream gene recombination fragments of the L-lactate dehydrogenase gene for constructing the gene deletion plasmid are obtained by adding 20-25 bp template plasmid head-tail connection fragments into the following three pairs of primers through PCR.

The PCR reaction system is as follows: the usage amount of the upper, middle and downstream primers 1 is 0.4 mu L respectively, the usage amount of the primers 2 is 0.4 mu L, the usage amount of bacterial liquid or plasmid is 1 mu L, the usage amount of prime star is 5 mu L (efficiency 10 s/kb), the usage amount of sterile water is 3.2 mu L, wherein the concentration of each primer is 10 mu M respectively.

The PCR reaction procedure was: pre-denaturation at 98℃for 2 min; denaturation at 98℃for 10s, annealing at 55℃for 10s, extension at 72℃for 30 min s to 2 min for 30s, cycle number 30; further extending at 72 ℃ for 2 min, and preserving heat at 12 ℃ for 20 min.

B. Linearization of template plasmid pPN and vector fragment recovery:

the pPN01 template plasmid was linearized and the primers and PCR conditions used were as follows:

primer 1: CTTACTCTAGCAGACTTGGCAATG the number of the individual pieces of the plastic,

primer 2: TCCATCTGTTTCGTTTGCCCTTTCC.

The PCR reaction system is as follows: primer 1 quantity 0.4 mu L, primer 2 quantity 0.4 mu L, bacterial liquid or plasmid quantity 1 mu L, prime star quantity 5 mu L (efficiency 10 s/kb), sterile water quantity 3.2 mu L. Wherein each primer concentration was 10. Mu.M.

The upper, middle and downstream gene recombination fragments are connected with a linearized template plasmid pPN by Gibson Assembly (30 ng for three recombination fragments, 30 ng,GibsonAssembly 5 mu L for linearized template plasmid pPN01, 10 mu L for sterile water, and 1 h for heat preservation at 50 ℃), then transferred into BL21 and coated on an ampicillin-containing rubber plate, and PCR screening and confirmation is carried out after bacterial colonies grow out.

The primers and conditions for PCR screening and confirmation are as follows:

primer 1: AGCGGTAAAAGTGAAGAAC; primer 2: TGGGCCCCTACTAAAATGA.

The PCR reaction system is as follows: primer 1 quantity 0.4 mu L, primer 2 quantity 0.4 mu L, bacterial liquid or plasmid quantity 1 mu L, prime star quantity 5 mu L (efficiency 10 s/kb), sterile water quantity 3.2 mu L, wherein each primer concentration is 10 mu M respectively. The PCR reaction procedure was: pre-denaturation at 98℃for 2 min; denaturation at 98℃for 10s, annealing at 55℃for 10s, extension at 72℃for 30s to 2 min for 30s, cycle number 30; further extending at 72 ℃ for 2 min, and preserving heat at 12 ℃ for 20 min.

The products after PCR amplification were subjected to agarose gel electrophoresis experiments (2% agarose, 110V, 30 min), and the positive verification gel pattern band size was 473 bp. Description of the recombinant plasmid PUC-ldhThe construction was successful. FIG. 1 is a recombinant plasmid PUC for knocking out the L-lactate dehydrogenase geneldhA map of, whereinldhUpstream of this is 967 and bp,ldhdownstream of this is 948 bp,ldhmidstream is 727 bp.

(2) Recombinant plasmid PUC-ldhIs transferred into (a)

Plasmid pPN01 by means of electrotransformationldhTransferring into clostridium thermocellum PN2102, and deleting target gene sequence in genome via homologous recombination and resistance screening. The operation steps are as follows:

1) Cell growth

After 50mL of the bacterial liquid was cultured until OD600 = 0.6-1, it was placed on ice for 20 minutes.

2) Cell collection and washing

Cells were collected by centrifugation at 6500 g at 4deg.C for 10 minutes, after centrifugation, the supernatant was carefully removed, wash buffer (purified water by reverse osmosis after steam sterilization) was carefully added to the centrifuge tube or flask without stirring the pellet during addition, and then again centrifuged at 6500 g at 4deg.C for 10 minutes, the supernatant was carefully removed, and the above steps were repeated twice.

3) Electric conversion

The collected cells were placed in an anaerobic chamber and gently resuspended with 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 electric beaker and thoroughly mixed. The electrotransformation conditions were set to a voltage of 1500V for a duration of 1.5. 1.5 ms for electrotransformation.

4) Incubating the electrotransformed cells

The cells after the electrotransformation were remixed with 1mL of CTFUD medium and incubated at 51 ℃ for 16 hours.

(3) Positive monoclonal selection and validation

1) Screening of transformed cells

The CTFUD solid medium was thawed and cooled to 55℃and 6 mg/mL of thiamphenicol 1mL was added to 20 mL of CTFUD solid medium with 50. Mu.L of the incubated electrotransformed cells and poured into a plate, the medium in the plate was allowed to solidify at room temperature for 30 minutes, and then the plate was allowed to stand at 55℃for 3 to 5 days.

2) Monoclonal colony is selected, and recombinant plasmid PUC (recombinant plasmid coding sequence) is verified by PCR (polymerase chain reaction)ldhWhether or not it has been transferred into Clostridium thermocellum competent cells. The PCR amplified products were subjected to a 2% agarose gel electrophoresis experiment (110V, 30 min), and the positive verification gel band size was 473 bp.

The PCR verification primer and the conditions are as follows:

primer 1: AGCGGTAAAAGTGAAGAAC;

primer 2: TGGGCCCCTACTAAAATGA.

The PCR reaction system is as follows: primer 1 quantity 0.4 mu L, primer 2 quantity 0.4 mu L, bacterial liquid or plasmid quantity 1 mu L, prime star quantity 5 mu L (efficiency 10 s/kb), sterile water quantity 3.2 mu L, each primer concentration is 10 mu M respectively.

The PCR reaction procedure was: pre-denaturation at 98℃for 2 min; denaturation at 98℃for 10s, annealing at 55℃for 10s, extension at 72℃for 30 min s to 2 min for 30s, cycle number 30; further extension at 72℃for 2 min and incubation at 12℃for 20 min, each primer concentration was 10. Mu.M.

The verified colony of monoclonal cells was inoculated into CTFUD liquid medium containing thiamphenicol (6 mg/mL) and cultured at 55℃for 1-2 days.

3) mu.L to 1mL of the culture broth was added to 20 mL CTFUD solid medium containing thiamphenicol (6 mg/L) and FUDR (10 mg/L), poured into a plate, and the medium in the plate was allowed to solidify at room temperature for 30 minutes, followed by culturing the plate at 55℃for 2 to 5 days.

4) Monoclonal colonies were selected and used as PCR-verified plasmid PUC-ldhIn (a) and (b)ldhUpstream andldhwhether midstream has been recombined onto the clostridium thermocellum genome. The primers and conditions for PCR verification were as follows:

primer 1: TCTTCCTCTGTCCTGGCT;

primer 2: GTATAAAGCCCATGCCTG.

The PCR reaction procedure was: pre-denaturation at 98℃for 2 min; denaturation at 98℃for 10s, annealing at 55℃for 10s, extension at 72℃for 30s to 2 min for 30s, cycle number 30; further extending at 72 ℃ for 2 min, and preserving heat at 12 ℃ for 20 min.

The PCR amplified products were subjected to a 2% agarose gel electrophoresis experiment (110V, 30 min), and the positive validation gel band size was 5493 bp.

The verified colony of monoclonal cells was added to 20 mL of CTFUD solid medium containing 8AZH (500 mg/L), poured into a plate, and the medium in the plate was allowed to solidify at room temperature for 30 minutes, followed by culturing the plate at 55℃for 2-5 days.

5) Monoclonal colony is selected and used for PCR verification of two recombinant plasmid genomesldhWhether downstream has recombined. The verified monoclonal cell colony is added to 5 mL culture medium for 1-2 days and then stored.

The primers and conditions for PCR verification were as follows:

primer 1: TCTTCCTCTGTCCTGGCT;

primer 2: GTATAAAGCCCATGCCTG.

The PCR amplified products were subjected to a 2% agarose gel electrophoresis experiment (110V, 30 min), and FIG. 3 is a DNA deletion proof gel diagram of the knocked-out L-lactate dehydrogenase gene. As shown in FIG. 3, the wild strain clostridium thermocellum PN2101 PCR amplified to obtain DNA fragment 2777 bp, and the strain with L-lactic dehydrogenase knocked out PCR obtained DNA fragment 1915 bp, the fragment size meets the design expectation, proves that the DNA fragment is on the clostridium thermocellum PN2102 genomeldhThe gene sequence SEQ ID NO.2 has been deleted to give Clostridium thermocellum PN 2102-ΔldhEngineering bacteria.

EXAMPLE 2 Clostridium thermocellum phosphoacetyltransferase/acetate kinase GeneptaacK deletion engineering bacterium PN 2102-Δ ldhΔptaackConstruction of (3)

Construction of phosphoacetyl transferase/acetate kinase GeneptaacK-knocked-out recombinant plasmid PUC-pta-ackTransformation of recombinant plasmids into Clostridium thermocellum PN2102 by electrotransformationΔldh. Knocked out phosphoacetyl transferase/acetate kinase geneptaacThe nucleotide sequence of k is shown in SEQ ID No. 3.

PCR amplification of target Gene

pPN01 is used as a template plasmid and inserted into the front section of the promoter P-gapDptaackRecombinant fragments of upstream and downstream genes, inhptGene back end insertionptaackA midstream gene recombination fragment. Phosphoacetyltransferase/acetate kinase gene for gene deletion plasmid constructionpta-ackThe upper, middle and downstream gene recombination fragments of (2) are obtained by adding 20-25 bp template plasmid head-tail connection fragments to the following three pairs of primers through PCR.

The PCR reaction system is as follows: the usage amount of the upper, middle and downstream primers 1 is 0.4 mu L respectively, the usage amount of the primers 2 is 0.4 mu L, the usage amount of bacterial liquid or plasmid is 1 mu L, the usage amount of prime star is 5 mu L (efficiency 10 s/kb), the usage amount of sterile water is 3.2 mu L, and the concentration of each primer is 10 mu M respectively.

B. Linearization of template plasmid pPN, construction of recombinant plasmid and recovery of vector fragment:

primer 2: TCCATCTGTTTCGTTTGCCCTTTCC.

The upper, middle and downstream gene recombination fragments are connected with a linearized plasmid template pPN by Gibson Assembly (three-segment recombination fragment 30 ng, linearized template plasmid pPN01 30 ng,Gibson Assembly5 [ mu ] L, sterile water is filled up to 10 [ mu ] L, heat preservation is carried out at 50 ℃ for 1 h), then the plasmid template is transferred into BL21 and coated on an ampicillin-containing rubber plate, and PCR screening and confirmation is carried out after bacterial colonies grow out. The PCR primer and the conditions are as follows:

primer 1: AGCGGTAAAAGTGAAGAAC;

primer 2: TGGGCCCCTACTAAAATGA.

The PCR amplified products were subjected to a 2% agarose gel electrophoresis experiment (110V, 30 min), and the positive verification gel band size was 473 bp. Description of the recombinant plasmid PUC-pta-ackThe construction was successful.

FIG. 2 is a recombinant plasmid PUC-ack with the phosphoacetyltransferase/acetate kinase gene knocked outpta-ackA map of, whereinptaackThe upstream of the probe is 911bp,ptaackthe downstream is 923bp,ptaackmidstream is 1028bp.

(2) Recombinant plasmid PUC-pta-ackIs transferred into (a)

Plasmid pPN01 by means of electrotransformationptaackTransfer into Clostridium thermocellum PN 2102-ΔldhIn engineering bacteria, target genes are subjected to homologous recombination and resistance screeningptaackSequences are deleted on the genome. The operation steps are as follows:

1) Cell growth

Culturing 50mL bacterial liquid to OD ₆₀₀ After 0.6-1, it was placed on ice for 20 minutes.

2) Cell collection and washing

3) Electric conversion

4) Incubating the electrotransformed cells

(3) Positive monoclonal selection and validation

1) Screening of transformed cells

CTFUD solid medium was thawed and cooled to 55deg.C, 6 mg/mL of thiamphenicol 1mL and 50. Mu.L of the incubated electrotransformed cells were added to 20 mL medium and poured into plates, the medium in the plates was allowed to solidify for 30 minutes at room temperature, and the plates were then allowed to incubate at 55deg.C for 3-5 days.

2) Monoclonal colonies were selected and used as PCR-verified plasmid PUC-pta-ackWhether or not the bacterium has been transferred into Clostridium thermocellum PN 2102-ΔldhEngineering bacteria cells. The PCR verification primer and the conditions are as follows:

primer 1: AGCGGTAAAAGTGAAGAAC;

primer 2: TGGGCCCCTACTAAAATGA.

The PCR amplified products were subjected to a 2% agarose gel electrophoresis experiment (110V, 30 min), and the positive verification gel band size was 473 bp.

4) Monoclonal colonies were selected and used as PCR-verified plasmid PUC-pta-ackIn (a) and (b)pta-ackUpstream andpta-ackwhether midstream has recombined onto the genome. The PCR verification primer and the conditions are as follows:

gene knockout verification primer 1: GGCAGACCTTCGGTTAAAA;

gene knockout verification primer 2: CGTCACCATACAACAAACC.

The PCR amplified products were subjected to a 2% agarose gel electrophoresis experiment (110V, 30 min), and the positive verification gel band size was 6755 bp.

The verified colony of monoclonal cells was added to 20 CTFUD solid medium containing 8AZH (500 mg/L) in mL, poured into a plate, and the medium in the plate was allowed to solidify at room temperature for 30 minutes, followed by culturing the plate at 55℃for 2-5 days.

9) Monoclonal colonies were selected and used as PCR-verified plasmid PUC-pta-ackTwo on the genomepta-ackWhether downstream has recombined. The PCR verification primer and the conditions are as follows:

gene knockout verification primer 1: GGCAGACCTTCGGTTAAAA;

gene knockout verification primer 2: CGTCACCATACAACAAACC.

The PCR amplified products were subjected to a 2% agarose gel electrophoresis experiment (110V, 30 min), and FIG. 4 is a DNA deletion proof gel diagram of knocked out phosphoacetyl transferase and acetate kinase genes. As shown in FIG. 4, wild type strain clostridium thermocellum PN2102 PCR amplified to obtain DNA fragment 4017 bp, phosphoacetyl transferase/acetate kinase gene ptaackThe DNA fragment obtained by PCR of the knocked-out strain is 1834bp, the fragment size accords with design expectation, and the demonstration on genome is provedptaackThe gene sequence SEQ ID NO.3 has been deleted to give Clostridium thermocellum PN 2102-ΔldhΔpta-ackEngineering bacteria.

The verified monoclonal cell colonies were added to 5 ml tfud medium for 1-2 days and stored.

EXAMPLE 3 fermentation culture of Clostridium thermocellum engineering bacteria

(1) Preparation of culture medium and deoxidization

The culture medium adopts MTC culture medium, and the culture medium is divided into five types of ABCDE liquid, wherein the liquid 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, 25.00 g/L Na ₂ SO ₄ 、25.00 g/L KH ₂ PO ₄ 、62.50 g/L NaHCO ₃ The method comprises the steps of carrying out a first treatment on the surface of the 250.00/g/L urea for C solution and 50.00/g/L MgCl for D solution 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 E solution is 1.00 g/L pyridoxamine dihydrochloride, 0.20 g/L para-aminobenzoic acid, 0.10 g/L biotin, 0.10 g/L VB ₁₂ . The specific formulation is shown in table 1.

After the preparation of the solution A is finished, sterilizing at 121 ℃ for 20 min; B. c, D, E liquid is prepared according to the table, is filled in an anaerobic bottle, is sealed by adding a rubber plug aluminum cover, is repeatedly vacuumized and filled with high-purity nitrogen for 3 times, and finally keeps positive pressure in the anaerobic bottle. The 5 culture solutions were injected into the sterilized anaerobic jar containing solution A in a total volume of not more than 40% of the volume of the anaerobic jar in a super clean bench using a disposable sterile syringe and a 0.22 μm sterile filter membrane at a ratio of 40:2:1:1 (v/v). Wherein, the carbon source (microcrystalline cellulose or straw fiber) in the solution A, microcrystalline cellulose Avicel PH105 is used in the seed culture medium, and the concentration is 5 g/L; straw fiber is used in the enzyme production of fermentation, and the concentration is 5 g/L.

TABLE 1 Components and concentrations of MTC Medium formulations

(2) Preparing seed liquid: placing the clostridium thermocellum strain preserved at-80deg.C in a refrigerator at 4deg.C for thawing, sucking 1mL strain under aseptic condition, injecting into seed culture medium, and culturing at 55deg.C and 200 rpm for 24 h; in the seed culture medium, microcrystalline cellulose (Avicel PH 105) with a carbon source of 5.00 g/L in the MTC culture medium A solution has a specific formula shown in Table 1.

(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 h. In the fermentation medium, the carbon source of the A liquid in the MTC medium is 5.00 g/L straw fiber, and the specific formula is shown in Table 1. Wherein, the preparation method of the straw fiber is a pretreatment step of lignocellulose in the application, see the step (1) of the example 4: the straw raw materials are washed and chopped, sodium hydroxide and sodium sulfite are adopted for hydrothermal reaction, lignin is fully removed, straw fibers are extracted, and residual alkali liquor is fully washed away after dehydration. Wherein the weight of sodium hydroxide: the absolute dry weight of the rice straw raw material is 1:6, and sodium sulfite: the weight ratio of sodium hydroxide is 1:3, and the treatment conditions are as follows: the reaction temperature was 150℃and the time was 2 h.

Example 4 application of Clostridium thermocellum in producing high quality reducing sugar with straw as raw material

(1) Pretreatment of lignocellulose

Cleaning and cutting the wheat straw raw material, and carrying out a hydrothermal reaction by adopting sodium hydroxide and sodium sulfite, wherein the absolute dry weight ratio of the sodium hydroxide to the wheat straw raw material is 1:6, and the sodium sulfite is as follows: the weight ratio of sodium hydroxide is 1:3, and the treatment conditions are as follows: the reaction temperature was 150℃and the hydrolysis time was 2 h. After lignin is fully removed, extracting wheat straw fiber, dehydrating and fully washing off residual alkali liquor;

(2) Hydrolysis of wheat straw fibers

The straw fiber is soaked in acetic acid-sodium acetate (or a similar buffer pair with a buffer effect) buffer solution, and clostridium thermocellum fermentation liquid is thrown in to hydrolyze the straw fiber, so that hydrolysis liquid is obtained. The hydrolysis conditions were as follows: pH 5, solid-liquid ratio 1:10 (the ratio of absolute dry weight of straw fiber to the volume of buffer solution added), the adding amount of clostridium thermocellum fermentation liquor of unit mass straw fiber is 40 mL, the adding amount of xylanase of unit mass straw fiber is 4 mg, the reaction temperature is 50 ℃, the reaction time is 48 h, the oscillation rate is 150 revolutions per minute, the glucose concentration in the hydrolysate is about 65.00 g/L, and the xylose concentration is 18.00 g/L when the hydrolysis is finished. Under the same conditions, when the commercial Trichoderma reesei cellulase is used for hydrolysis instead of clostridium thermocellum fermentation broth, the glucose concentration is about 42.00 g/L and the xylose concentration is 17.60 g/L when 48 h is hydrolyzed. When the clostridium thermocellum fermentation liquid is used for hydrolyzing the wheat straw fiber, the glucose concentration is improved by 54.8 percent compared with the commercial trichoderma reesei cellulase.

(3) Solid-liquid separation and concentration of hydrolysate

Solid-liquid separation of the hydrolysate: removing thallus and residue from the obtained hydrolysate by using hollow fiber microfiltration membrane, wherein the microfiltration membrane is purchased from Shandong gold membrane Tian-Limited liability company, and is made of MF1, polypropylene material, and has effective membrane area of 0.33 m ² The pore diameter was 0.2. Mu.m, the cross-flow rate was 0.052/m/s, the cell removal rate was 100%, and the total sugar recovery rate was 98%.

Concentrating: the concentration of the hydrolyzate was carried out at room temperature. Concentrating the hydrolysate by adopting a nanofiltration membrane DK1812C-34D of the membrane electric company, wherein the operation pressure is 17 bar, the flow is 5L/min, the operation time is 3 min/L, and the pH value is natural. The concentration of glucose and xylose in the hydrolysate after concentration is 6.0 times and 6.1 times that before concentration.

The foregoing detailed description of the embodiments of the application has been presented only to illustrate the preferred embodiments of the application and should not be taken as limiting the scope of the application. All equivalent changes and modifications within the scope of the present application are intended to be covered by the present application.

SEQUENCE LISTING

<110> su state poverty creature biotechnology limited

<120> engineering strain for producing high-quality reducing sugar, construction method and application thereof

<130>

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 1516

<212> DNA

<213> Clostridium thermocellum

<400> 1

agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaacacatg caagtcgagc 60

ggggatatac ggaaggttta ccggaagtat atcctagcgg cggacgggtg agtaacgcgt 120

gggtaaccta cctcatacag ggggataaca cagggaaacc tgtgctaata ccgcataata 180

taacggggcg gcatcgtcct gttatcaaag gagaaatccg gtatgagatg ggcccgcgtc 240

cgattagcta gttggtgagg taacggctca ccaaggcgac gatcggtagc cgaactgaga 300

ggttggtcgg ccacattggg actgagacac ggcccagact cctacgggag gcagcagtgg 360

ggaatattgc gcaatggggg aaaccctgac gcagcaacgc cgcgtgaagg aagaaggcct 420

tcgggttgta aacttctttg attggggacg aaggaagtga cggtacccaa agaacaagcc 480

acggctaact acgtgccagc agccgcggta atacgtaggt ggcgagcgtt gtccggaatt 540

actgggtgta aagggcgcgt aggcggggat gcaagtcaga tgtgaaattc cggggcttaa 600

ccccggcgct gcatctgaaa ctgtatctct tgagtgctgg agaggaaagc ggaattccta 660

gtgtagcggt gaaatgcgta gatattagga ggaacaccag tggcgaaggc ggctttctgg 720

acagtaactg acgctgaggc gcgaaagcgt ggggagcaaa caggattaga taccctggta 780

gtccacgccg taaacgatgg atactaggtg taggaggtat cgaccccttc tgtgccggag 840

ttaacacaat aagtatccca cctggggagt acggccgcaa ggttgaaact caaaggaatt 900

gacgggggcc cgcacaagca gtggagtatg tggtttaatt cgaagcaacg cgaagaacct 960

taccagggct tgacatccct ctgacagctc tagagatagg gcttcccttc ggggcagagg 1020

agacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1080

acgagcgcaa cccttgtcgt tagttgccag cacgttaagg tgggcactct agcgagactg 1140

ccggcgacaa gtcggaggaa ggtggggacg acgtcaaatc atcatgcccc ttatgtcctg 1200

ggctacacac gtactacaat ggctgctaca aagggaagcg ataccgcgag gtggagcaaa 1260

tccccaaaag cagtcccagt tcggattgca ggctgaaact cgcctgcatg aagtcggaat 1320

tgctagtaat ggcaggtcag catactgccg tgaatacgtt cccgggcctt gtacacaccg 1380

cccgtcacac catgagagtc tgcaacaccc gaagtcagta gtctaaccgc aaggagggcg 1440

ctgccgaagg tggggcagat gattggggtg aagtcgtaac aaggtagccg tatcggaagg 1500

tgcggctgga tcacct 1516

<210> 2

<211> 954

<212> DNA

<213> nucleotide of L-lactate dehydrogenase Gene ldh

<400> 2

atgaacaata acaaagtaat taaaaaagta accgtagttg gtgcaggctt tgtaggttcc 60

accacagctt atacattgat gctcagcgga cttatatctg aaattgtact gatagacata 120

aatgcaaaaa aagccgacgg agaagtcatg gacttaaatc acggcatgcc ttttgtaagg 180

cccgttgaaa tttatcgtgg tgactacaaa gactgtgccg gatccgacat agtaatcatt 240

accgccggtg ccaaccaaaa agaaggcgaa acgagaatag atcttgttaa aagaaacacg 300

gaagtattca aaaatatcat aaatgaaatt gtaaagtaca acaacgattg tattcttctg 360

gtagtcacaa atccggtgga tattttaacc tatgtaactt acaaactatc cggattcccg 420

aaaaacaaag taataggttc cggaacggtt ttggacacag ccaggttccg ttatctttta 480

agcgaacatg taaaagtgga tgcacgaaat gtacatgctt atattattgg cgaacacggt 540

gacaccgaag ttgcggcctg gagtcttgca aatattgcgg gaattcccat ggatcgctac 600

tgtgacgaat gccatcagtg cgaggagcag atttcccgga ataaaatata tgaaagtgtt 660

aaaaatgcag cttatgaaat catcaggaac aaaggtgcaa cctattatgc cgtagccctt 720

gccgtaagaa gaatcgttga agccattgta agaaatgaaa actccatcct taccgtttca 780

agccttttgg aaggacagta cggacttagc gatgtatgct taagtgttcc gacaatcgtg 840

ggtgtaaacg gtattgagga aatattgaac gtgcctttca acgatgaaga aattcagctt 900

ctcagaaagt ccggaaacac tctaaaagaa ataataaaaa cactagatat atga 954

<210> 3

<211> 2394

<212> DNA

<213> nucleotide of phosphoacetyl transferase/acetate kinase Gene pta-ack

<400> 3

gtgataatat atagttataa gtattacaaa tacagttttt atgataacag ttttgggatt 60

atgaaaggag aagaatttat gagttttttg gaacaaataa ttgaaagggc gaaatcagac 120

gtaaaaacca tagttttgcc ggaaagtacg gatctgaggg ttattaaagc cgcatccatg 180

atagtgaaaa agggaattgc aaaggttgta ctgataggca atgaaaagga gattaagagt 240

ctggcggggg atattgatct tgaaggagtg atgatagagg attccttaaa ttccgaaaaa 300

ttggaggatt atgcaaatac actgtatgag cttagaaaat cgaagggtat gactatagaa 360

gccgcaaggg aaacgatcaa agaccctctt tattatggag ttatgatggt aaaaaaaggt 420

gaagcggatg gtatggtggc gggtgctgtc aattccactg caaatacttt gagaccggct 480

ttgcagatat taaagacggc cccggggaca aaactcgtat catccttttt tgttatggtt 540

gtacccaact gtgaatatgg tcataacgga acctttgtat atgccgattg cggcttggtg 600

gaaaatccgg atgcagacca gctttctgaa attgcaatat ctgcatccaa atcttttgag 660

atgctggttg gagcaaaacc tcaggtggca atgctttctt attcttctta cggcagtgcc 720

aaaagtgagc tgaccgaaaa ggtaatcaag gcaacacagc ttgcaaagga aaaagctccc 780

caccttgcaa ttgacggaga acttcaggtg gatgccgcca ttgttccgga agtggcaaaa 840

tcgaaggcaa agggaagcag tgttgcagga aaggccaatg ttcttatttt cccggatctt 900

gatgccggaa atattgcata caagcttaca cagagattgg caaaagctga agcttacggc 960

ccgataacac aaggtttggc aagaccggta aatgagctgt cacgaggctg cagtgccgag 1020

gatatagtcg gggttgcggc aattactgcg gttcaggctc aatatgtcaa ggcataaatt 1080

ctgttaaaac cggacattga agaaggtgtt gcgcagcgtt taataaaaac atctgtttat 1140

cgaagtttag gaataggaaa attaaaaaaa acaagacggg agtgagtttt tgaaatgaat 1200

attttggtta ttaataccgg aagctcatca ctaaagtatc agctgattga catgacaaac 1260

gagtctgtgc ttgcaaaagg tgtgtgtgac agaattggtc ttgaacattc ctttttaaag 1320

catacaaaga ccggagggga aaccgtagtt atagaaaaag acctgtacaa tcacaagctt 1380

gccatacagg aggtaatttc ggctcttacg gatgaaaaaa tcggagtcat aaaaagcatg 1440

tcggaaattt ctgccgtcgg tcatcgtatt gttcacggcg gagagaagtt taaggaatct 1500

gccataattg atgaagatgt aatgaaagca atcagggatt gtgttgaact ggctccgctc 1560

cacaatccgt caaatataat cggaattgaa gcctgtaaac agatactgcc cgatgtgccg 1620

atggttgctg tgtttgacac agcttttcat cagacaatgc caaggcatgc atatatttat 1680

gccctccctt atgagatata tgagaagtat aaattgagaa aatacggatt ccacggaact 1740

tcccacaaat atgtggccca cagggcggct cagatgctgg gcaaacctat tgagagcctg 1800

aagctgataa cctgccatct tggaaacgga gcgagtattt gtgcggtaaa aggcggaaaa 1860

tccgttgaca cctcaatggg atttactcct ctgcaggggt tgtgcatggg taccagaagc 1920

ggcaatgttg accctgcggt tataacttat ttgatggaaa aggaaaaaat gaatattaac 1980

gatataaaca atttccttaa caagaaatca ggtgtgcttg gaatttcagg tgtaagcagt 2040

gatttcagag atgttcagga tgccgcagaa aagggagatg acagggcgca gctggcattg 2100

gatattttct gctatggtgt taggaaatat attggaaaat atattgcagt gctgaacggc 2160

gttgatgcgg tggtattcac tgcaggtatc ggcgaaaaca atgcttatat aagaagagaa 2220

gttttgaagg atatggactt tttcggaatt aaaatagatt tggataaaaa tgaagtgaaa 2280

ggcaaagaag cggatatcag tgctcccgat gcgaaagtaa agactttggt tatcccgaca 2340

aatgaggagc ttgagattgc aagggagact ttaagacttg taaaaaactt ataa 2394

Claims

1. A construction method of engineering strain for producing high-quality reducing sugar is characterized by knocking clostridium thermocellum by homologous recombinationClostridium thermocellum) The L-lactate dehydrogenase gene and the phosphoacetyl transferase/acetate kinase gene on the PN2102 genome to obtain engineering strain;

the clostridium thermocellum is [ ]Clostridium thermocellum) PN2102 is preserved on 2021, 07 and 09, and the preservation unit is China general microbiological culture Collection center, which is preservedThe collection number is CGMCC No.22869;

the nucleotide sequence of the L-lactate dehydrogenase gene is shown as SEQ ID No.2, and the nucleotide sequence of the phosphoacetyltransferase/acetate kinase gene is shown as SEQ ID No. 3.

2. An engineered strain of high quality reducing sugars produced by the construction method of claim 1.

3. Use of the engineered strain of high quality reducing sugars of claim 2 in efficient hydrolysis of fiber.