A modified strain of peptide endonuclease and its construction method and application

Abstract

The present application relates to the field of biotechnology, in particular to a kind of peptide endo-enzyme modified strain and its construction method and application, the strain is gene knockout clostridium saccharolyticum;The function of the peptide endo-enzyme in the gene knockout clostridium saccharolyticum is inactivated.The strain is in clostridium saccharolyticum Knockout the peptide endo-enzyme coding gene that has hydrolyzed bacterial cell wall peptidoglycan structure, thereby reducing cell autolysis activity, increasing anaerobic fermentation active cell density, relieving cell autolysis occurs, reducing the dissolution of intracellular protein and other contents, to strengthen solvent synthesis capacity such as ethanol, n-butanol, improve the concentration and yield of anaerobic fermentation solvent product, and significantly enhance the stress tolerance of clostridium saccharolyticum to n-butanol.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a peptide endonuclease-modified strain, its construction method, and its application. Background Technology

[0002] Solvogenic Clostridium, exemplified by *Clostridium propioni*, initiates solvent synthesis and spore development simultaneously. During the solvent-producing phase, cell growth slows, and the acid accumulated during the acid-producing phase is re-assimilated and metabolized to produce n-butanol, acetone, and ethanol. In the later stages of solvent production, a sharp decline in cell optical density and extensive autolysis typically occur. Autolysis releases spores into the environment, and intracellular proteins and other contents dissolve, thus affecting the cell's solvent synthesis capacity. Furthermore, the cell metabolite n-butanol has toxic inhibitory effects on cells. This toxicity can further exacerbate autolysis by inducing and releasing autolysins, leading to reduced or even premature cessation of solvent production in the later stages of anaerobic fermentation. Therefore, autolysins may play a crucial role in the physiological and metabolic activities of solvent-producing Clostridium.

[0003] In summary, there is an urgent need to develop a solvent-producing Clostridium with low autolysis activity and its construction method to reduce cell autolysis activity, increase the density of live cells in anaerobic fermentation, alleviate cell autolysis, reduce the dissolution of intracellular protein contents, and thus enhance the ability to synthesize solvents such as ethanol and n-butanol, effectively improving the concentration and yield of solvent products in anaerobic fermentation. Summary of the Invention

[0004] This invention addresses the problem of how to provide a solubilizing Clostridium species that regulates cell autolysis activity and its construction method.

[0005] To achieve the above objectives, the first aspect of the present invention provides a peptide endonuclease-modified strain, wherein the strain is a gene-knockout solvent-producing Clostridium.

[0006] The gene knockout lysosomal clostridium has inactivated the function of endopeptidase.

[0007] A second aspect of the present invention provides a method for constructing the above-mentioned peptide endonuclease-modified strain, comprising the following steps:

[0008] S1. Design overlapping extension PCR primers to modify the class II intron fragment on the pSY6 plasmid to obtain the expression vector;

[0009] S2. The expression vector obtained in S1 is transformed into E. coli DH10B to obtain a methylated expression vector;

[0010] S3. The methylated expression vector obtained in S2 is introduced into Clostridium solvogeneticum. The introns in the methylated expression vector are expressed in Clostridium solvogeneticum. Then, the introns are cleaved from the mRNA precursor and bind to the accessory protein LtrA. The introns can recognize and insert the endonuclease encoding gene. The introns are then reverse transcribed to synthesize a complementary strand and inserted into the target site in the form of double-stranded DNA, thereby achieving insertion inactivation of the endonuclease encoding gene. The strain modified with endonuclease is then obtained through culture and screening.

[0011] The third aspect of this invention provides the application of the above-mentioned endonuclease-modified strains or the method for constructing the above-mentioned endonuclease-modified strains in the production of short-chain alcohols.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] In a first aspect, the present invention knocks out the gene encoding a peptide bond endopeptidase that hydrolyzes the peptidoglycan structure of bacterial cell walls in Clostridium solubilizing, thereby reducing cell autolysis activity, increasing the density of viable cells in anaerobic fermentation, alleviating cell autolysis, reducing the dissolution of intracellular proteins and other contents, thereby enhancing the ability to synthesize solvents such as ethanol and n-butanol, improving the concentration and yield of anaerobic fermentation solvent products, and significantly enhancing the stress tolerance of Clostridium solubilizing to n-butanol.

[0014] In a second aspect, the strain provided by the present invention obtains a strain inactivated by knocking out the gene encoding a peptidase that hydrolyzes the peptidoglycan structure of bacterial cell walls, instead of using a strain overexpressing a peptidase, thus achieving a significant increase in solvent product concentration and yield. Attached Figure Description

[0015] Figure 1 This is the pSY6-EP plasmid map containing a class II intron vector.

[0016] Figure 2 This is a schematic diagram of type II intron insertion.

[0017] Figure 3 This is a diagram of the colony PCR verification results.

[0018] Figure 4 This is a comparison of the fermentation performance of the original strain and the strain inactivated by endopeptidase under natural pH conditions.

[0019] Figure 5 This describes the autolysis of the original strain and the strain inactivated by endopeptidase in sodium phosphate buffer.

[0020] Figure 6 This describes the changes in the content of extracellular soluble proteins during the fermentation process of the original strain and the strain with inactivated peptide endonuclease.

[0021] Figure 7This study compares the fermentation performance of the original strain and the strain inactivated by endopeptidase under conditions where pH > 5.0.

[0022] Figure 8 This study compares the fermentation performance of the original strain and the strain inactivated by endopeptidase under stress conditions with the addition of 10 g / L n-butanol inhibitor. Detailed Implementation

[0023] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] In this invention, the solvent-producing Clostridium is a type of Gram-positive, strictly anaerobic bacterium that can convert starch, cellulose, sugars, organic acids, and carbon dioxide substrates into ethanol, isopropanol, n-butanol, acetoin, formic acid, acetic acid, lactic acid, and butyric acid through a series of enzymatic reactions under anaerobic conditions.

[0025] Endopeptidases can disrupt the peptidoglycan structure of the cell wall of Gram-positive bacteria, leading to cell wall lysis, cell autolysis, dissolution of intracellular proteins and other contents, stagnation of cellular physiological and metabolic activities, or even cell death, thereby affecting the fermentation performance of solvent-producing Clostridium.

[0026] By using gene recombination, the gene encoding endopeptidase in Clostridium solubilizing agents was modified to obtain a strain with low autolysis activity. This effectively improved the concentration and yield of fermentation solvent products by reducing the autolysis activity of Clostridium solubilizing agents.

[0027] The first aspect of the present invention provides a peptide endonuclease-modified strain, wherein the strain is a gene-knockout solvent-producing Clostridium.

[0028] The gene knockout lysosomal clostridium has inactivated the function of endopeptidase.

[0029] In this invention, the strain exhibits decreased cell autolysis activity, increased viable cell number, reduced extracellular soluble protein, accelerated carbon source consumption, increased concentration and yield of ethanol and n-butanol solvent products, and enhanced tolerance to the toxic inhibitor n-butanol stress during the logarithmic fermentation phase.

[0030] According to the present invention, the solvent-producing Clostridium includes any one of Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, or Clostridium saccharobutylicum.

[0031] According to the present invention, the solvent-producing Clostridium is Clostridium propionidum, the gene knocked out is the endopeptidase-encoding gene cac1275, the nucleotide sequence of the endopeptidase-encoding gene cac1275 is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0032] The nucleotide sequence of the cac1275 gene encoding the peptide endonuclease is SEQ ID NO.1 as follows:

[0033] ATGAATAGTGTTAGAACTGACCTTGCAGTAGAGGCAAGAGAGATGTACTGTGAGAA

[0034] AACCGAAATGGTGATAATGGAGTAAGAGTAGATACAAAAAGAATAGAAGATATAG

[0035] AGATTACTACAGTCGATGTTTTAAATGATAAAGGCGAAGAAAGAATAAGGAAGCAA

[0036] AAAGGTACATATATAACACTAGATATACCAAAGGTTACCTTGTATGATTCTGAAGATA

[0037] TAGAAGAGATAAGTAAGGTGTTTGCTGATGAACTTTCGAAGATAATAAGTAAAGCA

[0038] AAGCTTGATAGCAGTATGACTGTTTTAGTAGTGGGACTTGGTAATTGGAACATAACA

[0039] CCAGATTCTTTAGGACCAAAGGTTATAGGAAAACTTATGGTGACAAGACATTTAAA

[0040] AAAATATATACCAGATAGTATTGATGAGGGAATACGTCCGGTTTGCGCTGTAGCACC

[0041] TGGAGTTCTTGGAATTACTGGTATGGAGACAGGGGAAATAATACGAGGAATAGTTC

[0042] AAAATATAAAACCCGATCTTATTGTATGCATAGATGCACTAGCCGCGAGAAAGATGG

[0043] GAAGAGTAAATTCTACTATTCAAATTGGCAACACTGGAATATCTCCTGGATCAGGTG

[0044] TAGGAAATATGAGGATGGAATTAAGCCAAAAGACTCTTGGAGTTCCAGTTATAGCA

[0045] GTTGGTGTTCCTACTGTAGTTGATGCTGCAACTATGGCAAATGATACTATTGATATAG

[0046] TTATTGAAAAAATGAAAGGCGAAGTTAGTGAAGATAGTAAATTTTATAGTTTGTTAA

[0047] AGGCAATAGATACAGAAGAAAAGTCTCAACTTATATATGAAGTACTTAATCCATATG

[0048] TTGGGGATTTACTTGTTACACCTAAAGAAGTGGATATGATTATGGAGACGCTTTCAA

[0049] AAATTATAGCATCAGGTATAAATATTGCACTTCAACCAGCACTTGAATTAAGTGAAA

[0050] TAAACAAATATGTTAATTAA。

[0051] The protein amino acid sequence of endopeptidase SEQ ID NO.2 is:

[0052] MNSVRTDLAVEAREMYCEKTENGDNGVRVDTKRIEDIEITTVDVLNDKGEERIRKQK

[0053] GTYITLDIPKVTLYDSEDIEEISKVFADELSKIISKAKLDSSMTVLVVGLGNWNITPDSL

[0054] GPKVIGKLMVTRHLKKYIPDSIDEGIRPVCAVAPGVLGITGMETGEIIRGIVQNIKPDLI

[0055] VCIDALAARKMGRVNSTIQIGNTGISPGSGVGNMRMELSQKTLGVPVIAVGVPTVVD

[0056] AATMANDTIDIVIEKMKGEVSEDSKFYSLLKAIDTEEKSQLIYEVLNPYVGDLLVTPK

[0057] EVDMIMETLSKIIASGINIALQPALELSEINKYVN*.

[0058] A second aspect of the present invention provides a method for constructing the above-mentioned peptide endonuclease-modified strain, comprising the following steps:

[0059] S1. Design overlapping extension PCR primers to modify the class II intron fragment on the pSY6 plasmid to obtain the expression vector;

[0060] S2. The expression vector obtained in S1 is transformed into E. coli DH10B to obtain a methylated expression vector;

[0061] S3. The methylated expression vector obtained in S2 is introduced into Clostridium solvogeneticum. The introns in the methylated expression vector are expressed in Clostridium solvogeneticum. Then, the introns are cleaved from the mRNA precursor and bind to the accessory protein LtrA. The introns can recognize and insert the endonuclease encoding gene. The introns are then reverse transcribed to synthesize a complementary strand and inserted into the target site in the form of double-stranded DNA, thereby achieving insertion inactivation of the endonuclease encoding gene. The strain modified with endonuclease is then obtained through culture and screening.

[0062] According to the present invention, in S3, the solvent-producing Clostridium includes any one of Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, or Clostridium saccharobutylicum.

[0063] According to the present invention, in S3, the solvent-producing Clostridium is Clostridium propionidum, and the endopeptidase encoding gene is the endopeptidase encoding gene cac1275, with the insertion position between 462bp and 463bp of the nucleotide sequence.

[0064] The nucleotide sequence SEQ ID NO.3 of the vector promoter ptb of the peptidase-modified strain of Clostridium propionidum is as follows:

[0065] TATAAAATATAAATAATTTTCTAAAAAAACTTAACTTCATGTGAAAAGTTTGTTAA

[0066] AATATAAATGAGCACGTTAATCATTTAACATAGATAATTAAATAGTAAAAGGGA

[0067] GTGTCGA.

[0068] According to the present invention, the culture is performed sequentially as activation culture, seed culture and fermentation culture.

[0069] In this invention, there are no particular limitations on the cultivation methods for activation culture, seed culture, and fermentation culture in S3, as long as the recombinant solvent-producing Clostridium can ferment normally. Specifically, the cultivation method and culture medium composition can be:

[0070] Step 1: Activation and Culture

[0071] The bacterial strain was inoculated into liquid activation medium at a rate of 5% (v / v), placed in an anaerobic dropper bottle, and incubated at 37°C and natural pH for 24 hours before being used for seed culture.

[0072] Step 2: Seed culture

[0073] The activated strain obtained in step 1 was inoculated into liquid seed culture medium at an inoculation rate of 10% (v / v), and placed in an anaerobic dropper bottle for shaking culture at a temperature of 37°C, a rotation speed of 150 rpm, and natural pH for 24 hours before being used for fermentation culture.

[0074] Step 3: Fermentation culture

[0075] The seed culture obtained in step 2 was inoculated into the liquid fermentation medium at an inoculation rate of 10% (v / v), and placed in an anaerobic fermenter for stirring and cultivation. The liquid fermentation medium volume was 1L, the cultivation temperature was 37℃, the stirring speed was 150rpm, and the pH was natural.

[0076] In step 1, the liquid activation culture medium contains 20 g / L glucose, 30 g / L tryptone, 10 g / L yeast extract, and natural pH.

[0077] In step 2, the liquid seed culture medium contains 70 g / L glucose, 3.22 g / L ammonium acetate, 2.0 g / L yeast extract, 0.2 g / L MgSO4·7H2O, 0.5 g / L KH2PO4, 0.5 g / L K2HPO4, 0.01 g / L FeSO4·7H2O, 0.01 g / L MnSO4·7H2O, 0.01 g / L biotin, and 0.01 g / L p-aminobenzoic acid.

[0078] In step 3, the liquid fermentation medium contains 70 g / L glucose, 3.22 g / L ammonium acetate, 2.0 g / L yeast extract, 0.2 g / L MgSO4·7H2O, 0.5 g / L KH2PO4, 0.5 g / L K2HPO4, 0.01 g / L FeSO4·7H2O, 0.01 g / L MnSO4·7H2O, 0.01 g / L biotin, and 0.01 g / L p-aminobenzoic acid.

[0079] In step 3, the pH of the fermentation broth is controlled to be greater than 5.0 by adding potassium hydroxide solution or ammonia solution.

[0080] Furthermore, the pH of the fermentation broth was controlled to be greater than 5.0 by adding a 15% (v / v) ammonia solution.

[0081] According to the present invention, in the fermentation culture, a toxicity inhibitor is added to the fermentation medium, the toxicity inhibitor including one or more products produced by Clostridium solvogeneticum that have toxic inhibitory effects on the metabolism of its own cells.

[0082] Preferably, the product is an organic acid and / or an alcohol.

[0083] According to the present invention, the toxicity inhibitor is n-butanol, and the addition amount is 5-20 g / L.

[0084] The third aspect of this invention provides the application of the above-mentioned endonuclease-modified strains or the method for constructing the above-mentioned endonuclease-modified strains in the production of short-chain alcohols.

[0085] The present invention will be described in detail below through embodiments.

[0086] Clostridium acetobutylicum ATCC 824 was purchased from the U.S. Collection of Typical Strains.

[0087] The solvent-producing Clostridium is Clostridium acetobutylicum ATCC 824, hereinafter referred to as the original strain; the strain named Clostridium acetobutylicum EP, hereinafter referred to as the endopeptidase-inactivated strain. The modified strain expressing endopeptidases with inactivated peptide chains was constructed using the original strain.

[0088] LB liquid medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, for Escherichia coli culture.

[0089] LB solid medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar powder 15g / L, for single colony culture of Escherichia coli.

[0090] Liquid activation medium: 30 g / L tryptone, 20 g / L glucose, 10 g / L yeast extract, for Clostridium propionidum activation culture.

[0091] Liquid seed culture medium: glucose 70 g / L, ammonium acetate 3.22 g / L, yeast extract 2.0 g / L, M-gSO4·7H2O 0.2 g / L, KH2PO4 0.5 g / L, K2HPO4 0.5 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·7H2O 0.01 g / L, biotin 0.01 g / L, p-aminobenzoic acid 0.01 g / L, for Clostridium propionidum seed culture.

[0092] Liquid fermentation medium: glucose 70 g / L, ammonium acetate 3.22 g / L, yeast extract 2.0 g / L, MgSO4·7H2O 0.2 g / L, KH2PO4 0.5 g / L, K2HPO4 0.5 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·7H2O 0.01 g / L, biotin 0.01 g / L, p-aminobenzoic acid 0.01 g / L, used for Clostridium propionidum fermentation culture.

[0093] Solid agar medium: glucose 20 g / L, yeast extract 2 g / L, tryptone 4 g / L, KH₂PO₄ 0.5 g / L, K₂HPO₄ 1 g / L, ammonium sulfate 2 g / L, agar powder 15 g / L, and a mineral mixture. The mineral mixture consists of: MgSO₄·7H₂O 0.2 g / L, FeSO₄·7H₂O 0.015 g / L, CaCl₂·2H₂O 0.015 g / L, MnSO₄·7H₂O 0.01 g / L, cobalt chloride 0.02 g / L, and zinc sulfate 0.002 g / L, used for single colony culture and counting of Clostridium propionitum.

[0094] n-Butanol-stressed fermentation medium: Based on the above liquid fermentation medium, add 5-20 g / L n-butanol for Clostridium propionidium stress fermentation culture. The concentration of n-butanol can be 5 g / L, 10 g / L, 14 g / L, 18 g / L, 20 g / L, or any value between the above concentrations.

[0095] Unless otherwise specified in the following examples and comparative examples, all conditions were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0096] Example 1

[0097] S1. Use the pSY6 plasmid containing class II introns as the vector, as per the instructions. Figure 1 The intron fragments are modified using overlap extension PCR to specifically recognize the target gene and are inserted into the target site to block the expression of the target gene, as shown in the instruction manual. Figure 2 Overlap extension PCR primers were designed using the Clostron website (www.clostron.com). The insertion site in the gene to be knocked out was predicted based on an algorithm published in the literature (Journal of Molecular Biology, 2004, 336:421-439). Primer sequences are shown in Table 1. In the first round of PCR, primers IBS and EBSuniversal were used to amplify intron fragment 1, and primers EBS1d and EBS2 were used to amplify intron fragment 2. In the second round of PCR, intron fragments 1 and 2 were used as templates to amplify the entire intron fragment. The PCR products were purified using the OMEGA Clean Kit. The purified PCR products and pSY6 plasmid were digested with BsrGI and XhoI restriction endonucleases. The pSY6 plasmid digestion product was purified using the OMEGA DNA Gel Extraction Kit. The purified plasmid fragments and intron fragments were ligated using ligase.

[0098] S2. The ligation product was introduced into E. coli for amplification, and the plasmid was extracted using the OMEGA plasmid DNA mini kit. Sequencing was performed to verify the recombinant plasmid construction. The recombinant plasmid was then transformed into E. coli DH10B competent cells for methylation and plasmid extraction for later use.

[0099] Table 1 Primer sequences used in Example 1

[0100]

[0101] S3. 400 μL of the original Clostridium acetobutylicum ATCC 824 bacterial culture stored at -80℃ was streaked onto a solid agar plate and incubated anaerobicly at 37℃ for 24 h. A single colony was picked and inoculated into liquid activation medium and incubated anaerobicly at 37℃ for 16 h. The activated bacterial culture was then transferred to liquid seed medium and incubated anaerobicly at 37℃ until the OD value reached 0.6. The obtained bacterial cells were centrifuged at 4500 rpm for 10 min, the supernatant was removed, and the cells were resuspended in 30 mL of ice-cold ETM buffer (280 mM sucrose, 0.6 mM Na2HPO4, 4.4 mM NaH2PO4, 9 mM MgCl2). The cells were incubated for 10 min, centrifuged at 4000 rpm for 10 min, the supernatant was removed, and 2.5 mL of ET buffer (280 mM sucrose, 0.6 mM Na2HPO4, 4.4 mM MgCl2) was added. Competent cells were prepared by resuspending the cells in NaH2PO4. 190 μL of competent cells were added to the methylated plasmid obtained in S2, mixed thoroughly, and incubated on ice. The mixture of competent cells and plasmid was then transferred to a 0.2 cm electroporation vessel for electroporation. Liquid seed culture medium was then added and mixed thoroughly. The mixture was then added to another equal volume of culture medium and incubated at 37°C. 100 μL of the culture medium was then spread onto a solid agar plate containing erythromycin resistance. Single colonies were picked for colony PCR verification. Primer sequences are shown in Table 2. Mutant bacteria with intron insertions were screened. The PCR product size of strains without intron insertions was approximately 1500 bp, while the PCR product size of strains with intron insertions was approximately 2400 bp, as per the instructions. Figure 3 As shown. Positive colonies were then passaged on antibiotic-free solid agar plates until resistance was lost, yielding an antibiotic-free, endopeptidase-inactivated strain, *Clostridium acetobutylicum* EP, with a peptidoglycan structure that hydrolyzes bacterial cell walls. Single colonies were inoculated into liquid seed culture medium (containing erythromycin resistance) and statically cultured at 37°C for 24 hours under anaerobic conditions. They were then transferred to corn mash medium and anaerobically expanded at 37°C for 36 hours. Afterward, the culture was mixed with an equal volume of 40% glycerol solution, aliquoted, and frozen at -80°C in the laboratory for later use.

[0102] Table 2 Primer sequences used in Example 1

[0103]

[0104] Comparative Example 1

[0105] Original strain.

[0106] Comparative Example 2

[0107] Construction of strains overexpressing peptide endonuclease

[0108] S1. According to the NCBI search information (https: / / www.ncbi.nlm.nih.gov / gene), the sequence information of the Clostridium acetobutylicum endopeptidase encoding gene involved is shown in SEQ ID NO.1. The gene fragments linked by these two genes were synthesized by Genewiz Biotechnology (Beijing) Co., Ltd. The restriction site BamHI / KpnI was selected. The endopeptidase shown in SEQ ID NO.1 was ligated into the vector plasmid pIMP1 [Mermelstein LD, Welker NE, Bennett G.N., Papoutsakis ET Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824. Nature Biotechnology, 1992, 10(2): 190-5.] at the 5' end of the nucleotide sequence of SEQ ID NO.1 is linked to the promoter shown in SEQ ID NO.4. The correct expression vector was named pIMP1-EP after verification by restriction enzyme digestion, PCR and sequencing.

[0109] The nucleotide sequence of the promoter gene thl of the overexpression strain vector is SEQ ID NO.4.

[0110] TTTTTAACAAAATATATTGATAAAAATAATAATAGTGGGTATAATTAAGTTGTTA

[0111] GAGAAAACGTATAAATTAGGGATAAACTATGGAACTTATGAAATAGATTGAAAT

[0112] GGTTTATCTGTTACCCCGTATCAAAATTTAGGAGGTTAGTTAGA.

[0113] S2. The expression vector pIMP1-EP constructed above was transformed into E. coli DH10B competent cells. After transformation, rejuvenation, and single colony culture, the methylated expression vector pIMP1-EP was extracted.

[0114] S3. The original strain stored at -80℃ was streaked on a solid plate medium and incubated statically at 37℃ for 24 hours under anaerobic conditions. Single colonies were picked and inoculated into liquid activation medium and incubated statically at 37℃ for 16 hours under anaerobic conditions. The activated bacterial solution was then transferred to liquid seed medium and incubated statically at 37℃ under anaerobic conditions until the OD value reached 0.6. The obtained bacterial cells were pre-cooled at 4℃ and used to prepare Clostridium propionidum competent cells. The methylated expression vector pIMP1-EP was electroporated into the prepared Clostridium proteoglycans competent cells. After rejuvenation, the cells were plated on solid agar plates containing erythromycin resistance and cultured statically at 37°C for 48 hours. The resulting single colonies were strains enhanced by overexpression or synthesis of endopeptidases. The single colonies were inoculated into liquid seed culture medium (containing erythromycin resistance) and cultured statically at 37°C for 24 hours. Then, they were transferred to corn mash medium and cultured anaerobicly at 37°C for 48 hours. After that, they were mixed with an equal volume of 40% glycerol solution, dispensed, and frozen in the laboratory at -80°C for later use.

[0115] The original strain, the strain inactivated by endopeptidase, and the comparative strain overexpressing endopeptidase were compared under different conditions.

[0116] Comparative Example 1

[0117] Comparison of fermentation processes of the original strain, strain overexpressing endonuclease, and strain inactivated by endonuclease under natural pH conditions.

[0118] Step 1: Activation and Culture

[0119] The original strain, the endopeptidase overexpressing strain, and the endopeptidase inactivated strain from Example 1 were inoculated into liquid activation medium at an inoculation rate of 5% (v / v), placed in anaerobic dropper bottles, and cultured statically at 37°C and natural pH for 24 hours before being used for seed culture.

[0120] Step 2: Seed culture

[0121] The activated strain obtained in step 1 was inoculated into liquid seed culture medium at an inoculation rate of 10% (v / v), and placed in an anaerobic dropper bottle for shaking culture at a temperature of 37℃, a rotation speed of 150 rpm, natural pH, and shake culture for 24 h before being used for fermentation culture.

[0122] Step 3: Fermentation culture

[0123] The seed culture obtained in step 2 was inoculated into the liquid fermentation medium at an inoculation rate of 10% (v / v), and placed in an anaerobic fermenter (T&J-Minibox61.5*8Itelli-Ferm) for stirring and culture. The liquid fermentation medium volume was 1L, the culture temperature was 37℃, the stirring speed was 150rpm, the pH was natural, and the fermentation culture was carried out for 72h.

[0124] According to the instruction manual Figure 4 To compare the fermentation performance of the original strain and the strain inactivated by endopeptidase under natural pH conditions, the original strain had a viable cell count of 0.91 × 10⁻⁶ after 12 hours of fermentation. 8 The cfu / mL concentration reached a peak viable cell count of 1.74 × 10⁻⁶ cells after 16 hours of fermentation. 8 The total alcohol product concentration during fermentation was 13.0 g / L, the sugar alcohol conversion rate was 0.209 g / g, and the total solvent product concentration and yield were 20.0 g / L and 0.321 g / g, respectively. In contrast, the endopeptidase-inactivated strain showed a 2-fold increase in viable cell count after 12 h of fermentation compared to the original strain. After 16 h of fermentation, the maximum viable cell count of the endopeptidase-inactivated strain was more than 5-fold higher than that of the original strain. The total alcohol product concentration and sugar alcohol conversion rate were increased by 15% and 19%, respectively, while the solvent product concentration and yield were increased by 14% and 17%, respectively. Compared to the original strain and the endopeptidase-inactivated strain, the fermentation efficiency of the endopeptidase-overexpressing strain was significantly reduced. After 24 h of fermentation, the OD of the endopeptidase-overexpressing strain was only 1.6, a decrease of 59% and 47% compared to the endopeptidase-inactivated strain and the original strain, respectively. The endopeptidase overexpressing strain consumed only 13.5 g / L of glucose, synthesized only 1.4 g / L of alcohol, and had a total solvent concentration of only 2 g / L after 24 h of fermentation, representing reductions of 64%, 84%, and 85% respectively compared to the original strain, and 70%, 85%, and 86% respectively compared to the endopeptidase inactivated strain. After 48 h of fermentation, the alcohol concentration and total solvent concentration of the endopeptidase overexpressing strain were only 6.8 g / L and 10.1 g / L respectively, representing reductions of 48% respectively compared to the original strain, and 50% and 51% respectively compared to the endopeptidase inactivated strain.

[0125] Comparative Example 1 shows that by inactivating the expression of endopeptidase-encoding genes in Clostridium difficile and constructing an endopeptidase-inactivated strain to weaken cell autolysis, compared with the original strain, it can effectively improve the anaerobic fermentation cell density (number of live cells) and the concentration and yield of solvent products such as ethanol and n-butanol under natural pH conditions.

[0126] Comparative Example 2

[0127] Comparison of autolysis activity among the original strain, strain overexpressing endopeptidase, and strain inactivated by endopeptidase.

[0128] The activation culture and seed culture were carried out according to the steps in Comparative Example 1.

[0129] Fermentation culture:

[0130] The seed culture obtained from seed culture was inoculated into liquid fermentation medium at a rate of 10% (v / v) and placed in an anaerobic fermenter (T&J-Minibox 61.5*8 Itelli-Ferm) with stirring. The liquid fermentation medium volume was 1L, the incubation temperature was 37℃, the stirring speed was 150rpm, the pH was set naturally, and the culture was shaken for 16h. After that, the OD was collected. 600 The cells were in the exponential phase of 2.0. They were centrifuged at 4000 rpm / min for 15 min at 4°C. The cell pellet was collected, the supernatant was discarded, and the residual growth medium was removed with a cotton swab.

[0131] Cell autolysis activity assay:

[0132] The cell pellet collected after centrifugation during fermentation was washed once with 0.1 mol / L sodium phosphate buffer (pH 6.3). The washed pellet was then diluted to the initial OD value. 600 Add 1 to a 0.1 mol / L sodium phosphate buffer (pH 6.3) pre-warmed to 37°C. Monitor the OD in the buffer every 0.5 h in an anaerobic environment at 37°C. 600 The change was defined with the initial test time point as 0h.

[0133] Instruction manual attached Figure 5 To assess cell autolysis in the original strain and the strain inactivated by endopeptidase in sodium phosphate buffer, the OD value was compared to the initial test point at 0 h in the cell autolysis activity assay. 600 At 0.5 h, the autolysis rate of cells in the endopeptidase-inactivated strain was less than 2%, while the autolysis rate of the original strain was as high as 9%, and the autolysis rate of the endopeptidase-overexpressing strain was as high as 14%. At 1.5 h, the autolysis rate of cells in the endopeptidase-inactivated strain was 8%, the autolysis rate of cells in the original strain was as high as 16%, and the autolysis rate of cells in the endopeptidase-overexpressing strain was as high as 30%. At 3 h, the autolysis rate of cells in the original strain was as high as 25%, the autolysis rate of cells in the endopeptidase-overexpressing strain was as high as 40%, while the autolysis rate of cells in the endopeptidase-inactivated strain was only 15%. Within the same time period, the endopeptidase-inactivated strain provided by this invention showed significantly reduced cell autolysis activity compared with the original strain. Throughout the detection process, the proportion of surviving cells in the endopeptidase-inactivated strain was always higher than that in the original strain, demonstrating lower cell autolysis activity.

[0134] Comparative Example 2 shows that inactivating the expression of endopeptidase-encoding genes in Clostridium solvogeneticum and constructing endopeptidase-inactivated strains to weaken cell autolysis levels can significantly reduce cell autolysis activity compared to the original strain.

[0135] Comparative Example 3

[0136] Extracellular soluble proteins during fermentation by strains with inactivated peptide endopeptidase.

[0137] The activation culture, seed culture, and fermentation culture were carried out according to the steps in Comparative Example 1.

[0138] Representative time points were selected at the initial fermentation time of 0h, the logarithmic growth period of 12h and 24h, and the fermentation decline period of 48h and 72h. Fermentation broth was collected, and the supernatant was collected by centrifugation at 7000rpm for 15min.

[0139] Extracellular soluble protein assay: The supernatant collected after centrifugation in the fermentation culture was diluted to an appropriate concentration gradient with PBS buffer from the BCA kit, and then the protein content was measured using the BCA kit, repeated at least three times.

[0140] Instruction manual attached Figure 6 The changes in extracellular soluble protein content during fermentation of the original strain and the strain with inactivated peptide chain restriction enzymes were shown. The overall trends of extracellular soluble protein content in both strains were consistent: at the initial fermentation time (0 h), due to the presence of protein-containing nitrogen sources such as yeast extract in the culture medium, the extracellular soluble protein content was highest and the values ​​were consistent; as fermentation progressed to 24 h, cell autolysis led to the dissolution of a large amount of intracellular protein contents, resulting in an increase in extracellular soluble protein content; at 48 h and 72 h, due to the degradation of soluble proteins in the solution and the significant increase in the number of autolyzed cells in the later stages of fermentation, the extracellular protein content showed a trend of first decreasing and then increasing. At 24 h, 48 h, and 72 h, the extracellular soluble protein content of the original strain was consistently higher than that of the strain with inactivated peptide chain restriction enzymes, with the latter showing a decrease of 25%, 23%, and 27%, respectively, compared to the original strain.

[0141] Comparative Example 3 shows that inactivating the expression of endopeptidase-encoding genes in Clostridium solventis and constructing an endopeptidase-inactivated strain to weaken the level of cell autolysis significantly reduced the content of extracellular soluble protein compared with the original strain, playing a certain role in protein quality control. This further verifies that the level of cell autolysis of the endopeptidase-inactivated strain is significantly reduced compared with the original strain.

[0142] Comparative Example 4

[0143] Comparison of fermentation between the original strain and the strain inactivated by endopeptidase under pH > 5.0 conditions.

[0144] The activation culture, seed culture, and fermentation culture followed the steps in Comparative Example 1, except that the pH of the fermentation broth was controlled to be greater than 5.0 by adding 15% (v / v) ammonia solution to the fermentation medium.

[0145] Instruction manual attached Figure 7To compare the fermentation performance of the original strain and the endopeptidase-inactivated strain under controlled pH > 5.0 conditions, the original strain was monitored throughout fermentation, with a maximum OD of 3.7, an alcohol product concentration of 15.0 g / L, and a sugar alcohol conversion rate of 0.216 g / g. In contrast, the endopeptidase-inactivated strain was monitored throughout fermentation, with a maximum OD of 4.4, representing a 20% and 23% increase in alcohol product concentration and sugar alcohol conversion rate, respectively, and a 13% and 17% increase in total solvent product concentration and yield.

[0146] Comparative Example 4 shows that by inactivating the expression of endopeptidase-encoding genes in Clostridium solubilization and constructing an endopeptidase-inactivated strain to weaken the level of cell autolysis, the production indicators such as the total solvent product concentration and yield of anaerobic fermentation can still be effectively improved compared with the original strain under the condition of controlling pH > 5.0.

[0147] Comparative Example 5

[0148] Comparison of fermentation between the original strain and the strain inactivated by endopeptidase under n-butanol inhibitor stress.

[0149] The activation culture and seed culture were carried out according to the steps in Comparative Example 1.

[0150] Fermentation culture:

[0151] The seed culture obtained from the seed culture was inoculated into the liquid fermentation medium at an inoculum rate of 10% (v / v), and then placed in an anaerobic dropper bottle for shaking culture at 37℃, stirring speed of 150 rpm, natural pH, for 72 h. 5 g / L, 10 g / L, and 14 g / L of n-butanol were added to the fermentation medium as toxicity inhibitors.

[0152] Instruction manual attached Figure 8To compare the fermentation performance of the original strain and the endopeptidase-inactivated strain under stress with 10 g / L n-butanol inhibitor, under the condition of adding 5 g / L n-butanol, the original strain showed a maximum OD of 2.0 throughout the fermentation process, a sugar alcohol conversion rate of 0.173 g / g, and total solvent product concentration and yield of 14.1 g / L and 0.304 g / g, respectively. In contrast, the endopeptidase-inactivated strain showed a maximum OD of 2.4 throughout the fermentation process, which was 20% higher than that of the original strain, and the sugar alcohol conversion rate, total solvent product concentration, and yield were increased by 37%, 22%, and 28%, respectively, compared with the original strain. Under the condition of adding 10 g / L n-butanol, the original strain showed a maximum OD of 1.9 throughout fermentation, a sugar alcohol conversion rate of 0.186 g / g, and a total solvent product yield of 0.290 g / g. In contrast, the endopeptidase-inactivated strain showed a maximum OD of 2.5 throughout fermentation, which was 32% higher than the original strain. The sugar alcohol conversion rate, total solvent product concentration, and yield were increased by 43%, 37%, and 40%, respectively, compared to the original strain. Under the condition of adding 14 g / L n-butanol, the physiological metabolic activity of the original strain was basically stopped. Compared with the original strain, the endopeptidase-inactivated strain provided by this invention maintained its physiological metabolic function, and the sugar alcohol conversion rate was more than 9 times higher than that of the original strain.

[0153] Compared with the original strain, the endopeptidase-inactivated strain provided by this invention shows a 20% and 23% increase in alcohol product concentration and sugar alcohol conversion rate, respectively; a 13% and 17% increase in total solvent product concentration and yield, respectively; and a more than 5-fold increase in the maximum viable cell count during the logarithmic growth phase. At 24h, 48h, and 72h of fermentation, the extracellular soluble protein content of the endopeptidase-inactivated strain is reduced by 25%, 23%, and 27% compared to the original strain, respectively. In the cell autolysis activity assay, from the initial 0h to 0.5h, the autolysis rate of cells in the endopeptidase-inactivated strain is less than 2%, while the autolysis rate of the original strain is as high as 9%. At 1.5h, the endopeptidase-inactivated strain shows a significantly higher rate of cell autolysis. The autolysis rate of the endopeptidase-inactivated strain was only 8%, while the autolysis rate of the original strain was as high as 16%. Within the same time period, the endopeptidase-inactivated strain provided by this invention showed significantly reduced cell autolysis activity compared with the original strain. Under 10 g / L n-butanol stress, the endopeptidase-inactivated strain provided by this invention increased the sugar alcohol conversion rate, total solvent product concentration, and yield by 43%, 37%, and 40%, respectively, compared with the original strain. Under 14 g / L n-butanol stress, the physiological metabolic activity of the original strain was basically stopped, while the physiological metabolic function of the endopeptidase-inactivated strain provided by this invention could be maintained, and the sugar alcohol conversion rate was more than 9 times higher than that of the original strain.

[0154] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A strain modified with endopeptidase, characterized in that, The strain is a gene-knockout solubilizing Clostridium. The solvent-producing Clostridium is Clostridium propionidum (Clostridium butyricum). Clostridium acetobutylicum The gene knocked out is the endopeptide-encoding gene cac1275, the nucleotide sequence of the endopeptide-encoding gene cac1275 is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

2. A method for constructing the endopeptidase-modified strain according to claim 1, characterized in that, Includes the following steps: S1. Design overlapping extension PCR primers to modify the class II intron fragment on the pSY6 plasmid to obtain the expression vector; S2. Transfer the expression vector obtained in S1 into... E. coli In DH10B, methylated expression vectors were obtained; S3. The methylated expression vector obtained in S2 is introduced into Clostridium solvogeneticum. The introns in the methylated expression vector are expressed in Clostridium solvogeneticum. Then, the introns are cleaved from the mRNA precursor and bind to the accessory protein LtrA. The introns can recognize and insert the endopeptide-encoding gene. The introns are then reverse transcribed to synthesize a complementary strand and inserted into the target site in the form of double-stranded DNA, thereby achieving insertion inactivation of the endopeptide-encoding gene. The strains modified with endopeptide-encoding gene are then obtained through culture and screening. In S3, the solvent-producing Clostridium is Clostridium propionidum (Clostridium spp.) Clostridium acetobutylicum The endopeptidase encoding gene is cac1275, and the insertion position is between 462 bp and 463 bp of the nucleotide sequence.

3. The construction method according to claim 2, characterized in that, The culture process consists of activation culture, seed culture, and fermentation culture, in that order.

4. The construction method according to claim 3, characterized in that, In the fermentation culture, a toxicity inhibitor is added to the fermentation medium, which includes one or more products produced by Clostridium solvogeneticum that have toxic inhibitory effects on its own cell metabolism.

5. The construction method according to claim 4, characterized in that, The toxicity inhibitor is n-butanol, and the addition amount is 5-20 g / L.

6. The application of the peptide endonuclease-modified strain according to claim 1 in the production of short-chain alcohols.

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