Clostridium wall teichoic acid modified to improve cell density of anaerobic fermentation and construction method and application thereof

Abstract

The application discloses a cell density-increasing Clostridium wall teichoic acid reformed strain and a construction method and application thereof, and belongs to the technical field of biology. The Clostridium wall teichoic acid reformed strain is a recombinant solventogenic Clostridium with overexpressed wall teichoic acid synthetase, and specifically, the wall teichoic acid synthetase coding gene nucleotide sequence is SEQ ID NO. 1, and the amino acid sequence of the protein coded by the wall teichoic acid synthetase is SEQ ID NO. 2. The cell density-increasing Clostridium wall teichoic acid reformed strain provided by the application is used for anaerobic fermentation to produce butanol and other alcohol solvents, the logarithmic growth period active cell number is significantly increased, the carbon source consumption rate and utilization rate are improved, the butanol and other alcohol solvent concentration, yield and production rate are increased, the formic acid inhibitor and high-temperature stress tolerance are enhanced, and high-density anaerobic fermentation production of biological butanol and other solvents is realized.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a Clostridium perfringens modified with teichoic acid that can increase the cell density of anaerobic fermentation, its construction method, and its application. Background Technology

[0002] Anaerobic fermentation of solvent-producing Clostridium acetone-butanol-producing bacteria can produce solvents such as biobutanol. Butanol, as a novel renewable liquid fuel, exhibits superior low-carbon emission reduction characteristics compared to bioethanol. It has a closer energy density to gasoline, better compatibility, lower hygroscopicity and corrosiveness, and better compatibility with existing refined oil transportation and storage facilities. However, during anaerobic fermentation, solvent-producing Clostridium cells undergo large-scale autolysis, lysis, and death during the late logarithmic growth phase and stationary phase, making it difficult to achieve or maintain high cell density and viable cell count. Furthermore, the toxic stress from environmental factors such as butanol inhibitors severely restricts production indicators such as cell biomass, carbon source utilization, solvent concentration, yield, and productivity. These problems urgently need to be addressed!

[0003] In fact, microorganisms, represented by Gram-positive bacteria, achieve autolysis through autolysins, which are specific hydrolytic enzymes produced by Gram-positive bacteria that hydrolyze the peptidoglycan structure of the cell wall. During the logarithmic growth phase of bacteria, autolysins are precisely located on the dividing septum, regulating cell elongation, division, cell wall flipping, flagellar movement, and chemotaxis. During the stationary phase of bacterial growth, the expression and activity of extracellular autolysins increase, leading to large-scale autolysis and lysis of the cell, the dissolution of cell contents and inhibitory factors, and the cell entering the death phase.

[0004] It is noteworthy that teichoic acid components in bacterial cell walls play a crucial role in regulating autolysin activity. Teichoic acid is divided into wall teichoic acid (WTA) and lipoteichoic acid (LTA). The former does not penetrate the plasma membrane, while the latter crosses the peptidoglycan layer. When bacteria are in their natural state, such as the logarithmic growth phase, both teichoic acid and autolysin are electronegative. They bind through interionic interactions mediated by divalent cations, causing autolysin to adsorb onto the teichoic acid anionic polymer, resulting in decreased autolysin hydrolytic activity and cell autolysis rate. When bacteria transition from the logarithmic growth phase to the stationary phase or are under stress, the expression level of teichoic acid in the cell wall decreases, the interionic interaction between autolysin and teichoic acid weakens, autolysin is released and its hydrolytic activity increases, leading to an increased cell autolysis rate and gradual progression into the death phase.

[0005] Therefore, based on the structural and functional characteristics of teichoic acid in Gram-positive bacteria, teichoic acid-modified Clostridium can be constructed. By enhancing teichoic acid synthesis, extracellular autolysin activity can be regulated, autolysin hydrolysis activity and cell autolysis rate can be reduced, weakening or alleviating cell autolysis and increasing fermentation cell density or viable cell count. Therefore, developing teichoic acid-modified Clostridium and its construction method to improve anaerobic fermentation cell density and achieve efficient fermentation production using solvents such as biobutanol has potential theoretical research significance and practical application value in the field of industrial microbiology. Summary of the Invention

[0006] The purpose of this invention is to provide a *Clostridium difficile* strain modified with teichoic acid (TBEA) that can improve cell density in anaerobic fermentation, along with its construction method and applications. By overexpressing the TBEA synthase-encoding gene in *Clostridium difficile*, a recombinant TBEA-modified TBEA strain was constructed. This enhanced TBEA synthesis reduces the activity of the extracellular hydrolase autolysin, increasing anaerobic fermentation cell density or viable cell count, thereby improving the concentration, yield, and productivity of solvents such as ethanol and butanol. This invention, for the first time in strictly anaerobic microorganisms like *Clostridium difficile*, overexpresses the TBEA synthase-encoding gene, promoting TBEA synthesis and expression in the cell wall. This addresses a series of practical problems in existing TBEA technologies, including low cell density or viable cell count during anaerobic fermentation, low carbon source consumption rate and utilization, long fermentation cycles, and low concentrations, yields, and productivity of solvents such as ethanol and butanol.

[0007] The technical solution of this invention is as follows:

[0008] In a first aspect, the present invention provides a celloteichoic acid-modified Clostridium that can increase the cell density of anaerobic fermentation, which is a recombinant solubilizing Clostridium. This is achieved by constructing an expression vector overexpressing celloteichoic acid synthase and introducing it into wild-type or modified solubilizing Clostridium, including Clostridium acetobutylicum, to obtain a celloteichoic acid-modified recombinant solubilizing Clostridium.

[0009] Furthermore, the nucleotide sequence of the gene encoding celloteichoic acid synthase is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0010] SEQ ID NO.1:

[0011] ATGAATAAAGTAGCAAAAAATTTCTTTTCAATGGGATTTTCTACACTTGTAT

[0012] CACAATTTTTAACCTTTTTTACAGGAACATATGCTGCTCATGTTTTAGGTAA

[0013] ATCTGGTTTCGGAGATATAACAACTGTTCAGGCAATCACATTATATTTTTCTA

[0014] TTGTGGTGCTTTTTGGACTTCAAACCTATGGTACAAGAGAGATTTCTAAAA

[0015] ATAAGGAAAATATTAAAGATAGTTGGTGAGATAACGCTTTTTAGAATAAT

[0016] TGTTTTTGGCTTTAGTTTCATTGTTATATTATATTGGCTTTAGTGCTGTCATT

[0017] TAACTCAAGAAGTATAGCTTGGCTTCTAGTTTTGTATAGCTTAACTTTGCTG

[0018] ACGAATGCATTGTGTATAGACTGGGTGTATAATGGACTTCAGGAGATGAAA

[0019] TATAATGCTTTATACAACATTTAAAGTCTAATACCGTTTGTTTTAATAGTA

[0020] TTATTCTTGAAAAATCCATCACAGGTTAACTATTATGCTGTTTTTACAGTTTT

[0021] AGGACTTGCAGTTGCAACAATTTATTCAGTTTTATATATTTCTTTCGTGAAA

[0022] AATTAAAATCAACTTTAAAATAAGCTTTGATGAAATAAAAAATACTCTTT

[0023] ATACGGGTGGCCATTTTTACTGGCAGGAGTTCTTGCTACAATAAATGTTAAT

[0024] GTTGATAGAATTGTAATAAGATTTTCCTGGGGAAGTTCAATGGCAGGTATAT

[0025] ATGCCTCTGCATATTACGTTCATATCCTTTTAACCAATATAGTTACTATAATAT

[0026] TTGGAGTTGTATTTCCTCTTATGATAAGTTACTTTAATGAAAAAAATATGGCT

[0027] TCTCTGCATGGACTAATTAAAAATGTTTCCAAGGTAATTATAGCTGTAATTG

[0028] TTCCTATAGTTTTAGGTGGAATTATTCTATCTAAAGAGATAATAATACTCCTA

[0029] TTTGGAAAAGCATATGAAAGTGCATATATGCCTTTTTCAATACTTTTAATATA

[0030] TAGTTTAATATTATTCATTAGAGAGATATATGCATATGGTTTAAATGCATGGA

[0031] ACATGGAAAAAAAATATTTAAGAGCAGTAGTAATTTCTTCCTTGTTTAACTT

[0032] AATTATAAATTTGCTTCTTACACCCAAGTTTGGTATGAATGTAGCGGCCATTA

[0033] TTACAGTGTTATCTGAAGTTATTACAATTTTGATTATGAAATACTATTCGGAC

[0034] AAAATAATTAAAGTTTCAAAGTATACTCTGTTTTTAAAAATATCAATTCCGT

[0035] GTATTGGCATGGGAATAATAACGTGTACTTTAAAATATTATAACGTAAATATT

[0036] ATTTTTAATATAGTTCTAAGTGCACTTTGCTATGTAACTTTTGTGATTTTATTT

[0037] AAATATTTTACTATAGAGGATATAAAAAAAATTTGCTGTGAGAAAGAGTTGASEQ ID NO.2:

[0038] MNKVAKNFFSMGFSTLVSQFLTFFTGTYAAHVLGKSGFGDITTVQAITLYFSIV

[0039] VLFGLQTYGTREISKNKENIKDIVGEITLFRIIVFGFSFIVILILALVLSFNSRSIA

[0040] WLLVLYSLTLLTNALCIDWVYNGLQEMKYNALYNIIKSLIPFVLIVLFLKNPSQ

[0041] VNYYAVFTVLGLAVATIYQFYIYFFREKLKINFKISFDEIKKYSLYGWPFLLAG

[0042] VLATINVNVDRIVIRFSWGSSMAGIYASAYYVISFLTNIVTIIFGVVFPLMISYFN

[0043] EKNMASLHGLIKNVSKVIIAVIVPIVLGGIILSKEIIILLFGKAYESAYMPFSILLI

[0044] YSLILFIREIYAYGLNAWNMEKKYLRAVVISSLFNLIINLLLTPKFGMNVAAIIT

[0045] VLSEVITILIMKYYSDKIIKVSKYTLFLKISIPCIMGGITCTLKYYNVNIIFNIVL

[0046] SALCYVTFVILFKYFTIEDIKKFAVRKS

[0047] Furthermore, the constructed Clostridium tumefaciens modified with teichoic acid exhibits increased viable cell number during the logarithmic growth phase of anaerobic fermentation, improved carbon source consumption rate and utilization, increased concentration, yield, and production of solvents such as butanol during fermentation, and enhanced tolerance to toxicity inhibitors and high-temperature stress.

[0048] Secondly, the present invention provides a method for constructing the above-mentioned Clostridium perfringens modified with teichoic acid, comprising the following steps:

[0049] 1) The nucleotide sequence of the celloteichoic acid synthase encoding gene shown in SEQ ID NO.1 was ligated into the vector plasmid pIMP1 by enzyme digestion and ligation method, wherein the 5' end of the nucleotide sequence of SEQ ID NO.1 was connected to the promoter shown in SEQ ID NO.3 (nucleotide sequence of promoter gene thl) to obtain the expression vector pIMP1-WTA;

[0050] SEQ ID NO.3:

[0051] TTTTTAACAAAATATATTGATAAAAATAATAATAGTGGGTATAATTAAGTTGT

[0052] TAGAGAAAACGTATAAATTAGGGATAAACTATGGAACTTATGAAATAGATTG

[0053] AAATGGTTTATCTGTTACCCCGTATCAAAATTTAGGAGGTTAGTTAGA

[0054] 2) The expression vector pIMP1-WTA obtained in step 1) was transformed into E. coli DH10B to obtain the methylated expression vector pIMP1-WTA;

[0055] 3) The methylated expression vector pIMP1-WTA obtained in step 2) was introduced into Clostridium solvogeneticum, and the recombinant Clostridium solvogeneticum modified with celloteichoic acid was obtained by culturing and screening.

[0056] Thirdly, the present invention also provides an application of Clostridium tumefaciens modified with teichoic acid for the fermentation of alcohols, which utilizes the aforementioned Clostridium tumefaciens modified with teichoic acid or the recombinant solvent-producing Clostridium obtained by the aforementioned construction method, comprising the following steps:

[0057] 1) Activation culture: Inoculate the bacterial strain into the liquid activation culture medium at an inoculation rate of 5% (v / v), place it in an anaerobic drop bottle and incubate statically at 37℃ and natural pH. After static activation culture for 18-24 hours, it can be used for seed culture.

[0058] 2) Seed culture: The activated strain obtained in step 1) is inoculated into liquid seed culture medium at an inoculation rate of 10% (v / v), and placed in an anaerobic drop bottle for shaking culture at a temperature of 37℃, a rotation speed of 100-250 rpm, natural pH, and shake culture for 18-24 hours before being used for fermentation culture.

[0059] 3) Fermentation culture: Inoculate the seed culture obtained in step 2) into the liquid fermentation medium at an inoculation rate of 10% (v / v), place it in an anaerobic fermenter and stir to culture at a temperature of 30-45℃, a stirring speed of 100-250 rpm, a pH of 4.0-6.5, and ferment for 24-72 hours to produce alcohols.

[0060] In step 1), the liquid activation culture medium contains 20 g / L glucose, 30 g / L tryptone and 10 g / L yeast extract, at natural pH.

[0061] 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, 0.01 g / L p-aminobenzoic acid, and 5 g / L glycerol, at natural pH.

[0062] 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, 0.01 g / L p-aminobenzoic acid, and 5 g / L glycerol.

[0063] In step 3), the pH of the fermentation broth after inoculation is controlled within the range of 4.0-6.5 by adding dilute sulfuric acid solution, potassium hydroxide solution, or adding the pH buffer calcium carbonate.

[0064] In a specific implementation plan, the pH of the fermentation broth is controlled to be >5.0 or >5.5 by adding 2 mol / L potassium hydroxide solution, and the pH of the fermentation broth is controlled to be in the range of 4.5-6.0 by adding 4 g / L calcium carbonate.

[0065] In step 3), high-temperature stress fermentation is achieved by increasing the temperature to 40-45℃.

[0066] Further, in step 3), the fermentation medium can be a synthetic medium with added toxicity inhibitors or a toxic hydrolysate of lignocellulose raw material. The toxicity inhibitors added to the synthetic medium are derived from one or more of the toxic hydrolysate inhibitors of lignocellulose raw material (organic acids, furans, phenols, etc.). The hydrolysate is obtained by pretreating lignocellulose biomass through physical, chemical, biological or combined pretreatment and enzymatic hydrolysis with cellulase.

[0067] In one specific implementation, 0.45 g / L formic acid is added to the fermentation medium as a toxicity inhibitor.

[0068] Furthermore, the lignocellulose raw material is derived from one or more of the following: corn stalks, corn cobs, wheat straw, and rice straw.

[0069] In summary, this invention provides the above-mentioned Clostridium perfringens modified with teichoic acid that can increase the cell density of anaerobic fermentation, the method for constructing the above-mentioned recombinant solvent-producing Clostridium perfringens modified with teichoic acid, and the practical application of using the above-mentioned modified Clostridium perfringens for anaerobic fermentation to produce solvents such as butanol.

[0070] The beneficial effects of this invention are as follows: This invention provides a Clostridium difficile modified with teichoic acid that can increase the cell density of anaerobic fermentation, its construction method, and its application. This invention constructs a recombinant teichoic acid-modified Clostridium difficile by overexpressing the gene encoding teichoic acid synthase in a solubilizing Clostridium. By enhancing teichoic acid synthesis and reducing the activity of the extracellular hydrolase autolysin, it increases the cell density or number of viable cells in anaerobic fermentation, thereby improving the concentration, yield, and productivity of alcohol solvents such as ethanol and butanol. Under different pH conditions, compared with the original strain, the recombinant strain provided by this invention can increase the number of viable cells in the logarithmic growth phase of fermentation by up to nearly 21 times, and the concentrations of butanol and total solvent, yield, and production rate can be increased by up to 33.3%, 28.2%, 28.6%, and 85.0%, respectively. Under formic acid inhibitor stress, compared with the original strain, the utilization rate of glucose as the fermentation carbon source of the recombinant strain provided by this invention increases from 34.3% to 100%, and the concentrations of butanol and solvent, yield, and production rate increase by 275.0%, 304.8%, 39.1%, and 305.8%, respectively. Under high temperature stress, compared with the original strain, the utilization rate of glucose as the fermentation carbon source of the recombinant strain provided by this invention increases from 84.3% to 91.4%, and the concentrations of butanol and solvent, yield, and production rate increase by 16.8%, 14.3%, 5.3%, and 14.3%, respectively. Therefore, this invention provides a Clostridium perfringens modified with teichoic acid that can improve the cell density of anaerobic fermentation, effectively solving a series of practical problems in the prior art, such as low cell density or number of viable cells in solvent-producing Clostridium anaerobic fermentation, low carbon source consumption rate, long fermentation production cycle, low concentration of solvents such as butanol, and low yield and production rate. Attached Figure Description

[0071] Figure 1 Map of the constructed expression vector pIMP1-WTA containing the gene encoding celloteichoic acid synthase. Detailed Implementation

[0072] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments, but this is not intended to limit the invention. Specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or functions as the reagents and instruments described below can be used to implement the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0073] Experimental materials

[0074] 1. Involved bacterial species

[0075] The solvent-producing Clostridium used in this invention is Clostridium acetobutylicum ATCC 824 (purchased from the American Type Culture Collection), hereinafter referred to as the original strain; the modified Clostridium acetobutylicum overexpressing cell wall teichoic acid synthase constructed using the original strain is Clostridium acetobutylicum WTA, hereinafter referred to as the recombinant strain.

[0076] 2. Main culture medium

[0077] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, natural pH, for Escherichia coli culture.

[0078] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder, natural pH, for single colony culture of Escherichia coli.

[0079] Liquid activation medium: tryptone 30g / L, glucose 20g / L, yeast extract 10g / L, natural pH, for Clostridium propionidum activation culture.

[0080] Liquid seed culture 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, glycerol 5 g / L, natural pH, for Clostridium propionidum seed culture.

[0081] 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, glycerol 5 g / L, pH 4.0-6.5, used for Clostridium propionidium fermentation culture.

[0082] Solid 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, glycerol 5 g / L, agar powder 20 g / L, natural pH, used for single colony culture and counting of Clostridium propionidum.

[0083] Formic acid stress fermentation medium: formic acid 0.45 g / L, 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, glycerol 5 g / L, natural pH, for Clostridium propionidium stress fermentation culture.

[0084] Example 1: Construction of expression vector and recombinant strain

[0085] 1. Synthesis of the gene fragment encoding celloteichoic acid synthase and construction of the expression vector

[0086] According to the NCBI search information (https: / / www.ncbi.nlm.nih.gov / gene), the sequence information of the Clostridium acetobutylicum cell wall teichoic acid synthase encoding gene involved is shown in SEQ ID NO.1. The gene fragment linked by the two was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the restriction site BamHI / KpnI was selected and ligated into the vector plasmid pIMP1 [Mermelstein LD, Welker NE, Bennett GN, Papoutsakis ET Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC824. Nature Biotechnology, 1992, 10(2): 190-5.]. The 5' end of the nucleotide sequence of SEQ ID NO.1 is linked to the promoter as shown in SEQ ID NO.3. The correct expression vector was named pIMP1-WTA after verification by restriction enzyme digestion, PCR and sequencing. Figure 1 Map of the constructed expression vector pIMP1-WTA containing the gene encoding celloteichoic acid synthase.

[0087] 2. Construction of recombinant strains

[0088] The original bacterial strain stored at -80℃ was streaked on a solid fermentation medium plate and incubated statically at 37℃ under anaerobic conditions for 24-36 hours. Single colonies were picked and inoculated into liquid activation medium and incubated statically at 37℃ under anaerobic conditions for 12-18 hours. The activated bacterial solution was then transferred to liquid seed medium and incubated under shaking at 37℃ under anaerobic conditions until the OD value reached 0.6-1.0. The obtained bacterial cells were pre-cooled at 4℃ and used to prepare Clostridium propionidum competent cells.

[0089] The constructed expression vector pIMP1-WTA was transformed into E. coli DH10B competent cells. [Mermelstein LD & Papoutsakis ET In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T Imethyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC824. Applied and Environmental] Microbiology, 1993, 59(4), 1077-1081.] After transformation, rejuvenation, and single colony culture, the methylated expression vector pIMP1-WTA was extracted; the above methylated expression vector pIMP1-WTA was then electroporated into the prepared Clostridium proteoglycan competent cells, and after rejuvenation, it was spread on solid fermentation medium containing erythromycin resistance. After static culture at 37℃ for 36h, the obtained single colonies were the recombinant strains overexpressed with teichoic acid synthase or enhanced with teichoic acid synthesis; the single colonies were inoculated into liquid seed medium (containing erythromycin resistance), and static cultured at 37℃ for 16-24h, and then transferred to corn mash medium. After anaerobic expansion at 37℃ for 40-48h, it was mixed with an equal volume of 40% glycerol solution, dispensed and frozen in the laboratory at -80℃ for later use.

[0090] Example 2: Comparison of fermentation between the original strain and the recombinant strain under natural pH conditions

[0091] Specifically, this embodiment includes the following steps:

[0092] 1) Activation culture: The original strain and recombinant strain from Example 1 were inoculated into liquid activation culture medium at an inoculation rate of 5% (v / v), placed in anaerobic drop bottle and statically cultured at 37°C and natural pH for 18-24 hours before being used for seed culture.

[0093] 2) Seed culture: The activated strain obtained in step 1) is inoculated into liquid seed culture medium at an inoculation rate of 10% (v / v), and placed in an anaerobic drop bottle for shaking culture at a temperature of 37℃, a rotation speed of 150 rpm, natural pH, and shake culture for 18-24 hours before being used for fermentation culture.

[0094] 3) Fermentation culture: The seed culture obtained in step 2) is inoculated into the liquid fermentation medium at an inoculation rate of 10% (v / v) and placed in an anaerobic fermenter (Biotec-3BG-4, Shanghai Baoxing Biotechnology) for stirring and culture. The liquid fermentation medium volume is 1L, the culture temperature is 37℃, the stirring speed is 150rpm, the pH is natural, and the fermentation culture is carried out for 24-72h.

[0095] Table 1 compares the fermentation performance of the original strain and the recombinant strain under natural pH conditions. The original strain, after 16 hours of fermentation, had a viable cell count of 0.6 × 10⁶. 8 CFU / mL, after 20 hours of fermentation, the number of viable cells increased to 1.0 × 10⁻⁶. 8 The concentration of cfu / mL was 12.0 g / L at the fermentation endpoint, with total solvent concentration, yield, and production rate of 19.2 g / L, 0.302 g / g, and 0.320 g / L / h, respectively. In contrast, the recombinant strain fermented for 16 h had a viable cell count of 6.9 × 10⁻⁶. 8 The cfu / mL concentration was increased by 10.5 times compared to the original strain, and the highest number of viable cells reached 8.4 × 10⁻⁶ after 20 hours of fermentation. 8 The concentration of cfu / mL was 7.4 times higher than that of the original strain. The concentration of butanol product at the fermentation endpoint was 13.0 g / L, which was 8.3% higher than that of the original strain. The total solvent concentration, yield, and production rate were 21.3 g / L, 0.332 g / g, and 0.592 g / L / h, respectively, which were 10.9%, 9.9%, and 85.0% higher than those of the original strain.

[0096] Table 1 Comparison of fermentation performance between the original strain and the recombinant strain under natural pH conditions.

[0097]

[0098] In summary, the experimental results of this embodiment fully demonstrate that overexpressing the cell membrane teichoic acid synthase encoding gene in solvent-producing Clostridium and constructing a cell membrane teichoic acid-modified Clostridium can effectively increase the cell density or number of viable cells in the logarithmic growth phase of anaerobic fermentation under natural pH conditions compared with the original strain, and improve production indicators such as the concentration of solvents such as butanol, yield, and production rate.

[0099] Example 3: Comparison of fermentation between the original strain and the recombinant strain under controlled pH > 5.0 conditions

[0100] Specifically, this embodiment includes the following steps:

[0101] 1) Activation culture: Same as the activation culture steps in Example 2;

[0102] 2) Seed culture: Same as the seed culture steps in Example 2;

[0103] 3) Fermentation culture: The fermentation culture steps are the same as in Example 2, except that the pH of the fermentation broth is controlled to be greater than 5.0 by adding 2 mol / L potassium hydroxide solution.

[0104] Table 2 compares the fermentation performance of the original strain and the recombinant strain under pH > 5.0 conditions. The original strain, after 16 hours of fermentation, had a viable cell count of 0.5 × 10⁻⁶. 8 CFU / mL, when fermented for 20 hours, the number of viable cells increased to 1.4 × 10⁻⁶. 8 The concentration of cfu / mL was 12.5 g / L at the fermentation endpoint, with total solvent concentration, yield, and production rate of 20.3 g / L, 0.290 g / g, and 0.338 g / L / h, respectively. In contrast, the recombinant strain achieved a viable cell count of 1.1 × 10⁻⁶ cells after 16 h of fermentation. 9 The cfu / mL concentration was nearly 21 times higher than that of the original strain, and the number of viable cells reached a maximum of 3.0 × 10⁻⁶ after 20 hours of fermentation. 9 The cfu / mL concentration was 20.4 times higher than that of the original strain. The fermentation endpoint butanol concentration was 13.5 g / L, which was 8.0% higher than that of the original strain. The total solvent concentration, yield, and production rate were 21.1 g / L, 0.301 g / g, and 0.586 g / L / h, respectively, which were 3.9%, 3.8%, and 73.4% higher than those of the original strain.

[0105] Table 2 Comparison of fermentation performance between the original strain and the recombinant strain under pH > 5.0 conditions.

[0106]

[0107] In summary, the experimental results of this embodiment fully demonstrate that overexpressing the cell teichoic acid synthase encoding gene in solvent-producing Clostridium and constructing a cell teichoic acid-modified Clostridium can still effectively increase the cell density or number of viable cells in the logarithmic growth phase of anaerobic fermentation, and improve production indicators such as the concentration, yield, and production rate of solvents such as butanol, under controlled pH > 5.0 conditions, compared with the original strain.

[0108] Example 4: Comparison of fermentation between the original strain and the recombinant strain under pH > 5.5 conditions

[0109] Specifically, this embodiment includes the following steps:

[0110] 1) Activation culture: Same as the activation culture steps in Example 2;

[0111] 2) Seed culture: Same as the seed culture steps in Example 2;

[0112] 3) Fermentation culture: The fermentation culture steps are the same as in Example 2, except that the pH of the fermentation broth is controlled to be greater than 5.5 by adding 2 mol / L potassium hydroxide solution.

[0113] Table 3 compares the fermentation performance of the original strain and the recombinant strain under pH > 5.5 conditions. The original strain, after 16 hours of fermentation, had a viable cell count of 0.5 × 10⁻⁶. 8 CFU / mL, and after 20 hours of fermentation, the number of viable cells continued to increase to 0.9 × 10⁻⁶. 8 The fermentation endpoint was achieved with cfu / mL butanol concentration of 7.2 g / L, total solvent concentration, yield, and production rate of 10.3 g / L, 0.147 g / g, and 0.215 g / L / h, respectively. In contrast, the recombinant strain achieved a viable cell count of 5.5 × 10⁻⁶ cells after 16 h of fermentation. 8 The cfu / mL concentration was nearly 10 times higher than that of the original strain, and the number of viable cells reached a maximum of 1.0 × 10⁻⁶ after 20 hours of fermentation. 9 The concentration of cfu / mL was nearly 10 times higher than that of the original strain. The fermentation endpoint butanol concentration was 9.6 g / L, which was 33.3% higher than that of the original strain. The total solvent concentration, yield, and production rate were 13.2 g / L, 0.189 g / g, and 0.367 g / L / h, respectively, which were 28.2%, 28.6%, and 70.7% higher than those of the original strain.

[0114] Table 3 Comparison of fermentation performance between the original strain and the recombinant strain under pH > 5.5 conditions.

[0115]

[0116] In summary, the experimental results of this embodiment fully demonstrate that overexpressing the cell teichoic acid synthase encoding gene in solvent-producing Clostridium and constructing a cell teichoic acid-modified Clostridium can still effectively increase the cell density or number of viable cells in the logarithmic growth phase of anaerobic fermentation, and improve production indicators such as the concentration, yield, and production rate of solvents such as butanol, under controlled pH > 5.5 conditions, compared with the original strain.

[0117] Example 5: Comparison of fermentation between the original strain and the recombinant strain under the condition of adding pH buffer calcium carbonate

[0118] Specifically, this embodiment includes the following steps:

[0119] 1) Activation culture: Same as the activation culture steps in Example 2;

[0120] 2) Seed culture: Same as the seed culture steps in Example 2;

[0121] 3) Fermentation culture: The fermentation culture steps are the same as in Example 2, except that the pH of the fermentation broth is controlled in the range of 4.5-6.0 by adding 4g / L calcium carbonate.

[0122] Table 4 compares the fermentation performance of the original strain and the recombinant strain under the condition of adding the pH buffer calcium carbonate. After 16 hours of fermentation, the original strain had only 0.4 × 10⁻⁶ viable cells. 8CFU / mL, after 20 hours of fermentation, the number of viable cells increased to 0.9 × 10⁻⁶. 8 The concentration of cfu / mL was 13.2 g / L at the fermentation endpoint, with total solvent concentration, yield, and production rate of 20.4 g / L, 0.291 g / g, and 0.340 g / L / h, respectively. In contrast, the recombinant strain achieved a viable cell count of 4.1 × 10⁻⁶ cells after 16 h of fermentation. 8 The cfu / mL concentration was increased by 9.3 times compared to the original strain, and the number of viable cells reached a maximum of 6.8 × 10⁻⁶ after 20 hours of fermentation. 9 The cfu / mL concentration was 6.6 times higher than that of the original strain. The fermentation endpoint butanol concentration was 16.3 g / L, which was 23.5% higher than that of the original strain. The total solvent concentration, yield, and production rate were 24.7 g / L, 0.349 g / g, and 0.515 g / L / h, respectively, which were 21.1%, 19.9%, and 51.5% higher than those of the original strain.

[0123] Table 4 Comparison of fermentation performance between the original strain and the recombinant strain under the condition of adding pH buffer calcium carbonate.

[0124]

[0125] In summary, the experimental results of this embodiment fully demonstrate that by overexpressing the cell teichoic acid synthase encoding gene in solvent-producing Clostridium, a cell teichoic acid-modified Clostridium was constructed. Compared with the original strain, it can still effectively increase the cell density or number of viable cells in the logarithmic growth phase of anaerobic fermentation, and improve production indicators such as the concentration of solvents such as butanol, yield, and production rate, even with the addition of the pH buffer calcium carbonate.

[0126] Example 6: Comparison of fermentation between the original strain and the recombinant strain under formic acid inhibitor stress.

[0127] Specifically, this embodiment includes the following steps:

[0128] 1) Activation culture: Same as the activation culture steps in Example 2;

[0129] 2) Seed culture: Same as the seed culture steps in Example 2;

[0130] 3) Fermentation culture: The fermentation culture steps are the same as in Example 2, except that 0.45 g / L formic acid is added to the fermentation culture medium as a toxicity inhibitor.

[0131] Table 5 compares the fermentation performance of the original and recombinant strains under formic acid inhibitor stress. The original strain consumed only 24 g / L glucose throughout fermentation, with a carbon source utilization rate of only 34.3%, a maximum OD of 1.4, and a butanol concentration of only 4.0 g / L at the fermentation endpoint. The total solvent concentration, yield, and production rate were 6.2 g / L, 0.258 g / g, and 0.086 g / L / h, respectively. In contrast, the recombinant strain consumed 70 g / L glucose, restored 100% carbon source utilization, and achieved a maximum OD of 2.3, a 64.3% increase compared to the original strain. The butanol concentration at the fermentation endpoint was 15.0 g / L, a 275.0% increase compared to the original strain. The total solvent concentration, yield, and production rate were 25.1 g / L, 0.359 g / g, and 0.349 g / L / h, respectively, representing increases of 304.8%, 39.1%, and 305.8% compared to the original strain.

[0132] Table 5 Comparison of fermentation performance between the original strain and the recombinant strain under formic acid inhibitor stress.

[0133]

[0134] In summary, the experimental results of this embodiment fully demonstrate that overexpressing the cell teichoic acid synthase encoding gene in solvent-producing Clostridium and constructing a cell teichoic acid-modified Clostridium can significantly improve fermentation performance under formic acid inhibitor stress compared with the original strain, enhance the cell's tolerance to formic acid stress, promote the smooth progress of anaerobic fermentation, and greatly improve the fermentation carbon source glucose consumption rate and utilization rate, cell concentration, solvent concentration such as butanol, yield and production indicators.

[0135] Example 7: Comparison of fermentation between the original strain and the recombinant strain under high temperature stress

[0136] Specifically, this embodiment includes the following steps:

[0137] 1) Activation culture: Same as the activation culture steps in Example 2;

[0138] 2) Seed culture: Same as the seed culture steps in Example 2;

[0139] 3) Fermentation culture: The fermentation culture steps are the same as in Example 2, but the fermentation temperature is increased to 40℃.

[0140] Table 6 compares the fermentation performance of the original strain and the recombinant strain under high-temperature stress. The original strain consumed only 59 g / L of glucose throughout the fermentation process, with a carbon source utilization rate of 84.3%, a maximum OD of 2.0, and a butanol concentration of 12.5 g / L at the fermentation endpoint. The total solvent concentration, yield, and production rate were 21.0 g / L, 0.356 g / g, and 0.350 g / L / h, respectively. In contrast, the recombinant strain consumed 64 g / L of glucose throughout the fermentation process, with a carbon source utilization rate increased to 91.4%. Although the maximum OD was only 1.6, the butanol concentration at the fermentation endpoint reached 14.6 g / L, an increase of 16.8% compared to the original strain. The total solvent concentration, yield, and production rate were 24.0 g / L, 0.375 g / g, and 0.400 g / L / h, respectively, representing increases of 14.3%, 5.3%, and 14.3% compared to the original strain.

[0141] Table 6 Comparison of fermentation performance between the original strain and the recombinant strain under high temperature stress.

[0142]

[0143] In summary, the experimental results of this embodiment fully demonstrate that overexpressing the cell teichoic acid synthase encoding gene in solvent-producing Clostridium and constructing a cell teichoic acid-modified Clostridium can effectively improve anaerobic fermentation performance, enhance cell tolerance to high temperature stress, and significantly improve production indicators such as the rate and utilization of fermentation carbon source glucose, cell concentration, concentration of solvents such as butanol, yield, and production rate under high temperature stress conditions compared with the original strain.

Claims

1. A Clostridium perfringens modified with teichoic acid that can increase cell density in anaerobic fermentation, characterized in that, The aforementioned Clostridium clavatum modified with clavatin is a recombinant solubilizing Clostridium clavatum; by constructing an expression vector for overexpressing clavatin synthase, and introducing it into wild or modified solubilizing Clostridium clavatum, recombinant solubilizing Clostridium clavatum modified with clavatin is obtained. The amino acid sequence of the cell wall teichoic acid synthase is shown in SEQ ID NO.2; The solvent-producing Clostridium is Clostridium propioni, and the solvent is an alcohol.

2. The Clostridium perfringens modified with teichoic acid according to claim 1, characterized in that, The nucleotide sequence of the cell wall teichoic acid synthase encoding gene is shown in SEQ ID NO.

1.

3. A method for constructing Clostridium perfringens modified with teichoic acid as described in claim 2, characterized in that, Includes the following steps: 1) The nucleotide sequence encoding the celloteichoic acid synthase gene as shown in SEQ ID NO.1 was ligated into the vector plasmid pIMP1 by an enzyme digestion and ligation method, wherein the 5' end of the nucleotide sequence of SEQ ID NO.1 was connected to the promoter as shown in SEQ ID NO.3, so as to obtain the recombinant plasmid pIMP1-WTA; 2) The recombinant plasmid pIMP1-WTA obtained in step 1) was transformed into E. coli DH10B to obtain the methylated recombinant plasmid pIMP1-WTA; 3) The methylated recombinant plasmid pIMP1-WTA obtained in step 2) was introduced into Clostridium solvogeneticum, and the recombinant Clostridium solvogeneticum modified with celloteichoic acid was obtained by culturing and screening.

4. The application of the Clostridium perfringens modified with phosphatidylcholine as described in any one of claims 1-2 for alcohol fermentation.

5. The application according to claim 4, characterized in that, Includes the following steps: 1) Activation culture: Inoculate the bacterial strain into the liquid activation culture medium at a volume ratio of 5%, place it in an anaerobic drop bottle and incubate statically at 37℃ and natural pH. After static activation culture for 18-24 hours, it can be used for seed culture. 2) Seed culture: The activated strain obtained in step 1) is inoculated into liquid seed culture medium at a volume ratio of 10%, and placed in an anaerobic drop bottle for shaking culture at a temperature of 37℃, a rotation speed of 100-250 rpm, natural pH, and shake culture for 18-24 hours before being used for fermentation culture. 3) Fermentation culture: Inoculate the seed culture obtained in step 2) into the liquid fermentation medium at a volume ratio of 10%, place it in an anaerobic fermenter and stir to culture at a temperature of 30-45℃, a stirring speed of 100-250 rpm, a pH of 4.0-6.5, and ferment for 24-72 hours to produce alcohols.

6. The application according to claim 5, characterized in that, In step 3), the fermentation medium is a synthetic medium with added toxicity inhibitors or a toxic hydrolysate of lignocellulose raw material. The toxicity inhibitors added to the synthetic medium are derived from one or more of the toxic hydrolysate inhibitors of lignocellulose raw material. The hydrolysate is obtained by pretreating lignocellulose biomass through physical, chemical, biological or combined pretreatment and enzymatic hydrolysis with cellulase.

7. The application according to claim 6, characterized in that, The lignocellulose raw material is derived from one or more of the following: corn stalks, corn cobs, wheat straw, and rice straw.

8. The application according to claim 5, characterized in that, In step 1), the liquid activation culture medium contains 20 g / L glucose, 30 g / L tryptone and 10 g / L yeast extract, at natural pH. In step 2), the liquid seed culture medium comprises 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, 0.01 g / L p-aminobenzoic acid, and 5 g / L glycerol, at natural pH. 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, 0.01 g / L p-aminobenzoic acid, and 5 g / L glycerol.

9. The application according to claim 5, characterized in that, In step 3), the pH of the fermentation broth after inoculation is controlled within the range of 4.0-6.5 by adding dilute sulfuric acid solution, potassium hydroxide solution, or adding the pH buffer calcium carbonate.

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