A membrane teichoic acid modified strain for producing short-chain alcohol and a construction method and application thereof

Abstract

The application discloses a membrane teichoic acid modified strain for producing short-chain alcohol and a construction method and application thereof, and belongs to the technical field of biology. The membrane teichoic acid modified strain is a recombinant Clostridium autoethanogenum with overexpressed membrane teichoic acid synthetase, and specifically, the nucleotide sequence of a membrane teichoic acid synthetase coding gene is SEQ ID NO. 1, and the amino acid sequence of a protein coded by the membrane teichoic acid synthetase coding gene is SEQ ID NO. 2. The membrane teichoic acid modified strain for producing short-chain alcohol provided by the application is used for anaerobic fermentation to produce short-chain alcohol products, the number of living cells in a logarithmic growth phase is increased, substrate utilization is accelerated, the concentration, yield and production rate of ethanol and butanol products are improved, the tolerance to toxic inhibitors and high-temperature stress is enhanced, and efficient production of biological ethanol and butanol and other short-chain alcohol products is realized.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a membrane teichoic acid modified strain for producing short-chain alcohols, its construction method, and its application. Background Technology

[0002] Acetone can be produced by anaerobic fermentation of Clostridium solvogeneticum. A cetone), butanol B utanol), ethanol E Solvent-producing Clostridium difficile produces solvents such as ethanol (butanol), hence the process is also known as ABE fermentation. It is one of the oldest industrial fermentation methods to date, second only to Saccharomyces cerevisiae ethanol fermentation in scale. Solvent-producing Clostridium difficile is a type of Gram-positive, strictly anaerobic bacteria that, under anaerobic conditions, can convert starchy, cellulose, and sugary substrates into short-chain alcohols such as bioethanol and butanol through a series of enzymatic reactions.

[0003] Gram-positive bacteria, including Clostridium solvogeneticum, have relatively thick cell walls, consisting of 15-50 layers, each about 1 nm thick, for a total thickness of approximately 20-80 nm. These walls are rich in peptidoglycan components, with teichoic acid comprising about 50-80% of the cell's dry weight. Teichoic acid (TA) is a polymer composed of ribitol or glycerol residues linked together by phosphate double bonds. There are two types of teichoic acid: wall teichoic acid (WTA) and membrane teichoic acid (MTA). The former is linked to the N-acetylmuroic acid of the peptidoglycan in the cell wall, while the latter is linked to the cell membrane, with the other end of both types remaining free outside the cell wall. Teichoic acid is highly antigenic and is an important surface antigen of Gram-positive bacteria. In some bacteria, teichoic acid can adhere to the surface of human cells, functioning similarly to pili, which may be related to pathogenicity.

[0004] In fact, teichoic acid plays a crucial regulatory role in the autolysis of Gram-positive bacteria. When Gram-positive bacteria are in their natural state, such as the logarithmic growth phase, both teichoic acid and autolysins are electronegative. They bind through interionic interactions mediated by divalent cations, adsorbing autolysins—a type of hydrolytic enzyme that directly causes cell autolysis—into the teichoic acid anionic polymer, thus reducing autolysin hydrolytic activity and cell autolysis rate. However, when bacteria transition from the logarithmic growth phase to the stationary phase, teichoic acid expression levels decrease, and the interionic interaction between autolysins and teichoic acid weakens, leading to the release of large amounts of autolysins and enhanced hydrolytic activity. This results in an increased cell autolysis rate and a gradual decline into the apoptosis phase, thereby affecting key production indicators such as the concentration, yield, and productivity of ABE fermentation products.

[0005] Therefore, based on the structural and functional characteristics of teichoic acid in the cell wall membrane of Gram-positive bacteria, a recombinant solubilizing Clostridium could be constructed using genetic engineering strategies to enhance teichoic acid synthesis in the cell membrane, adsorb and inhibit autolysin activity, weaken or alleviate cell autolysis, increase the density of viable fermentation cells (number of viable cells), and thus improve the concentration, yield, and productivity of ethanol and butanol products. Therefore, developing effective teichoic acid-modified strains for the production of short-chain alcohols and their construction methods to achieve efficient fermentation production of bioethanol and butanol has potential theoretical research significance and practical application value in the field of bioenergy. Summary of the Invention

[0006] The purpose of this invention is to provide a modified strain of Clostridium solventiformis for producing short-chain alcohols, its construction method, and its applications. By overexpressing the gene encoding membrane teichoic acid synthase in Clostridium solventiformis, a modified strain of membrane teichoic acid is constructed, namely a recombinant Clostridium solventiformis with enhanced membrane teichoic acid synthesis. This weakens or alleviates cell autolysis, increases the density of viable fermentation cells (number of viable cells), and thus improves the concentration, yield, and productivity of ethanol and butanol short-chain alcohols. This invention is the first to overexpress the gene encoding membrane teichoic acid synthase in the strictly anaerobic microorganism Clostridium solventiformis, promoting the expression and synthesis of membrane teichoic acid in the cell wall, thereby solving the practical problems of long anaerobic fermentation cycles, low viable cell density (number of viable cells), and low concentration, yield, and productivity of ethanol and butanol products in existing technologies of Clostridium solventiformis.

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

[0008] In a first aspect, the present invention provides a membrane teichoic acid-modified strain for producing short-chain alcohols, which is a recombinant solvogenic Clostridium. By constructing an expression vector overexpressing membrane teichoic acid synthase, and introducing it into wild-type or modified solvogenic Clostridium, including Clostridium acetobutylicum, a recombinant solvogenic Clostridium for producing short-chain alcohols is obtained.

[0009] Furthermore, the nucleotide sequence of the membrane teichoic acid synthase encoding gene 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] ATGAGATTTTCTGCAATATACAAAAATACCTTGTCAGAGAAGACAAGGAA

[0012] AATATTAAATTATGGTGATATTATAGTATTTTTTATAATCTTAGTTTTTAA

[0013] ATTGATTACTTATGGAAAAGATTTCACCGGAATATTTTGATAGTTCGG

[0014] TTATAACACCAATAATTGCTTCAGTATTAATAATTTTAGTTTCTCTTTTTTT

[0015] ATTCAAGGGTAAAAAAAGAACAGCGTATTTACTTATAATTTGATTTGATAA

[0016] TAAGCCTCATTCTCATATCGGATATAGCTTATTTTAGGTATAATAAAGATA

[0017] TAATAACGGTTTCAGCTGTTCAAACAGCAAAACTTTTGGTTGGTGTAAAA

[0018] TCGAGTGTGGCTGATGTTCTAAGCATTAAGGATTTTATGTTTTTATTAGAT

[0019] ATTATTATACTTGTTCCAACTTTTATGTTAAAAGCATAAATAAAAT

[0020] ACTATTAAGTTAAAGTTTAGATTAGTTTTATTTTTAGCTGTCTTTATATTCG

[0021] GAGCAGCTTTAGATGCACAACAATTCATAAAGTTAATGTGGAACAACCA

[0022] ACACTGCTTACTGCAATGAGTAATAGAATTTTACCTAACAAAGATGATAGG

[0023] AAATTAAATTTTCATGTGGTAGATGCCTATAATTTTACAAGCACAAAGG

[0024] TGAAAATTCAAAGTTGCCTGAAGCAAGGAAGAATGCAATTAATAA

[0025] CTATCTTAAGAATAATAACTCTATTCCTGGTGTTAATTCAAATCAGAAG

[0026] GTTCAGGAAAAAATCTCATAATGATTCAAGTTGAGGCTCTTCAAGGATTT

[0027] GTTGTGAACAAAAGTATAAATGGAAAGAAATAACACCTATTTGAATA

[0028] AATGGATAAATAAAAGCTTATATTTTGATAATTATTCTATCAAGTAGCTC

[0029] AAGGAAATACATCTGATGCTGAGTTTTATGTCATTGAATTCATTGTATCCTG

[0030] CCCAATCTGGAGCTGCATATTATACATATGCAGGAAATACTTTTAATTCTA

[0031] TTCCTAAAGATTTAGGCGACAAAGGATACTATACTGCAGCGCTTCATGGA

[0032] TATAATGAGGGGTTCTGGAATAGAAATGTAATGTATAAATCAGAAAAATT

[0033] TGATGATTTTTATGGTCAGACTAGTTATAAAAAATGATGAGGTAGTTGGTA

[0034] TGGGATTAAGTGATGAGTCATTTTTAAATCAGTCATTGGCTAAAATGAAA

[0035] ACTTTTAAACAACCATATTTTTCTTTCCTTATAACTTTAAGTAGTCACTATTC

[0036] CTTTTAACGACACGGCTGGATATGAAAAGTATACTATGAATAAGTTAGAT

[0037] GTTGGTGAGTATAAAGATACTCTTTTAGGAAATTACTTTGAAGGAATACA

[0038] TTATACAGATGAACAAATTAGGTAAGTTCCTGGACAAACTTGATAAAGAAG

[0039] GATTAACTAAGAATTCAATAATTGTATTATATGGAGATCATTTTGCTATAC

[0040] CAAAAGAAAATATAAATCAACTTTATAGTTTTGAGGGGATAAAAGATCCT

[0041] ACTGATCTTACGTGGTATGAATATCAAAGGTACCTATGTTTATTCATTTT

[0042] CCAGATGACGAACATAAGGGAGTGAATCATACTTATTCAGGGCAGATGG

[0043] ATATTTATCCCTACAATAGCTAACTTATATAACATAAGTAATAATTATATGT

[0044] TTGGTGAAGATATATTAAATGTAAAAGATGGTGATAGTACCAATGTTACA

[0045] TTTAGAAATGGATCGTTTACAGATGGTAAGATATTTTATGTGTCATGGACT

[0046] AACACTTATTATGATATAGCTACAGGTAAAGATAAAGGAGACACCTG

[0047] AACTTAATAAGAAGAAAGAAGCTGCGACAACTGAATTAGAGTATTCTGA

[0048] CGATCTACTAAATCATAATTTAATTAAAACTTTTTTGAAATAA

[0049] SEQ ID NO.2:

[0050] MRFSAIYKNTLSEKTRKILNYGDIIVFFIILVFKLITYGKEISPEYFDSSVITPIIAS

[0051] VLIIFSFSFLFKGKKRTAYLLIIDLIISLILISDIAYFRYNKDIITVSAVQTAKLLV

[0052] GVKSSVADVLSIKDFMFLLDIIILVPTFIYVKSINKNTIKLKFRLVLFLAVFIFGA

[0053] ALDAQQFHKVNVEQPTLLTAMSNRIYLTKMIGNLNFHVVDAYNFTSTKVKN

[0054] SKKLPEARKNAINNYLKNNNSIPGVKFKSEGSGKNLIMIQVEALQGFVVNKSI

[0055] NGKEITPNLNKWINKSLYFDNYFYQVAQGNTSDAEFMSLNSLYPAQSGAAY

[0056] YTYAGNTFNSIPKDLGDKGYYTAALHGYNEGFWNRNVMYKSEKFDDFYGQ

[0057] TSYKNDEVVGMGLSDESFLNQSLAKMKTFKQPYFSFLITLSSHYPFNDTAGY

[0058] EKYTMNKLDVGEYKDTLLGNYFEGIHYTDEQLGKFLDKLDKEGLTKNSIIVL

[0059] YGDHFAIPKENINQLYSFEGIKDPTDLTWYEYQKVPMFIHFPDDEHKGVNHT

[0060] YSGQMDIYPTIANLYNISNNYMFGEDILNVKDGDSTNVTFRNGSFTDGKIFYV

[0061] SWTNTYYDIATGKKIKETPELNKKKEAATTELEYSDDLLNHNLIKTFLK

[0062] Furthermore, the constructed membrane teichoic acid modified strain for producing short-chain alcohols exhibits increased viable cell count during the logarithmic growth phase, accelerated substrate utilization, improved concentration, yield, and production of ethanol and butanol products, and enhanced tolerance to toxicity inhibitors and high-temperature stress.

[0063] Secondly, the present invention provides a method for constructing the above-mentioned membrane teichoic acid modified strain for producing short-chain alcohols, comprising the following steps:

[0064] 1) The nucleotide sequence encoding the membrane teichoic 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 shown in SEQ ID NO.3 (nucleotide sequence of promoter gene thl) to obtain the expression vector pIMP1-MTA;

[0065] SEQ ID NO.3:

[0066] TTTTTAACAAAATATATTGATAAAAATAATAATAGTGGGTATAATTAAGT

[0067] TGTTAGAGAAAACGTATAAATTAGGGATAAACTATGGAACTTATGAAATAG

[0068] ATTGAAATGGTTTATCTGTTACCCCGTATCAAAATTTAGGAGGTTAGTTAGA

[0069] 2) The expression vector pIMP1-MTA obtained in step 1) was transformed into E. coli DH10B to obtain the methylated expression vector pIMP1-MTA;

[0070] 3) The methylated expression vector pIMP1-MTA obtained in step 2) was introduced into Clostridium solvogeneticum, and recombinant Clostridium solvogeneticum producing short-chain alcohols was obtained through culture and screening.

[0071] Thirdly, the present invention also provides a short-chain alcohol fermentation application, which utilizes the above-mentioned membrane teichoic acid modified strain or the recombinant solvent-producing Clostridium obtained by the above-mentioned construction method, comprising the following steps:

[0072] 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.

[0073] 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.

[0074] 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 for stirring and culture. The culture temperature is 30-42℃, the stirring speed is 100-250rpm, the pH is 4.0-6.5, and the fermentation culture is carried out for 24-72h to produce short-chain alcohol products such as ethanol and butanol.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] In step 3), high-temperature stress fermentation is achieved by increasing the temperature to 40-42℃.

[0081] 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.

[0082] In one specific implementation, 0.45 g / L formic acid is added to the fermentation medium as a toxicity inhibitor.

[0083] Furthermore, the lignocellulose raw material is derived from one or more of the following: corn stalks, corn cobs, wheat straw, and rice straw.

[0084] In summary, this invention provides the above-mentioned membrane teichoic acid modified strain for producing short-chain alcohols, the above-mentioned method for constructing the above-mentioned recombinant solvent-producing Clostridium, and the practical application of using the above-mentioned recombinant solvent-producing Clostridium to produce short-chain alcohols such as ethanol and butanol through anaerobic fermentation.

[0085] The beneficial effects of this invention are as follows: This invention provides a membrane teichoic acid modified strain for producing short-chain alcohols, its construction method, and its application. This invention overexpresses the membrane teichoic acid synthase encoding gene in Clostridium solubilizing, constructing a membrane teichoic acid modified strain, namely, a recombinant clostridium solubilizing with enhanced membrane teichoic acid synthesis, to weaken or alleviate cell autolysis, increase the density of viable fermentation cells (number of viable cells), and thus improve the concentration, yield, and productivity of ethanol and butanol short-chain alcohol products. This invention is the first to overexpress the membrane teichoic acid synthase encoding gene in the strictly anaerobic microorganism Clostridium solubilizing, promoting the expression and synthesis of membrane teichoic acid in the cell wall, thereby solving the practical problems of long anaerobic fermentation cycles, low viable cell density (number of viable cells), and low concentration, yield, and productivity of ethanol and butanol products in existing technologies of Clostridium solubilizing. 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 6.2 times, and the concentration, yield, and production rate of total solvent products such as ethanol and butanol can be increased by up to 27.2%, 26.5%, and 117.7%, respectively. Under formic acid inhibitor stress, compared with the original strain, the recombinant strain of this invention increases the concentration, yield, and production rate of total solvent products such as ethanol and butanol by 314.5%, 42.2%, and 211.6%, respectively. Under high temperature stress, compared with the original strain, the recombinant strain of this invention increases the concentration, yield, and production rate of total solvent products such as ethanol and butanol by 21.0%, 8.1%, and 20.9%, respectively. Therefore, this invention provides a membrane teichoic acid-modified strain for producing short-chain alcohols, achieving efficient fermentation production of short-chain alcohols such as ethanol and butanol. Attached Figure Description

[0086] Figure 1 Map of the constructed expression vector pIMP1-MTA containing the gene encoding membrane teichoic acid synthase. Detailed Implementation

[0087] 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.

[0088] Experimental materials

[0089] 1. Involved bacterial species

[0090] 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 strain that overexpresses membrane teichoic acid synthase using the original strain is Clostridium acetobutylicum MTA, hereinafter referred to as the recombinant strain.

[0091] 2. Main culture medium

[0092] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, natural pH, for Escherichia coli culture.

[0093] 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.

[0094] Liquid activation medium: tryptone 30g / L, glucose 20g / L, yeast extract 10g / L, natural pH, for Clostridium propionidum activation culture.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] Example 1: Construction of expression vector and recombinant strain

[0100] 1. Synthesis of the gene fragment encoding membrane teichoic acid synthase and construction of the expression vector

[0101] According to the NCBI search information (https: / / www.ncbi.nlm.nih.gov / gene), the sequence information of the Clostridium acetobutylicum membrane 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-MTA after verification by restriction enzyme digestion, PCR and sequencing. Figure 1 Map of the constructed expression vector pIMP1-MTA containing the gene encoding membrane teichoic acid synthase.

[0102] 2. Construction of recombinant strains

[0103] 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.

[0104] The constructed expression vector pIMP1-MTA 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-MTA was extracted; the above methylated expression vector pIMP1-MTA 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 colony was the recombinant strain that overexpressed membrane teichoic acid synthase or produced short-chain alcohols; the single colony was inoculated in liquid seed medium (containing erythromycin resistance), and statically 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.

[0105] Example 2: Comparison of fermentation between the original strain and the recombinant strain under natural pH conditions

[0106] Specifically, this embodiment includes the following steps:

[0107] 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.

[0108] 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.

[0109] 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.

[0110] Table 1 compares the fermentation performance of the original strain and the recombinant strain under natural pH conditions. The original strain showed a maximum OD of 4.2 throughout the fermentation process, and a maximum viable count of 1.8 × 10⁻⁶ during the logarithmic growth phase. 8 The concentrations of acetone, ethanol, and butanol products were 5.9 g / L, 1.3 g / L, and 12.0 g / L, respectively. Therefore, the total solvent product concentrations, yields, and production rates of ethanol and butanol were 19.2 g / L, 0.302 g / g, and 0.320 g / L / h, respectively. In contrast, the recombinant strain showed a maximum OD of 3.8 and a maximum viable count of 4.1 × 10⁻⁶ cells / h during the entire fermentation process. 8 The cfu / mL concentration was increased by 1.3 times compared to the original strain. The product concentrations of acetone, ethanol, and butanol were 6.9 g / L, 1.6 g / L, and 13.5 g / L, respectively. Therefore, the total solvent product concentrations, yields, and production rates of ethanol and butanol were 22.0 g / L, 0.356 g / g, and 0.611 g / L / h, respectively, which were increased by 14.6%, 17.9%, and 90.9% compared to the original strain.

[0111] Table 1 Comparison of fermentation performance between the original strain and the recombinant strain under natural pH conditions.

[0112]

[0113] In summary, the experimental results of this embodiment fully demonstrate that overexpressing the membrane teichoic acid synthase encoding gene in solvent-producing Clostridium and constructing a recombinant strain to enhance membrane teichoic acid synthesis can effectively improve production indicators such as live cell density (number of live cells), total solvent product concentrations such as ethanol and butanol, yield and production rate in anaerobic ABE fermentation under natural pH conditions compared with the original strain.

[0114] Example 3: Comparison of fermentation between the original strain and the recombinant strain under controlled pH > 5.0 conditions

[0115] Specifically, this embodiment includes the following steps:

[0116] 1) Activation culture: Same as the activation culture steps in Example 2;

[0117] 2) Seed culture: Same as the seed culture steps in Example 2;

[0118] 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.

[0119] Table 2 compares the fermentation performance of the original strain and the recombinant strain under pH > 5.0 conditions. The original strain showed a maximum OD of 4.1 and a maximum viable count of 1.8 × 10⁻⁶ during the entire fermentation process. 8 The concentrations of acetone, ethanol, and butanol products were 6.2 g / L, 1.6 g / L, and 12.5 g / L, respectively. Therefore, the total solvent product concentrations, yields, and production rates of ethanol and butanol were 20.3 g / L, 0.290 g / g, and 0.338 g / L / h, respectively. In contrast, the maximum OD of the recombinant strain throughout fermentation was 4.1, and the maximum viable count during the logarithmic growth phase was 8.6 × 10⁻⁶. 8 The cfu / mL concentration was increased by 3.8 times compared to the original strain. The concentrations of acetone, ethanol, and butanol products were 7.2 g / L, 2.0 g / L, and 14.0 g / L, respectively. Therefore, the total solvent product concentration, yield, and production rate of ethanol and butanol were 23.2 g / L, 0.329 g / g, and 0.644 g / L / h, respectively, which were increased by 14.3%, 13.4%, and 90.5% compared to the original strain.

[0120] Table 2 Comparison of fermentation performance between the original strain and the recombinant strain under pH > 5.0 conditions.

[0121]

[0122] In summary, the experimental results of this embodiment fully demonstrate that overexpressing the membrane teichoic acid synthase encoding gene in solvent-producing Clostridium and constructing a recombinant strain to enhance membrane teichoic acid synthesis can still effectively improve production indicators such as live cell density (number of live cells), total solvent product concentrations such as ethanol and butanol, yield and production rate in anaerobic ABE fermentation under controlled pH > 5.0 conditions, compared with the original strain.

[0123] Example 4: Comparison of fermentation between the original strain and the recombinant strain under pH > 5.5 conditions

[0124] Specifically, this embodiment includes the following steps:

[0125] 1) Activation culture: Same as the activation culture steps in Example 2;

[0126] 2) Seed culture: Same as the seed culture steps in Example 2;

[0127] 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.

[0128] Table 3 compares the fermentation performance of the original strain and the recombinant strain under pH > 5.5 conditions. The original strain showed a maximum OD of 3.2 and a maximum viable count of 3.2 × 10⁻⁶ throughout the fermentation process. 8 The concentrations of acetone, ethanol, and butanol products were 1.9 g / L, 1.2 g / L, and 7.2 g / L, respectively. Therefore, the total solvent product concentrations, yields, and production rates of ethanol and butanol were 10.3 g / L, 0.147 g / g, and 0.215 g / L / h, respectively. In contrast, the recombinant strain showed a maximum OD of 3.7 and a maximum viable count of 5.3 × 10⁻⁶ cells / h during the entire fermentation process. 8 The concentration of cfu / mL was increased by 65.6% compared to the original strain. The concentrations of acetone, ethanol, and butanol were 3.3 g / L, 1.4 g / L, and 8.4 g / L, respectively. Therefore, the total solvent product concentration, yield, and production rate of ethanol and butanol were 13.1 g / L, 0.186 g / g, and 0.468 g / L / h, respectively, which were increased by 27.2%, 26.5%, and 117.7% compared to the original strain.

[0129] Table 3 Comparison of fermentation performance between the original strain and the recombinant strain under pH > 5.5 conditions.

[0130]

[0131] In summary, the experimental results of this embodiment fully demonstrate that overexpressing the membrane teichoic acid synthase encoding gene in solvent-producing Clostridium and constructing a recombinant strain to enhance membrane teichoic acid synthesis can effectively improve production indicators such as live cell density (number of live cells), total solvent product concentrations such as ethanol and butanol, yield and production rate in anaerobic ABE fermentation under controlled pH > 5.5 conditions, compared with the original strain.

[0132] Example 5: Comparison of fermentation between the original strain and the recombinant strain under the condition of adding pH buffer calcium carbonate

[0133] Specifically, this embodiment includes the following steps:

[0134] 1) Activation culture: Same as the activation culture steps in Example 2;

[0135] 2) Seed culture: Same as the seed culture steps in Example 2;

[0136] 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.

[0137] Table 4 compares the fermentation performance of the original strain and the recombinant strain under the condition of adding the pH buffer calcium carbonate. The original strain showed a maximum OD of 5.4 and a maximum viable count of 1.8 × 10⁻⁶ during the entire fermentation process. 8The concentrations of acetone, ethanol, and butanol products were 6.2 g / L, 1.0 g / L, and 13.2 g / L, respectively. Therefore, the total solvent product concentrations, yields, and production rates of ethanol and butanol were 20.4 g / L, 0.291 g / g, and 0.340 g / L / h, respectively. In contrast, the recombinant strain showed a maximum OD of 5.6 throughout the fermentation process, and a maximum viable count of 1.3 × 10⁻⁶ during the logarithmic growth phase. 9 The cfu / mL concentration was 6.2 times higher than that of the original strain. The concentrations of acetone, ethanol, and butanol were 8.8 g / L, 1.6 g / L, and 14.5 g / L, respectively. The concentrations, yields, and production rates of total solvents such as ethanol and butanol were 24.9 g / L, 0.354 g / g, and 0.692 g / L / h, respectively, which were 22.1%, 21.6%, and 103.5% higher than those of the original strain.

[0138] Table 4 Comparison of fermentation performance between the original strain and the recombinant strain under the condition of adding pH buffer calcium carbonate.

[0139]

[0140] In summary, the experimental results of this embodiment fully demonstrate that overexpressing the membrane teichoic acid synthase encoding gene in solvent-producing Clostridium and constructing a recombinant strain to enhance membrane teichoic acid synthesis can significantly improve production indicators such as live cell density (number of live cells), total solvent product concentrations such as ethanol and butanol, yield, and production rate in anaerobic ABE fermentation under the condition of adding the pH buffer calcium carbonate, compared with the original strain.

[0141] Example 6: Comparison of fermentation between the original strain and the recombinant strain under formic acid inhibitor stress.

[0142] Specifically, this embodiment includes the following steps:

[0143] 1) Activation culture: Same as the activation culture steps in Example 2;

[0144] 2) Seed culture: Same as the seed culture steps in Example 2;

[0145] 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.

[0146] Table 5 compares the fermentation performance of the original strain and the recombinant strain under formic acid inhibitor stress. The original strain consumed only 24 g / L of glucose during fermentation, with a glucose utilization rate of only 34.3%. The maximum OD value was 1.4, and the concentrations of acetone, ethanol, and butanol were 1.9 g / L, 0.3 g / L, and 4.0 g / L, respectively. Consequently, the total solvent product concentrations, yields, and production rates of ethanol and butanol 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 of glucose during fermentation, with a glucose utilization rate of 100%. The maximum OD value was 1.9, and the concentrations of acetone, ethanol, and butanol were 8.6 g / L, 2.0 g / L, and 15.1 g / L, respectively. Consequently, the total solvent product concentrations, yields, and production rates of ethanol and butanol were 25.7 g / L, 0.367 g / g, and 0.268 g / L / h, respectively, representing increases of 314.5%, 42.2%, and 211.6% compared to the original strain.

[0147] Table 5 Comparison of fermentation performance between the original strain and the recombinant strain under formic acid inhibitor stress.

[0148]

[0149] In summary, the experimental results of this embodiment fully demonstrate that overexpressing the membrane teichoic acid synthase encoding gene in Clostridium solubilizing and constructing a recombinant strain to enhance membrane teichoic acid synthesis significantly improves ABE fermentation performance under formic acid inhibitor stress compared to the original strain. This enhances the cell's formic acid stress tolerance, promotes the smooth progress of anaerobic ABE fermentation, and greatly improves production indicators such as substrate utilization, total solvent product concentrations (ethanol and butanol), yield, and production rate.

[0150] Example 7: Comparison of fermentation between the original strain and the recombinant strain under high temperature stress

[0151] Specifically, this embodiment includes the following steps:

[0152] 1) Activation culture: Same as the activation culture steps in Example 2;

[0153] 2) Seed culture: Same as the seed culture steps in Example 2;

[0154] 3) Fermentation culture: The fermentation culture steps are the same as in Example 2, but the fermentation temperature is increased to 40℃.

[0155] 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 during fermentation, with a glucose utilization rate of 84.3%. The maximum OD value was 2.0, and the concentrations of acetone, ethanol, and butanol were 6.9 g / L, 1.6 g / L, and 12.5 g / L, respectively. Consequently, the total solvent product concentrations, yields, and production rates of ethanol and butanol were 21.0 g / L, 0.356 g / g, and 0.350 g / L / h, respectively. In contrast, the recombinant strain consumed 66 g / L of glucose during fermentation, with a glucose utilization rate increased to 94.3%. The maximum OD value was 2.1, and the concentrations of acetone, ethanol, and butanol were 8.4 g / L, 2.0 g / L, and 15.0 g / L, respectively. Consequently, the total solvent product concentrations, yields, and production rates of ethanol and butanol were 25.4 g / L, 0.385 g / g, and 0.423 g / L / h, respectively, representing increases of 21.0%, 8.1%, and 20.9% compared to the original strain.

[0156] Table 6 Comparison of fermentation performance between the original strain and the recombinant strain under high temperature stress.

[0157]

[0158] In summary, the experimental results of this embodiment fully demonstrate that overexpressing the membrane teichoic acid synthase encoding gene in Clostridium difficile and constructing a recombinant strain to enhance membrane teichoic acid synthesis can effectively improve the anaerobic ABE fermentation performance, enhance the cell's tolerance to high temperature stress, and improve production indicators such as substrate utilization, total solvent product concentrations (ethanol and butanol), yield, and production rate under high temperature stress compared to the original strain.

Claims

1. A membrane phospholipid remodeled strain for producing short-chain alcohol, characterized by, The membrane teichoic acid modified strain is a recombinant Clostridium saccharolyticum; the membrane teichoic acid modified recombinant Clostridium saccharolyticum is obtained by introducing an expression vector for overexpression of membrane teichoic acid synthase into wild or modified Clostridium saccharolyticum. The Clostridium that produces solvent is Clostridium Clostridium acetobutylicum ATCC 824; The nucleotide sequence of the membrane teichoic acid synthase encoding gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is SEQ ID NO.

2.

2. The method for constructing a membrane phospholipid-remodeled strain for producing a short-chain alcohol according to claim 1, characterized by, The method comprises the following steps: 1) The nucleotide sequence of the membrane teichoic acid synthase encoding gene shown as SEQ ID NO. 1 is connected into the vector plasmid pIMP1 by enzyme digestion and ligation, wherein the 5' end of the nucleotide sequence of SEQ ID NO. 1 is connected with the promoter shown as SEQ ID NO. 3 to obtain the recombinant plasmid pIMP1-MTA; 2) The recombinant plasmid pIMP1-MTA obtained in step 1) is transformed into E. coli DH10B to obtain the methylated recombinant plasmid pIMP1-MTA; 3) The methylated recombinant plasmid pIMP1-MTA obtained in step 2) is introduced into Clostridium saccharolyticum, and the recombinant Clostridium saccharolyticum for producing short-chain alcohol is obtained by culture and screening.

3. The application of the membrane teichoic acid modified strain for producing short-chain alcohol in claim 1 to short-chain alcohol fermentation.

4. Use according to claim 3, characterized in that, The method comprises the following steps: 1) Activation culture: inoculate the strain into a liquid activation culture medium at a volume ratio of 5%, and place it in an anaerobic drop bottle for static culture; the culture temperature is 37℃, the natural pH, and the static activation culture is used for seed culture after 18-24h; 2) Seed culture: inoculate the activated strain obtained in step 1) into a liquid seed culture medium at a volume ratio of 10%, and place it in an anaerobic drop bottle for shake flask culture; the culture temperature is 37℃, the rotation speed is 100-250rpm, the natural pH, and the shaking culture is used for fermentation culture after 18-24h; 3) Fermentation culture: inoculate the seed strain obtained in step 2) into a liquid fermentation culture medium at a volume ratio of 10%, and place it in an anaerobic fermentation tank for stirring culture; the culture temperature is 30-42℃, the stirring rotation speed is 100-250rpm, the pH is 4.0-6.5, and the fermentation culture is used to obtain the short-chain alcohol product after 24-72h.

5. Use according to claim 4, characterized in that, In step 3), the fermentation culture medium is a synthetic culture medium added with toxic inhibitors or a toxic hydrolysate of lignocellulosic raw materials; the toxic inhibitors added in the synthetic culture medium are derived from one or more than two kinds of mixed inhibitors in the toxic hydrolysate of lignocellulosic raw materials; and the hydrolysate is obtained after physical, chemical, biological or combined pretreatment and cellulase enzymolysis of lignocellulosic biomass.

6. Use according to claim 5, characterized in that, The lignocellulosic raw materials are derived from one or more than two kinds of mixed corn stalks, corn cobs, wheat stalks and rice stalks.

7. Use according to claim 4, characterized in that, In step 1), the liquid activation culture medium comprises 20g / L of glucose, 30g / L of tryptone and 10g / L of yeast powder, and the natural pH. In step 2), the liquid seed culture medium comprises glucose 70 g / L, ammonium acetate 3.22 g / L, yeast powder 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 and glycerol 5 g / L, natural pH; In step 3), the liquid fermentation medium comprises glucose 70 g / L, ammonium acetate 3.22 g / L, yeast powder 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 and glycerol 5 g / L.

8. Use according to claim 4, characterized in that, In step 3), the pH of the inoculated fermentation broth is controlled in the range of 4.0-6.5 by feeding dilute sulfuric acid solution, potassium hydroxide solution or adding pH buffer calcium carbonate.

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