Clostridium perfringens and its construction method and application

Abstract

The application provides a Clostridium permanentis engineering bacterium and a construction method and application thereof, belongs to the technical field of biology, and compared with a wild type strain, the Clostridium permanentis engineering bacterium can only produce acetic acid under the condition of low pH environment and CO2 and H2 as raw materials, without the generation of by-products such as ethanol, lactic acid and 2,3-butanediol. In addition, the fermentation liquor of the Clostridium permanentis engineering bacterium can be directly used as an acidic substrate to adjust the pH of a downstream yeast fermentation liquor, carbon source automatic addition is realized, inorganic salt zero addition in the cascade conversion process from CO2, H2 to oil or yeast protein is realized, and the economic efficiency and environmental friendliness of the overall process are improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an engineered strain of Clostridium yongdarii, its construction method, and its application. Background Technology

[0002] Microbial manufacturing, with its advantages of not relying on arable land, short production cycles, and high production efficiency, is widely considered one of the important technological pathways to address future food and energy crises. Currently, industrial microbial fermentation mainly relies on starch-based raw materials, while my country imports large quantities of starch for fermentation annually, indicating insufficient self-sufficiency in raw materials. In addition, pre-treating and enzymatically hydrolyzing lignocellulosic raw materials such as plant straw to produce fermentable sugars is a feasible alternative, but its large-scale industrial application is limited by high pre-treatment costs and expensive cellulase.

[0003] In recent years, biosynthesis strategies using CO / CO2 as a carbon source and H2 as an energy source have gradually gained attention. my country has abundant CO2 resources, and the rapid iteration of renewable energy technologies such as photovoltaics and wind power, coupled with the continuous decline in the cost of hydrogen production through water electrolysis, provides a solid foundation for the implementation of this strategy. Currently, a typical conversion pathway using CO / CO2 and H2 as direct raw materials involves first using acetic acid-producing bacteria to convert CO / CO2 and H2 into acetic acid, and then using acetic acid as a carbon source to produce single-cell proteins or other biochemicals through fermentation by yeast and other microorganisms. For example, the invention patent with publication number "CN115491395A" provides a two-step fermentation method for producing oils. First, acetic acid is produced using acetic acid-producing bacteria, and then the acetic acid is converted into the unsaturated fatty acid DHA by Schizochytrium fungi, achieving the conversion from CO / CO2 to DHA. This will provide a large amount of inexpensive carbon source for the production of DHA as animal feed, and has significant application prospects. However, the above-mentioned conversion pathway still faces significant technical bottlenecks: the optimal growth pH for acetic acid-producing bacteria is typically around 7, requiring continuous addition of alkali to maintain a neutral environment during fermentation; subsequently, in the fermentation of yeast and other microorganisms, the utilization of acetic acid by these microorganisms causes the pH to rise, necessitating further acid neutralization to maintain the normal growth of the acetic acid-producing bacteria. This repeated pH adjustment not only complicates the operation but also introduces large amounts of inorganic salts, affecting product purity and increasing the burden of post-processing. Although some naturally occurring acetic acid-producing bacteria possess a certain degree of acid tolerance and can ferment at lower pH levels, the production of acetic acid is often accompanied by the accumulation of byproducts such as ethanol and butyric acid.

[0004] Yeast lipolyticis is an unconventional yeast with significant industrial application value. It can utilize acetic acid as a carbon source to ferment and produce yeast proteins and lipids. Its lipid synthesis and storage capabilities are extremely strong, and its lipid content is far higher than that of common oil-producing microorganisms (such as Saccharomyces cerevisiae and oleogenetic microalgae). It exhibits significant advantages in oil production efficiency, substrate adaptability, lipid quality, and industrialization potential, making it a current research and application hotspot. In addition, Candida utilis and Kluyveromyces martensii can also utilize acetic acid as a carbon source to ferment and produce yeast proteins, possessing important application value in the industrial and biotechnology fields. Summary of the Invention

[0005] This invention provides an engineered strain of Clostridium yongdarii, its construction method, and its application.

[0006] The technical solution of this invention is as follows: An engineered strain of Clostridium yongdar contains an alcohol dehydrogenase mutant with the amino acid sequence shown in SEQ ID NO.2, wherein the mutant is formed by mutating aspartic acid at position 485 to glycine and proline at position 516 to leucine in the amino acid sequence shown in SEQ ID NO.1.

[0007] Preferably, the Clostridium yongdarii is Clostridium yongdarii DSM 13528.

[0008] The method for constructing the engineered Clostridium yongdarella involves controlling the expression of the SpCas9 gene through a propylene glycol-inducible promoter in Clostridium yongdarella, thereby editing the alcohol dehydrogenase gene in Clostridium yongdarella.

[0009] The application of the engineered Clostridium yongdarii strain in the production of acetic acid.

[0010] The *Clostridium yongdarii* engineered strain produces acetic acid using CO2 and H2 as raw materials.

[0011] The application of the engineered Clostridium yongdarii strain in yeast fermentation, wherein the yeast uses acetic acid as a carbon source for fermentation.

[0012] Preferably, the yeast includes Yersinia lipolytica, Candida utilis, and Kluyveromyces masculinii Bulgaria.

[0013] Preferably, the application method includes the following steps: fermenting the engineered Clostridium yongdarii bacteria in an environment with a pH of 5.1 to 5.5, and then transferring it to an environment where the pH is not controlled. When the pH of the fermentation broth is 4.3 to 4.7, the fermentation broth is transferred to a yeast fermentation system.

[0014] A further preferred application method includes the following steps: fermenting the *Clostridium yongdarii* engineered bacteria in an environment with a pH of 5.3, and then transferring it to an environment where the pH is not controlled for fermentation. When the pH of the fermentation broth reaches 4.5, the fermentation broth is transferred to a yeast fermentation system.

[0015] An alcohol dehydrogenase mutant, the amino acid sequence of which is shown in SEQ ID NO.2, wherein the mutant is formed by mutating aspartic acid at position 485 to glycine and proline at position 516 to leucine in the amino acid sequence shown in SEQ ID NO.1.

[0016] A genetically engineered bacterium containing the alcohol dehydrogenase mutant.

[0017] Beneficial effects: (1) The present invention provides a strain of Clostridium yongdariensis engineered strain, which can produce only acetic acid under low pH conditions and with CO2 and H2 as raw materials, without the generation of by-products such as ethanol, lactic acid, and 2,3-butanediol.

[0018] (2) The present invention provides the application of the above-mentioned Clostridium yongdar engineered strain in yeast fermentation. The fermentation broth of the engineered strain can be directly used as an acidic substrate to adjust the pH of the downstream yeast fermentation broth. At the same time, the carbon source is automatically added, realizing zero addition of inorganic salts in the cascade conversion process from CO2, H2 to oil or yeast protein, thereby improving the economy and environmental friendliness of the overall process. Attached Figure Description

[0019] Figure 1 This is the result of enzyme activity assay; Figure 2 Fermentation process of wild-type Clostridium yongdarii strain at pH 6.0; Figure 3 Fermentation process of wild-type Clostridium yongdarii strain at pH 5.7; Figure 4 Fermentation process of wild-type Clostridium yongdarii strain at pH 5.3; Figure 5 The fermentation process of the engineered Clostridium yongdarii strain under pH conditions of 5.7 and 5.3; Figure 6 The fermentation process of Yersinia lipophila after adding acidic solutions with pH values ​​of 5.3 and 4.5. Detailed Implementation

[0020] The following description is based on specific embodiments: Explanation of the source of experimental materials: Clostridium yongdarii ( Clostridium ljungdahlii DSM 13528: Purchased from the German Center for the Preservation of Microorganisms and Cell Cultures.

[0021] Yarrowia lipolytica ( Yarrowia lipolytic a): MATa, leu2-270, ura3-302::URA3, xpr2-3, purchased from Yeeastern Biotech Co., Ltd.

[0022] Candida utilis ( Candida utilis CGMCC 2.615, purchased from China General Microbiological Culture Collection Center.

[0023] Kluyveromyces masculinii Bulgarian variant ( Kluyveromyces marxianus var. bulgaricus CGMCC 2.2281, purchased from China General Microbiological Culture Collection Center.

[0024] Escherichia coli ( E. coli BL21 (DE3): Purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0025] plasmid pMTL83151-P PD -pyrE: Recorded in the document "Inducible promoters of bacterialmicrocompartments improve the CRISPR / Cas9 tools for efficient metabolic engineering of Clostridium ljungdahlii DOI:10.1128 / aem.02183-24".

[0026] pMTLcas-pyrE: Recorded in the literature "Huang H, Chai C, Li N, et al. Crispr / cas9-based efficient genome editing in Clostridium ljungdahlii, an autotrophicgas-fermenting bacterium. ACS Synthetic Biology, 2016."

[0027] Example 1: Construction of engineered Clostridium yongdarii strain Propylene glycol-inducible promoters were used to control SpCas9 gene expression for gene editing of Clostridium yongdarii DSM 13528. The alcohol dehydrogenase AdhE1 underwent a D485G+P516L point mutation. The specific steps are as follows: I. pMTL83151-P PD Construction of -adhE1MU plasmid The plasmid pMTL83151-P was double-digested with restriction endonucleases SalI and XhoI. PD -pyrE, recovers a fragment of approximately 9000bp, which is the plasmid backbone pMTL83151-P PD .

[0028] A guide RNA was designed targeting the DSM 13528 insertion site of Clostridium yongdarii. The guide RNA sequence is TCTTTTATCAATTAGGTTATG. Primers adhE1MU-sgRNA-F and adhE1MU-sgRNA-R were designed, with nucleotide sequences shown in SEQ ID NO. 3-4, respectively. Using pMTLcas-pyrE as a template and adhE1MU-sgRNA-F and adhE1MU-sgRNA-R as primers, the sgRNA scaffold was obtained through PCR, which yielded the adhE1MU-sgRNA fragment.

[0029] Primers LHA-F, LHA-R, RHA-F, and RHA-R, with nucleotide sequences shown in SEQ ID NO. 5-8, were designed to amplify the homologous arms flanking the point mutation. Primers adhE1MU-F and adhE1MU-R, with nucleotide sequences shown in SEQ ID NO. 9-10, were designed to introduce the point mutation. Using the Clostridium yongdarii DSM 13528 genome as a template, and using primers LHA-F and LHA-R, the upstream homologous arm LHA was obtained by PCR. Using the Clostridium yongdarii DSM 13528 genome as a template, and using primers RHA-F and RHA-R, the downstream homologous arm RHA was obtained by PCR. Using the Clostridium yongdarii DSM 13528 genome as a template, and using primers adhE1MU-F and adhE1MU-R, the point mutation adhE1MU was obtained by PCR.

[0030] The adhE1MU-sgRNA fragment and its upstream homologous arm LHA were assembled by fusion PCR to obtain the sgRNA-LHA fragment. The fusion PCR reaction system was as follows: 20 μL of 2×fastpfu Mix; 1 μL each of adhE1MU-sgRNA-F and LHA-R primers (10 μM); 30 ng of the adhE1MU-sgRNA / LHA fragment (molar ratio 1:1); and water was added to a final volume of 40 μL.

[0031] Similarly, the adhE1MU fragment was assembled with downstream homologous RHA via fusion PCR to obtain the adhE1MU-RHA fragment.

[0032] The plasmid backbone pMTL83151-P was assembled using 2× Fusein Mix cloning recombinase.PD fragments sgRNA-LHA and adhE1MU-RHA were used to obtain pMTL83151-P. PD -adhE1MU plasmid.

[0033] II. Construction of the engineered strain adhE1MU of Clostridium yongdarii The frozen Clostridium yongdarii DSM 13528 was activated on ice, then subcultured twice in YTF liquid medium and cultured at 37°C until the OD of the bacterial culture was reached. 600 The concentration should be between 0.35 and 0.4. Add 0.1 volume of 2M sucrose solution and 0.15 volume of 1.25M glycine solution to the bacterial culture and incubate at 37°C for 2 hours. Centrifuge at 10,000 rpm for 10 minutes at 4°C, collect the cell pellet, wash the cell pellet twice with SMP buffer and resuspend it to obtain competent cells for later use.

[0034] Electroporation (the electroporation process is performed in an anaerobic chamber): Take 200 μL of the above competent cells and mix with 4 μg of the above pMTL83151-P PD The -adhE1MU plasmid was mixed and incubated on ice for 1.5 min; the mixture was then transferred to a 2 mm electroporation cuvette, and the electroporation parameters were set to 1.0 kV, 200 Ω, and 50 μF to obtain the conversion solution.

[0035] Resuspend the transformation solution in 5 mL of YTF liquid medium and incubate at 37°C for 14 h. Centrifuge 2.5 mL of the resuspended solution and spread the precipitate onto YTF solid medium containing 5 μg / mL clarithromycin. Incubate at 37°C for approximately 3 days until single colonies appear. Pick a single colony and inoculate it into YTF liquid medium containing 5 μg / mL thiamphenicol. Incubate at 37°C for approximately 36 h until the culture becomes turbid. Inoculate the culture at a 5% (v / v) ratio into YTF liquid medium containing 5 μg / mL thiamphenicol and 3 g / L propylene glycol. Incubate at 37°C for 48 h. Streak the culture onto YTF solid medium and incubate at 37°C for approximately 48 h until single colonies appear.

[0036] Single colonies were selected, and the fragment containing the point mutation was amplified by PCR using primers adhE1MU-test-F and adhE1MU-test-R, with nucleotide sequences shown in SEQ ID NO.11~12, respectively. The amplified products were sequenced using primer adhE1MU-seq-F, with nucleotide sequences shown in SEQ ID NO.13. Positive strains were screened, which were identified as the engineered Clostridium yongdarii strain adhE1MU.

[0037] Example 2: Determination of the activity of alcohol dehydrogenase AdhE1 in engineered Clostridium yongdarii strains I. Recombinant Expression of Alcohol Dehydrogenase AdhE1 The genomes of Clostridium yongdarii DSM 13528 and the engineered bacterium adhE1MU were amplified by PCR using primers Adhe1-F and Adhe1-R, respectively. The PCR products were recovered by gel electrophoresis. The nucleotide sequences of Adhe1-F and Adhe1-R are shown in SEQ ID NO. 14-15, respectively.

[0038] The amplification products were ligated into the expression vector pEASY-Blunt E1 and transformed into trans1-T1 competent cells, and positive clones were screened; the positive clones were then transformed into Escherichia coli (E. coli). E. coli In BL21(DE3), the culture was expanded, and IPTG was added to induce expression. Subsequently, nickel column purification was performed to obtain alcohol dehydrogenase ADH (derived from Clostridium yongdarii DSM 13528, concentration 0.0647 g / L) and ADH Mutation (derived from the engineered Clostridium yongdarii strain adhE1MU, concentration 0.403 g / L), which were then used for further enzyme activity assays.

[0039] II. Enzyme Activity Assay The enzyme activation reaction system is shown in Table 1: Table 1. Enzyme activity reaction system

[0040] The absorbance A of the above reaction system at 340 nm was measured using a UV spectrophotometer. 340nm The measurement was performed continuously for 30 minutes, and the results were as follows: Figure 1 As shown. By Figure 1 It can be seen that, compared with the ADH group, the absorbance values ​​in the ADH Mutation group and the Control group remained basically unchanged. This indicates that the enzyme was inactivated in the reaction system and did not react with the substrate. That is, the ethanol dehydrogenase of the Clostridium yongdarii engineered bacteria lost its activity due to amino acid mutation.

[0041] Example 3: Fermentation of *Clostridium yongdarii* engineered strains under conditions using CO2 and H2 as raw materials. I. Fermentation of wild-type Clostridium yongdarii strain Clostridium yongdar DSM 13528 was inoculated into PETC medium and fermented using CO2 and H2 as raw materials at 37℃ and pH 6.0, 5.7 and 5.3, respectively. The fermentation products and their contents were detected by high performance liquid chromatography and differential refractive index display.

[0042] Fermentation results at pH 6.0 are as follows: Figure 2 As shown. By Figure 2It can be seen that at pH 6.0, the final fermentation products of Clostridium yongdar DSM13528 (around 240 h) include acetic acid, ethanol, lactic acid and 2,3-butanediol, of which: the yield of acetic acid is 33 g / L, the yield of ethanol is 5 g / L, the yield of lactic acid is 0.4 g / L and the yield of 2,3-butanediol is 0.15 g / L.

[0043] Fermentation results at pH 5.7 are as follows: Figure 3 As shown. By Figure 3 It can be seen that at pH 5.7, the final fermentation products of Clostridium yongdar DSM13528 (around 132 h) include acetic acid, ethanol, lactic acid and 2,3-butanediol, of which: the yield of acetic acid is 36 g / L, the yield of ethanol is 11 g / L, the yield of lactic acid is 0.07 g / L and the yield of 2,3-butanediol is 0.12 g / L.

[0044] The fermentation results at pH 5.3 are as follows: Figure 4 As shown. By Figure 4 It can be seen that at pH 5.3, the final fermentation products of Clostridium yongdar DSM13528 (around 180 h) include acetic acid, ethanol, lactic acid and 2,3-butanediol, of which: the yield of acetic acid is 32 g / L, the yield of ethanol is 19 g / L, the yield of lactic acid is 0.05 g / L and the yield of 2,3-butanediol is 0.15 g / L.

[0045] II. Fermentation of *Clostridium yongdarii* engineered strain The engineered Clostridium yongdarii strain adhE1MU was inoculated into PETC medium and fermented using CO2 and H2 as raw materials at 37°C and pH 5.7 and 5.3, respectively. The fermentation products and their contents were detected using the same method as in step (I).

[0046] The results are as follows Figure 5 As shown. By Figure 5 It can be seen that at pH 5.7 or 5.3, the final fermentation product of the engineered Clostridium yongdar is only acetic acid, with acetic acid yields of 31 g / L and 33 g / L, respectively, and no other byproducts are produced.

[0047] Example 3: Application of engineered Clostridium yongdarii strain in Yersinia lipophila fermentation I. Fermentation of Yeast Extract Yersinia lipolyticis was activated in YPD medium and then transferred to fermentation medium for fermentation at 28°C and 180 rpm.

[0048] The fermentation medium consists of the following components: sodium acetate 30.0 g / L, yeast powder 6.0 g / L, ammonium sulfate 12.0 g / L, magnesium sulfate heptahydrate 1.5 g / L, potassium dihydrogen phosphate 6.0 g / L, and disodium hydrogen phosphate dodecahydrate 3.0 g / L. It is sterilized at 115℃ for 20 min. After sterilization and cooling, trace elements and vitamins are added.

[0049] The trace elements (1000×) include the following components: citric acid 15.0 g / L, calcium chloride dihydrate 3.0 g / L, boric acid 1.0 g / L, potassium iodide 0.2 g / L, ferric chloride 0.4 g / L, copper sulfate 0.08 g / L, manganese sulfate 0.8 g / L, sodium molybdate 0.4 g / L, zinc sulfate 0.8 g / L, ferrous sulfate heptahydrate 20.0 g / L, and cobalt chloride 0.4 g / L; sterilized by filtration.

[0050] The vitamin (1000×) comprises the following components: biotin (vitamin B7) 0.05 g / L, calcium pantothenate 0.8 g / L, folic acid (vitamin B9) 0.004 g / L, inositol 4.0 g / L, niacin (vitamin B3) 0.8 g / L, para-aminobenzoic acid 0.4 g / L, pyridoxine hydrochloride (vitamin B6) 0.8 g / L, riboflavin (vitamin B2) 0.4 g / L, and thiamine hydrochloride (vitamin B1) 1.5 g / L; filtered for sterilization.

[0051] II. Application of *Clostridium yongdarii* engineered bacterial fermentation broth in *Yarrowia lipolytica* fermentation (1) Fermentation under different pH control conditions This experiment was conducted in an automated fermenter control system. The engineered *Clostridium yongdarii* strain adhE1MU was inoculated into PETC medium and subjected to gaseous fermentation using CO2 and H2 as raw materials at 37°C and pH values ​​of 5.3 and 4.5, respectively, to obtain the *Clostridium yongdarii* engineered strain fermentation broth. The fermentation broth obtained from gaseous fermentation was directly used as the acid solution for downstream *Yarrowia lipolytica* fermentation. Specifically, when *Yarrowia lipolytica* utilized acetic acid, causing the fermentation pH to rise, the automated fermenter control system would add acid solution into the *Yarrowia lipolytica* fermentation system. The fermentation volume (1.5 L) of the *Yarrowia lipolytica* fermentation system was maintained constant by controlling the discharge rate of the fermentation broth. After fermentation stabilized, fermentation parameters such as the residual acetic acid content in the *Yarrowia lipolytica* fermentation broth were measured.

[0052] The experimental procedure was as follows: At the start of *Yarrowia lipolytica* fermentation, *Clostridium yongdarii* engineered bacterial fermentation broth (i.e., acidic solution) with a pH of 5.3 was first added. After fermentation stabilized, i.e., at 144 hours of *Yarrowia lipolytica* fermentation, acetic acid was added to adjust the pH of the *Clostridium yongdarii* engineered bacterial fermentation broth to 4.5 and then added to the *Yarrowia lipolytica* fermentation system. The change in acetic acid content in the fermentation broth during the *Yarrowia lipolytica* fermentation process was detected. The results are as follows: Figure 6As shown, when the added acid solution has a pH of 5.3, Yersinia lipolytica can stably utilize 16 g / L of acetic acid; when an acid solution with a pH of 4.5 is added at 144 h of fermentation, Yersinia lipolytica can stably utilize 23 g / L of acetic acid.

[0053] (2) Construction of a continuous fermentation system of Clostridium yongdariensis engineered strain and Yersinia lipophila fermentation system The specific operating procedure is as follows: The engineered *Clostridium yongdarii* strain adhE1MU is inoculated into fermenter A containing PETC medium. Fermentation is carried out using CO2 and H2 as raw materials at 37℃ and pH 5.3. When the acetic acid product content reaches 20 g / L, 80% by volume of the fermentation broth is transferred to fermenter B (pH not controlled) for continued fermentation. When the pH of the fermentation broth in fermenter B naturally decreases to 4.5, the fermentation broth in fermenter B is then transferred to the *Yarrowia lipolytica* fermentation system. Fresh PETC medium is continuously added to fermenter A to maintain continuous fermentation of both the engineered *Clostridium yongdarii* strain and *Yarrowia lipolytica*.

[0054] After fermentation stabilized, the biomass, yeast cell content (dry weight), and lipid content of *Yarrowia lipolytica* during fermentation were measured. Among these: Biomass assay: The Yersinia lipolytica fermentation broth was appropriately diluted (until the measured value was within the confidence interval), and the OD was measured using a UV spectrophotometer. 600 The value is 36.

[0055] Yeast cell content (dry weight) detection: Yeast lipophilic yeast fermentation broth was placed into a dried and weighed empty EP tube; deionized water was added, and the tube was centrifuged at 10000 rpm for 6 min. The supernatant was discarded, and this step was repeated 3 times to wash away residual culture medium components. The tube was then dried in a 65℃ constant temperature drying oven until constant weight was achieved. Each sample was weighed in triplicate, and the average value was taken as the dry weight. The yeast cell content (dry weight) was calculated as: (dry weight - empty EP tube weight) g / 0.001 L. Using this method, the yeast cell content (dry weight) was determined to be 5.5 g / L.

[0056] Oil content detection (performed in a fume hood): Yeast fermentation broth was collected by centrifugation, the cells were added with HCl for acid hydrolysis, and oil was obtained by chloroform / methanol co-extraction. The oil content was then detected, and the result was 1.1 g / L.

[0057] Example 4: Application of engineered Clostridium yongdarii strain in Candida utilis fermentation Construction of a continuous fermentation system of *Clostridium yongdarii* engineered strains and *Candida utilis* fermentation system The specific operating procedure is as follows: The engineered *Clostridium yongdarii* strain adhE1MU is inoculated into fermenter A containing PETC medium. Fermentation is carried out using CO2 and H2 as raw materials at 37°C and pH 5.3. When the acetic acid product content reaches 20 g / L, 80% by volume of the fermentation broth is transferred to fermenter B (pH not controlled) for continued fermentation. When the pH of the fermentation broth in fermenter B naturally decreases to 4.5, the fermentation broth in fermenter B is then transferred to the *Candida utilis* fermentation system. Fresh PETC medium is continuously added to fermenter A to maintain the continuous fermentation of both the engineered *Clostridium yongdarii* strain and *Candida utilis*.

[0058] After fermentation stabilized, the biomass, yeast cell content (dry weight), and SCP (single-cell protein) content of *Candida utilis* during fermentation were measured. The methods for measuring biomass and yeast cell content (dry weight) were the same as for *Yarrowia lipolytica*, while the SCP content was determined using the Kjeldahl method. The results were: OD 600 The value is 28, the yeast cell content (dry weight) is 4.5 g / L, and the SCP content is 42%.

[0059] Example 5: Application of engineered Clostridium yunnanense in the fermentation of Kluyveromyces bulgaria. Construction of a continuous fermentation system of *Clostridium yongdarii* engineered strains and *Kluyveromyces bulgaria* fermentation strain The specific operating procedure is as follows: The engineered *Clostridium yongdarii* strain adhE1MU is inoculated into fermenter A containing PETC medium. Fermentation is carried out using CO2 and H2 as raw materials at 37°C and pH 5.3. When the acetic acid product content reaches 20 g / L, 80% (by volume) of the fermentation broth is transferred to fermenter B (without pH control) for further fermentation. When the pH of the fermentation broth in fermenter B naturally decreases to 4.5, the fermentation broth in fermenter B is then transferred to the *Kluyveromyces brevis* Bulgarian variant fermentation system. Fresh PETC medium is continuously added to fermenter A to maintain continuous fermentation of the engineered *Clostridium yongdarii* strain and the *Kluyveromyces brevis* Bulgarian variant.

[0060] After fermentation stabilized, the biomass, yeast cell content (dry weight), and SCP (single-cell protein) content of *Kluyveromyces martensii* var. *bulgaricus* during fermentation were measured using the same methods as for *Candida utilis*. The results were: OD... 600 The value is 43, the yeast cell content (dry weight) is 6.9 g / L, and the SCP content is 52%.

Claims

1. An engineered strain of Clostridium yongdarii, characterized in that, The product contains an alcohol dehydrogenase mutant with the amino acid sequence shown in SEQ ID NO.2, wherein the mutant is formed by mutating aspartic acid at position 485 to glycine and proline at position 516 to leucine in the amino acid sequence shown in SEQ ID NO.1; the host bacterium of the engineered Clostridium yongdar is Clostridium yongdar DSM13528.

2. The method for constructing the engineered Clostridium yongdarii strain according to claim 1, characterized in that, Gene editing of alcohol dehydrogenase in Clostridium yongdar was achieved by controlling the expression of the SpCas9 gene through the propylene glycol-inducible promoter in Clostridium yongdar.

3. The application of the engineered Clostridium yongdarii strain described in claim 1 in the production of acetic acid.

4. The application as described in claim 3, characterized in that, The engineered Clostridium yongdarii strain produces acetic acid using CO2 and H2 as raw materials.

5. The application of the engineered Clostridium yunnanense strain according to claim 1 in yeast fermentation, wherein the yeast uses acetic acid as a carbon source for fermentation; wherein the yeast is one or more of Yersinia lipolytica, Candida utilis, and Kluyveromyces masculinii Bulgaria.

6. The application as described in claim 5, characterized in that, The application method includes the following steps: fermenting the engineered Clostridium yongdarii bacteria in an environment with a pH of 5.1 to 5.5, and then transferring it to an environment without pH control for fermentation. When the pH of the fermentation broth is 4.3 to 4.7, the fermentation broth is transferred to a yeast fermentation system.

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