Clostridium yangdaei engineering bacterium as well as construction method and application thereof

Abstract

The invention provides a clostridium endophyllum engineering bacterium as well as a construction method and application thereof, and belongs to the technical field of biology, compared with a wild type strain, the clostridium endophyllum engineering bacterium can only produce acetic acid in a low-pH environment under the condition of taking CO2 and H2 as raw materials, and does not generate by-products such as ethanol, lactic acid, 2, 3-butanediol and the like. Besides, the fermentation liquor of the clostridium endophyllum engineering bacteria can be directly used as an acidic substrate to adjust the pH value of downstream yeast fermentation liquor, meanwhile, automatic addition of a carbon source is achieved, zero addition of inorganic salt in the cascade conversion process from CO2 and H2 to grease or yeast protein is achieved, and the economical efficiency and environmental friendliness of the whole process are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a Clostridium perfringens engineering strain and a construction method and application thereof. BACKGROUND

[0002] Microbial manufacturing has the advantages of not relying on arable land, short production cycle, high production efficiency, etc., and is widely considered as one of the important technical paths to cope with future food and energy crises. At present, industrial microbial fermentation mainly relies on starch raw materials, and China needs to import a large amount of starch for fermentation every year, and the self-supporting capacity of raw materials is insufficient. In addition, the generation of fermentable sugar by pretreatment and enzymolysis of plant straw and other lignocellulose raw materials is also a feasible alternative path, but due to the high cost of pretreatment and the high price of cellulase, this path has not yet been realized on a large scale.

[0003] In recent years, the biosynthetic strategy of CO / CO2 as carbon source and H2 as energy has gradually attracted attention. China has abundant CO2 sources, combined with the rapid iteration of renewable energy technologies such as photovoltaic and wind power, and the cost of hydrogen production by water electrolysis continues to decline, providing a good foundation for the implementation of this strategy. At present, a typical conversion path using CO / CO2 and H2 as direct raw materials is to first convert CO / CO2 and H2 into acetic acid using acetogenic bacteria, and then use acetic acid as a carbon source to produce single-cell protein or other bio-chemicals through microbial fermentation. For example, the invention patent with publication number "CN115491395A" provides a method for producing oil through two-step fermentation, which first produces acetic acid using acetogenic bacteria, and then converts the acetic acid into unsaturated fatty acid DHA through Schizochytrium, realizing the conversion from CO / CO2 to DHA. This will provide a large amount of cheap carbon source for the production of DHA as animal feed, which has important application prospects. However, the above conversion path still has significant technical bottlenecks: the optimal growth pH of acetogenic bacteria is usually around 7, and alkali solution needs to be continuously added during fermentation to maintain a neutral environment; while in the subsequent fermentation of microorganisms such as yeast, the use of acetic acid by microorganisms will cause the pH to rise, and acid needs to be added again to neutralize it to maintain the normal growth of acetogenic bacteria. This repeated pH adjustment not only makes the operation very complex, but also introduces a large amount of inorganic salts, affecting the purity of the product and increasing the burden of post-processing. Although some natural acetogenic bacteria have certain acid tolerance and can ferment at lower pH, they often produce ethanol, butyric acid and other by-products at the same time.

[0004] Yarrowia lipolytica is a very unconventional yeast with great industrial value, which can use acetic acid as carbon source to produce yeast protein and oil, and has strong ability of oil synthesis and storage, and the oil content is much higher than that of ordinary oil-producing microorganisms (such as Saccharomyces cerevisiae and oil-producing microalgae), which shows significant advantages in oil production efficiency, substrate adaptability, oil quality and industrialization potential, and has become a research and application hotspot. In addition, Candida utilis and Kluyveromyces marxianus can also use acetic acid as carbon source to produce yeast protein, which has important application value in the field of industry and biotechnology. SUMMARY

[0005] The application provides an engineered Clostridium permanentre and a construction method and application thereof.

[0006] The technical scheme of the application is as follows: The engineered Clostridium permanentre comprises an ethanol dehydrogenase mutant with an amino acid sequence shown as SEQ ID NO. 2, wherein the aspartic acid at the 485th position of the amino acid sequence shown as SEQ ID NO. 1 is mutated into glycine, and the proline at the 516th position is mutated into leucine.

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

[0008] The construction method of the engineered Clostridium permanentre controls the expression of the SpCas9 gene through a propylene glycol inducible promoter in the Clostridium permanentre, and performs gene editing on the ethanol dehydrogenase in the Clostridium permanentre.

[0009] The application of the engineered Clostridium permanentre in producing acetic acid.

[0010] Preferably, the engineered Clostridium permanentre produces acetic acid by taking CO2 and H2 as raw materials.

[0011] The application of the engineered Clostridium permanentre in yeast fermentation, wherein the yeast utilizes acetic acid as carbon source for fermentation.

[0012] Preferably, the yeast comprises Yarrowia lipolytica, Candida utilis, and Kluyveromyces marxianus var. bulgaricus.

[0013] Preferably, the application method comprises the following steps: fermenting the engineered Clostridium permanentre in an environment with a pH of 5.1-5.5, and then transferring the fermentation liquor into an environment without pH control, and when the pH of the fermentation liquor is 4.3-4.7, transferring the fermentation liquor into a yeast fermentation system.

[0014] Further preferably, the application method comprises the following steps: fermenting the Clostridium Ijungdahlii engineering bacteria in an environment with pH of 5.3, and then transferring into an environment without pH control, and when the pH of the fermentation liquor is 4.5, transferring the fermentation liquor into a yeast fermentation system.

[0015] An ethanol dehydrogenase mutant, the amino acid sequence of which is shown as SEQ ID NO. 2, wherein the aspartic acid at position 485 of the amino acid sequence shown as SEQ ID NO. 1 is mutated into glycine, and the proline at position 516 is mutated into leucine.

[0016] An engineered bacterium, comprising the ethanol dehydrogenase mutant.

[0017] Beneficial effects: (1) The application provides a Clostridium Ijungdahlii engineering bacterium, which can produce only 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.

[0018] (2) The application provides the application of the Clostridium Ijungdahlii engineering bacterium in yeast fermentation, and the fermentation liquor of the engineering bacterium can be directly used as an acidic substrate to adjust the pH of the downstream yeast fermentation liquor, and at the same time, the automatic addition of carbon source is realized, the inorganic salt is zero-added in the cascade conversion process from CO2 and H2 to oil or yeast protein, and the economic efficiency and environmental friendliness of the overall process are improved. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the result of enzyme activity determination; Figure 2 is the fermentation process of the wild-type Clostridium Ijungdahlii strain under the condition of pH 6.0; Figure 3 is the fermentation process of the wild-type Clostridium Ijungdahlii strain under the condition of pH 5.7; Figure 4 is the fermentation process of the wild-type Clostridium Ijungdahlii strain under the condition of pH 5.3; Figure 5 is the fermentation process of the Clostridium Ijungdahlii engineering bacterium under the condition of pH 5.7 and 5.3, respectively; Figure 6 is the fermentation process of Yarrowia lipolytica after adding acid liquor with pH of 5.3 and 4.5, respectively. DETAILED DESCRIPTION

[0020] The following will be described in combination with specific embodiments: Explanation of sources of experimental materials: Clostridium Ijungdahlii (C. Ijungdahlii) Clostridium ljungdahlii DSM 13528: purchased from the German Collection 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 Plasmid pMTL83151-P was digested with restriction enzymes Sail and Xhol PD -pyrE, and a fragment of about 9000 bp was recovered, which was the plasmid backbone pMTL83151-P PD .

[0028] A guide RNA targeting the insertion site of Clostridium permanentre DSM 13528 was designed, and the sequence of the guide RNA was TCTTTATCAATTAGGTTATG. Primers adhE1MU-sgRNA-F and adhE1MU-sgRNA-R were designed, and the nucleotide sequences were shown in SEQ ID NO. 3-4. The sgRNA scaffold was obtained by PCR reaction using pMTLcas-pyrE as the template and adhE1MU-sgRNA-F and adhE1MU-sgRNA-R as the primers, which was fragment adhE1MU-sgRNA.

[0029] Primers LHA-F, LHA-R and RHA-F, RHA-R were designed, and the nucleotide sequences were shown in SEQ ID NO. 5-8, which were used to amplify the homologous arms on both sides of the point mutation; primers adhE1MU-F, adhE1MU-R were designed, and the nucleotide sequences were shown in SEQ ID NO. 9-10, which were used to introduce the point mutation. The upstream homologous arm LHA was obtained by PCR reaction using Clostridium permanentre DSM 13528 genome as the template and LHA-F and LHA-R as the primers; the downstream homologous arm RHA was obtained by PCR reaction using Clostridium permanentre DSM 13528 genome as the template and RHA-F and RHA-R as the primers; the point mutation adhE1MU was obtained by PCR reaction using Clostridium permanentre DSM 13528 genome as the template and adhE1MU-F and adhE1MU-R as the primers.

[0030] The fragments adhE1MU-sgRNA and the upstream homologous arm LHA were assembled by fusion PCR to obtain the fragment sgRNA-LHA. In the fusion PCR reaction system, 2x fastpfu Mix was 20 μL; adhE1MU-sgRNA-F / LHA-R primers (10 μM) were 1 μL each; adhE1MU-sgRNA / LHA fragments were 30 ng (molar ratio 1:1) in total; and water was added to 40 μL.

[0031] The fragments adhE1MU and the downstream homologous arm RHA were assembled by fusion PCR in the same way to obtain the fragment adhE1MU-RHA.

[0032] The plasmid backbone pMTL83151-P was assembled using 2x Fusein MixPD , fragment sgRNA-LHA, fragment adhE1MU-RHA, obtain pMTL83151-P PD -adhE1MU plasmid.

[0033] II. Construction of Clostridium Hungatei engineered strain adhE1MU The frozen Clostridium Hungatei DSM 13528 was activated on ice, then subcultured twice in YTF liquid medium, and cultured at 37°C until the OD 600 The OD of the bacterial liquid was between 0.35 and 0.4. 0.1 volume of 2M sucrose solution and 0.15 volume of 1.25M glycine solution were added to the bacterial liquid, and incubated at 37°C for 2h. The cell precipitate was collected by centrifugation at 10000rpm for 10min at 4°C, washed twice with SMP buffer, resuspended, and prepared into competent cells for standby use.

[0034] Electroporation (the electroporation process was operated in an anaerobic box): 200μL of the above competent cells were mixed with 4μg of the above pMTL83151-P PD -adhE1MU plasmid on ice for 1.5min; the mixture was transferred to a 2mm electroporation cup, and the electroporation parameters were set to 1.0kV, 200Ω, 50μF, and the transformation liquid was obtained.

[0035] The transformation liquid was resuspended with 5mL of YTF liquid medium, incubated at 37°C for 14h, 2.5mL of the resuspension was centrifuged, the precipitate was plated on YTF solid medium containing 5μg / mL of clarithromycin, and incubated at 37°C for about 3 days until single colonies grew. Single colonies were picked and inoculated into YTF liquid medium containing 5μg / mL of thiostrepton, and incubated at 37°C for about 36h until the bacterial liquid was turbid. The bacterial liquid was inoculated into YTF liquid medium containing 5μg / mL of thiostrepton and 3g / L of propylene glycol at a ratio of 5% volume percentage, and incubated at 37°C for 48h. The culture liquid was streaked on YTF solid medium, and incubated at 37°C for about 48h until single colonies grew.

[0036] Single colonies were picked, and the point mutation fragment was amplified by PCR reaction, using adhE1MU-test-F and adhE1MU-test-R as the primers, and the nucleotide sequences were shown in SEQ ID NO. 11-12. The amplified product was sequenced using adhE1MU-seq-F as the primer, and the nucleotide sequence was shown in SEQ ID NO. 13. Positive strains were screened, and the Clostridium Hungatei engineered strain adhE1MU was obtained.

[0037] Example 2: Enzyme activity determination of ethanol dehydrogenase AdhE1 in Clostridium Hungatei engineered strain I. Recombinant expression of ethanol 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 comprises 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, disodium hydrogen phosphate dodecahydrate 3.0 g / L, sterilization at 115°C for 20 min, after sterilization and cooling, trace elements and vitamins are added.

[0049] The trace elements (1000x) comprise 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, cobalt chloride 0.4 g / L; sterilization by filtration.

[0050] The vitamins (1000x) comprise the following components: biotin (Vitamin B7) 0.05 g / L, calcium pantothenate 0.8 g / L, folic acid (Vitamin B9) 0.004 g / L, myo-inositol 4.0 g / L, nicotinic acid (Vitamin B3) 0.8 g / L, p-aminobenzoic acid 0.4 g / L, pyridoxine hydrochloride (Vitamin B6) 0.8 g / L, riboflavin (Vitamin B2) 0.4 g / L, thiamine hydrochloride (Vitamin B1) 1.5 g / L; sterilization by filtration.

[0051] II. Application of Clostridium Ijungdahlii engineering strain fermentation broth in fermentation of Yarrowia lipolytica (1) Fermentation under different pH control conditions The experiment was carried out in the automatic control system of the fermentation tank. The Clostridium Ijungdahlii engineering strain adhE1MU was inoculated into the PETC medium, CO2 and H2 were used as raw materials, and gas fermentation was carried out at 37°C and pH of 5.3 and 4.5, respectively, to obtain the fermentation broth of the Clostridium Ijungdahlii engineering strain; the fermentation broth obtained by gas fermentation was directly used as the acid liquid in the downstream Yarrowia lipolytica fermentation, that is, when the use of acetic acid by Yarrowia lipolytica caused the pH of the fermentation to rise, the automatic control system of the fermentation tank added the acid liquid into the Yarrowia lipolytica fermentation system, and by controlling the discharge rate of the Yarrowia lipolytica fermentation broth, the fermentation volume (1.5 L) of the Yarrowia lipolytica fermentation system was kept unchanged; after the fermentation was stabilized, the acetic acid residual amount and other fermentation parameters of the Yarrowia lipolytica fermentation broth were determined.

[0052] The experimental process was as follows: at the beginning of the Yarrowia lipolytica fermentation, the Clostridium Ijungdahlii engineering strain fermentation broth (i.e. acid liquid) with pH of 5.3 was first added, and after the fermentation was stabilized, i.e. at the 144th hour of the Yarrowia lipolytica fermentation, the Clostridium Ijungdahlii engineering strain fermentation broth was adjusted to pH of 4.5 by adding acetic acid and added to the Yarrowia lipolytica fermentation system, and the change of the acetic acid content in the fermentation broth during the Yarrowia lipolytica fermentation was detected. The results are shown in Table 1. Figure 6As shown, when the pH of the added acid solution is 5.3, Yarrowia lipolytica can stably utilize 16 g / L of acetic acid; when the pH of the added acid solution is 4.5 at the 144th hour of Yarrowia lipolytica fermentation, Yarrowia lipolytica can stably utilize 23 g / L of acetic acid.

[0053] (2) Construction of Clostridium Ijungdahlii engineering bacteria continuous fermentation-Yarrowia lipolytica fermentation system The specific operation method is as follows: the Clostridium Ijungdahlii engineering bacteria adhE1MU is inoculated into the A fermentation tank containing the PETC medium, CO2 and H2 are used as raw materials, and fermentation is carried out at 37°C and pH 5.3; when the content of acetic acid product reaches 20 g / L, 80% of the fermentation broth is transferred into the B fermentation tank without pH control for continuous fermentation; when the pH of the fermentation broth in the B fermentation tank naturally decreases to 4.5, the fermentation broth in the B fermentation tank is transferred into the Yarrowia lipolytica fermentation system. Among them, fresh PETC medium is continuously supplemented into the A tank to maintain the continuous fermentation of Clostridium Ijungdahlii engineering bacteria and Yarrowia lipolytica.

[0054] After fermentation stabilization, the biomass, yeast cell content (dry weight) and oil content in the Yarrowia lipolytica fermentation process are detected. Among them: Biomass detection: after appropriate dilution of the Yarrowia lipolytica fermentation broth (diluted to the value within the confidence interval), the OD value is measured by using the ultraviolet spectrophotometer, and the result is 36. 600

[0055] Yeast cell content (dry weight) detection: the Yarrowia lipolytica fermentation broth is taken into the dried and weighed empty EP tube; deionized water is added, centrifuged at 10000 rpm for 6 min, and the supernatant is discarded, this step is repeated 3 times to wash the residual medium components; put into a constant temperature drying oven at 65°C and dry to constant weight, take out and weigh, 3 repeats for each sample, take the average value, which is the weight after drying. The yeast cell content (dry weight) is calculated as: (weight after drying-empty EP tube weight) g / 0.001 L. The yeast cell content (dry weight) measured by this method is 5.5 g / L.

[0056] Oil content detection (in a fume hood): take the Yarrowia lipolytica fermentation broth, centrifuge to collect the bacteria, add HCl for acid hydrolysis, and cooperatively extract by chloroform / methanol solution to obtain oil, and detect its content, the result is 1.1 g / L.

[0057] Example 4: Application of Clostridium Ijungdahlii engineering bacteria in Candida utilis fermentation Construction of Clostridium Ijungdahlii engineering bacteria continuous fermentation-Candida utilis fermentation system ​The specific operation method is as follows: the Clostridium ljungdahlii engineering bacteria adhE1MU is inoculated into the A fermentation tank containing PETC medium, CO2 and H2 are used as raw materials, and fermentation is carried out at 37℃ and pH 5.3; when the content of acetic acid product is 20g / L, 80% of the fermentation broth is transferred into the B fermentation tank without pH control for continuous fermentation; when the pH of the fermentation broth in the B fermentation tank naturally reduces to 4.5, the fermentation broth in the B fermentation tank is transferred into the Candida utilis fermentation system. Among them, fresh PETC medium is continuously supplemented into the A tank to maintain the continuous fermentation of Clostridium ljungdahlii engineering bacteria and Candida utilis.

[0058] After fermentation is stabilized, the biomass, yeast cell content (dry weight) and SCP (single cell protein) content in the Candida utilis fermentation process are detected. Among them, the detection method of biomass and yeast cell content (dry weight) is the same as that of Yarrowia lipolytica, and the SCP content is determined by Kjeldahl nitrogen determination method. The detection result is: OD 600 value is 28, the yeast cell content (dry weight) is 4.5g / L, and the SCP content is 42%.

[0059] Example 5: Application of Clostridium ljungdahlii engineering bacteria in the fermentation of Kluyveromyces marxianus var. bulgaricus Construction of Clostridium ljungdahlii engineering bacteria continuous fermentation-Kluyveromyces marxianus var. bulgaricus fermentation system The specific operation method is as follows: the Clostridium ljungdahlii engineering bacteria adhE1MU is inoculated into the A fermentation tank containing PETC medium, CO2 and H2 are used as raw materials, and fermentation is carried out at 37℃ and pH 5.3; when the content of acetic acid product is 20g / L, 80% of the fermentation broth is transferred into the B fermentation tank without pH control for continuous fermentation; when the pH of the fermentation broth in the B fermentation tank naturally reduces to 4.5, the fermentation broth in the B fermentation tank is transferred into the Candida utilis fermentation system. Among them, fresh PETC medium is continuously supplemented into the A tank to maintain the continuous fermentation of Clostridium ljungdahlii engineering bacteria and Candida utilis.

[0060] After fermentation is stabilized, the biomass, yeast cell content (dry weight) and SCP (single cell protein) content in the Candida utilis fermentation process are detected. Among them, the detection method of biomass and yeast cell content (dry weight) is the same as that of Yarrowia lipolytica, and the SCP content is determined by Kjeldahl nitrogen determination method. The detection result is: OD 600 value is 28, the yeast cell content (dry weight) is 4.5g / L, and the SCP content is 42%.

Claims

1. An engineered Clostridium perfringens bacterium, characterized in that, The ethanol dehydrogenase mutant comprises an amino acid sequence as shown in SEQ ID NO. 2, wherein the aspartic acid at position 485 and the proline at position 516 in the amino acid sequence as shown in SEQ ID NO. 1 are mutated to glycine and leucine, respectively.

2. The engineered bacterium of claim 1, wherein, The Clostridium permanentre is Clostridium permanentre DSM 13528.

3. The method for constructing engineered Clostridium perfringens bacteria according to claim 1, wherein, The expression of the SpCas9 gene is controlled by a propylene glycol inducible promoter in Clostridium permanentre, and the ethanol dehydrogenase in Clostridium permanentre is genetically edited.

4. The Clostridium permanentre engineered bacterium of claim 1 is applied to produce acetic acid.

5. The use according to claim 4, wherein the compound is ###0002### The Clostridium permanentre engineered bacterium uses CO2 and H2 as raw materials to produce acetic acid.

6. The Clostridium permanentre engineered bacterium of claim 1 is applied to ferment yeast, and the yeast uses acetic acid as a carbon source for fermentation.

7. Use according to claim 6, wherein The yeast includes Yarrowia lipolytica, Candida utilis, and Kluyveromyces marxianus var. bulgaricus.

8. The use according to claim 6, wherein The application method comprises the following steps: fermenting the Clostridium permanentre engineered bacterium in an environment with a pH of 5.1-5.5, and then transferring the bacterium to an environment without pH control, and when the pH of the fermentation broth is 4.3-4.7, transferring the fermentation broth to a yeast fermentation system.

9. An alcohol dehydrogenase mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO. 2, wherein the aspartic acid at position 485 and the proline at position 516 in the amino acid sequence as shown in SEQ ID NO. 1 are mutated to glycine and leucine, respectively.

10. A genetically engineered bacterium, characterized in that, The ethanol dehydrogenase mutant of claim 9 is comprised.

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