Clostridium permanent gene engineering bacteria and application thereof in fermentation of one-carbon gas to synthesize ethanol, lactic acid and 2,3-butanediol

Abstract

This invention belongs to the field of microbial and genetic engineering technology, specifically relating to a genetically engineered *C. yongdar* strain and its application in the synthesis of ethanol, lactic acid, and 2,3-butanediol from one-carbon gas fermentation. This invention proposes a strategy for metabolic modification of *C. yongdar* based on the lactate cycle. Three engineered strains were constructed by knocking out lactate dehydrogenase (LDH) producing lactate, knocking out lactate dehydrogenase oxidizing lactate, and overexpressing LDH producing lactate dehydrogenase based on the knockout of lactate dehydrogenase oxidizing lactate. This modification strategy can increase the yield of 2,3-butanediol, ethanol, and lactic acid in the one-carbon gas autotrophic fermentation of *C. yongdar*, providing a feasible metabolic modification technology for the syngas fermentation of acetic acid-producing bacteria to produce high-value compounds, thus possessing good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial and genetic engineering technology, specifically relating to a Clostridium yongdar genetically engineered strain and its application in the fermentation of one-carbon gas to synthesize ethanol, lactic acid and 2,3-butanediol. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] With the global energy crisis and environmental problems becoming increasingly severe, developing biomanufacturing technologies based on carbon monoxide and carbon dioxide to convert them into high-value chemicals offers both environmental and economic benefits. Aerobic Clostridium (such as Clostridium yunnanense) can utilize syngas or industrial waste gas as carbon and energy sources for growth, converting it to acetic acid via the Wood-Ljungdahl pathway, making it an ideal microbial substrate for carbon gas conversion. However, the natural metabolic networks of wild-type aerobic Clostridium typically preferentially support growth, limiting their ability to synthesize target chemicals. Therefore, reshaping the metabolic flux of strains through metabolic engineering to enable the targeted and efficient synthesis of high-value chemicals such as ethanol, lactic acid, and 2,3-butanediol has become a key research focus in this field.

[0004] The inventors found that although some progress has been made in the metabolic engineering of Clostridium yongdarii, existing research mainly focuses on the synthesis of compounds such as ethanol, 2,3-butanediol and acetone, while there is relatively little research on lactic acid synthesis. Summary of the Invention

[0005] The purpose of this invention is to provide a genetically engineered *Clostridium yongdar* strain and its application in the fermentation of one-carbon gas to synthesize ethanol, lactic acid, and 2,3-butanediol. Specifically, this invention unexpectedly discovered a hidden metabolic behavior, the lactic acid cycle, in *Clostridium yongdar*. This metabolic pattern not only affects the accumulation of lactic acid during one-carbon gas fermentation but also influences overall energy metabolism and metabolic flux. Based on this, this invention metabolically modifies *Clostridium yongdar* to improve its ability to synthesize high-value chemicals such as ethanol, lactic acid, and 2,3-butanediol from one-carbon gas, thereby increasing its alcohol-acid ratio. Based on the above research results, this invention is thus completed.

[0006] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a genetically engineered *Clostridium yongdar* strain, wherein the *Clostridium yongdar* strain is deficient in or overexpresses lactate cycle-related genes.

[0007] The lactate cycle-related genes encode the single-subunit lactate dehydrogenases LdhA and Ldh / EtfAB_1 complex in Clostridium yunnanense; LdhA catalyzes the reduction of pyruvate to lactate by NADH, and Ldh / EtfAB_1 is an electron-disproportionating lactate dehydrogenase that catalyzes the ferroreductase Fd red 2- and NAD + Lactic acid oxidation is dependent on lactate oxidation.

[0008] Furthermore, the lactic acid cycle-related gene is located at position 3524789..3525769 in the GCF_000143685.1 sequence of the Clostridium yunnanense genome. ldhA The (CLJU_RS15865) gene and its location at 2356527..2360164 ldh / etfAB _1 (CLJU_RS10590~10600) gene.

[0009] A second aspect of the present invention provides the application of the above-mentioned Clostridium yongdar genetically engineered strain in the synthesis of high-value chemicals based on one-carbon gas.

[0010] A third aspect of the present invention provides a method for fermenting and producing high-value chemicals, the method comprising: performing one-carbon gas fermentation culture on the above-mentioned Clostridium yongdarii genetically engineered strain.

[0011] It should be noted that although this invention uses *Clostridium yongdarii* as an example for metabolic modification, the above-mentioned metabolic modification strategy is also applicable to other acetic acid-producing bacteria with similar lactic acid cycles, such as... Clostridium autoethanogenum , Clostridium luticellarii , Clostridium coskatii and Eubacterium limosum This increases the yield of its fermentation products. Therefore, other genetically engineered lactic acid bacteria obtained based on the metabolic modification strategy of this invention are also within the scope of protection of this invention.

[0012] The beneficial technical effects of one or more of the above technical solutions are as follows: The above-mentioned technical solution significantly affects the metabolic flux of *Clostridium yongdarense* by metabolically modifying the lactate cycle pathway. Under small-volume closed-batch fermentation conditions (with the expectation of achieving higher yields through continuous culture in optimized fermenters), it knocks out the lactate-producing single-subunit lactate dehydrogenase. ldhA mutant strain Clj In strain 15865, the production of 2,3-butanediol increased to 2.3 times that of the starting strain. Meanwhile, the elimination of oxidized lactic acid... ldh / etfAB After _1, the mutant strain Clj Lactic acid production in strain 10590 was more than 10-fold higher than that of the original strain, while the production of acetic acid and ethanol was also altered, with the alcohol-acid ratio increasing from 3.7:1 to 2.1:1. In the mutant strain Clj... Further overexpression based on 10590 ldhA This further increased lactic acid production to 2.5 mM. This metabolic modification strategy provides a new direction for the metabolic modification of Clostridium yongdarii to produce lactic acid, ethanol, 2,3-butanediol and other compounds, and has broad practical application value. Attached Figure Description

[0013] Figure 1 This refers to the lactic acid cycle in Clostridium yongdarii in this invention.

[0014] Figure 2 It is the mutant strain Clj in this invention. 15865. Gel image validated by PCR. Lane 1 is Clostridium yongdarii wild-type, lanes 2 and 3 are Clj. 15865 knockout bacteria, ck is a negative control, and M is a 5K marker.

[0015] Figure 3 It is the mutant strain Clj in this invention. Figure 15865 shows the growth and metabolic changes in 100% CO2 fermentation medium. The figures represent OD values. 600nm Curves showing the changes in pH (A), acetic acid and ethanol (B), and lactic acid and 2,3-butanediol (C) over time. Clj_WT is the wild type of Clostridium yongdarii. 15865 is ldhA _15865 gene knockout strain.

[0016] Figure 4 It is the mutant strain Clj in this invention. 10590 gel images validated by PCR. Lane 1 is wild-type bacteria, lanes 2 and 3 are Clj. 10590 knockout bacteria, ck is a negative control, and M is a 5K marker.

[0017] Figure 5 It is the mutant strain Clj in this invention. Figure 10590 shows the growth and metabolic changes in 100% CO2 fermentation medium. The figures represent OD values. 600nm Curves showing the changes in pH (A), acetic acid and ethanol (B), and lactic acid and 2,3-butanediol (C) over time. Clj_WT is the wild type of Clostridium yongdarii. 10590 is ldh _10590 gene knockout strain.

[0018] Figure 6It is the overexpression in this invention ldhA The level of lactic acid accumulation in 100% CO2 fermentation medium. Clj_WT is the wild-type Clostridium yongdarii. 10590 is ldh _10590 gene knockout bacteria, Clj 10590::blank is in Clj Based on 10590, the pMTL83151-blank plasmid was expressed, Clj 10590::P 01440 - ldhA It is in Clj Based on 10590, the pMTL83151-P01440-ldhA plasmid was expressed. Detailed Implementation

[0019] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0020] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0021] In a typical embodiment of the present invention, a Clostridium yongdar genetically engineered strain is provided, wherein the Clostridium yongdar genetically engineered strain lacks or overexpresses lactate cycle-related genes.

[0022] The lactate cycle-related genes encode the single-subunit lactate dehydrogenases LdhA and Ldh / EtfAB_1 complex in Clostridium yunnanense; LdhA catalyzes the reduction of pyruvate to lactate by NADH, and Ldh / EtfAB_1 is an electron-disproportionating lactate dehydrogenase that catalyzes the ferroreductase Fd red 2- and NAD + Lactic acid oxidation is dependent on lactate oxidation.

[0023] Furthermore, the lactic acid cycle-related gene is located at position 3524789..3525769 in the GCF_000143685.1 sequence of the Clostridium yunnanense genome. ldhA The (CLJU_RS15865) gene and its location at 2356527..2360164ldh / etfAB _1 (CLJU_RS10590~10600) gene.

[0024] In this invention, gene deletion can be mediated by CRISPR / Cas gene editing tools, ClosTron class II intron-mediated gene inactivation, and homologous recombination techniques, thereby mediated by knockdown, deletion, or insertion of any one or more lactate dehydrogenase-related genes in the lactate cycle of Clostridium yongdarii. No specific limitations are imposed here.

[0025] The overexpression may use a constitutive or inducible promoter suitable for Clostridium yunnanense; further including P pta P thl P 01440 P iLacP This leads to an increase in the expression level of the lactate dehydrogenase gene, with the preferred promoter P... 01440 .

[0026] In one specific embodiment of the present invention, a genetically engineered *Clostridium yongdarii* strain A is provided, wherein the genetically engineered *Clostridium yongdarii* strain A is a genetically engineered strain of the starting strain *Clostridium yongdarii*. ldhA (CLJU_RS15865) was knocked out, and this genetically engineered bacterium A can effectively increase the yield of 2,3-butanediol.

[0027] In one specific embodiment of the present invention, a genetically engineered strain B of Clostridium yunnanense is provided, wherein the genetically engineered strain B is a genetically engineered strain of Clostridium yunnanense from which the starting strain was derived. ldh / etfAB_1 (CLJU_RS10590~10600) was knocked out. This genetically engineered bacterium B can effectively increase the yield of lactic acid and the alcohol-acid ratio.

[0028] In one specific embodiment of the present invention, a *Clostridium yongdar* genetically engineered bacterium C is provided, wherein the *Clostridium yongdar* genetically engineered bacterium C is based on *Clostridium yongdar* genetically engineered bacterium B. ldhA The gene is overexpressed, thereby further increasing lactic acid production.

[0029] In this invention, the starting strain *Clostridium yongdar* can specifically be *Clostridium yongdar* DSM 13528. *Clostridium yongdar* DSM 13528, as the type strain of *Clostridium yongdar*, has been extensively studied and reported.

[0030] In another specific embodiment of the present invention, the application of the above-mentioned Clostridium yongdar genetically engineered strain in the synthesis of high-value chemicals based on carbon gas is provided.

[0031] The carbon gas can be any one or more of CO, CO2, and H2, and the high-value chemicals include, but are not limited to, ethanol, lactic acid, and 2,3-butanediol.

[0032] In another specific embodiment of the present invention, a method for producing high-value chemicals by fermentation is provided, the method comprising: performing one-carbon gas fermentation culture on the above-mentioned Clostridium yongdarii genetically engineered strain.

[0033] The high-value chemicals include, but are not limited to, ethanol, lactic acid, and 2,3-butanediol. The carbon gas includes, but is not limited to, any one or more of CO, CO2, and H2; preferably, the carbon gas is 100% CO, and fermentation is carried out at a partial pressure of 0.15 MPa.

[0034] It should be noted that although this invention uses *Clostridium yongdarii* as an example for metabolic modification, the above-mentioned metabolic modification strategy is also applicable to other acetic acid-producing bacteria with similar lactic acid cycles, such as... Clostridium autoethanogenum , Clostridium luticellarii , Clostridium coskatii and Eubacterium limosum This increases the yield of its fermentation products. Therefore, other genetically engineered lactic acid bacteria obtained based on the metabolic modification strategy of this invention are also within the scope of protection of this invention.

[0035] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the materials, reagents, instruments, and methods used in the following examples are all conventional materials, reagents, instruments, and methods in the art and are commercially available.

[0036] Materials and Methods Strains and plasmids: The strain was *Clostridium yunnanense* DSM 13528, purchased from the German Collection of Microorganisms and Cell Cultures. The plasmid was PMTL83151. Clostridium – E. coli Shuttle plasmid, preserved in our laboratory.

[0037] Culture media and buffers: CO medium (1L) contains 0.9 g NH4Cl, 0.9 g NaCl, 0.4 g MgSO4·6H2O, 0.75 g KH2PO4, 1.5 g K2HPO4, 2.5 mg FeCl3·3H2O, 1 g yeast extract, 5 g fructose, 0.75 g Cys-HCl, 1 ml 0.1% resazurite, 10 ml trace metal stock solution, and 10 mL vitamin stock solution, pH 6.0. YTF medium (1L): 10 g yeast extract, 16 g peptone, 10 g fructose, 0.2 g NaCl, 0.75 g Cys-HCl, 1 ml 0.1% resazurite, 10 ml trace metal stock solution, and 10 ml vitamin stock solution, pH 6.0. Trace metal element mother liquor: Nitrilotriacetic acid 1.5 g, MgSO4·7H2O 3 g, MnSO4·H2O 0.5 g, NaCl1 g, FeSO4·7H2O 0.1 g, CoSO4·7H2O 0.18 g, CaCl2·2H2O 0.1 g, ZnSO4·7H2O 0.18g, CuSO4·5H2O 0.01 g, KAl(SO4)2·12H2O 0.02 g, NiCl·6H2O 0.03 g, Na2SeO3·5H2O0.3 mg, Na2WO4·5H2O 0.4 mg. Vitamin stock solution: Biotin 2 mg, Folic acid 2 mg, Pyridoxine 10 mg, Thiamine 5 mg, Riboflavin 5 mg, Niacin 5 mg, Calcium pantothenate 5 mg, Vitamin B12 0.1 mg, Para-hydroxybenzoic acid 5 mg, Alpha-lipoic acid 5 mg.

[0038] SMP buffer: 270 mM sucrose, 1 mM MgCl2, 1 mM NaH2PO4, containing 15% glycerol.

[0039] Product analysis method: High performance liquid chromatography (HPLC). The chromatographic column used was NanoChrom ChromCore Sugar-10H (7.8 mm × 300 mm, 6 μm); mobile phase: 5 mM sulfuric acid; injection volume: 20 μL; flow rate: 0.8 mL / min; column temperature: 65℃; differential refractive index detector was used; the program for each sample was 20 min.

[0040] The PCR primers used in each example are listed in Table 1 and were synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd.

[0041] Table 1 Primers used in the gene knockout plasmids and overexpression plasmids constructed in the examples.

[0042] The crRNA sequences used in the examples are listed in Table 2 and were synthesized by Qingke Biotechnology Co., Ltd.

[0043] Table 2. crRNA sequences of different knockout plasmids

[0044] The underlined part was designed by the CRISPOR (https: / / crispor.gi.ucsc.edu / ) online website.

[0045] Example 1: Mutant strain Clj Gas fermentation experiment 15865 (1) Construction of knockout plasmid The pMTL83151-icas12a-10590 knockout plasmid (construction method of this plasmid is detailed in Example 2) was double-digested using restriction endonucleases BamHI and PaeI to obtain the plasmid backbone. ldhA _15865 designed crRNA and synthesized it directly using Beijing Ruiboxingke Biotechnology Co., Ltd. Primers P13 / P14 and P15 / P16 were used to obtain... ldhA The homologous arms HA1 and HA2 flanking the _15865 gene were extracted. HA1 was then overlapped with crRNA using primers P17 / P14 to obtain 15865-crRNA-HA1. The plasmid backbone was then homologously ligated with the target fragments 15865-crRNA-HA1 and HA2 using Novizan's ClonExpress MultiS one-step cloning kit C113. The product was then transformed into *E. coli* DH5α competent cells. After PCR screening and sequencing verification, the complete pMTL83151-iCas12a-15865 knockout plasmid was obtained.

[0046] (2) Mutant strain Clj Construction of 15865 Preparation of competent cells and other materials: fresh C. ljungdahliiThe seed culture was inoculated into 50 mL of fructose medium. When the OD value reached approximately 0.4, a cell wall weakening agent (1.25% glycine and 0.3 M sucrose) was added and treated for 1 h. The bacterial culture was then placed on ice for about 10 min, followed by centrifugation at 6000 rpm and 4°C for 10 min. After discarding the supernatant, an appropriate amount of SMP buffer (270 mM sucrose, 1 mM MgCl2, 15% glycerol, 1 mM phosphate) was added to resuspend the bacterial cells. The culture was then centrifuged at 6000 rpm and 4°C for 10 min, and the supernatant was discarded. This process was repeated once. Finally, 1 mL of SMP buffer was added to the centrifuge tube to resuspend the bacterial cells, and the tube was placed on ice for later use. The plasmid pMTL83151-iCas12a-15865 constructed in step (1) was extracted from Escherichia coli in advance for later use.

[0047] Electroporation: Pre-cool the cleaned 2 mm electroporation cuvette on ice, then take 200 μL of prepared competent cells, add 4 μg of plasmid, pre-cool on ice for 5 min, and transfer them together into the electroporation cuvette. The electroporation conditions are 1000 V, 50 μF, and 100 Ω.

[0048] Resuscitation: After electroporation, quickly add 1 mL of fructose medium and transfer the bacterial cells to an anaerobic vial using a syringe. Resuscitate the bacteria in a 37°C incubator for 12-18 h. Once the medium in the anaerobic vial becomes turbid, spread all the bacterial cells onto YTF plates containing thiamphenicol (Tm) resistance and place them in a 37°C anaerobic incubator for further culture.

[0049] mutant strain Clj Validation of 15865: After about three days of incubation, pale yellow colonies grew on the plate. The colony surface was not smooth, and there was a noticeable bulge in the center. Single clones were selected for PCR, and primers P28 / P29 were used to verify whether the plasmid transformation was successful. Then, strains that were successfully transformed with the plasmid were selected, and PCR was performed using primers P26 / P27 for verification. The PCR product was then sequenced to verify whether the knockout was successful.

[0050] All experimental steps described above, except for centrifugation, are conducted in an anaerobic chamber. All experimental consumables must be placed in the anaerobic chamber beforehand to replace the oxygen.

[0051] (3) CO gas fermentation The obtained mutant strain Clj 15865 was inoculated at a 2% inoculum into a 1L anaerobic flask containing 250 mL of CO2 medium. The headspace was then evacuated to -0.1 MPa using a vacuum pump and maintained for 5 minutes. Next, 0.15 MPa of 100% CO2 gas was introduced. The purged flask was then placed horizontally in a 37°C incubator for static incubation, with no further CO2 gas added during the incubation process. Samples were taken every 2-3 days during the incubation period to measure OD. 600 Changes in pH and metabolites in the fermentation broth.

[0052] The results showed that knockout ldhA The _15865 gene had little effect on the changes in acetic acid and ethanol production, but it increased 2,3-butanediol production by 2.3 times compared to the wild type. This is because the metabolic flux that was originally directed to lactate synthesis was redirected to the 2,3-butanediol synthesis pathway.

[0053] Example 2: Mutant strain Clj 10590 gaseous fermentation experiment (1) Construction of knockout plasmid The specific construction method of the pMTL83151-iCas12a-10590 knockout plasmid is as follows: Select from C. perfringens The lactose-inducible promoter bgaR-PbgaL (iLacP for short) has been shown in the following literature: Banerjee A, Leang C, Ueki T, Nevin KP, Lovley DR. Lactose-inducible system for metabolic engineering of Clostridium ljungdahlii . Appl Environ Microbiol. 2014 Apr;80(8):2410-6.) Controlling cas12a expression, selecting from C. ljungdahlii strong promoter P 01440 Controls the expression of crRNA (this promoter is...) C.ljungdahlii The protein encoding ferroreductase is 1000 mmol / L. The promoter of the gene. This has been shown in the following literature: Zhao, R., Liu, Y., Zhang, H., Chai, C., Wang, J., Jiang, W., Gu, Y., 2019. CRISPR-Cas12a-mediated gene deletion and regulationin Clostridium ljungdahliiand Its application in carbon flux redirection in synthesis gas fermentation. ACS Synth. Biol. 8, 2270-2279.). The nucleic acid sequence of iLacP was synthesized by Qingke Biotechnology Co., Ltd., and then the target fragment iLacP was amplified using primers P1 / P2; using pFnCpf1_full plasmid (a gift from Professor Liang Quanfeng) as a template, the cas12a fragment was amplified using primers P3 / P4. C. ljungdahlii Using the genome as a template, P was amplified using primers P5 / P6 to obtain P. 01440 The fragments cas12a, iLacP, and P01440 were connected by overlap to obtain the icas12a-PP fragment. Then, using... C. ljungdahlii Using the genome as a template, the upstream and downstream homologous arm fragments HA1 and HA2 of the gene were amplified via P7 / P8 and P9 / P10. The crRNA sequence was designed using the online software CRISPOR (https: / / crispor.gi.ucsc.edu / ), synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd., and amplified using primers P11 / P12. The synthesized crRNA fragment was overlapped with the HA fragment to obtain 10590-crRNA-HA. The pMTL83151 plasmid vector was double-digested with BamHI and HindIII to obtain a linearized vector backbone. The vector backbone, icas12a-PP fragment, and 10590-crRNA-HA fragment were then ligated using the Novizan ClonExpressMultiS One-Step Cloning Kit C113 for homologous recombination, and the product was transformed into *E. coli* DH5α competent cells. After PCR screening and sequencing verification, the complete pMTL83151-iCas12a-10590 knockout plasmid was obtained.

[0054] (2) Mutant strain Clj Construction of 10590 The pMTL83151-iCas12a-10590 knockout plasmid constructed in step (1) was transformed into the target cell via electroporation. C. ljungdahliiIn wild-type fungi (experimental methods refer to Example 1). After about three days of cultivation, pale yellow colonies grew on the plates. The colony surface was not smooth, and there was a noticeable bulge in the center. Single clones were selected for PCR, and primers P28 / P29 were used to verify whether the plasmid transformation was successful. Then, strains that had successfully transformed with the plasmid were selected, and PCR was performed using primers P24 / P25 for verification. The PCR product was then sequenced to verify whether the knockout was successful. In the above experimental steps, except for centrifugation, all procedures were performed in an anaerobic chamber. All experimental consumables were placed in the anaerobic chamber beforehand to replace the oxygen.

[0055] (3) CO gas fermentation The obtained mutant strain Clj 10590 was inoculated at a 2% inoculum into a 1L anaerobic flask containing 250 mL of CO2 medium. The headspace was then evacuated to -0.1 MPa using a vacuum pump and maintained for 5 minutes. Then, 0.15 MPa of 100% CO2 gas was introduced. The purged flask was placed horizontally in a 37°C incubator for static incubation, with no further CO2 gas added during the incubation process. Samples were taken every 2-3 days during the incubation period to measure OD. 600 Changes in pH and metabolites in the fermentation broth.

[0056] The results showed that ldh The knockout of _10590 would affect C. ljungdahlii It had a significant impact on growth and total metabolite yield in CO fermentation. Among them, Clj 10590 produced 0.45 mM lactic acid on day 36 of CO fermentation, more than 10 times higher than wild-type bacteria, achieving a transformation "from nothing to something". Furthermore, Clj Ethanol production in 10590 was 1.4 times that of the wild type, while acetic acid production decreased by about 21% compared to the wild type. The production of acetic acid and ethanol changed inversely, causing the ethanol / acetic acid ratio to change from 1:3.7 to 1:2.1.

[0057] Example 3 Overexpression ldhA Conducting gas fermentation experiments (1) Construction of overexpression plasmid Use P 01440 Strong promoters are enhanced using the pMTL83151 plasmid. ldhA Gene expression. First, P was amplified using primers P20 / P21 to obtain P. 01440 The promoter was amplified using primers P22 / P23 to obtain a single-subunit lactate dehydrogenase. ldhA Genes, then the two fragments are overlapped to obtain P 01440 - ldhAThe pMTL83151 plasmid was digested with restriction endonucleases BamHI and HindIII to obtain the plasmid backbone. Then, the plasmid backbone was coupled with the target fragment P using E. coli GB05-dir. 01440 - ldhA pMTL83151-P was obtained through homologous recombination. 01440 - ldhA Furthermore, constructing a system containing only the promoter P... 01440 It does not contain ldhA The plasmid pMTL83151-blank was used as a control. P was amplified using primers P18 / P19 to obtain P. 01440 The promoter, consisting of two segments containing homologous fragments on a 40 bp plasmid backbone, was then used to bind the plasmid backbone to the target fragment P using E. coli GB05-dir. 01440 pMTL83151-blank was obtained by homologous recombination.

[0058] (2) Engineered bacteria Clj 10590::P 01440 - ldhA Construction The pMTL83151-P01440- constructed in step (1) ldhA The pMTL83151-blank plasmid was transformed into Clj via electroporation. In 10590, after about three days of incubation, pale yellow colonies grew on the plate. The colony surface was not smooth, and there was a noticeable bulge in the center. Single clones were picked and PCR was performed using universal primers M13F / R to verify whether the plasmid transformation was successful.

[0059] (3) CO gas fermentation Will C. ljungdahlii Wild mushroom WT, mutant strain Clj 10590, Clj 10590::blank and Clj 10590::P 01440 - ldhA Inoculate 2% of the culture medium into a 1L anaerobic flask containing 250 mL of CO2 medium. Then, use a vacuum pump to evacuate the headspace to -0.1 MPa and maintain this position for 5 minutes. Next, purge with 0.15 MPa of 100% CO2 gas. Place the purged flask horizontally in a 37°C incubator for static incubation; no further CO2 gas will be added during the incubation process. After fermentation, detect the differences in lactic acid content among different strains.

[0060] The results showed that in Clj Based on the 10590 mutant strain, using P 01440The promoter was overexpressed in the pMTL83151 plasmid. ldhA This further increased the production of lactic acid, Clj 10590::P01440- ldhA It produced 2.5 mM lactic acid, compared to the strain containing the blank control plasmid (Clj). Lactic acid production increased 2.0 times (10590::blank, 0.83 mM). Compared with the original strain of Clostridium yongda, Clj 10590::P01440- ldhA The lactic acid production increased by more than 20 times, demonstrating the potential for improving the lactic acid cycle of Clostridium yongdarii.

[0061] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention. It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions for the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A genetically engineered Clostridium yongdarii bacterium, characterized in that, The genetically engineered Clostridium yongdarii strain lacks or overexpresses lactate cycle-related genes; The lactate cycle-related genes encode the single-subunit lactate dehydrogenases LdhA and Ldh / EtfAB_1 complex in Clostridium yongdarii.

2. The *Clostridium yongdarii* genetically engineered bacterium as described in claim 1, characterized in that, The lactic acid cycle-related gene is located at position 3524789..3525769 in the GCF_000143685.1 sequence of the Clostridium yunnanense genome. ldhA The (CLJU_RS15865) gene and its location at 2356527..2360164 ldh / etfAB _1 (CLJU_RS10590~10600) gene.

3. The *Clostridium yongdarii* genetically engineered bacterium as described in claim 1, characterized in that, Gene deletion was performed using CRISPR / Cas gene editing tools, ClosTron class II intron-mediated gene inactivation and homologous recombination; Overexpression using a constitutive or inducible promoter suitable for Clostridium yunnanense; further including P pta P thl P 01440 P iLacP The preferred promoter is P. 01440 .

4. The *Clostridium yongdarii* genetically engineered bacterium as described in claim 1, characterized in that, The genetically engineered *Clostridium yongdar* strain is *Clostridium yongdar* strain A, which is a genetically engineered strain of the original strain *Clostridium yongdar*. ldhA (CLJU_RS15865) was obtained after knocking it out; Alternatively, the genetically engineered *Clostridium yongdar* strain may be *Clostridium yongdar* strain B, wherein *Clostridium yongdar* strain B is a genetically engineered strain of the originating strain *Clostridium yongdar*. ldh / etfAB_1 (CLJU_RS10590~10600) was obtained after knocking out the following: Alternatively, the *Clostridium yongdar* genetically engineered strain is *Clostridium yongdar* genetically engineered strain C, which is a modified version of *Clostridium yongdar* genetically engineered strain B. ldhA The gene is overexpressed.

5. The *Clostridium yongdarii* genetically engineered bacterium as described in any one of claims 1-4, characterized in that, The starting strain, Clostridium yongdar, is specifically Clostridium yongdar DSM 13528.

6. The application of the Clostridium yongdar genetically engineered strain according to any one of claims 1-5 in the synthesis of high-value chemicals based on one-carbon gas.

7. The application as described in claim 6, characterized in that, The carbon gas is any one or more of CO, CO2, and H2, and the high-value chemicals include ethanol, lactic acid, and 2,3-butanediol.

8. A method for producing high-value chemicals through fermentation, characterized in that, The method includes: performing one-carbon gas fermentation culture on the Clostridium yongdar genetically engineered strain according to any one of claims 1-5.

9. The method as described in claim 8, characterized in that, The high-value chemicals include ethanol, lactic acid, and 2,3-butanediol.

10. The method as described in claim 8, characterized in that, The carbon gas includes any one or more of CO, CO2, and H2.

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