Gene influencing performance of clostridium in synthesizing ethanol by using carbon gas and application of gene

Abstract

The invention discloses an application of some functional genes in improving the performance of clostridium for synthesizing ethanol by utilizing one-carbon gas fermentation, wherein overexpression genes NCBI (National Center for Biotechnology Information) are CLJUc04490, CLJUc25060, CLJUc06990, CLJUc22650 and / or CLJUc07720, and expression of inhibition genes CLJUc09160 and / or CLJUc23840 can be used for improving the autotrophic growth capability and the ethanol synthesis yield of clostridium Yangdall when the clostridium Yangdall is fermented by utilizing C1 gas; therefore, the economical efficiency of the clostridium yangdall fermentation is improved.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, relates to genes that affect the performance of Clostridium in synthesizing ethanol using one-carbon gases and their applications, and also relates to a method for improving the performance of Clostridium ljungdahlii in synthesizing ethanol using one-carbon gases (CO2, CO). Background Art

[0002] Carbon (C1) gases such as carbon dioxide (CO2) and carbon monoxide (CO) can be absorbed and utilized by certain microorganisms, such as Clostridium aerovorum, potentially alleviating environmental challenges while converting them into high-value chemicals and generating economic benefits. CO, CO2, and H2 are the main components of industrial syngas, which primarily originates from exhaust gases emitted by coal, steel, and chemical companies. The CO and CO2 in these gases serve as carbon sources for C. aerovorum's fermentation, while the H2 provides energy for this process.

[0003] Acetogens are an important class of chemocarbon-fixing microorganisms capable of fixing and converting CO2 and CO through the Wood-Ljungdahl (WL) pathway. Compared to other chemocarbon-fixing microorganisms, such as nitrifying bacteria and iron-sulfur bacteria, acetogens have higher conversion efficiencies for one-carbon gases, and laboratory isolation, purification, and cultivation techniques for these strains are more mature. Therefore, using acetogens as a base cell, achieving efficient conversion and utilization of CO2 and CO through metabolic engineering and fermentation process optimization has become a research hotspot in recent years. Gas-fermenting Clostridia is a major acetogen that can rapidly propagate using CO2 and CO and has the ability to synthesize ethanol, making it an autotrophic microorganism with industrial applications. Ethanol is a highly important biofuel with a huge market demand. However, wild-type Clostridia has a low ability to utilize CO2 and CO for ethanol production, necessitating the development of engineered strains of C. ljungdahlii with improved performance for gas fermentation and ethanol production.

[0004] In order to efficiently discover functional genes related to gas utilization and ethanol synthesis in Clostridium jundal for use in strain modification, we used transposon insertion sequencing (Tn-seq) technology to achieve high-throughput screening. This technology uses transposon mutagenesis to construct a random mutant library of the target strain, and then uses specific screening pressure and high-throughput sequencing to quickly discover genes related to specific functions, thereby providing potential gene targets for genetic modification of the strain. For example, in 2022, Woods et al. used transposon sequencing technology to screen important genes for autotrophic growth of Clostridium autoethanogenum (WOODS C, HUMPHREYS CM, TOMI-ANDRINO C, et al. 2022. Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum. Appl Environ Microbiol [J], 88: e0247921.). In view of this, we developed Tn-seq technology suitable for Clostridium jundal in previous studies, and carried out subsequent functional gene discovery and identification based on this technology. Summary of the Invention

[0005] Based on a transposon mutant library and Tn-seq data from C. ljungdahlii, we screened a number of genes. These genes were then either knocked down using CRISPR interference (CRISPRi) technology or cloned into plasmids for overexpression in wild-type C. ljungdahlii DSM 13528. The resulting knockdown and overexpression strains were then fermented with syngas, and ethanol production was measured. The experiment found that compared with the control strain, the ethanol production of five engineered bacteria (numbered OE-04490, OE-25060, OE-06990, OE-22650 and OE-07720, respectively) that overexpressed the CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650 and CLJU_c07720 genes alone, as well as the engineered bacteria (numbered ddCas12a-09160 and ddCas12a-23840, respectively) that inhibited the CLJU_c09160 and CLJU_c23840 genes alone, was significantly increased.

[0006] Furthermore, we introduced the above CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, and CLJU_c07720 genes into another high-ethanol-producing Clostridium jondahlii engineered strain, Clj-EtOH21, previously constructed by our research group (this strain had the 2,3-butanediol synthesis genes CLJU_c23220, CLJU_c25840, and CLJU_c25810 knocked out, and overexpressed the ethanol synthesis pathway genes CLJU_c11960 and CLJU_c16510, as well as the acid recycling pathway gene CLJU_c20110). We found that overexpression of the CLJU_c04490 and CLJU_c25060 genes further improved the ethanol synthesis capacity of the engineered strain Clj-EtOH21. Based on the above research results, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides the use of one or more genes selected from the following group in improving the performance of Clostridium in fermenting ethanol using one-carbon gas: NCBI accession numbers CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, CLJU_c07720, CLJU_c09160 and CLJU_c23840.

[0008] As an application mode, the Clostridium is caused to overexpress one or more genes selected from the following group A: CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, CLJU_c07720; and / or

[0009] Inhibit the expression of genes CLJU_c09160 and / or CLJU_c23840 in the Clostridium genome.

[0010] Preferably, the above-mentioned Clostridium is Clostridium aerovorum capable of converting and utilizing C1 gas, preferably Clostridium ljungdahlii or Clostridium autoethanogenum, for example, the original strain of Clostridium ljungdahlii DSM 13528;

[0011] The carbon-containing gas may be synthesis gas, such as steel mill tail gas rich in CO2 / CO.

[0012] The above-mentioned inhibition refers to blocking or downregulating the expression of gene CLJU_c09160 and / or CLJU_c23840.

[0013] In one embodiment, the above-mentioned inhibition can be achieved by blocking the gene CLJU_c09160 and / or CLJU_c23840 through gene editing technology.

[0014] The gene editing technology can be selected from the following groups: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system or CRISPR-Cas12 system, CRISPR-BEST system, MuGENT.

[0015] Overexpression can be achieved through plasmid transformation, which includes the following steps: subcloning the target gene into a plasmid vector suitable for expression in Clostridium, and transforming the recombinant plasmid into Clostridium aerovorans host cells via heat shock, chemical transformation, or electroporation. The resulting transformants can be used to express the target gene.

[0016] In one embodiment, the plasmid suitable for expression in Clostridium can be selected from the group consisting of pMTL 82151, pMTL 83151, pMTL 84151, pMTL 85151, pMTL 82251, pMTL 82254, pMTL 83251, pMTL 83254, pMTL 84251, pMTL 84254, pMTL 85251, pMTL 85254, and pIMP1. Preferably, the plasmid is pMTL83151.

[0017] In one embodiment, the promoter driving the expression of the target gene in the above plasmid can be selected from the following groups: pta 、P ptb 、P 01440 、P 1339 and P thl , preferably a strong promoter P pta Or a strong constitutive promoter P 1339 , more preferably promoter P pta Experiments have found that compared with other common promoters, this promoter is more suitable for the overexpression of the above five genes.

[0018] As an application method, the above genes can be used for metabolic engineering of Clostridium aerovorum to construct industrial Clostridium aerovorum with improved performance.

[0019] In a specific embodiment, the above-mentioned genes are used in combination with one or more genes selected from the following group B: 2,3-butanediol synthesis gene NCBI accession numbers CLJU_c23220, CLJU_c25840 and CLJU_c25810; ethanol synthesis pathway genes CLJU_c11960 and CLJU_c16510; acid recycling pathway gene CLJU_c20110.

[0020] Preferably, the 2,3-butanediol synthesis genes CLJU_c23220, CLJU_c25840 and CLJU_c25810 in the Clostridium genome can be knocked out; and the ethanol synthesis pathway genes CLJU_c11960 and CLJU_c16510 and the acid recycling pathway gene CLJU_c20110 can be overexpressed.

[0021] As a specific application of the above gene, the present invention provides a method for improving the ability of Clostridium aerovorum to synthesize ethanol using one-carbon gas, comprising the following steps:

[0022] A. overexpressing one or more genes selected from the group A: CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, CLJU_c07720; and / or

[0023] B. Blocking or downregulating the expression of gene CLJU_c09160 and / or CLJU_c23840, wherein the blocking is knocking out or inactivation.

[0024] The second aspect of the present invention provides an engineered bacterium of Clostridium aerovorum capable of utilizing one-carbon gas, which is constructed according to the above method.

[0025] The third aspect of the present invention provides the use of the engineered Clostridium aerovorans bacteria in the biological utilization of one-carbon gas, wherein the engineered Clostridium aerovorans bacteria is used for fermentation to convert the one-carbon gas into ethanol.

[0026] Experimental results show that the functional genes CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, CLJU_c07720, CLJU_c09160 and CLJU_c23840 discovered in the present invention can affect the ethanol biosynthesis of Clostridium Jungdalar, and can therefore be used to construct engineered Clostridium Jungdalar, and can effectively improve the syngas utilization efficiency and ethanol production of Clostridium Jungdalar by changing the metabolic engineering strategy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A bar graph comparing ethanol production from syngas fermentation of engineered strains expressing CLJU_c09160 and CLJU_c23840 genes with that of wild-type strain C. ljungdahlii DSM 13528 (control). (A) Comparison of ethanol production from syngas fermentation of engineered strain ddCas12a-09160 and wild-type strain; (B) Comparison of ethanol production from syngas fermentation of engineered strain ddCas12a-23840 and wild-type strain.

[0028] Figure 2 A bar chart comparing ethanol production from syngas fermentation by engineered strains overexpressing five genes and the wild-type strain C. ljungdahlii DSM13528 (control) is shown. A compares ethanol production from syngas fermentation by engineered strain OE-04490 and the wild-type strain; B compares ethanol production from syngas fermentation by engineered strain OE-25060 and the wild-type strain; C compares ethanol production from syngas fermentation by engineered strain OE-06690 and the wild-type strain; D compares ethanol production from syngas fermentation by engineered strain OE-22650 and the wild-type strain; and E compares ethanol production from syngas fermentation by engineered strain OE-07720 and the wild-type strain.

[0029] Figure 3 Schematic diagrams of the engineered bacteria's plasmids and syngas fermentation phenotypes are shown. A is a schematic diagram of the plasmid overexpressing the CLJU_c04490 and CLJU_c25060 genes; B is a graph showing the syngas fermentation growth curves of the engineered bacteria Clj-EtOH24 and Clj-EtOH25, as well as the control strain Clj-EtOH21; and C is a bar chart comparing ethanol production from syngas fermentation of the engineered bacteria Clj-EtOH24, Clj-EtOH24, and the control strain Clj-EtOH21. DETAILED DESCRIPTION

[0030] Our research group has identified several unreported functional genes in some important industrial microorganisms, Clostridium jondahlii, that can affect the syngas fermentation performance of the strain, including NCBI accession numbers CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, CLJU_c07720, etc., and applied them to the metabolic engineering modification of the strain. The research results can be used to guide the construction of industrial Clostridium oxysporum with better performance.

[0031] By enhancing the expression of the above functional genes alone or over-expressing these genes in combination with other functional genes in Clostridium Jungdalar, the autotrophic growth ability and ethanol synthesis yield of Clostridium Jungdalar during gas fermentation can be effectively improved, and the economic efficiency of Clostridium Jungdalar fermentation can be improved accordingly.

[0032] Even when used in engineered Clostridium aerovorum strains that already have the ability to synthesize ethanol from one-carbon gases, some of these genes can still play an important role in promoting the process. For example, studies have found that overexpression of the CLJU_c04490 and CLJU_c25060 genes can further enhance the ethanol synthesis capacity of the engineered strain Clj-EtOH21.

[0033] After database and literature research, we discovered that the gene CLJU_c04490, encoding glutamate-1-semialdehyde aminomutase, plays a crucial role in the synthesis of 5-aminolevulinic acid. 5-aminolevulinic acid is a universal precursor for the synthesis of tetrapyrroles, which can then be further converted to porphyrins and heme. Genomic data indicate that C. ljungdahlii also possesses a porphyrin biosynthesis pathway, but lacks a heme biosynthesis pathway. Porphyrins bind to [4Fe-4S] clusters to form porphyrin iron-sulfur proteins (CoFeSPs), which play a crucial role in methyl transfer and acetyl-CoA synthesis in the Wood-Ljungdahl pathway. Furthermore, the molybdenum cofactor biosynthesis protein encoded by the gene CLJU_c25060 is potentially closely related to carbon, nitrogen, and sulfur metabolism. Molybdenum cofactor-dependent enzymes primarily include dimethyl sulfoxide reductase (DMSOR), xanthine oxidase, and nitrite oxidase. These enzymes participate in redox reactions typically involving electron and proton transfer. In particular, most members of the DMSOR family act as terminal electron acceptors in anaerobic respiratory metabolism. C. ljungdahlii growth and ethanol synthesis during gas fermentation require substantial amounts of energy and reducing power, potentially mediated by redox reactions involving molybdenum cofactors. However, enhanced expression of these two genes has not been reported to enhance CO / CO2 utilization and product synthesis in C. aerovorans.

[0034] The above genes CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, CLJU_c07720, CLJU_c09160 and CLJU_c23840 can be used alone or in combination of two or more.

[0035] In the application embodiment of the present invention, overexpression of genes CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650 and / or CLJU_c07720 can be implemented alone or together with the inhibition of genes CLJU_c09160 and / or CLJU_c23840, thereby effectively improving the autotrophic growth ability and product ethanol yield of Clostridium vulgaris, such as Clostridium jundal, when fermenting using one-carbon gas, thereby improving the economic efficiency of Clostridium jundal fermentation.

[0036] As used herein, the terms "(performance of one-carbon gas utilization efficiency / ethanol production) is improved", "enhance" or "enhance" can mean an increase of at least 10% compared to a reference level (such as a wild-type Clostridium / starting strain), for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level.

[0037] In the description of the technical solutions of the present invention, the term "and / or" used in phrases such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0038] The terms "recombinant bacteria" and "(genetically) engineered bacteria (strains)" herein have the same meaning, and both refer to strains that have been genetically modified to improve the ability of wild-type Clostridium aerovorum or starting strains to ferment and utilize one-carbon gas to synthesize ethanol.

[0039] To investigate the technical solution of the present invention, engineered bacteria were used to ferment syngas (also known as C1 gas) using simulated steel mill tail gas as a carbon source (CO-CO2-H2-N2, with the components in a volume ratio of 56% / 20% / 9% / 15%). Analysis of the ethanol content in the fermentation products confirmed that recombinant bacteria overexpressing genes CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, and CLJU_c07720 all produced higher ethanol production than the original strain. Furthermore, recombinant bacteria suppressing expression of genes CLJU_c09160 and / or CLJU_c23840 also produced higher ethanol production than the original strain. This indicates that the recombinant bacteria's ability to ferment ethanol from C1 gas has been significantly enhanced. These experimental results confirm that the proposed solution has achieved its intended effect and provide new ideas and strategies for metabolic engineering of Clostridium aerovorum.

[0040] The present invention will be further described in detail below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0041] Example

[0042] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned therein, unless otherwise specified, refer to the percentage by mass.

[0043] Materials and methods

[0044] The molecular biology experiments herein, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were primarily performed with reference to Molecular Cloning: A Laboratory Manual (3rd edition), edited by J. Sambrook and D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the competent cell transformation method and competent cell preparation method were performed with reference to Chapter 1, page 96 of Molecular Cloning: A Laboratory Manual (3rd edition). Specific experimental conditions can be determined through simple experiments if necessary.

[0045] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. These conditions can be adjusted through simple experiments if necessary.

[0046] Main culture medium:

[0047] LB medium: 5g / L yeast extract, 10g / L tryptone, 10g / L sodium chloride. (Add 20g / L agar powder to LB solid medium.)

[0048] YTF (yeast extract, tryptone, fructose) medium: 10 g / L yeast extract, 16 g / L tryptone, 10 g / L fructose, 0.2 g / L sodium chloride, 0.3 g / L cysteine, 500 μL / L resazurin stock solution, 2 mL / L trace element stock solution, 1 mL / L vitamin stock solution, dissolved in double-distilled water, and adjusted to pH 6.0 with hydrochloric acid.

[0049] Trace element storage solution: 10.0 g / L nitrilotriacetic acid, 5.0 g / L manganese sulfate, 4.0 g / L ferrous ammonium sulfate hexahydrate, 1.0 g / L cobalt chloride hexahydrate, 1.0 g / L zinc sulfate heptahydrate, 0.1 g / L cupric chloride dihydrate, 0.1 g / L nickel chloride hexahydrate, 0.1 g / L sodium molybdate dihydrate, 0.1 g / L sodium stannate, 0.1 g / L sodium tungstate dihydrate, dissolved in double distilled water.

[0050] Vitamin storage solution: 100 mg / L vitamin B6, 50.0 mg / L thiamine, 50.0 mg / L vitamin B2, 50.0 mg / L calcium pantothenate, 50.0 mg / L lipoic acid, 50.0 mg / L p-aminobenzoic acid, 50.0 mg / L nicotinic acid, 50.0 mg / L vitamin B12, 20.0 mg / L biotin, 20.0 mg / L folic acid, dissolved in double distilled water.

[0051] Resazurin stock solution: 1g / L resazurin, dissolved in double distilled water.

[0052] PETC 1754 Medium:

[0053] 30 mL / L mineral element stock solution, 1 mL / L vitamin stock solution, 2 mL / L trace element stock solution, 0.5 g / L yeast extract, 5 g / L 2-morpholineethanesulfonic acid, 5 mL / L reducing agent stock solution, 500 μL / L resazurin stock solution, dissolved in double distilled water, and adjusted to pH 6.0 with NaOH.

[0054] Mineral element storage solution: 80 g / L sodium chloride, 100 g / L ammonium chloride, 10 g / L potassium chloride, 10 g / L dipotassium hydrogen phosphate, 20 g / L magnesium sulfate heptahydrate, 4 g / L calcium chloride dihydrate, dissolved in double distilled water.

[0055] Reducing agent stock solution: 90 g / L sodium hydroxide, 40 g / L cysteine ​​hydrochloride, 40 g / L sodium sulfide nonahydrate, dissolved in double distilled water.

[0056] SMP buffer: 270 mM sucrose, 4.4 mM NaH2PO4, 0.6 mM Na2HPO4, 1 mM MgCl2·6H2O.

[0057] Clostridium ljungdahlii DSM 13528 was purchased from the German Culture Collection (DSMZ).

[0058] The plasmids used to express genes CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, and CLJU_c07720, the gene editing plasmids used to inhibit the expression of genes CLJU_c09160 and / or CLJU_c23840, and the strain Clj-EtOH21 in the examples were constructed and preserved by the research group of Jiang Weihong at the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences. Any unit or individual may obtain the plasmids for verification of the present invention, but may not be used for other purposes, including development, utilization, scientific research, and teaching, without the permission of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences.

[0059] The PCR primers used in the examples are listed in Table 1 and were synthesized by Boshang Biotechnology (Shanghai) Co., Ltd.

[0060] Table 1 Primers used in constructing recombinant plasmids in the examples

[0061]

[0062]

[0063]

[0064] In Table 1, "-F" in the name stands for forward direction; "-R" stands for reverse direction.

[0065] The provider of the simulated steel mill tail gas (CO-CO2-H2-N2, with the volume ratio of each component being 56% / 20% / 9% / 15%) is Shanghai Haoqi Gas Co., Ltd.

[0066] Detection method of ethanol and / or acetic acid in fermentation broth:

[0067] The ethanol and acetic acid contents in the fermentation broth were determined using gas chromatography. The instrument and detection method were as follows: an Agilent 7890 gas chromatograph, a flame ionization detector (FID), an Alltech ECTM-WAX capillary column, an oven temperature ramped from 85°C to 200°C, a column pressure of 10 psi to 30 psi, an injector temperature of 250°C, and a detector temperature of 300°C. The carrier gases were nitrogen at a flow rate of 25 mL / min, hydrogen at a flow rate of 30 mL / min, and air at a flow rate of 400 mL / min. Internal standards included isobutanol, isobutyric acid, and hydrochloric acid (for acidification). The ethanol and acetic acid contents were calculated using the internal standard method.

[0068] It should be noted that, for the sake of convenience of description, in the embodiments, the plasmid number and the corresponding strain number may share the same number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0069] Example 1: Discovery and identification of functional genes affecting the performance of Clostridium jundal based on Tn-seq technology

[0070] Based on the Tn-seq technology suitable for Clostridium lndall, some functional genes affecting the performance of Clostridium lndall were discovered and identified, including CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, CLJU_c07720, CLJU_c09160 and CLJU_c23840. The method is briefly described as follows.

[0071] First, a plasmid containing the transposase Himar1C9 was constructed and introduced into wild-type Clostridium jondahlii via electroporation. Upon expression, the transposase cleaves double-stranded DNA containing the "TA" site, exposing sticky ends. The resulting transposon sequence can then jump across the chromosome and randomly insert into other "TA" sites in the genome. A genome-wide mutant library was constructed using these transposon insertion mutagenesis. This mutant library was then subcultured under syngas fermentation conditions for appropriate passages. Subsequently, genomes from both the original and subcultured bacterial colonies were extracted to construct sequencing libraries. Finally, by comparing the sequencing results of the two colony genomes, functional genes related to gas fermentation were predicted. The screening thresholds were set at log2 (fold change) > 1 or log2 (fold change) < -1. Based on these two screening thresholds, 368 genes were found to be upregulated in the Tn-seq dataset, while 538 genes were found to be downregulated. We selected 43 genes with the most significant down-regulated abundance (log2FC < -4.8) and 22 genes with the most significant up-regulated abundance (log2FC > 2.5) for further functional analysis. pta The promoter was overexpressed, and 22 genes were individually repressed using a dCas12a-based CRISPRi approach. The 65 constructed plasmids were electroporated into wild-type Clostridium jundaloids, and the resulting engineered strains were subjected to syngas fermentation to measure ethanol production.

[0072] After the above steps, some genes affecting the ability of Clostridium to synthesize ethanol using one-carbon gas were screened out, including CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650 and CLJU_c07720 with positive regulatory effects, and CLJU_c09160 and CLJU_c23840 with negative regulatory effects.

[0073] Example 2: Construction of a gene expression inhibitory plasmid

[0074] In order to verify the relationship between the significantly upregulated genes CLJU_c09160 and CLJU_c23840 in the transposon sequencing results and the utilization of syngas and product synthesis by Clostridium ljungdahlii, we constructed plasmids that inhibit the expression of genes CLJU_c09160 and CLJU_c23840, respectively. The dCas12a-based CRISPRi method was used to inhibit gene expression. The plasmid backbone used was pZG-ddCas12a, which is a common gene expression plasmid in Clostridium ljungdahlii (ZHAO R, LIU Y, ZHANG H, et al. 2019. CRISPR-Cas12a-Mediated Gene Deletion and Regulation in Clostridium ljungdahlii and Its Application in Carbon Flux Redirection in Synthesis Gas Fermentation. ACS Synth Biol [J], 8: 2270-2279.). One inhibition site was selected for each gene for plasmid construction.

[0075] For example, the method for constructing a recombinant plasmid for inhibiting the expression of gene CLJU_c09160 includes the following steps.

[0076] 1) Construct a plasmid to inhibit target gene expression. First, locate the first "TTN" sequence after the start codon of the gene CLJU_c09160. Select the 24 bp following the "TTN" sequence as the crRNA for plasmid construction. In the first round of PCR, use the two primers CLJU_c09160-F and CLJU_c09160-R as templates for PCR amplification to generate a double-stranded DNA template. In the second round of PCR, use the first-round PCR product as a template and primers pth1-F and fdx-NcoI-R for amplification. After two rounds of PCR, a DNA fragment targeting the gene is obtained. Subsequently, the pZG-ddCas12a plasmid (ZHAO R, LIU Y, ZHANG H, et al. 2019. CRISPR-Cas12a-Mediated Gene Deletion and Regulation in Clostridiumljungdahlii and Its Application in Carbon Flux Redirection in Synthesis Gas Fermentation. ACS Synth Biol [J], 8: 2270-2279.) previously constructed in this laboratory was used as a backbone, and the pZG-ddCas12 plasmid was double-digested with restriction endonucleases BamHI and NcoI to obtain a linearized vector. Finally, the DNA fragment targeting the CLJU_c09160 gene and the linearized vector were assembled into the target plasmid ddCas12a-09160 using a non-ligase-dependent single-fragment one-step cloning kit (Novozyme (Nanjing) Biotechnology Co., Ltd., product number: C112-02).

[0077] 2) PCR reaction procedure: 98°C pre-denaturation for 5 min; 30 cycles of denaturation at 98°C for 30 s, annealing at 55°C for 30 s, and extension at 68°C for 2 min; extension at 68°C for 10 min, and storage at 16°C. High-fidelity DNA polymerase was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., product number: KFX-101. The PCR reaction system was prepared according to the manufacturer's instructions. Amplified products were recovered by electrophoresis on a 2.0% (w / v) agarose gel. A PCR product recovery kit was purchased from AXYGEN (Shanghai) Co., Ltd., product number: AP-GX-250.

[0078] 3) Preparation of competent strains. Inoculate the Clostridium jundal stored in 30% glycerol at ultra-low temperature into 5 mL YTF liquid medium and activate the culture at 37°C. 600When the OD reaches 0.8-1.0, use an inoculation loop to take a small amount of bacterial liquid, streak it on YTF solid medium, and culture it anaerobically at 37°C. Pick a single colony grown on the plate and inoculate it into 5mL YTF liquid medium. Wait until it grows to OD 600 When the OD value is 0.8-1.0, the bacterial solution is transferred to 200mL YTF liquid medium at a 5% (v / v) inoculum for expansion. 600 When the pH is 0.4-0.6, add sucrose solution (final concentration 0.3 M) and glycine solution (final concentration 1.25%, plasmid volume ratio w / v). Incubate for approximately 2 hours. Place on ice for 20 minutes and collect the cells by centrifugation (4°C, 4500 rpm, 10 minutes). Carefully remove the supernatant and wash the cells 2-3 times with pre-chilled SMP buffer, then completely discard the supernatant. Finally, resuspend the cells in 2-3 mL of a 9:1 SMP / DMSO mixture and store at -80°C until needed.

[0079] 4) Electrotransformation conditions. Mix the circular plasmid and competent cells and place them on ice for 20 minutes. Set the parameters of the electroporator: voltage 1000V, capacitance 50μF, resistance 200Ω. Add the plasmid and competent mixture after the ice bath to the electroporation cup, wipe off the water stains on the surface of the electroporation cup with absorbent paper, and place it in the electroporation tank. Next, click the shock button of the electroporator. After the shock, transfer the mixture to 1mL of YTF liquid culture medium and resuscitate in an anaerobic environment at 37°C for 20 hours. Finally, spread the revived 1mL bacterial liquid on a YTF solid culture medium containing 5μg / mL chloramphenicol resistance for culture.

[0080] The same method was used to construct a recombinant plasmid (named ddCas12a-23840) that inhibits the expression of the gene CLJU_c23840.

[0081] Example 3: Construction of overexpression plasmid

[0082] To verify the relationship between the significantly downregulated genes CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, and CLJU_c07720 in transposon sequencing results and syngas utilization and product synthesis in Clostridium ljungdalar, we constructed plasmids overexpressing CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, and CLJU_c07720. The plasmid backbone used was pMTL83151, a common gene expression plasmid in Clostridium ljungdalar (ZHANG L, LIU Y, ZHAO R, et al. 2020. Interactive Regulation of Formate Dehydrogenase during CO2 Fixation in Gas-Fermenting Bacteria. mBio[J], 11.). The promoter used to drive gene expression was P pta , which has also been reported as a strong promoter that can be used for gene overexpression in Clostridium lndall (JONES SW, FAST AG, CARLSON ED, et al. 2016. CO2 fixation by anaerobicnon-photosynthetic mixotrophy for improved carbon conversion. Nat Commun[J], 7: 12800.).

[0083] For example, the method for constructing a recombinant plasmid for overexpressing gene CLJU_c04490 includes the following steps.

[0084] 1) Construction of gene overexpression plasmid. Using the C. ljungdahlii genome as a template, primers CLJU_c04490-F and CLJU_c04490-R were used to amplify the gene fragment. Subsequently, the above gene fragment was used as a template and primers P pta-F and universal primer Primer-R were used for the second PCR to amplify the DNA fragment containing the homologous recombination region. Next, the pMTL83151 plasmid (ZHANG L, LIU Y, ZHAO R, et al. 2020. Interactive Regulation of Formate Dehydrogenase during CO2 Fixation in Gas-Fermenting Bacteria. mBio[J], 11.) was enzymatically digested with KpnI and NcoI restriction endonucleases to obtain a linearized vector. Finally, the DNA fragment containing the homologous recombination region and the linearized vector were assembled into the target plasmid OE-04490 using a non-ligase-dependent single-fragment one-step cloning kit (Novozymes (Nanjing) Biotechnology Co., Ltd., product number: C112-02).

[0085] 2) PCR reaction procedure: 98°C denaturation for 5 min; 30 cycles of denaturation at 98°C for 30 s, annealing at 55°C for 1 min, and extension at 68°C for 2 min; extension at 68°C for 10 min, followed by storage at 16°C. High-fidelity DNA polymerase was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., product number: KFX-101. The PCR reaction system was prepared according to the manufacturer's instructions. Amplified products were recovered by electrophoresis on a 2.0% (w / v) agarose gel. A PCR product recovery kit was purchased from AXYGEN (Shanghai) Co., Ltd., product number: AP-GX-250.

[0086] 3) The preparation of competent cells of Clostridium jundal and the electroporation conditions were the same as those in Example 2.

[0087] The same method was used to construct a recombinant plasmid overexpressing the CLJU_c25060 gene (named OE-25060); a recombinant plasmid overexpressing the CLJU_c06990 gene (named OE-06990); a recombinant plasmid overexpressing the CLJU_c22650 gene (named OE-22650); and a recombinant plasmid overexpressing the CLJU_c07720 gene (named OE-07720).

[0088] Example 4: Construction of Clostridium Ljungdahl engineered strains Clj-EtOH24 and Clj-EtOH25

[0089] Our research group previously constructed a high-ethanol-producing engineered Clostridium jondahlii strain, Clj-EtOH21, in which the 2,3-butanediol synthesis genes CLJU_c23220, CLJU_c25840, and CLJU_c25810 were knocked out, and the ethanol synthesis pathway genes CLJU_c11960 and CLJU_c16510 and the acid recycling pathway gene CLJU_c20110 were overexpressed.

[0090] In order to further improve the ethanol synthesis ability of the strain, we constructed recombinant plasmids overexpressing genes CLJU_c04490 and CLJU_c25060 (see Figure 3 A) was used to transform into strain Clj-EtOH21 for gene overexpression. The plasmid backbone used was pMTL83151, a plasmid commonly used for gene expression in Clostridium jundal. The promoter driving the expression of gene CLJU_c04490 or CLJU_c25060 used in the recombinant plasmid was P 01440 , which has also been reported as a strong promoter that can be used for gene overexpression in Clostridium lndellii.

[0091] The construction process of the engineered strain overexpressing gene CLJU_c04490 or CLJU_c25060 is briefly described as follows.

[0092] In the early stage, in order to improve the ethanol production of Clostridium jundal, we knocked out the 2,3-butanediol biosynthesis genes CLJU_c23220, CLJU_c25840 and CLJU_c25810 in the wild-type Clostridium jundal. We used this as the chassis strain and introduced the promoter P pta and plasmids P containing ald (CLJU_c11960), adhE1 (CLJU_c16510), and aor1 (CLJU_c20110) genes pta -ald-adhE1-aor1 to achieve overexpression of these three genes. The resulting engineered strain Clj-EtOH21 can produce 2.0 g / L of ethanol through syngas fermentation, showing a strong ethanol synthesis ability. In order to further improve the ethanol synthesis ability of the bacteria, we overexpressed CLJU_c04490 and CLJU_c25060 in the above-mentioned engineered strain Clj-EtOH21. The plasmids for overexpressing genes CLJU_c04490 and CLJU_c25060 were constructed with plasmid P pta -ald-adhE1-aor1 was used as the skeleton, and the promoter selected was P 01440, which is a strong promoter that has been reported to be used for gene overexpression in Clostridium ljungdahlii (ZHAO R, LIU Y, ZHANG H, et al. 2019. CRISPR-Cas12a-Mediated Gene Deletion and Regulation in Clostridium ljungdahlii and Its Application in Carbon Flux Redirection in Synthesis Gas Fermentation. ACS Synth Biol[J], 8: 2270-2279.).

[0093] For example, the method for constructing a recombinant plasmid for overexpressing gene CLJU_c04490 in the engineered strain Clj-EtOH21 includes the following steps.

[0094] 1) Construction of gene overexpression plasmid. Using the C. ljungdahlii genome as template, primers 04490-1440-F and 04490-R were used to amplify the CLJU_c04490 gene fragment, and primers 1440-R and 1440-F were used to amplify the promoter P 01440 Subsequently, the amplified gene fragment and promoter fragment were used as templates and primers 1440-F and 04490-R were used to perform overlapping PCR, thereby connecting the gene fragment and the promoter into a single DNA fragment (P 01440 -CLJU_c04490). Next, based on the above P pta -ald-adhE1-aor1 plasmid was digested with restriction endonuclease XhoI, and after 3 hours, FastAP (dephosphorylase) was added and treated for 30 minutes (heated in a water bath at 37°C) to obtain a linearized vector. Finally, the above DNA fragment ((P 01440 -CLJU_c04490) and the linearized vector to form the target plasmid P pta -ald-adhE1-aor1-P 01440 -04490 (see Figure 3 Middle A).

[0095] 2) PCR reaction procedure: 98°C denaturation for 5 min; 30 cycles of denaturation at 98°C for 30 s, annealing at 55°C for 1 min, and extension at 68°C for 2 min; extension at 68°C for 10 min, followed by storage at 16°C. High-fidelity DNA polymerase was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., product number: KFX-101. The PCR reaction system was prepared according to the manufacturer's instructions. Amplified products were recovered by electrophoresis on a 2.0% (w / v) agarose gel. A PCR product recovery kit was purchased from AXYGEN (Shanghai) Co., Ltd., product number: AP-GX-250.

[0096] 3) Preparation of competent strains. The knockout bacteria (Clostridium jundalii with knockout of 2,3-butanediol synthesis genes CLJU_c23220, CLJU_c25840, and CLJU_c25810) stored in 30% glycerol were inoculated into 5 mL of YTF liquid medium and activated at 37°C. 600 When the OD reaches 0.8-1.0, use an inoculation loop to take a small amount of bacterial liquid, streak it on YTF solid medium, and culture it anaerobically at 37°C. Pick a single colony grown on the plate and inoculate it into 5mL YTF liquid medium. Wait until it grows to OD 600 When the OD value is 0.8-1.0, the bacterial solution is transferred to 200 mL YTF liquid medium with a 5% (v / v) inoculum for expansion. 600 When the pH is 0.4-0.6, add sucrose solution (final concentration 0.3 M) and glycine solution (final concentration 1.25%, plasmid volume ratio w / v). Incubate for approximately 2 hours. Place on ice for 20 minutes and collect the cells by centrifugation (4°C, 4500 rpm, 10 minutes). Carefully remove the supernatant and wash the cells 2-3 times with pre-chilled SMP buffer, then completely discard the supernatant. Finally, resuspend the cells in 2-3 mL of a 9:1 SMP / DMSO mixture and store at -80°C until needed.

[0097] 4) The electroporation conditions were the same as those in Example 2.

[0098] The above recombinant plasmids were transformed into strain Clj-EtOH21 to obtain Clj-EtOH24 and Clj-EtOH25 overexpressing the CLJU_c04490 or CLJU_c25060 gene.

[0099] Example 5: Preparation of electrocompetent Clostridium jundal

[0100] C. ljungdahlii (wild-type strain C. ljungdahlii DSM13528 or engineered strain Clj-EtOH21) stored at -80°C in 30% glycerol was inoculated into 5 mL YTF liquid medium and activated at 37°C. 600 When the OD reaches 0.8-1.0, use an inoculation loop to take a small amount of bacterial liquid, streak it on YTF solid medium, and culture it anaerobically at 37°C. Pick a single colony grown on the plate and inoculate it into 5mL YTF liquid medium. Wait until it grows to OD 600 When the OD value is 0.8-1.0, the bacterial solution is transferred to 200 mL YTF liquid medium with a 5% (v / v) inoculum for expansion. 600 When the concentration of the plasmid is 0.4-0.6, add sucrose solution (final concentration 0.3 M) and glycine solution (final concentration 1.25%, plasmid volume ratio w / v) for approximately 2 hours. Place the cells on ice for 20 minutes and collect the cells by centrifugation (4°C, 4500 rpm, 10 minutes). Carefully remove the supernatant and wash the cells 2-3 times with pre-chilled SMP buffer, then completely discard the supernatant. Finally, resuspend the cells in 2-3 mL of SMP / DMSO mixture (9:1 volume ratio) to obtain competent Clostridium ljungdahlii (wild-type strain C. ljungdahlii DSM 13528 or engineered strain Clj-EtOH21) and store them at -80°C until use.

[0101] Example 6: Electroporation transformation of Clostridium jundal

[0102] The recombinant plasmid for inhibiting the expression of a certain gene constructed in Example 2 or the recombinant plasmid for overexpressing a certain gene constructed in Example 3 was respectively introduced into the competent state of Clostridium ljungdahlii (wild-type strain C.ljungdahlii DSM 13528 or engineered strain Clj-EtOH21) prepared in Example 5 by electroporation transformation. The specific steps are: take 4 μg of plasmid and mix it with 200 μL of competent state, incubate on ice for 10 minutes, and then transfer it to a pre-cooled 2mm electroporation cup. Set the electroporation parameters to 1000v, 200Ω, and 50μF, place the electroporation cup containing the mixed solution on the electroporator, electroshock, and then quickly add 800 μL of YTF liquid culture medium as a resuscitation solution, resuscitate for 6-20 hours, and then spread it on a YTF solid plate with resistance to chloramphenicol (working concentration is 5 mg / L). After anaerobic culture at 37°C for 3-4 days, the monoclonal clones grown on the plate were verified by colony PCR sequencing to obtain the correctly sequenced engineered strain. Among them,

[0103] The engineered bacteria that inhibited the expression of the CLJU_c09160 gene were named ddCas12a-09160;

[0104] The engineered bacteria that inhibit the expression of the CLJU c23840 gene were named ddCas12a-23840;

[0105] The engineered bacteria overexpressing CLJU_c04490 were named OE-04490;

[0106] The engineered bacteria overexpressing CLJU_c25060 were named OE-25060;

[0107] The engineered bacteria overexpressing CLJU_c06990 were named OE-06990;

[0108] The engineered bacteria overexpressing CLJU_c22650 were named OE-22650;

[0109] The engineered bacteria overexpressing CLJU_c07720 were named OE-07720;

[0110] The engineered strain overexpressing CLJU_c04490 using strain Clj-EtOH21 as the base strain was named Clj-EtOH24;

[0111] The engineered bacterium overexpressing CLJU_c25060 using strain Clj-EtOH21 as the chassis was named CljEtOH25.

[0112] Subsequently, we performed syngas fermentation verification on the overexpression engineered strain and the strain containing only the pMTL83151 empty plasmid (no gene was overexpressed), or on the engineered strain for inhibiting gene expression and the strain containing only the pZG-ddCas12a (without crRNA) plasmid (no gene was inhibited).

[0113] Example 7: Investigation of ethanol production by strains using syngas fermentation

[0114] Syngas fermentation was performed on each strain obtained in Example 6. The fully synthetic culture medium used for the fermentation was PETC1754, and the gas used was simulated steel mill exhaust gas (gas composition: CO:CO2:H2:N2 = 56%:20%:9%:15%). Fermentation was carried out in a 120 mL serum bottle with a 30 mL fermentation system. The atmospheric pressure in the fermentation bottle was 0.2 MPa, and the fermentation system was cultured in a shaking incubator at 37°C at a speed of 120 rpm. All culture media and fermentation serum bottles were sterilized in advance at high temperature and high pressure at 121°C for 20 min. The specific operating steps are as follows:

[0115] In the preparation stage of the first-level seed solution, a single bacterium from the plate was inoculated into 5 mL of YTF medium and cultured at 37°C for about 24 hours until the OD of the bacterial solution reached 600 Greater than 1.0.

[0116] Secondary seed solution preparation stage: aspirate 1mL of primary seed solution to measure OD 600 , according to OD 600 Calculate the amount of secondary seeds to be transferred, the transfer amount is about 500 / OD 600 μL, culture at 37℃ for 12-16h.

[0117] Fermentation: Pipette 0.5mL of secondary seed liquid and measure OD 600 , when OD 600 Fermentation can begin when the pH is between 1.0 and 1.2. First, pipette 1.5 mL of secondary seed solution into a serum bottle. Using a graduated cylinder, measure 28.5 mL of PETC 1754 medium (supplemented with 5 μg / mL Tm) and place it into the serum bottle. Press the cap securely and aerate the bottle. The aeration conditions are to replace the gas in the bottle with syngas at 0.2 MPa pressure for 2 minutes, followed by aeration for 1 minute 30 seconds. After aeration, incubate the bottle in a shaker at 37°C at 100 rpm.

[0118] Gas exchange sampling: Gas fermentation requires gas replacement in the fermentation bottle every 24 hours. The replacement conditions are 0.2MPa atmospheric pressure synthesis gas exchange for 2 minutes and aeration for 1 minute 30 seconds. Before each gas exchange, a 1mL sterile syringe is used to sample, and the sample is measured with a visible spectrophotometer for OD. 600 , draw a growth curve, and then use gas chromatography to determine the ethanol production in the fermentation broth.

[0119] Product determination: The fermentation broth samples were centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was taken for determination of ethanol content. The results are shown below.

[0120] Results and Discussion

[0121] 1. Inhibiting the expression of CLJU_c09160 and CLJU_c23840 genes can increase the ethanol production of Clostridium jondahlii gas fermentation.

[0122] See also Figure 1 The ethanol production of the engineered bacteria (ddCas12a-09160) that inhibited the expression of the CLJU_c09160 gene increased by 57% compared with the control strain (wild-type Clostridium jundal, control). Figure 1 The engineered bacteria (ddCas12a-23840) that inhibited the expression of the CLJU_c23840 gene could synthesize more ethanol (0.365 g / L), which was 1.8 times that of the control strain (wild-type Clostridium jundal). Figure 1 Middle B).

[0123] 2. Overexpression of CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650 and CLJU_c07720 genes can increase the ethanol production of syngas fermentation by Clostridium Yongdal.

[0124] See also Figure 2 Compared with the control strain (wild-type Clostridium jundal, control), the ethanol production of strain OE-04490 overexpressing CLJU_c04490 increased by 118.2% ( Figure 2 The ethanol production of strain OE-25060 overexpressing CLJU_c25060 increased by 9.7% ( Figure 2 Middle B); the ethanol production of strain OE-06990 overexpressing CLJU_c06990 increased by 37.9% ( Figure 2 C); the ethanol production of strain OE-22650 overexpressing CLJU_c22650 increased by 14.1% ( Figure 2 D); the ethanol production of strain OE-07720 overexpressing CLJU_c07720 increased by 17% ( Figure 2 Middle E).

[0125] 3. Overexpression of CLJU_c04490 and CLJU_c25060 genes in the engineered Clostridium jundal strain Clj-EtOH21 further improved ethanol production.

[0126] See also Figure 3 Engineered strains Clj-EtOH24 and CljEtOH25 were used for syngas fermentation, and their growth and ethanol production were compared with those of the control strain Clj-EtOH21. The results showed that while Clj-EtOH24 showed no significant growth difference compared to the control strain (Clj-EtOH21), its ethanol yield (2.5 g / L) was 50% higher than the control. CljEtOH25 grew more slowly than the control strain, but its ethanol yield reached 3.64 g / L, a 119% increase compared to the control. These results further confirm the positive regulatory role of genes CLJU_c04490 and CLJU_c25060 in the ethanol biosynthesis pathway.

[0127] The above experimental results show that genes CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, and CLJU_c07720 can promote ethanol synthesis in Clostridium, while genes CLJU_c09160 and CLJU_c23840 inhibit or downregulate ethanol synthesis in Clostridium. These genes can be used to modify the metabolic pathways of Clostridium aerovorum to improve the strain's ability to synthesize ethanol using one-carbon gas.

[0128] It should be noted that the listing and discussion of previously disclosed documents in this specification should not be regarded as an admission that the documents are prior art or common knowledge.

Claims

1. Use of one or more genes selected from the following group in improving the performance of Clostridium in fermenting ethanol using one-carbon gas: NCBI accession numbers CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, CLJU_c07720, CLJU_c09160 and CLJU_c23840.

2. The use according to claim 1, characterized in that Overexpressing one or more genes selected from the group A: CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, CLJU_c07720; and / or Inhibit the expression of genes CLJU_c09160 and / or CLJU_c23840 in the Clostridium genome.

3. The use according to claim 1 or 2, characterized in that The Clostridium is a Clostridium aerogenes capable of converting and utilizing C1 gas, preferably Clostridium ljungdahlii or Clostridium autoethanogenum; The one-carbon gas is synthesis gas.

4. The use according to claim 2, characterized in that The inhibition refers to blocking or downregulating the expression of gene CLJU_c09160 and / or CLJU_c23840; The overexpression is implemented by plasmid transformation, which includes the following steps: subcloning the target gene into a plasmid vector suitable for expression in Clostridium, and transforming the recombinant plasmid into Clostridium aerovorum.

5. The use according to claim 1, characterized in that The gene is used for metabolic engineering transformation of Clostridium aerovorum to construct industrial Clostridium aerovorum with improved performance.

6. The use according to claim 1, wherein It is used in combination with one or more genes selected from the following group B: 2,3-butanediol synthesis gene NCBI accession number CLJU_c23220, CLJU_c25840 and CLJU_c25810; ethanol synthesis pathway genes CLJU_c11960 and CLJU_c16510; acid recycling pathway gene CLJU_c20110.

7. The use according to claim 1 or 6, characterized in that The 2,3-butanediol synthesis genes CLJU_c23220, CLJU_c25840 and CLJU_c25810 in the Clostridium genome were knocked out; the ethanol synthesis pathway genes CLJU_c11960 and CLJU_c16510 and the acid recycling pathway gene CLJU_c20110 were overexpressed.

8. A method for improving the ability of Clostridium aerovorum to synthesize ethanol using one-carbon gas, characterized in that: The steps include: A. overexpressing one or more genes selected from the group A as described in claim 2: CLJU_c04490, CLJU_c25060, CLJU_c06990, CLJU_c22650, CLJU_c07720; and / or B. Blocking or downregulating the expression of genes CLJU_c09160 and / or CLJU_c23840.

9. An engineered bacterium of Clostridium aerovorum capable of synthesizing ethanol from a carbon-containing gas, characterized in that: The method according to claim 8 is constructed.

10. Use of the engineered Clostridium aerovorum according to claim 9 in the bioutilization of mono-carbon gases.

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