Method for improving utilization of clostridium synthesis gas

Abstract

The invention discloses a method for improving utilization of synthesis gas of clostridium. The method comprises the following steps: blocking or down-regulating expression of a transcription factor NrdR; and / or enhancing the expression of a transcription factor DeoR. The method provided by the invention effectively improves the utilization and conversion efficiency of the clostridium aerovorans on the carbon gas such as synthesis gas, and has popularization and application values.

Description

Technical Field

[0001] The invention belongs to the field of metabolic engineering and relates to a method for improving the utilization of clostridial synthesis gas, and specifically to a method for improving the ability of Clostridium ljungdahlii to utilize one-carbon gas (CO2, CO). Background Art

[0002] In recent years, the bioconversion and utilization technology of carbon dioxide (CO2) and other carbon gases has attracted widespread attention. Absorbing and utilizing carbon gases such as CO2 through biological fermentation and converting them into various high-value-added chemicals and fuels will not only help alleviate the environmental problems caused by large-scale CO2 emissions, but also contribute to the formation and development of a new generation of biomanufacturing industry using CO2 as a resource.

[0003] Clostridium ljungdahlii is a chemocarbon fixation microorganism that was first isolated from chicken farm waste (Tanner et al., 1993, doi:10.1099 / 00207713-43-2-232). It can use carbon dioxide (CO2) and carbon monoxide (CO) as the only carbon source, converting it into acetyl-CoA through its own reductive acetyl-CoA pathway (also known as the Wood-Ljungdahl pathway), and further synthesizing products such as acetic acid and ethanol (Bengelsdorf et al., 2018, doi:10.1016 / bs.aambs.2018.01.002). Among the currently known natural biological carbon fixation pathways, the Wood-Ljungdahl pathway has the fewest reaction steps and the lowest energy consumption (Gong et al., 2016, doi:10.1007 / s11427-016-0304-2). However, the utilization efficiency of one-carbon gas fermentation by Clostridium Yongdal is still relatively low compared to organic carbon sources such as sugars, and needs to be further improved to meet higher industrial application needs.

[0004] Microorganisms usually contain a large number of transcription factor encoding genes. Transcription factors are a class of regulatory proteins that can regulate the transcription and expression of a large number of genes in microorganisms, thus significantly affecting cell functions and participating in the regulation of a large number of physiological and metabolic processes in cells. Therefore, we hope to identify transcription factors from C. ljungdahlii that can affect its ability to utilize one-carbon gases such as CO2, and optimize the gas fermentation performance of the strain by modifying the transcription factors.

[0005] Since 2022, the inventors have proposed the following idea: Using C. ljungdahlii as the research object, all possible transcription factor encoding genes in Clostridium ljungdahlii are knocked out through the CRISPR-Cas9 genome editing system (Huang et al., 2016, doi:10.1021 / acssynbio.6b00044) developed in the laboratory in the early stage, and these transcription factor encoding genes are overexpressed using a plasmid overexpression system. Secondly, the above-mentioned gene knockout or overexpression strains are fermented in syngas (a mixture of carbon monoxide, carbon dioxide, hydrogen and nitrogen), and the changes in strain growth and product synthesis ability are observed to discover important transcription factors. Summary of the invention

[0006] We knocked out and overexpressed the possible transcription factor encoding genes in Clostridium ljungdahlii one by one, and fermented the mutant strains with syngas, comparing the growth and product synthesis ability of the mutant strains and the original strains. During the identification process, it was found that after knocking out the gene CLJU_c12350 encoding the transcription factor NrdR, the growth and product (acetic acid, ethanol) production of the mutant strain during syngas fermentation were significantly improved compared with the original strain. In further studies, the above-predicted transcription factor encoding genes were overexpressed one by one, and the obtained engineered strains were also cultured in syngas, and the growth and product synthesis ability of the engineered strains and the original strains were compared. It was found that the overexpression of the transcription factor DeoR encoding gene CLJU_c20610 significantly improved the product (acetic acid and ethanol) synthesis ability of the engineered strains compared with the original strains. Next, the expression of the transcription factor DeoR encoding gene CLJU_c20610 was optimized to improve the gas fermentation performance of the C. ljungdahlii strain. By using promoters of different strengths P pta , P ptb , P 01440 and P thl To drive the expression of CLJU_c20610 gene, the corresponding engineered bacteria were cultured in syngas. The results showed that the promoter P thl The overexpression gene CLJU_c20610 has the best effect, which can significantly improve the growth of the strain and the product (acetic acid and ethanol) yield during syngas fermentation. Based on the above research results, the present invention provides the following technical solutions:

[0007] A method for improving the ability of Clostridium to utilize one-carbon gas comprises the following steps:

[0008] A. blocking or down-regulating the expression of the transcription factor NrdR (NCBI accession number CLJU_c12350, GenBank: ADK14303.1); and / or

[0009] B. Enhanced expression of transcription factor DeoR (NCBI accession number CLJU_c20610, GenBank: ADK15123.1).

[0010] Those skilled in the art will readily understand that methods of blocking protein expression include, but are not limited to, methods such as knocking out or inactivating the coding gene to cause it to lose its original function.

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

[0012] Preferably, the Clostridium is a Clostridium aerovorans capable of converting and utilizing C1 gas, preferably Clostridium ljungdahlii, such as Clostridium ljungdahlii DSM 13528.

[0013] In one embodiment, in the above step A, the expression of the gene encoding the transcription factor NrdR is blocked by gene editing technology.

[0014] The above-mentioned gene editing technology is selected from the following groups: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-BEST system, MuGENT.

[0015] In one embodiment, the above step B is to activate the activity of the transcription factor DeoR or enhance the expression of the transcription factor DeoR using one or more copies of a regulatory element.

[0016] The above regulatory element may be a promoter selected from the group consisting of: pta , P ptb , P 01440 , P 1339 and P thl , preferably the promoter P thl Although in terms of strength, among these promoters, P thl It is not the strongest, but experimental comparison found that the transcription factor DeoR in the promoter P thl Regulated expression can most effectively promote the growth of the strain and the production of acetic acid and ethanol, indicating that the two have the best adaptability in Clostridium and can significantly improve the growth and product (acetic acid and ethanol) production of the strain during syngas fermentation.

[0017] In one embodiment, the regulatory element, ie, the promoter, can be integrated into the upstream of the transcription factor DeoR gene in the genome of the host cell by gene editing technology, thereby placing the transcription factor DeoR gene under the regulation of the promoter.

[0018] The above-mentioned gene editing technology can be selected from the following groups: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-BEST system, MuGENT.

[0019] In another embodiment, step B can be to subclone the coding gene of the transcription factor DeoR into a plasmid vector suitable for expression in Clostridium, and then transfer the recombinant plasmid into the host cell by heat shock method, chemical transformation method or electroporation method, so as to overexpress the transcription factor DeoR in Clostridium.

[0020] Preferably, the above plasmid vector is selected from the following group: pMTL 82151, pMTL 83151, pMTL 84151, pMTL85151, pMTL 82251, pMTL 82254, pMTL 83251, pMTL 83254, pMTL 84251, pMTL 84254, pMTL85251, pMTL 85254, and pIMP1.

[0021] The above recombinant plasmid comprises a gene encoding the transcription factor DeoR and a promoter selected from the following group for regulating its expression: pta , P ptb , P 01440 , P 1339 and P thl .

[0022] The second aspect of the present invention provides a recombinant plasmid for expressing the transcription factor DeoR in Clostridium aerovorum, which is selected from the following group: a P having a nucleotide sequence of SEQ ID NO: 1; 1339 -deoR, P with a nucleotide sequence of SEQ ID NO: 2 pta -deoR, P with a nucleotide sequence of SEQ ID NO: 3 ptb -deoR, P with a nucleotide sequence of SEQ ID NO: 4 01440 -deoR and P having a nucleotide sequence of SEQ ID NO: 5 thl -deoR.

[0023] The third aspect of the present invention provides an engineered (aerotrophic) Clostridium bacterium capable of utilizing one-carbon gas, which is constructed according to the above method.

[0024] The fourth aspect of the present invention provides the use of the above-mentioned engineered Clostridium bacteria in the utilization of one-carbon gases such as synthesis gas.

[0025] Preferably, the engineered Clostridium aerovorum bacteria can convert a carbon gas such as synthesis gas into ethanol and / or acetic acid with industrial value through fermentation.

[0026] The present invention first discovered two important transcription factors NrdR (NCBI accession number CLJU_c12350) and DeoR (NCBI accession number CLJU_c20610) in Clostridium, which have an important influence on the conversion and utilization of one-carbon gas by Clostridium to synthesize ethanol and / or acetic acid, among which the transcription factor NrdR has a negative effect, while DeoR has a promoting effect on the metabolic pathway. Experiments have found that the metabolic engineering strategy of blocking NrdR expression and / or strengthening DeoR expression of the present invention can effectively improve the syngas utilization efficiency of Clostridium jundal. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The graph shows the growth of the original strain of C. ljungdahlii DSM 13528 and the engineered strain with the NrdR encoding gene CLJU_c12350 knocked out in syngas fermentation, as well as the synthesis of acetic acid and ethanol. Among them, Figure A is a comparison of the growth of the mutant strain and the original strain; Figure B is the synthesis of acetic acid and ethanol of the mutant strain and the original strain; *, P < 0.05, **, P < 0.01, ***, P < 0.001; WT, original strain of C. ljungdahlii; △ 12350, mutant strain with the NrdR encoding gene knocked out.

[0028] Figure 2 The original strain of C. ljungdahlii DSM 13528 and the promoter P are shown. 1339 Comparison curves of the growth of the engineered strain with regulated DeoR overexpression in syngas fermentation and the synthesis of acetic acid and ethanol. Figure A is a comparison of the growth of the mutant strain and the original strain; Figure B is the synthesis of acetic acid and ethanol of the mutant strain and the original strain; ***, P < 0.001; WT / P, Clostridium jundal transformed with empty plasmid pMTL83151; P 1339 -deoR, containing the overexpression plasmid P 1339 -deoR mutant strain of Clostridium juncea.

[0029] Figure 3 Different promoters are shown pta , P ptb , P 1339 , P 01440 and P thl Comparison of the growth of the engineered bacteria of Yondahl with regulated DeoR overexpression in syngas fermentation and the production of acetic acid and ethanol. 01440 , P 1339 , P pta , Pptb and P thl Schematic diagram of the overexpression of the transcription factor DeoR; Figure B is the growth of the strain overexpressing the transcription factor DeoR; Figure C is the ethanol production of the strain overexpressing the transcription factor DeoR; Figure D is the acetic acid production of the strain overexpressing the transcription factor DeoR; *, P < 0.05, **, P < 0.01; P 1339 -deoR, containing the overexpression plasmid P 1339 -deoR Clostridium jundal; P pta -deoR, containing the overexpression plasmid P pta -deoR Clostridium jundal; P ptb -deoR, containing the overexpression plasmid P ptb -deoR Clostridium jundal; P 01440 -deoR, containing the overexpression plasmid P 01440 -deoR Clostridium jundal; P thl -deoR, containing the overexpression plasmid P thl -deoR of Clostridium junceum. DETAILED DESCRIPTION

[0030] The present invention provides a strain metabolic engineering transformation strategy for improving the syngas fermentation performance of Clostridium aerovorum, so as to obtain industrial Clostridium aerovorum with better performance.

[0031] In general, by blocking the expression of one (or more) transcription factors, such as NrdR, and / or enhancing the expression of another (or more) transcription factors, such as DeoR, in the strain, the autotrophic growth ability, product synthesis rate and yield of Clostridium perfringens, such as Clostridium jundal, during syngas fermentation can be effectively improved, thereby improving the economic efficiency of Clostridium jundal fermentation.

[0032] The metabolic engineering strategy of the present invention effectively improves the utilization performance / efficiency of clostridium to one carbon gas, such as syngas. The term "(one carbon gas utilization performance / efficiency) improves", "promotes" or "enhances" 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 an increase of 100%, or any increase between 10%-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.

[0033] In the description of the technical solutions of the present invention, the term "and / or" used in terms such as "A and / or B", "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).

[0034] One metabolic engineering strategy is to block the expression of transcription factor NrdR. For example, by knocking out the transcription factor NrdR encoding gene CLJU_c12350 in the Clostridium genome through gene editing technology, a recombinant bacterium that does not express NrdR is obtained.

[0035] 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 to ferment and utilize carbon dioxide. Wild-type Clostridium aerovorum may also be referred to as the "original strain", and "recombinant bacteria" and "engineered bacteria" may be referred to as "mutant strains".

[0036] Another metabolic engineering strategy is to enhance the expression of DeoR, for example, by cloning the gene encoding the transcription factor DeoR, CLJU_c20610, into a suitable plasmid vector and placing it under the control of a suitable promoter to construct a recombinant plasmid expressing DeoR, such as P thl -deoR and transferred into Clostridium cells to obtain recombinant bacteria overexpressing DeoR, which can be referred to as "overexpression strains".

[0037] Another metabolic engineering strategy is to combine the first two strategies, that is, to express DeoR in the above-mentioned recombinant plasmid, such as P thl -deoR was transferred into the recombinant bacterial cells that did not express NrdR, thus obtaining the recombinant bacteria that overexpressed DeoR but did not express NrdR.

[0038] In order to optimally express the transcription factor DeoR in the clostridial engineering bacteria, the expression gene of the transcription factor DeoR can be codon optimized. Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Different organisms usually show a special preference for one of some codons encoding the same amino acid due to mutation tendency and natural selection. For example, in fast-growing microorganisms, the optimization codon reflects the composition of its respective genome tRNA library. Therefore, in fast-growing microorganisms, the low-frequency codons of amino acids can be used for the same amino acids but the high-frequency codon replacement. Therefore, the expression of the optimized DNA sequence is improved in fast-growing microorganisms.

[0039] In this article, for the sake of simplicity, a certain protein, such as the transcription factor DeoR, is sometimes used interchangeably with the name of its coding gene (DNA), deoR. Those skilled in the art should understand that they represent different types of substances in different description occasions. Those skilled in the art can easily understand their meanings based on the context. For example, for DeoR, when used to describe the function or category of a transcription factor, it refers to the protein; when described as a gene, it refers to the coding gene deoR of the protein.

[0040] In order to investigate the metabolic engineering strategy of the present invention, simulated steel mill tail gas was used as a carbon source (CO-CO2-H2-N2, with a volume ratio of 56% / 20% / 9% / 15%) as syngas (also known as C1 gas) to carry out syngas fermentation of engineered bacteria. By analyzing the content of acetic acid and ethanol in the fermentation product, it was confirmed that the fermentation cell density and acetic acid + ethanol synthesis of the recombinant bacteria overexpressing DeoR and the recombinant bacteria not expressing NrdR were higher than those of the wild-type strain, indicating that the gas fermentation performance of the recombinant bacteria, i.e., the ability to utilize syngas, has been improved to a certain extent. The experimental results verify that the metabolic engineering strategy of the present invention has achieved the expected effect.

[0041] We further optimized the regulatory elements for expressing DeoR in the recombinant bacteria, using promoter P pta , P ptb , P 01440 , P 1339 and P thl Regulate the expression of DeoR. thl The gene encoding the transcription factor DeoR had the best adaptability in Clostridium jundal, as evidenced by the highest growth and product (acetic acid and ethanol) production during syngas fermentation of the engineered strain.

[0042] The above research results provide new ideas and strategies for the metabolic engineering transformation of such microorganisms that can absorb and utilize one-carbon gases, and are expected to more effectively improve the fermentation performance of the strains.

[0043] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0044] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned here refer to the mass percentages unless otherwise specified.

[0045] Example

[0046] Materials and methods

[0047] The molecular biology experiments in this article, including plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., are mainly carried out with reference to "A Guide to Molecular Cloning Experiments" (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). For example, the competent cell transformation method and the competent cell preparation method are all carried out with reference to "A Guide to Molecular Cloning Experiments" (3rd edition), Chapter 1, page 96. If necessary, the specific experimental conditions can be determined by simple experiments.

[0048] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0049] Main culture medium:

[0050] LB medium: 5g / L yeast extract, 10g / L tryptone, 10g / L sodium chloride. (LB solid medium plus 20g / L agar powder.)

[0051] YTF (yeast extract, tryptone, fructose) medium: 10g / L yeast extract, 16g / L tryptone, 10g / L fructose, 0.2g / L sodium chloride, 0.3g / L cysteine, 500μL / L resazurin storage solution, 2mL / L trace element storage solution, 1mL / L vitamin storage solution, dissolved in double distilled water, and adjusted to pH 6.0 with hydrochloric acid. Among them,

[0052] Trace element storage solution: 10.0g / L nitrilotriacetic acid, 5.0g / L manganese sulfate, 4.0g / L ferrous ammonium sulfate hexahydrate, 1.0g / L cobalt chloride hexahydrate, 1.0g / L zinc sulfate heptahydrate, 0.1g / L cupric chloride dihydrate, 0.1g / L nickel chloride hexahydrate, 0.1g / L sodium molybdate dihydrate, 0.1g / L sodium stannate, 0.1g / L sodium tungstate dihydrate, dissolved in double distilled water.

[0053] 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.

[0054] Resazurin stock solution: 1g / L Resazurin, dissolved in double distilled water.

[0055] PETC 1754 Medium:

[0056] 30mL / L mineral element stock solution, 1mL / L vitamin stock solution, 2mL / L trace element stock solution, 0.5g / L yeast extract, 5g / L 2-morpholineethanesulfonic acid, 5mL / L reducing agent stock solution, 500μL / L resazurin stock solution, dissolved in double distilled water, and adjusted to pH 6.0 with NaOH.

[0057] Mineral element storage solution: 80g / L sodium chloride, 100g / L ammonium chloride, 10g / L potassium chloride, 10g / L dipotassium hydrogen phosphate, 20g / L magnesium sulfate heptahydrate, 4g / L calcium chloride dihydrate, dissolved in double distilled water.

[0058] 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.

[0059] SMP buffer: 270 mM sucrose, 4.4 mM NaH2PO4, 0.6 mM Na2HPO4, 1 mM MgCl2·6H2O.

[0060] Clostridium ljungdahlii DSM 13528 was purchased from the German Culture Collection (DSMZ).

[0061] In the embodiment, the plasmid expressing the gene deoR is P thl -deoR、P pta -deoR、P ptb -deoR、P 01440 -deoR and P 1339 -deoR, plasmids used for gene editing operations such as pMTLcas-pta (CRISPR / Cas9-Based Efficient Genome Editing in Clostridium ljungdahlii, an Autotrophic Gas-Fermenting Bacterium. He Huang, et al., 2016. ACS Synthetic Biology. doi: 10.1021 / acssynbio.6b00044.) are constructed and preserved by the research group of Jiang Weihong, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences. Any unit or individual can obtain the plasmid for verification of the present invention, but it shall not be used for other purposes, including development and utilization, scientific research and teaching, without the permission of Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences.

[0062] The PCR primers used in the examples are listed in Table 1 and were synthesized by Boshang Biotechnology (Shanghai) Co., Ltd.

[0063] Table 1. Primers used in constructing the recombinant plasmids in the examples

[0064]

[0065] In Table 1, "-F" in the name stands for forward direction; "-R" stands for reverse direction.

[0066] The provider of simulated steel plant exhaust gas (CO-CO2-H2-N2, with the volume ratio of each component being 56% / 20% / 9% / 15%) is Shanghai Haoqi Gas Co., Ltd.

[0067] Detection method of ethanol and / or acetic acid in fermentation broth:

[0068] The content of acetic acid and ethanol in the fermentation broth was determined by gas chromatography. The gas chromatograph machine and detection method are as follows: Agilent 7890 gas chromatograph, the detector is a hydrogen flame ionization detector (hydrogen flame detector), the chromatographic column is an Alltech ECTM-WAX capillary column, the column box temperature is gradually increased from 85°C to 200°C; the column pressure is 10psi to 30psi; the injector temperature is 250°C; the detector temperature is 300°C; the carrier gas is nitrogen, the flow rate is 25mL / min; the hydrogen flow rate is 30mL / min; the air flow rate is 400mL / min. The internal standards are isobutanol, isobutyric acid and hydrochloric acid (for acidification). The internal standard method is used to calculate the content of acetic acid and ethanol.

[0069] 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.

[0070] Example 1: Discovery of the transcription factor NrdR that can affect syngas utilization by Clostridium jundal

[0071] Experimental steps:

[0072] 1) Take out the C.ljungdahlii DSM 13528 strain stored in the -80℃ freezer, inoculate it in a test tube containing 5mL YTF medium at a 10% (v / v) inoculum in an anaerobic glove box, and culture it statically until the logarithmic growth phase. After that, take 10μL of the bacterial solution and streak it on a non-antibiotic YTF plate (containing 2.0% agar), and culture it anaerobically at 37℃ for 2-4 days until a single colony visible to the naked eye grows on the plate.

[0073] 2) Gene knockout plasmid was constructed in E. coli DH5α. Using the C. ljungdahlii genome as a template, the upstream homology arm and downstream homology arm fragments were amplified using primers 12350-UpArm-F / 12350-UpArm-R and 12350-DownArm-F / 12350-DownArm-R respectively; using the pMTLcas-pta previously constructed in the laboratory (see He Huang, et al. 2016. ACS Synthetic Biology. doi: 10.1021 / acssynbio.6b00044.) as a template, the sgRNA fragment was amplified using primers 12350-gRNA-F / gRNA-R, and then Overlap PCR was used to assemble the three fragments into a long fragment. The pMTLcas-pta plasmid was double-digested with restriction endonucleases SalI and XhoI to obtain a linearized vector. Finally, the long fragment and the linearized vector were assembled into a circular plasmid pMTLcas-nrdR using a non-ligase-dependent single-fragment one-step cloning kit (Novogene (Nanjing) Biotechnology Co., Ltd., product number: C112-02).

[0074] PCR reaction program: 98°C pre-denaturation for 5 min; 98°C denaturation for 30 s, 55°C annealing for 30 s, 68°C extension for 2 min, 30 cycles; 68°C extension for 10 min, and storage at 16°C. High-fidelity DNA polymerase was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., product number: KFX-101, and the PCR reaction system was prepared according to the instructions.

[0075] The amplified product was recovered by 2.0% (w / v) agarose gel electrophoresis. The PCR product recovery kit was purchased from AXYGEN (Shanghai) Company, product number: AP-GX-250.

[0076] 3) Preparation of competent strains. First, inoculate a single colony of C. ljungdahlii into 5 ml of liquid YTF medium and culture at 37°C for 24 hours. Then, inoculate the cultured bacterial liquid into 200 ml of liquid YTF medium at an inoculum volume of 1‰ v / v and culture at 37°C for about 16 hours. 600 When the pH reaches 0.4-0.6, ice-bathe the bacterial solution for 20 minutes, and then centrifuge at 4500rpm and 4℃ for 10 minutes to collect the bacteria. Next, place the centrifuged bacterial solution on ice, pour out the supernatant in the anaerobic box, add 30mL of SMP solution, gently resuspend the bacterial solution, and centrifuge at 4500rpm and 4℃ for 10 minutes to collect the bacteria, pour out the supernatant in the anaerobic box, and resuspend and wash twice. Finally, add 3.6mL of SMP solution and 400μL of DMSO, mix well, and then divide into portions and store at -80℃ for later use.

[0077] 3) Electrotransformation conditions. Mix the circular plasmid and competent cells mentioned above 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 into 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, transfer the mixture to 1mL of YTF liquid culture medium after the shock, and resuscitate in an anaerobic environment at 37℃ for 20 hours. Finally, spread the revived 1mL bacterial solution on the YTF solid culture medium containing 5μg / mL chloramphenicol methyl sulfone resistance for culture.

[0078] 4) Identification of knockout strains. After the electroporated plate was cultured in an anaerobic chamber at 37°C for 5 days, colony identification was performed. The identification primers 12350-ko-identify-F / 12350-ko-identify-R were designed outside the upstream and downstream homology arms of the genome. A single colony was dipped into a toothpick as a template, and the identification primers were added to the KOD FX DNA polymerase amplification system for amplification. The amplified product was run on a gel for identification.

[0079] 5) Fermentation conditions of knockout strains. A single bacterium was inoculated into 5 mL YTF liquid medium and cultured at 37°C for 24 h. 600 After reaching 0.8-1.0, draw 1.5mL of bacterial solution and inoculate it into a serum bottle containing 30mL of PETC 1754 optimized liquid culture medium and seal it with an aluminum cap. Connect the sealed serum bottle containing the culture to a synthetic gas cylinder equipped with a pressure-dividing valve through a plastic hose. The connecting end of the plastic hose and the sealed serum bottle is equipped with a sterile disposable syringe needle. When inflating, open the main valve of the cylinder, adjust the pressure of the pressure-dividing valve to 0.2MPa, insert the needle at one end of the plastic hose into the serum bottle through the sealed port, and inflate for 1min. Thereafter, culture the serum bottle in a shaker at 37°C and a speed of 100rpm. Inflate once every 24h according to the above steps.

[0080] 6) Determination of fermentation growth (biomass) of the strain. The fermentation sample was appropriately diluted with ultrapure water and the bacterial solution concentration (OD 600 ) and draw the growth curve of the strain accordingly.

[0081] 7) Determination and analysis of the fermentation products of the strains. The fermentation samples were centrifuged at 12000 rpm for 10 min, and the supernatant was taken to determine the contents of acetic acid and ethanol in the fermentation broth using a gas chromatograph.

[0082] Experimental results:

[0083] See also Figure 1When the transcription factor NrdR encoding gene CLJU_c12350 was inactivated, the mutant strain (denoted as △12350) and the original strain (WT) were fermented for 72 hours under the conditions of PETC 1754 fermentation medium with syngas as the carbon source. The maximum biomass (OD 600 ) is about 15.5% higher than the latter ( Figure 1 A), the product (acetic acid and ethanol combined) yield increased by 17.9% ( Figure 1 Middle B), demonstrating that knocking out the nrdR gene can improve the gas fermentation ability of Clostridium Yongdal.

[0084] Example 2: Discovery of the transcription factor DeoR that can affect syngas utilization by Clostridium jundal

[0085] Experimental steps:

[0086] 1) First, the C. ljungdahlii genome was used as a template and primers P 1339 -F / P 1339 -R amplified the promoter fragment, primer G20610 (P 1339 )-F / G20610-R amplified CLJU_c20610 gene fragment. Then, the promoter fragment and gene fragment were amplified by overlap PCR to obtain P 1339 -DeoR fragment. Then, the pMTL83151 plasmid was double-digested with restriction endonucleases NdeI and HindIII to obtain a linearized vector. Finally, the fragment P was cloned into the pMTL83151 vector using a ligase-independent single-fragment one-step cloning kit (Novizan (Nanjing) Biotechnology Co., Ltd., product number: C112-02). 1339 -DeoR recombines with the linearized vector to form a circular plasmid P 1339 -deoR.

[0087] 2) The plasmid P obtained above 1339 -deoR was electrotransformed into C. ljungdahlii competent cells, and then spread on YTF solid medium containing 5 μg / mL chloramphenicol methylsulfone resistance and cultured for 5 days. The preparation of C. ljungdahlii competent cells and electrotransformation conditions refer to Example 1.

[0088] 3) Fermentation conditions of overexpression strains. The plasmid overexpression strain was inoculated into 5 mL YTF liquid culture medium and cultured at 37°C for 24 h. 600After reaching 0.8-1.0, the pipette draws 1.5mL of bacterial solution and inoculates it into a serum bottle containing 30mL of PETC 1754 optimized liquid culture medium and seals it with an aluminum cap. The sealed serum bottle containing the culture is connected to a synthetic gas cylinder equipped with a pressure-dividing valve through a plastic hose. The connecting end of the plastic hose and the sealed serum bottle is equipped with a sterile disposable syringe needle. When inflating, open the main valve of the cylinder, adjust the pressure of the pressure-dividing valve to 0.2MPa, insert the needle at one end of the plastic hose into the serum bottle through the sealed port, and inflate for 1min. Thereafter, the serum bottle is cultured in a shaker at 37°C and a speed of 100rpm. Inflate once every 24h according to the above steps.

[0089] 4) Determination of fermentation growth (biomass) of the strain. The fermentation sample was appropriately diluted with ultrapure water and the bacterial solution concentration (OD 600 ) and draw the growth curve of the strain accordingly.

[0090] 5) Determination and analysis of the fermentation products of the strains. The fermentation samples were centrifuged at 12000 rpm for 10 min, and the supernatant was taken out. The contents of acetic acid and ethanol in the fermentation broth were determined by gas chromatography.

[0091] Experimental results:

[0092] See also Figure 2 , use P 1339 The promoter drives the overexpression of the DeoR encoding gene CLJU_c20610 in Clostridium jundal. When deoR is overexpressed, the overexpression strain (P 1339 -deoR) and the original strain (WT / P) after 72 hours of fermentation, the growth of the former had no significant difference with the latter ( Figure 2 A), but the product (acetic acid and ethanol) yield increased by 23.9% ( Figure 2 Middle B), demonstrating that overexpression of the deoR gene can improve the syngas utilization ability of Clostridium Yongdal.

[0093] Example 3: Optimizing DeoR expression to further improve the syngas fermentation performance of Clostridium jundal

[0094] Use promoter P pta , P ptb , P 01440 , P 1339 and P thl To regulate the expression of DeoR, the experimental steps are as follows:

[0095] 1) Plasmid P pta-deoR was constructed as an example. First, the C. ljungdahlii genome was used as a template and primers P pta -F / P pta -R amplified the promoter fragment, primer G20610 (P pta )-F / G20610-R amplified CLJU_c20610 gene fragment. Then, the promoter fragment and gene fragment were amplified by overlap PCR to obtain P pta -DeoR fragment. Then, the pMTL83151 plasmid was double-digested with restriction endonucleases NdeI and HindIII to obtain a linearized vector. Finally, the fragment P was cloned into the pMTL83151 vector using a ligase-independent single-fragment one-step cloning kit (Novizan (Nanjing) Biotechnology Co., Ltd., product number: C112-02). pta -DeoR recombines with the linearized vector to form a circular plasmid P pta -deoR (SEQ ID NO: 2). Using the above plasmid construction strategy, plasmid P ptb -deoR (SEQ ID NO: 3), P 01440 -deoR (SEQ ID NO: 4), P 1339 -deoR (SEQ ID NO: 1) and P thl -deoR (SEQ ID NO: 5).

[0096] 2) The plasmid obtained above was electrotransformed into C. ljungdahlii competent cells, and then spread on YTF solid medium containing 5 μg / mL chloramphenicol methyl sulfone resistance and cultured for 5 days. The preparation of C. ljungdahlii competent cells and electrotransformation conditions refer to Example 1.

[0097] 3) Fermentation conditions of overexpression strains. The plasmid overexpression strain was inoculated into 5 mL YTF liquid culture medium and cultured at 37°C for 24 h. 600 After reaching 0.8-1.0, the pipette draws 1.5mL of bacterial solution and inoculates it into a serum bottle containing 30mL of PETC 1754 optimized liquid culture medium and seals it with an aluminum cap. The sealed serum bottle containing the culture is connected to a synthetic gas cylinder equipped with a pressure-dividing valve through a plastic hose. The connecting end of the plastic hose and the sealed serum bottle is equipped with a sterile disposable syringe needle. When inflating, open the main valve of the cylinder, adjust the pressure of the pressure-dividing valve to 0.2MPa, insert the needle at one end of the plastic hose into the serum bottle through the sealed port, and inflate for 1min. Thereafter, the serum bottle is cultured in a shaker at 37°C and a speed of 100rpm. Inflate once every 24h according to the above steps.

[0098] 4) Determination of the fermentation growth (biomass) of the strain. pta , P ptb , P 01440 , P 1339 and P thl The fermentation samples of the promoter overexpressing the transcription factor DeoR were appropriately diluted with ultrapure water, and the bacterial concentration (OD 600 ), and draw the growth curve of the overexpression strain.

[0099] 5) Determination and analysis of the fermentation products of the strain. pta , P ptb , P 01440 , P 1339 and P thl The fermentation samples of the promoter overexpressing the transcription factor DeoR were centrifuged at 12000 rpm for 10 min, and the supernatant was taken. The contents of acetic acid and ethanol in the fermentation broth were determined by gas chromatography.

[0100] Experimental results:

[0101] See also Figure 3 In order to further improve the syngas fermentation capacity of the strain, different promoter strengths (P pta , P ptb , P 01440 , P 1339 and P thl ) to optimize the regulation of deoR gene expression ( Figure 3 In the fermentation medium of PETC 1754 with syngas as carbon source, the newly constructed overexpression strain (P pta -deoR、P ptb -deoR、P 01440 -deoR and P thl -deoR) and the above Figure 2 Medium 1339 The strain overexpressing the deoR gene (P 1339 -deoR) were compared. The results showed that the overexpression strain P thl -deoR grew faster than the control strain P 1339 -deoR has significantly improved ( Figure 3 At the end of fermentation (72 hours), the overexpressing strain P 01440 -deoR had a higher ethanol yield than the control strain P 1339 -deoR increased by 2 times, and there was no significant difference in acetic acid production; while the overexpression strain P thl -deoR also produced more ethanol than the control strain P 1339 -deoR increased by 3.17 times, and there was no significant difference in the production of acetic acid ( Figure 3 C and D), indicating that the promoter P thl It has a significant promoting effect on the expression of deoR gene.

[0102] 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 document is prior art or common knowledge.

Claims

1. A method for improving the ability of Clostridium to utilize one-carbon gas, characterized in that: The steps include: A. blocking or down-regulating the expression of the transcription factor NrdR (NCBI accession number CLJU_c12350); and / or B. Enhanced expression of transcription factor DeoR (NCBI accession number CLJU_c20610).

2. The method according to claim 1, characterized in that The Clostridium is Clostridium aerogenes capable of converting and utilizing one-carbon gas, preferably Clostridium ljungdahlii.

3. The method according to claim 1 or 2, characterized in that In step A, the expression of the gene encoding the transcription factor NrdR is blocked by gene editing technology.

4. The method according to claim 1, characterized in that Step B is to activate the activity of the transcription factor DeoR or enhance the expression of the transcription factor DeoR using one or more copies of the regulatory element; or The regulatory element is integrated into the upstream of the transcription factor DeoR gene in the genome of the host cell by gene editing technology, thereby placing the transcription factor DeoR gene under the regulation of the promoter.

5. The method according to claim 4, characterized in that The regulatory element is a promoter selected from the group consisting of: pta , P ptb , P 01440 , P 1339 and P thl , preferably the promoter P thl .

6. The method according to claim 4, characterized in that Step B is to clone the coding gene of the transcription factor DeoR into a plasmid vector suitable for expression in Clostridium, and then transfer the recombinant plasmid into the host cell, so as to overexpress the transcription factor DeoR in Clostridium.

7. The method according to claim 6, characterized in that The plasmid vector is selected from the following group: pMTL 82151, pMTL83151, pMTL 84151, pMTL 85151, pMTL 82251, pMTL 82254, pMTL 83251, pMTL 83254, pMTL84251, pMTL 84254, pMTL 85251, pMTL 85254, and pIMP1.

8. A recombinant plasmid for expressing transcription factor DeoR, which is selected from the following group: P with a nucleotide sequence of SEQ ID NO: 1 1339 -deoR, P with a nucleotide sequence of SEQ ID NO: 2 pta -deoR, P with a nucleotide sequence of SEQ ID NO: 3 ptb -deoR, P with a nucleotide sequence of SEQ ID NO: 4 01440 -deoR and P having a nucleotide sequence of SEQ ID NO: 5 thl -deoR.

9. An engineered Clostridium bacterium capable of utilizing a carbon gas, characterized in that: The method is constructed according to any one of claims 1 to 7.

10. Use of the engineered Clostridium sp. as claimed in claim 9 in bioutilization of a carbon-containing gas, for example, the engineered Clostridium sp. converts the carbon-containing gas into ethanol and / or acetic acid by fermentation.

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