Recombinant expression vector, clostridium aerovorans engineering bacterium with micro-aerobic tolerance and construction method and application of clostridium aerovorans engineering bacterium

Abstract

The invention relates to the technical field of biology, in particular to a recombinant expression vector, clostridium aerovorans engineering bacteria with micro-aerobic tolerance and a construction method and application of the clostridium aerovorans engineering bacteria. Functional elements of the recombinant vector are all from clostridium ethanolicum, the recombinant expression vector comprises a first recombinant expression vector and a second recombinant expression vector, and the first recombinant expression vector comprises a molecular chaperone CaDnaK as shown in SEQ ID NO.1 and a carbon monoxide dehydrogenase mutant as shown in SEQ ID NO.2 which are expressed in series; the second recombinant expression vector comprises formate dehydrogenase CaFdhF as shown in SEQ ID NO.3 and a hydrogenase mutant as shown in SEQ ID NO.4, wherein the formate dehydrogenase CaFdhF and the hydrogenase mutant are expressed in series; the recombinant expression vector is introduced into clostridium aerovorans, so that the viability of the strain and the conversion efficiency of a gas substrate in a micro-aerobic fermentation environment can be comprehensively improved, the industrial fermentation deoxygenization cost is reduced, and finally, the synthesis gas fermentation income is increased.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a recombinant expression vector, an engineered bacterium of Clostridium aeruginosa with micro-oxygen tolerance, and a construction method and application thereof. Background Art

[0002] Synthesis gas is a mixture of CO2, CO and H2. The raw materials of synthesis gas are widely available. It can be produced by gasification of solid fuels such as fossil fuels such as coal or coke, or by gasification of biomass such as agricultural and forestry wastes. At present, the production of bio-based alcohol chemicals by biofermentation using synthesis gas as raw material is a very valuable synthesis gas conversion pathway. The metabolic pathway for synthesizing organic acids and organic alcohols such as acetic acid and ethanol in this conversion pathway mainly depends on the Wood-Ljugdahl pathway (WLP). The WLP pathway is also called the reductive acetyl-CoA pathway (Acetyl-CoA pathway). It is the metabolic pathway for Syngas Clostridium to fix CO and CO2 to synthesize organic acids and organic alcohols such as acetic acid and ethanol. It is also the most effective carbon fixation pathway known to date with the fewest reaction steps. However, in the WLP pathway, carbon monoxide dehydrogenase (CODH), formate dehydrogenase (FDH) and hydrogenase (H2ase) are the key enzymes that limit the conversion of CO, CO2 and H2. Among these key enzymes, carbon monoxide dehydrogenase first provides additional reducing equivalents for the conversion of CO to CO2, and then formate dehydrogenase reduces CO2 to formate. The subsequent formate generated is synthesized into tetrahydrofolate by synthase, and the generated formyltetrahydrofolate then enters the methyl branch of the WLP pathway, and finally formsyltetrahydrofolate is synthesized into acetyl-CoA under the action of the bifunctional enzyme carbon monoxide dehydrogenase / acetyl-CoA synthase (CODH / ACS). From this process, we can know that the utilization of CO as an electron donor is largely affected by the expression level of the WLP pathway enzymes. Therefore, in the process of synthetic biology modification, it is generally necessary to overexpress various enzymes of the WLP pathway to enhance the utilization efficiency of CO.

[0003] However, syngas is usually doped with some oxygen. Existing studies have shown that oxygen can not only increase the redox potential of the growth medium, but also affect the activity and stability of key enzymes such as carbon monoxide dehydrogenase, hydrogenase, and formate dehydrogenase in the WLP pathway. Therefore, oxygen is one of the most toxic gases in the syngas fermentation process. Since most of these key enzymes contain transition metals, such as molybdenum, tungsten, and non-heme iron, these key enzymes also contain ferredoxin (Fd), and the redox active center composed of them is extremely sensitive to oxygen. Existing studies have shown that the D-cluster is the key site of irreversible oxidative damage in Desulfovibrio vulgaris carbon monoxide dehydrogenase Dv-CODH, among which the presence of [2Fe-2S]D-cluster determines the oxygen tolerance of carbon monoxide dehydrogenase. This shows that the oxygen contained in the syngas will seriously affect the normal growth and metabolism of syngas Clostridium, thereby affecting the production efficiency of syngas fermentation products and restricting the industrial development of syngas fermentation.

[0004] The syngas fermentation Clostridium currently used in industry is a strict anaerobic bacteria. It is extremely sensitive to oxygen. Short-term continuous exposure to an aerobic environment will significantly inhibit the growth performance and metabolic activity of the strain, causing the strain to stop growing and metabolizing, or even die completely. Therefore, at this stage, in order to ensure the normal physiological metabolism of Clostridium aerogenes, it is necessary to deoxygenate the syngas used in the syngas fermentation culture process, and also to isolate the fermentation culture process from oxygen. In addition, it is necessary to add inorganic or organic deoxygenators to the culture medium during the syngas fermentation process to eliminate the hazards of oxygen, which undoubtedly increases the production cost of syngas fermentation. Summary of the invention

[0005] The present application provides a recombinant expression vector, an engineered bacterium of Clostridium aerovorum with micro-oxygen tolerance, and a construction method and application thereof, in order to solve the following technical problem: how to improve the oxygen tolerance of Clostridium aerovorum.

[0006] In a first aspect, the present application provides a recombinant expression vector, comprising a first recombinant expression vector and a second recombinant expression vector, wherein the first recombinant expression vector and the second recombinant expression vector comprise a plurality of functional elements derived from ethanol Clostridium, wherein the functional elements of the first recombinant expression vector comprise a tandemly expressed molecular chaperone CaDnaK and a carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H; and the second recombinant expression vector comprises a tandemly expressed formate dehydrogenase CaFdhF and a hydrogenase mutant CaHydA2_C183D-A311L;

[0007] The gene fragment encoding the molecular chaperone CaDnaK has a nucleotide sequence as shown in SEQ ID NO.1;

[0008] The gene fragment encoding the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H has a nucleotide sequence as shown in SEQ ID NO.2;

[0009] The gene fragment encoding the formate dehydrogenase CaFdhF has a nucleotide sequence as shown in SEQ ID NO.3;

[0010] The gene fragment encoding the hydrogenase mutant CaHydA2_C183D-A311L has a nucleotide sequence as shown in SEQ ID NO.4.

[0011] Optionally, the expression product of the molecular chaperone CaDnaK has an amino acid sequence as shown in SEQ ID NO.5;

[0012] The carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H includes a *401L mutation site and a W556H mutation site, and the expression product of the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H has an amino acid sequence as shown in SEQ ID NO.6;

[0013] The expression product of the formate dehydrogenase CaFdhF has an amino acid sequence as shown in SEQ ID NO.7;

[0014] The hydrogenase mutant CaHydA2_C183D-A311L includes a C183D mutation site and an A311L mutation site, and the expression product of the hydrogenase mutant CaHydA2_C183D-A311L has an amino acid sequence as shown in SEQ ID NO.8.

[0015] In a second aspect, the present application provides a method for constructing the recombinant expression vector described in the first aspect, the method comprising:

[0016] Amplifying a first gene fragment encoding a molecular chaperone CaDnaK and a second gene fragment encoding a carbon monoxide dehydrogenase CaAcsA using a first amplification primer pair and a second amplification primer, respectively, to obtain a first amplification product encoding a molecular chaperone CaDnaK and a second amplification product encoding a carbon monoxide dehydrogenase CaAcsA, respectively;

[0017] The first amplification product encoding the molecular chaperone CaDnaK and the second amplification product encoding the carbon monoxide dehydrogenase CaAcsA are sequentially connected to the expression vector to obtain a first construction vector containing CaDnaK-CaAcsA;

[0018] Constructing a mutant vector of carbon monoxide dehydrogenase CaAcsA of the first construction vector, and then screening carbon monoxide dehydrogenase activity to obtain a first recombinant expression vector containing the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H;

[0019] Amplifying a third gene fragment encoding formate dehydrogenase CaFdhF and a fourth gene fragment encoding hydrogenase CaHydA2 using a third amplification primer pair and a fourth amplification primer respectively, to obtain a third amplification product encoding formate dehydrogenase CaFdhF and a fourth amplification product encoding hydrogenase CaHydA2;

[0020] The third amplification product encoding formate dehydrogenase CaFdhF and the fourth amplification product encoding hydrogenase CaHydA2 are sequentially connected to the expression vector to obtain a second construction vector containing CaFdhF-CaHydA2;

[0021] Constructing a mutation vector of the hydrogenase CaHydA2 of the second construction vector, and then performing hydrogenase activity screening to obtain a second recombinant expression vector containing the hydrogenase mutant CaHydA2_C183D-A311L;

[0022] Collecting the first recombinant expression vector and the second recombinant expression vector to obtain a recombinant expression vector;

[0023] Wherein, the first amplification primer pair is used to amplify the first gene fragment, the upstream primer of the first amplification primer pair is shown as SEQ ID NO.9, and the downstream primer of the first amplification primer pair is shown as SEQ ID NO.10;

[0024] The second amplification primer pair is used to amplify the second gene fragment, the upstream primer of the second amplification primer pair is shown as SEQ ID NO.11, and the downstream primer of the second amplification primer pair is shown as SEQ ID NO.12;

[0025] The third amplification primer pair is used to amplify the third gene fragment, the upstream primer of the third amplification primer pair is shown as SEQ ID NO.13, and the downstream primer of the third amplification primer pair is shown as SEQ ID NO.14;

[0026] The fourth amplification primer pair is used to amplify the fourth gene fragment, the upstream primer of the fourth amplification primer pair is shown as SEQ ID NO.15, and the downstream primer of the fourth amplification primer pair is shown as SEQ ID NO.16.

[0027] Optionally, the construction of the mutant vector of carbon monoxide dehydrogenase CaAcsA of the first construction vector, followed by carbon monoxide dehydrogenase activity screening to obtain a first recombinant expression vector containing the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H, comprises the steps of:

[0028] In the case where the first construction vector is used as a parent, a mutation vector of the carbon monoxide dehydrogenase CaAcsA of the first construction vector is constructed using a site-directed mutagenesis method;

[0029] The carbon monoxide dehydrogenase activity of the mutant vector of the carbon monoxide dehydrogenase CaAcsA is screened using a gas environment containing a first preset oxygen volume content to obtain a first recombinant expression vector containing the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H.

[0030] Optionally, the volume content V1 of the first preset oxygen and the total gas volume V2 of the gas environment satisfy the relationship: V1:V2≤0.5%.

[0031] Optionally, the construction of the second construction vector of the hydrogenase CaHydA2 mutant vector, followed by hydrogenase activity screening to obtain a second recombinant expression vector containing the hydrogenase mutant CaHydA2_C183D-A311L, comprises the steps of:

[0032] In the case where the second construction vector is used as a parent, a mutation vector of the hydrogenase CaHydA2 in the second construction vector is constructed using a site-directed mutagenesis method;

[0033] The hydrogenase activity of the mutant vector of the hydrogenase CaHydA2 is screened using a gas environment containing a second preset oxygen volume content to obtain a second recombinant expression vector containing the hydrogenase mutant CaHydA2_C183D-A311L.

[0034] Optionally, the volume content V3 of the second preset oxygen and the total gas volume V2 of the gas environment satisfy the relationship: V3:V2≤0.5%.

[0035] In a third aspect, the present application provides an engineered bacterium of Clostridium aerovorum having microaerobic tolerance, wherein the engineered bacterium of Clostridium aerovorum comprises the recombinant expression vector described in the first aspect or the recombinant expression vector obtained by the method described in the second aspect, wherein the Clostridium aerovorum comprises at least one of the following:

[0036] Clostridium alcoholicum, Clostridium ljungdahlii, Clostridium carboxyvorans, Clostridium kluyveri, and Clostridium ragsdalei.

[0037] In a fourth aspect, the present application provides a method for constructing the engineered bacterium Clostridium aerovorum described in the third aspect, the method comprising:

[0038] Synchronously introducing the first recombinant expression vector and the second recombinant expression vector in the recombinant expression vector of the first aspect into Clostridium aeruginosa to obtain an engineered Clostridium aeruginosa bacterium with microaerobic tolerance;

[0039] The engineered Clostridium aerovorum bacteria were verified by sequencing positive clones to obtain the engineered Clostridium aerovorum bacteria that co-expressed the first recombinant expression vector and the second recombinant expression vector.

[0040] In a fifth aspect, the present application provides an application of an engineered bacterium of Clostridium aerovorum having micro-oxygen tolerance, wherein the engineered bacterium of Clostridium aerovorum described in the third aspect is applied to anaerobic syngas fermentation under an atmosphere containing oxygen with a volume concentration of ≤0.5%.

[0041] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0042] A recombinant expression vector provided in an embodiment of the present application is split into a first recombinant expression vector and a second recombinant expression vector, and the first recombinant expression vector and the second recombinant expression vector include multiple functional elements derived from ethanol Clostridium, and these functional elements can show better effects in adverse fermentation environments after long-term stress strengthening in industrial environments; in addition, the molecular chaperone CaDnaK in the first recombinant expression vector can improve the tolerance of the engineered bacteria of Clostridium aerogenes to stress, and assist in the correct folding of the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H protein in the first recombinant expression vector, and the correctly folded carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H protein can improve the catalytic activity and tolerance to oxygen of the engineered bacteria containing the recombinant expression vector; at the same time, the mutants CaAcsA_*401L-W556H and CaHydA2_C obtained by protein engineering and enzyme activity test screening of carbon monoxide dehydrogenase CaAcsA and hydrogenase CaHydA2 in a microaerobic environment. 183D-A311L further improved the tolerance of carbon monoxide dehydrogenase and hydrogenase to oxygen and the catalytic activity in a microaerobic environment; in addition, the second recombinant expression vector contained the tandemly expressed formate dehydrogenase CaFdhF and the hydrogenase mutant CaHydA2_C183D-A311L, and the tandemly expressed formate dehydrogenase CaFdhF and the hydrogenase mutant CaHydA2_C183D-A311L not only improved the expression of formate dehydrogenase CaFdhF, but also shortened the hydrogenase mutant The distance of CaHydA2_C183D-A311L electron transfer improves the energy metabolism level of the engineering bacteria containing the recombinant expression vector, so as to enhance the growth and metabolic activity of the engineering bacteria containing the recombinant expression vector, thereby improving the activity of formate dehydrogenase and hydrogenase in the engineering bacteria containing the recombinant expression vector while improving the tolerance of formate dehydrogenase and hydrogenase in the engineering bacteria to the micro-oxygen environment, thereby improving the conversion efficiency of the engineering bacteria containing the recombinant expression vector to the gas substrate in the syngas fermentation under the micro-oxygen environment. Therefore, by setting these functional elements on the first recombinant expression vector and the second recombinant expression vector, the tolerance of the engineering bacteria containing the recombinant expression vector to oxygen and the catalytic activity to the gas substrate can be comprehensively improved, and the yield and production efficiency of the fermentation product under the micro-oxygen environment can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0044] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0045] Figure 1 The plasmid map of the first recombinant expression vector pMTL83152-Pthl-CaDnaK-CaAcsA_*401L-W556H provided in the examples of the present application;

[0046] Figure 2 The plasmid map of the second recombinant expression vector pMTL82253-Pfdx-CaFdhF-CaHydA2_C183D-A311L provided in the examples of the present application;

[0047] Figure 3 A schematic diagram of a method for constructing a recombinant expression vector provided in an embodiment of the present application;

[0048] Figure 4 A detailed schematic diagram of a method for constructing a recombinant expression vector provided in an embodiment of the present application;

[0049] Figure 5 A schematic diagram of a method for constructing engineered bacteria provided in an embodiment of the present application;

[0050] Figure 6 A comparison chart of the survival rates of the engineered bacteria Clostridium ljungdahlii DSM13528 co-overexpressing CaDnaK-CaAcsA_*401L-W556H and CaFdhF-CaHydA2_C183D-A311L provided in the examples of the present application after aerobic exposure for 10 minutes and 30 minutes;

[0051] Figure 7 The growth and metabolic changes of wild Clostridium ljungdahlii DSM13528 (WT), CaDnaK-CaAcsA_*401L-W556H and CaFdhF-CaHydA2_C183D-A311L co-overexpressing engineered bacteria under microaerobic conditions provided in the examples of the present application, wherein: Figure 7 A is a comparison of biomass changes after 72h fermentation of wild Clostridium ljungdahlii DSM13528 (WT), CaDnaK-CaAcsA_*401L-W556H and CaFdhF-CaHydA2_C183D-A311L co-overexpressing engineered bacteria, Figure 7 B is wild strain of Clostridium ljungdahlii DSM13528 (WT), CaDnaK-CaAcsA_*401L-W556H and

[0052] Comparison of changes in ethanol and acetic acid metabolites after 72 hours of fermentation of CaFdhF-CaHydA2_C183D-A311L co-overexpressing engineered bacteria. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0054] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0055] In this document, the terms "including" and "comprising" mean "including but not limited to". Relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0056] It should be noted that, in view of the defects recorded in the background technology, the inventors of the present application have found that strengthening the activity of the key enzymes of the carbon fixation pathway of Clostridium carboxidivorans by synthetic biology technology can improve the carbon fixation efficiency of the strain and improve the synthesis intensity of the product. By overexpressing the carbon monoxide dehydrogenase from Clostridium carboxidivorans in acetobutylicum, the carbon monoxide dehydrogenase can be prompted to show excellent CO2 reduction and CO oxidation activities. In addition, studies have shown that overexpression of heat shock proteins can enhance the tolerance of bacteria to different environmental pressures. And DnaK is a Class I heat shock protein contained in syngas fermentation Clostridium, which assists the correct folding of newly synthesized proteins or the refolding, positioning and depolymerization of damaged proteins in cells, and plays an important role in the process of Gram-positive bacteria coping with extracellular stress. Overexpression of DnaK from Deinococcus wulumuqiensis R12 in Clostridium acetobutylicum can enhance the tolerance and fermentation performance of the engineered bacteria to stresses such as butanol, low pH and cellulose hydrolysate inhibitors.

[0057] Figure 1 The plasmid map of the first recombinant expression vector pMTL83152-Pthl-CaDnaK-CaAcsA_*401L-W556H provided in the examples of the present application is exemplarily shown;

[0058] Figure 2 The plasmid map of the second recombinant expression vector pMTL82253-Pfdx-CaFdhF-CaHydA2_C183D-A311L provided in the examples of the present application is exemplarily shown;

[0059] like Figure 1 and Figure 2 As shown, the embodiment of the present application provides a recombinant expression vector, the recombinant expression vector includes a first recombinant expression vector and a second recombinant expression vector, the first recombinant expression vector and the second recombinant expression vector include multiple functional elements derived from ethanol Clostridium, the functional elements of the first recombinant expression vector include a tandemly expressed molecular chaperone CaDnaK and a carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H; the functional elements of the second recombinant expression vector include a tandemly expressed formate dehydrogenase CaFdhF and a hydrogenase mutant CaHydA2_C183D-A311L;

[0060] The gene fragment encoding the molecular chaperone CaDnaK has a nucleotide sequence as shown in SEQ ID NO.1;

[0061] The gene fragment encoding the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H has a nucleotide sequence as shown in SEQ ID NO.2;

[0062] The gene fragment encoding the formate dehydrogenase CaFdhF has a nucleotide sequence as shown in SEQ ID NO.3;

[0063] The gene fragment encoding the hydrogenase mutant CaHydA2_C183D-A311L has a nucleotide sequence as shown in SEQ ID NO.4.

[0064] It should be noted that the vector plasmid used by the first recombinant expression vector and the second recombinant expression vector can be a pMLT83152-Pthl vector or a pMTL82253-Pfdx vector.

[0065] In some optional embodiments, the expression product of the molecular chaperone CaDnaK has an amino acid sequence as shown in SEQ ID NO.5;

[0066] The carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H includes a *401L mutation site and a W556H mutation site, and the expression product of the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H has an amino acid sequence as shown in SEQ ID NO.6;

[0067] The expression product of the formate dehydrogenase CaFdhF has an amino acid sequence as shown in SEQ ID NO.7;

[0068] The hydrogenase mutant CaHydA2_C183D-A311L includes a C183D mutation site and an A311L mutation site, and the expression product of the hydrogenase mutant CaHydA2_C183D-A311L has an amino acid sequence as shown in SEQ ID NO.8;

[0069] In these embodiments, the expression product of the molecular chaperone CaDnaK may have the amino acid sequence shown in SEQ ID NO.5, and the expression product of the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H may include a *401L mutation site and a W556H mutation site, and the expression product of the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H may have the amino acid sequence shown in SEQ ID NO.6, and the expression product of the formate dehydrogenase CaFdhF may have the amino acid sequence shown in SEQ ID NO.7, and the hydrogenase mutant

[0070] CaHydA2_C183D-A311L may include a C183D mutation site and an A311L mutation site, and the expression product of the hydrogenase mutant CaHydA2_C183D-A311L may have an amino acid sequence as shown in SEQ ID NO.8, and a CaDnaK-CaAcsA_*401L-W556H fusion protein with oxygen tolerance and improved activity may be expressed through a first recombinant expression vector, and a CaFdhF-CaHydA2_C183D-A311L fusion protein with oxygen tolerance and improved activity may be expressed through a second recombinant expression vector, thereby improving the oxygen tolerance of the engineered strain through these two types of recombinant expression vectors.

[0071] It should be noted that these two recombinant expression vectors, while improving the oxygen tolerance of the engineered strains, can also correspondingly increase the activity of the three corresponding gas utilization key enzymes in the engineered bacteria containing the recombinant expression vectors, so as to improve the energy supply of the engineered bacteria, thereby comprehensively enhancing the growth and metabolic level of the engineered bacteria containing the recombinant expression vectors in a microaerobic environment.

[0072] Figure 3 A schematic diagram of a method for constructing a recombinant expression vector provided in an embodiment of the present application is exemplarily shown;

[0073] Based on a general inventive concept, such as Figure 3 As shown, the present application embodiment provides a method for constructing the recombinant expression vector, the method comprising:

[0074] S1. using a first amplification primer pair and a second amplification primer to amplify a first gene fragment encoding a molecular chaperone CaDnaK and a second gene fragment encoding a carbon monoxide dehydrogenase CaAcsA, respectively, to obtain a first amplification product encoding a molecular chaperone CaDnaK and a second amplification product encoding a carbon monoxide dehydrogenase CaAcsA;

[0075] S2. The first amplified product encoding the molecular chaperone CaDnaK and the second amplified product encoding the carbon monoxide dehydrogenase CaAcsA are sequentially connected to the expression vector to obtain a first construction vector containing CaDnaK-CaAcsA;

[0076] S3. constructing a mutant vector of carbon monoxide dehydrogenase CaAcsA of the first construction vector, and then screening for carbon monoxide dehydrogenase activity to obtain a first recombinant expression vector containing the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H;

[0077] S4. using the third amplification primer pair and the fourth amplification primer to amplify the third gene fragment encoding formate dehydrogenase CaFdhF and the fourth gene fragment encoding hydrogenase CaHydA2, respectively, to obtain the third amplification product encoding formate dehydrogenase CaFdhF and the fourth amplification product encoding hydrogenase CaHydA2;

[0078] S5. The third amplified product encoding formate dehydrogenase CaFdhF and the fourth amplified product encoding hydrogenase CaHydA2 are sequentially connected to the expression vector to obtain a second constructed vector containing CaFdhF-CaHydA2;

[0079] S6. constructing a mutant vector of hydrogenase CaHydA2 of the second construction vector, and then performing hydrogenase activity screening to obtain a second recombinant expression vector containing the hydrogenase mutant CaHydA2_C183D-A311L;

[0080] S7. collecting the first recombinant expression vector and the second recombinant expression vector to obtain a recombinant expression vector;

[0081] Wherein, the first amplification primer pair is used to amplify the first gene fragment, the upstream primer of the first amplification primer pair is shown as SEQ ID NO.9, and the downstream primer of the first amplification primer pair is shown as SEQ ID NO.10;

[0082] The second amplification primer pair is used to amplify the second gene fragment, the upstream primer of the second amplification primer pair is shown as SEQ ID NO.11, and the downstream primer of the second amplification primer pair is shown as SEQ ID NO.12;

[0083] The third amplification primer pair is used to amplify the third gene fragment, the upstream primer of the third amplification primer pair is shown as SEQ ID NO.13, and the downstream primer of the third amplification primer pair is shown as SEQ ID NO.14;

[0084] The fourth amplification primer pair is used to amplify the fourth gene fragment, the upstream primer of the fourth amplification primer pair is shown as SEQ ID NO.15, and the downstream primer of the fourth amplification primer pair is shown as SEQ ID NO.16.

[0085] This method is a method for constructing the above-mentioned recombinant expression vector. The specific steps of the construction method can refer to the above-mentioned embodiment. Since the recombinant expression vector adopts part or all of the technical solutions of the above-mentioned embodiment, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiment, which will not be repeated here one by one.

[0086] Figure 4A detailed schematic diagram of a method for constructing a recombinant expression vector is exemplarily shown;

[0087] In some optional embodiments, such as Figure 4 As shown, the method of constructing a mutant vector of carbon monoxide dehydrogenase CaAcsA of the first construction vector, and then screening carbon monoxide dehydrogenase activity to obtain a first recombinant expression vector containing the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H comprises the steps of:

[0088] S301. In the case where the first construction vector is used as a parent, a mutant vector of carbon monoxide dehydrogenase CaAcsA of the first construction vector is constructed using a site-directed mutagenesis method;

[0089] S302. Using a gas environment containing a first preset oxygen volume content, the mutant vector of the carbon monoxide dehydrogenase CaAcsA is screened for carbon monoxide dehydrogenase activity to obtain a first recombinant expression vector containing the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H.

[0090] In these embodiments, mutant vectors of the carbon monoxide dehydrogenase CaAcsA in the first construction vector are constructed by a site-directed mutagenesis method, and then these constructed mutant vectors are screened in a gas environment containing a first preset oxygen volume content, so that the oxygen-tolerant carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H can be screened out to improve the oxygen tolerance of the first recombinant expression vector.

[0091] In some optional embodiments, the volume content V1 of the first preset oxygen and the total gas volume V2 of the gas environment satisfy the relationship: V1:V2≤0.5%;

[0092] In these embodiments, the volume content V1 of the first preset oxygen and the total gas volume V2 of the gas environment can satisfy the relationship: V1:V2≤0.5%. The mutant vector of carbon monoxide dehydrogenase CaAcsA is screened under the conditions of the first preset oxygen to obtain a first recombinant expression vector containing the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H with oxygen tolerance.

[0093] In some optional embodiments, such as Figure 4 As shown, the construction of the second construction vector of the hydrogenase CaHydA2 mutant vector, followed by hydrogenase activity screening to obtain a second recombinant expression vector containing the hydrogenase mutant CaHydA2_C183D-A311L, comprises the steps of:

[0094] S601. In the case where the second construction vector is used as a parent, a hydrogenase mutation vector of the second construction vector is constructed using a site-directed mutagenesis method;

[0095] S602. Using a gas environment containing a second preset oxygen volume content, the mutant vector of the hydrogenase CaHydA2 is screened for hydrogenase activity to obtain a second recombinant expression vector containing the hydrogenase mutant CaHydA2_C183D-A311L.

[0096] In these embodiments, a mutant vector of the hydrogenase CaHydA2 in the second construction vector is constructed by a site-directed mutagenesis method, and then these constructed mutant vectors are screened in a gas environment containing a second preset oxygen volume content, and the oxygen-tolerant hydrogenase mutant CaHydA2_C183D-A311L can be screened out to improve the oxygen tolerance of the second recombinant expression vector.

[0097] In some optional embodiments, the volume content V3 of the second preset oxygen and the total gas volume V2 of the gas environment satisfy the relationship: V3:V2≤0.5%;

[0098] In these embodiments, the volume content V3 of the second preset oxygen and the total gas volume V2 of the gas environment can satisfy the relationship: V3:V2≤0.5%. The mutant vector of the hydrogenase CaHydA2 is screened under the second preset oxygen condition to obtain a second recombinant expression vector containing the hydrogenase mutant CaHydA2_C183D-A311L with oxygen tolerance.

[0099] Based on a general inventive concept, an embodiment of the present application provides an engineered bacterium of Clostridium aerovorum with micro-oxygen tolerance, wherein the engineered bacterium of Clostridium aerovorum includes the recombinant expression vector or the recombinant expression vector obtained by the method.

[0100] The engineered bacteria is realized based on the above-mentioned recombinant expression vector or the above-mentioned method. The specific composition of the recombinant expression vector or the specific steps of the method can refer to the above-mentioned embodiments. Since the engineered bacteria adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here one by one.

[0101] In some optional embodiments, the Clostridium aerovorum comprises at least one of the following:

[0102] Clostridium alcoholicum, Clostridium ljungdahlii, Clostridium carboxyvorans, Clostridium kluyveri, and Clostridium ragsdalei;

[0103] In these embodiments, the aerotrophic Clostridium may include at least one of: ethanolic Clostridium, young's Clostridium, carbon monoxide-eating Clostridium, kluyveromyces and ragsdalei Clostridium, which may cover most of the aerotrophic Clostridium, thereby enabling the first recombinant expression vector and the second recombinant expression vector to enter the engineered bacteria.

[0104] Figure 5 A schematic diagram of a method for constructing an engineered bacterium of Clostridium aerovorum provided in an embodiment of the present application is shown as an example;

[0105] Based on a general inventive concept, such as Figure 5 As shown, the present application embodiment provides a method for constructing the engineered bacteria of Clostridium aerovorum, the method comprising:

[0106] S1. Synchronously introducing the first recombinant expression vector and the second recombinant expression vector in the recombinant expression vector into Clostridium aeruginosa to obtain engineered Clostridium aeruginosa;

[0107] S2. The engineered Clostridium aerovorum bacteria are verified by sequencing positive clones to obtain the engineered Clostridium aerovorum bacteria that co-express the first recombinant expression vector and the second recombinant expression vector.

[0108] This method is a method for constructing the above-mentioned engineered bacteria. The specific composition of the engineered bacteria can refer to the above-mentioned embodiment. Since this method adopts part or all of the technical solutions of the above-mentioned embodiment, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiment, which will not be repeated here one by one.

[0109] Based on a general inventive concept, an embodiment of the present application provides an application of an engineered Clostridium aerovorum with micro-oxygen tolerance, wherein the engineered Clostridium aerovorum is applied to anaerobic syngas fermentation under an atmosphere containing oxygen with a volume concentration of ≤0.5%.

[0110] This application is realized based on the above-mentioned engineered Clostridium aerovorum bacteria. The specific composition of the engineered Clostridium aerovorum bacteria can refer to the above-mentioned embodiments. Since this application adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here one by one.

[0111] The present application is further described below in conjunction with specific examples. The experimental methods in the following examples that do not specify specific conditions are usually measured according to industry standards; if there are no corresponding industry standards, they are carried out according to common international standards, conventional conditions, or conditions recommended by the manufacturer.

[0112] Example 1

[0113] 1. Construction of the first recombinant expression vector:

[0114] The genome of ethanolic Clostridium was extracted, and the first primer pair and the second primer pair were designed to amplify the molecular chaperone CaDnaK (CAETHG_2891) gene fragment and the carbon monoxide dehydrogenase CaAcsA (CAETHG_1621-1620) gene fragment, respectively, and they were sequentially connected to the pMLT83152-Pthl vector using the seamless cloning method. The constructed vector was transformed into Escherichia coli Top10 competent cells, and then verified by sequencing of positive clones to obtain the recombinant tandem expression vector pMLT83152-Pthl-CaDnaK-CaAcsA (the first constructed vector).

[0115] The recombinant tandem expression vector pMLT83152-Pthl-CaDnaK-CaAcsA was used as the parent, and the mutant vector of carbon monoxide dehydrogenase CaAcsA was constructed using the site-directed mutagenesis method, and the positive rate was detected using the colony PCR method. The mutant was transformed into Escherichia coli BL21 (DE3) competent cells, and the induced expression of the recombinant Escherichia coli was based on the strategy of micro-oxygen (oxygen content of 0.5%) and low temperature (temperature of 16°C). After the induction, the cells were collected, the cell wall was broken to obtain the crude protein solution, and the carbon monoxide dehydrogenase activity was tested under micro-oxygen conditions (oxygen content of 0.5%) to screen for mutants with excellent performance.

[0116] pMLT83152-Pthl-CaDnaK-CaAcsA_*401L-W556H (the first recombinant expression vector), wherein the gene fragment of the molecular chaperone CaDnaK is:

[0117] ATGTCAAAAATAATAGGTATTGATTTAGGAACAACTAATTCATGTGTTGCAGTTATGGAAGGTGG

[0118] AGATCCTGCAGTTATAGCAAATTCAGAAGGAGCAAGAACAACTCCATCAGTAGTATCATTCCAGG

[0119] CAAATGGAGAAAGATTGGTAGGTCAAGTTGCCAAAAGACAGGCAATAACAAATCCTGATAAGAC

[0120] AATAATGTCAATAAAAAGGCAAATGGGAACAGACCATAAAGTAAATATAGATGGAAAAGATTAT

[0121] ACACCACAGGAGATATCTGCGATGATACTCCAAAAAATAAAAGCAGATGCTGAAGCTTATTTAG

[0122] GAGAAACTGTAACTGAAGCAGTTATAACAGTACCAGCATATTTTAACGATAGTCAGAGACAGGC

[0123] AACTAAAGATGCAGGTAAGATTGCAGGATTAAATGTACGTAGAATAATAAATGAACCAACAGCT

[0124] GCATCACTTGCTTATGGACTTGATAAAACTGATACAAGTCAAAAGATATTTGTATATGACTTAGGT

[0125] GGAGGTACTTTTGATGTATCCATCTAGAACTTGGAGATGGAGTATTTGAAGTTAAAGCTACAAA

[0126] TGGTGATACTCATCTAGGTGGGAGATGACTTTGACCAGAAAGTTATGGACTATATAGCAGAAGATT

[0127] TCAAAGCTAAGAATGGTATAGATTTAAGAAATGACAAAATGGCACTTCAAAGATTAAAGGAAGC

[0128] AGCTGAAAGCAAAAATTGAACTTTCGGCATCTACTCAAACAAATATAAACTTACCATTTATTA

[0129] CAGCAGATGCAACTGGTCCAAAACATATAGATATGAATTTGACAAGAGCAAAATTTAATGAGTTG

[0130] ACTCAAGATCTAGTTGAAAGAACAATTGAACCTATGAGAAAGCATTAAATGATGCAGGACTTA

[0131] CAATAAATGATATAAATAAGATCATATTAGTTGGTGGTTCTACAAGAATACCAGCTGTTCAGGAA

[0132] GCAGTTAAGAATTTACTGGTAAAGATCCATCAAAGGGAGTTAACCCTGATGAATGTGTAGCTGT

[0133] AGGGGCTGCAATTCAGGCCGGAGTTTTAACTGGAGATGTAAAAGACGTATTACTCCTTGATGTTA

[0134] CACCTCTTACACTTGGAATTGAAACTTTAGGAGGAGTTGCCACTCCACTTATTGATAGAAATACTA

[0135] CAGTACCAACTAAGAAGAGTCAGGTATTTTCAACTGCAGCAGAATGGCCAGACTTCAGTTGAAATT

[0136] CATGTAGTTCAAGGTGAAAGAAAGATGGCTGCTGATAATAAAACTCTTGGAAGATTTACGCTTTC

[0137] AGGAATAGCTCCAGCTCCAAGGGGAATTCCTCAAATTGAAGTTACATTTGACATAGATGCCAACG

[0138] GTATAGTAAATGTATCTGCTAAAGATAAAGGAACAGGAAGAAGCTAATATAACAATTACAGC

[0139] TTCAACTAATTTAAGCGATGATGAAATAAACAAGGCAGTAGATGAAGCTAAAAGTTTGAAGAA

[0140] CAGGATAAAGAGAGAAGAATCCATAGACATAAAAAATAATGCAGATCAATCTGTATATCAGA

[0141] CAGAAAAGACATTAAAGGACTTAGGAGATAAAGTATCAGCTGAAGATAAGAAAACTGTAGAGGA

[0142] AAAAATTGAAGCTTTAAAGAAGATAAAAGATGGAGAAGATTTAGAGGCAATAAAGAAAGCTACT

[0143] GAAGATTTAACTCAAACTTTCTATGGAATTACATCTAAAATATATAGTCAGAATGCTCAAGCAGG

[0144] ACAAAATCCAGGAGCAGATCCAAATATGGGAGCAGGACAAAATCCAGGGGCAGGAGCAGGTTCT

[0145] CAAGGTGCATCAGAAAAAAAAGATGATAATGTAGTTGATGCGGATTACAAAGTAGATGATGATAAATAA(SEQ ID NO.1);

[0146] The amino acid sequence of the expression product of molecular chaperone CaDnaK is:

[0147] MSKIIGIDLGTTNSCVAVMEGGDPAVIANSEGARTTPSVVSFQANGERLVGQVAKRQAITNPDKTIMSI

[0148] KRQMGTDHKVNIDGKDYTPQEISAMILQKIKADAEAYLGETVTEAVITVPAYFNDSQRQATKDAGKI

[0149] AGLNVRRIINEPTAASLAYGLDKTDTSQKIFVYDLGGGTFDVSILELGDGVFEVKATNGDTHLGGDDF

[0150] DQKVMDYIAEDFKAKNGIDLRNDKMALQRLKEAAEKAKIELSASTQTNINLPFITADATGPKHIDMNL

[0151] TRAKFNELTQDLVERTIEPMRKALNDAGLTINDINKIILVGGSTRIPAVQEAVKNFTGKDPSKGVNPDE

[0152] CVAVGAAIQAGVLTGDVKDVLLLDVTPLTLGIETLGGVATPLIDRNTTVPTKKSQVFSTAADGQTSVEI

[0153] HVVQGERKMAADNKTLGRFTLSGIAPAPRGIPQIEVTFDIDANGIVNVSAKDKGTGKEANITITASTNLS

[0154] DDEINKAVDEAKKFEEQDKKRKESIDIKNNADQSVYQTEKTLKDLGDKVSAEDKKTVEEKIEALKKIK

[0155] DGEDLEAIKKATEDLTQTFYGITSKIYSQNAQAGQNPGADPNMGAGQNPGAGAGSQGASEKKDDNVVDADYKVDDDK(SEQ ID NO.5).

[0156] The gene fragment of the screened carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H is:

[0157] ATGGAAGAAAAAGCAAAATCAATTGATCAGGCTACTTTACAATTATTGGACAAGGCAAAACA

[0158] AGATGGGGTCGAAACAGTTTGGGATAGAAAAGCAGACATGAAGGTACAGTGTGGATTTGGATCA

[0159] GCAGGAGTTTGCTGTAGAAATTGCAGCATGGGCCCATGTAGAGTAAGTCCAGTGCCAGGAAAAG

[0160] GTGTAGAAAGAGGTATATGTGGAGCTACAGCAGATGTAATTGTATCTAGAAATTTTGCAAGAATGG

[0161] TTGCAGCAGGTACTGCAGCACACTCAGATCATGGTAGAAGTATAGCACTTAGCTTGTATCACACTA

[0162] GTAAAGATGGAGATATAAAAGTTAAAGATGAAAATAAATTGAAAGAAGTTGCAAAGAGCTTTAAT

[0163] GTTGAAACTGAGGGAAGAGATATATATGACATAGCTCATGATGTAGCAAAAGAAGGATTAAGTAAT

[0164] TATGGTAAACAGCTTGGAGAAGTTACTTTACCACCTTCTTTACCAGAAAAGAGAAAAGAATTGTG

[0165] GAGAAAATTAGGTGTATATCCAAGGGCAGTTGATAGAGAAATAGCTGCAGTTATGCATTCAACACA

[0166] TATAGGATGTAATGCAGATGCAGAAGCTATGATTAAAATGTCTATGAGATGTTCACTAACTGATGGA

[0167] TGGATGGGCTCATTCATGGGAACAGAATTCAGTGATATAATGTTTGGAACACCTCATTCCATTGATA

[0168] CAGAGGCAAATCTTGGAGTACTTGAAAAGAATTCTGTAAATGTAGTTTTACACGGACATGAACCA

[0169] CTTCTTTCAGAAATGGTAGTAGAAGCAGCATCAGATCCAGAGTTAGTTGAGTTGAACTTGCTAAATCAGT

[0170] AGGTGCTGATGGAATAAATTTATGTGGAATGTGCTGTACTGGAAATGAAGTTTCCATGAGACATGG

[0171] CATCAAAATAGCAGGAAACTTTATGCAGCAGGAATTGGCTGTAGTTACAGGAGCAGTAGATGGAC

[0172] TTATAGTTGATGTACAGTGTATTATGCCAGCACTAGCAAAATTGTCCAAGTCATATCATACTAAGTTT

[0173] ATAACAACTTCACCAAAGGCACACATCACAGATTCAATTTATATGGAATTTGATGAAGAAAACCCA

[0174] CTTGATTCAGCTAAGAAGATTCTAAGAGAAGCAATATTAAACTTTAAAAATAGAGATCAGAGCAA

[0175] AGTAATGATTCCTGAATTGAAA TTAAAGGCAATT TTGGGATACAGTGTTGAAGAAATTATAAATAA

[0176] ATTAGACAAGGTTGTAAATACACAAATAGGACCAATGCAAACTGTAAAACCTTTGGCAGATGTTTT

[0177] AGTATCAGGAGTATTAAGAGGTGCTGCAGCTGTGGTTGGATGTAATAATCCTAAAGTTGTACAAGA

[0178] TTCTGCACACATTGAAACTATAAAAGGATTAATAAAAAATGATGTAATTGTTGTTGTTACAGGTTGT

[0179] GCAGCTCAAGCAGCAGCAAAATATGGCTTATTACAAAAGAAGCAGCAGAAAAATATGCAGGAC

[0180] CAGGACTAGCTACTGTATGTAAACTTGTAGACATACCACCTGTACTTCATATGGGTTCTTGTGTTGA

[0181] TATAAGTCGTATATTAGATTTGGTTGGAAGAGTGGCTAATTTATTGGGCGTTGACATGAGTGACCTT

[0182] CCAGTTGCAGGTGTAGCACCTGAACATATGTCAGAAAAAGCCGTAGCAATAGGTACTTATGTAGT

[0183] AACTTCAGGTATAGATACTTGGCTTGGAGTAGCACCTCCAGTAACAGGCGGCCCAGAAGTTGTTG

[0184] ACATTCTTACTAATAAGATGGAAGACTGGGTAGGAGCTAAATTCTTTATAGAAACAGATCCTCATAAAGCAGTTGAACAAATTGTAAATAGGATGAATGAAAAACGTAAAAAAATTAGGTATCTAA(SEQ ID NO.2);

[0185] In the gene fragment of the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H, the bases marked with horizontal lines are the mutation sites of carbon monoxide dehydrogenase CaAcsA*401L, the bases marked with wavy lines are the mutation sites of carbon monoxide dehydrogenase CaAcsAW556H, and the bases marked with double horizontal lines are the coding sequence of the short peptide connecting carbon monoxide dehydrogenase CaAcsA CAETHG_1621 and CAETHG_1620.

[0186] The amino acid sequence of the expression product of the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H is: MEEKAKSIDQATLQLLDKAKQDGVETVWDRKADMKVQCGFGSAGVCCRNCSMGPCRVSPVPGKGVERGICGATADVIVSRNFARMVAAGTAAHSDHGRSIALSLYHTSKDGDIKVKDENKLKEVAKSFNVETEGRDIYDIAHDVAKEGLSNYGKQLGEVTLPPSLPEKRKELWRKLGVY PRAVDREIAAVMHSTHIGCNADAEAMIKMSMRCSLTDGWMGSFMGTEFSDIMFGTPHSIDTEANLGVLEKNSVNVVLHGHEPLLSEMVVEAASDPELVELAKSVGADGIN LCGMCCTGNEVSMRHGIKIAGNFMQQELAVVTGAVDGLIVDVQCIMPALAKLSKSYHTKFITTSPKAHITDSIYMEFDEENPLDSAKKILKEAILNFKNRDQSKVMIPELK LKAI MGYSVEEIINKLDKVVNTQIGPMQTVKPLADVLVSGVLRGAAAVVGCNNPKVVQDSAHIETIKGLIKNDVIVVVTGCAAQAAAKYGLLQKEAAEKYAGPGLATVCKLVDIPPVLHMGSCVDISRILDLVGRVANLLGVDMSDLPVAGVAPE H MSEKAVAIGTYVVTSGIDTWLGVAPPVTGGPEVVDILTNKMEDWVGAKFFIETDPHKAVEQIVNRMNEKRKKLGI (SEQ ID NO. 6).

[0187] In the expression product of the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H, the amino acids marked with horizontal lines are the mutation sites of carbon monoxide dehydrogenase CaAcsA*401L, the amino acids marked with wavy lines are the mutation sites of carbon monoxide dehydrogenase CaAcsAW556H, and the amino acids marked with double horizontal lines are the short peptide connecting carbon monoxide dehydrogenase CaAcsACAETHG_1621 and CAETHG_1620.

[0188] The upstream primer of the first primer pair used to amplify the molecular chaperone CaDnaK of Clostridium alcoholicum is:

[0189] 5'-AAATTTAGGAGGTTAGTTCAATGTCAAAAATAATAGGTATTGATTTAGGAAC-3' (SEQ IDNO.9);

[0190] The downstream primer of the first primer pair is:

[0191] 5'-CAATTGATTTTGCTTTTTCTCCATTTATTTATCATCATCTACTTTGTAATCCG-3' (SEQ ID NO. 10).

[0192] The upstream primer of the second primer pair used to amplify the carbon monoxide dehydrogenase CaAcsA of Clostridium alcoholicum is:

[0193] 5'-CGGATTACAAAGTAGATGATGATAAATAAATGGAAGAAAAAGCAAAATCAATT G-3' (SEQ ID NO. 11);

[0194] The downstream primer of the second primer pair is:

[0195] 5'-AATTCGTAATCATGGTCATATTAGATACCTAATTTTTTACGTTTTTCATTC-3' (SEQ ID NO. 12).

[0196] Example 2

[0197] 2. Construction of the Second Recombinant Vector:

[0198] The genome of ethanolic Clostridium was extracted, and the second and third primer pairs were designed to amplify the formate dehydrogenase CaFdhF (CAETHG_2790) gene fragment and the hydrogenase CaHydA2 (CAETHG_2798) gene fragment, respectively, and they were sequentially connected to the pMTL82253-Pfdx vector using the seamless cloning method. The constructed vector was transformed into Escherichia coli Top10 competent cells, and then verified by sequencing the positive clones to obtain the recombinant tandem expression vector.

[0199] pMTL82253-Pfdx-CaFdhF-CaHydA2 (second construction vector).

[0200]

[0201] The amino acid sequence of the expression product of formate dehydrogenase CaFdhF is:

[0202] MDKKVLTVCPYCGAGCNLYLHVKNGKIIKAEPANGRTNEGSLCLKGHFGWDFLNDPKILTSRIKHPMI

[0203] RKNGELEEVSWDEAISFTASRLSQIKEKYGPDSIMGTGCARGSGNEANYIMQKFMRAVIGTNNVDHC

[0204] ARVUHAPSVAGLAYVLGNGAMSNGIHEIDDTDLLLIFGYNGAASHPIVAKRIVRAKQKGAKVIVVDPR

[0205] ITESGRIADLWLPIKNGTNMVLVNTFANILINKQFYNKQYVEDHTVGFEEYRSIVENYTPEYAEKVTGIP

[0206] SEDIVEAMKMYSGAKNAMILYGMGVCQFAQAVDVVKGLASIALLTGNFGRPNVGIGPVRGQNNVQG

[0207] ACDMGALPNVYPGYQSVTDDAIRQKFEKAWGVKLPNKVGYHLTQVPELTLKEDKIKAYYIMGEDPV

[0208] QSDPDSNEMRETLDKMELVIVQDIFMNKTALHADVILPSTSWGEHEGVFSSADRGFQRFRKAVEPKG

[0209] DVKPDWEIISKIACAMGYNMHYNNTEEIWNELINLCPNFKGATYKRLEELGGIQWPPCSENHPGTSYL

[0210] YKGNKFNTPTGKANLFAAEWRPPVEQTDKDYPLVLSTVREVGHYSVRTMTGNCRALQQLADEPGYV

[0211] QVNPMDAKAKGIIDGELMRISSRRGSVVARALITERANKGAVYMTYQWWVGACNELTSNNLDPVSKTPELKYCAVKIEAIKDQKEAEKFIKDQYDLLKKKMNV(SEQ ID NO.7).

[0212] The gene fragment of the screened hydrogenase mutant CaHydA2_C183D-A311L is: ATGCCAACTAGTACTTCTATGATAAATATAGATGAAGAATTATGTACAGGCTGCAGACGATGTGCGGATGTCTGCCCTGTAGATGCTATAGAAGGTGAACAGGGTAAACCTCAGAAGATAAATACTGAAAAGTGTGTTATGTGCGGACAATGCATTCAAGTTTGTAAAGGCTATCAATCTGTATACGACGATGTTCCTACTCCAGTTAGCAAAAGGTTATTTGATAGAGGATTGTTAAAGGAAGTAGATGAACCATTATTTGCAGCATATAATAAAGGTCAGGTAAAGAGTGTTAAAGAAATTTTACAAAACAAAGATGTATTTAAAATTGTGCAATGTGCACCTGCTGTAAGAGTTGCTATAGGAGAGGATTTTGGAATGCCTCTTGGAACTTTAAGTGAAGGAAAAATGGCAGCTGCACTCAGAAAATTAGGATTTGACAAAGTATATGATACAAACTTTGGTGCAGATCTTACTATAATGGAAGAAGGTAGTGAGTTACTAAAAAGAGTAGCTGAAGGCGGAGTTTTGCCAATGTTTACTTCT GAT(SEQ ID NO.4).

[0213] In the gene fragment of the hydrogenase mutant CaHydA2_C183D-A311L, the bases marked with horizontal lines are the mutation sites of CaHydA2C183D, and the bases marked with wavy shapes are the mutation sites of CaHydA2 A311L.

[0214] The amino acid sequence of the expression product of the hydrogenase mutant CaHydA2_C183D-A311L is:

[0215] MPTSTSMINIDEELCTGCRRCADVCPVDAIEGEQGKPQKINTEKCVMCGQCIQVCKGYQSVYDD

[0216] VPTPVSKRLFDRGLLKEVDEPLFAAYNKGQVKSVKEILQNKDVFKIVQCAPAVRVAIGEDFGMPLGTL

[0217] SEGKMAAALRKLGFDKVYDTNFGADLTIMEEGSELLKRVAEGGVLPMFTS D CPAWVKYAEQTYPEL

[0218] LPHLSSCKSPNQMAGAIFKTYGAEINKVNPAKIYNVSVMPTCKEFESEREEMHDSGHRDVDAVITTR

[0219] ELAQLFKDADIDFNTIEEEQFDTPLGMYTGAGTIFGATGGVMEALLRTGYELYTKKTIPSIDLTMVRG

[0220] GEGFRTAEVDLGDIRLKVGVVSGLKNVKDVMESVKAGKCDLHFIEVMTCPQGCISGGGQPKVILDSDKEEAYNNRKKGLYNHDANLTYRKSHENPEIKKIYDEFLDKPLGAKSHELLHTKYISRKKES (SEQ ID NO. 8).

[0221] In the expression product of the hydrogenase mutant CaHydA2_C183D-A311L, the amino acid marked with a horizontal line is the hydrogenase CaHydA2 C183D mutation site, and the amino acid marked with a wavy line is the hydrogenase CaHydA2 A311L mutation site.

[0222] The upstream primer of the third primer pair used to amplify the formate dehydrogenase CaFdhF of Clostridium alcoholicum is:

[0223] 5'-TTTTAAGGAGGTGTGTTACAATGGATAAAAAAGTTTTAACTGTTTGTC-3' (SEQ ID NO. 13);

[0224] The downstream primer of the third primer pair is:

[0225] 5'-ATATTTATCATAGAAGTACTAGTTGGCATTTAAACATTCATCTTTTCTTTAAAA GATC-3' (SEQ ID NO. 14).

[0226] The upstream primer of the fourth primer pair used to amplify the Clostridium alcoholicum hydrogenase CaHydA2 is:

[0227] 5'-GATCTTTTAAAGAAAAAGATGAATGTTTAAATGCCAACTAGTACTTCTATGATA AATAT-3' (SEQ ID NO. 15);

[0228] The downstream primer of the fourth primer pair is:

[0229] 5'-AATTCGTAATCATGGTCATATTAACTCTCCTTTTTTCTTGAGATATATTTAG-3'

[0230] (SEQ ID NO.16).

[0231] Example 3

[0232] 3. Construction of wild strain DSM13528 of Clostridium ljungdahlii co-expressing CaDnaK-CaAcsA_*401L-W556H and CaFdhF-CaHydA2_C183D-A311L:

[0233] Wild strain Clostridium ljungdahlii DSM13528 was purchased from German Collection of Microorganisms and Cell Cultures DSMZ (https: / / www.dsmz.de / collection / catalogue / details / culture / DSM-13528).

[0234] The recombinant tandem expression vector pMLT83152-Pthl-CaDnaK-CaAcsA_*401L-W556H and

[0235] pMTL82253-Pfdx-CaFdhF-CaHydA2_C183D-A311L was simultaneously introduced into Clostridium ljungdahlii DSM13528, and the specific steps were as follows:

[0236] The recombinant tandem expression vectors pMLT83152-Pthl-CaDnaK-CaAcsA_*401L-W556H and pMTL82253-Pfdx-CaFdhF-CaHydA2_C183D-A311L were transformed into Escherichia coli sExpress competent cells, and then verified by positive clone sequencing to obtain Escherichia coli sExpress donor bacteria overexpressing the recombinant tandem expression vectors pMLT83152-Pthl-CaDnaK-CaAcsA_*401L-W556H and pMTL82253-Pfdx-CaFdhF-CaHydA2_C183D-A311L.

[0237] Take the frozen wild Clostridium ljungdahlii DSM13528 and quickly place it in an anaerobic operation box, inoculate it into YTF liquid culture medium without antibiotics at an inoculum size of 10% (the formula of YTF culture medium is: 1.0 g ammonium chloride, 0.2 g magnesium sulfate heptahydrate, 0.8 g sodium chloride, 0.1 g potassium dihydrogen phosphate, 0.02 g calcium chloride dihydrate, 10 g yeast extract powder, 16 g peptone, 10 g fructose, 0.61 g potassium sulfate, 0.3 g L-cysteine ​​hydrochloride, 0.50 mL resazurin sodium (0.1% w / v), then adjust the pH to 5.5 with phosphoric acid and make the volume to 1 L), and place it in a 37°C shaker for anaerobic shaking culture.

[0238] At the same time, take LB liquid culture medium and add the prepared chloramphenicol and kanamycin respectively, shake well and divide into shaking tubes. Inoculate the E. coli sExpress donor bacteria containing pMLT83152-Pthl-CaDnaK-CaAcsA_*401L-W556H into LB liquid culture medium containing chloramphenicol and kanamycin, place it in a 37°C shaker for shaking culture overnight. When the OD600 of the overnight cultured bacteria reaches 0.2-0.4, take 1mL of E. coli sExpress donor bacteria and centrifuge at 3000g for 3min, discard the supernatant and wash the precipitate twice with 500μL PBS buffer, centrifuge and discard the supernatant to obtain donor cells, and transfer them to an anaerobic operation box. Take the overnight cultured Clostridium youngdensii wild bacteria DSM13528 bacterial solution and quickly transfer it to an anaerobic operation box, resuspend the donor cells with 200μL of Clostridium youngdensii wild bacteria DSM13528 bacterial solution, and mix them upside down. The cell mixture was plated on YTF solid medium plates without antibiotics and anaerobically conjugated at 37°C overnight.

[0239] On the second day, the mixed bacteria were eluted from the overnight conjugation plate with 500 μL of PBS buffer and the transduction conjugates were collected. The transduction conjugates were coated on YTF solid culture medium plates containing thiamphenicol and D-cycloserine and cultured anaerobically at 37°C for 72 hours. Single clones were picked from the transformants transferred by conjugation of pMLT83152-Pthl-CaDnaK-CaAcsA_*401L-W556H Escherichia coli sExpress and Clostridium ljungdahlii DSM13528, and cultured anaerobically at 37°C for 72 hours using liquid YTF culture medium. Positive clones were screened for sequencing to obtain engineered Clostridium ljungdahlii DSM13528 bacteria overexpressing CaDnaK-CaAcsA_*401L-W556H. Using the engineered strain of Clostridium ljungdahlii DSM13528 overexpressing CaDnaK-CaAcsA_*401L-W556H as the starting strain, the above conjugation transfer process was repeated to introduce pMTL82253-Pfdx-CaFdhF-CaHydA2_C183D-A311L. The positive clones were sequenced and verified to finally obtain the engineered strain of Clostridium ljungdahlii DSM13528 co-overexpressing CaDnaK-CaAcsA_*401L-W556H and CaFdhF-CaHydA2_C183D-A311L.

[0240] Example 4

[0241] 4. Test of oxygen tolerance of engineered bacteria Clostridium youngdae DSM13528:

[0242] The engineered bacteria of Clostridium ljungdahlii DSM13528 constructed in Example 3 were inoculated into YTF liquid culture medium containing antibiotics at a 10% inoculation rate and placed in a 37°C shaker for anaerobic shaking culture. The bacterial solution after 24 hours of culture was gradiently diluted in an anaerobic operation box and coated with YTF solid plates containing antibiotics. The coated plates were taken out of the anaerobic operation box and placed in a 37°C incubator. After being exposed to air for 10 minutes and 30 minutes, they were returned to the anaerobic operation box for culture, and the growth of colonies on each plate was observed and counted.

[0243] The results are as follows Figure 6As shown, the introduction of CaDnaK-CaAcsA_*401L-W556H and CaFdhF-CaHydA2_C183D-A311L into Clostridium ljungdahlii can comprehensively improve the survival of the strain in a microaerobic fermentation environment. Compared with the strain not exposed to air, the survival rate of the engineered bacteria was above 35% after 10min and 30min of aerobic exposure. The oxygen tolerance test showed that the engineered bacteria of Clostridium ljungdahlii DSM13528 co-overexpressing CaDnaK-CaAcsA_*401L-W556H and CaFdhF-CaHydA2_C183D-A311L had a certain tolerance to oxygen, and its survival rate was greatly improved. After exposure to oxygen, the engineered bacteria Clostridium ljungdahlii DSM13528 co-overexpressing CaDnaK-CaAcsA_*401L-W556H and CaFdhF-CaHydA2_C183D-A311L can continue to grow when encountering suitable growth conditions.

[0244] Example 5

[0245] 5. Syngas fermentation test of Clostridium ljungdahlii DSM13528 engineered bacteria under microaerobic conditions:

[0246] A shake flask syngas fermentation test was carried out under microaerobic conditions using Clostridium ljungdahlii DSM13528 engineered bacteria co-overexpressing CaDnaK-CaAcsA_*401L-W556H and CaFdhF-CaHydA2_C183D-A311L.

[0247] The fermentation experiment was carried out in a 200 mL serum bottle containing 50 mL of culture medium, and the serum bottle was sealed with a rubber stopper. Wild strain DSM13528 of Clostridium ljungdahlii and co-overexpressing CaDnaK-CaAcsA_*401L-W556H and

[0248] The engineered bacteria of CaFdhF-CaHydA2_C183D-A311L were cultured in liquid YTF medium, and when the bacterial concentration reached 0.5 g / L, they were transferred to 50 mL of syngas fermentation medium according to 10% inoculation amount (the syngas fermentation medium contained 1.0 g of ammonium chloride, 0.61 g of potassium sulfate, 0.3 g of L-cysteine ​​hydrochloride, 0.50 mL of resazurin sodium (0.1% w / v), 10 mL of modified Wolin's mineral salts, 1 mL of Wolin's vitamin solution (10×), pH was adjusted to 5.5, and the volume was adjusted to 1 L. The modified Wolin's mineral salts contained 1.5 g of nitrilotriacetic acid, 3.0 g of magnesium sulfate heptahydrate, 0.5 g of manganese sulfate, 1.0 g of sodium chloride, 0.1 g of ferrous sulfate heptahydrate, 0.18 g of cobalt sulfate heptahydrate, and 0.2 g of chlorine dihydrate). 0.1 g calcium chloride, 0.18 g zinc sulfate heptahydrate, 0.01 g copper sulfate pentahydrate, 0.02 g potassium aluminum sulfate dodecahydrate, 0.01 g boric acid, 0.01 g sodium molybdate dihydrate, 0.03 g nickel chloride hexahydrate, 0.30 mg sodium selenate pentahydrate, 0.40 mg sodium tungstate dihydrate, adjust pH to 5.5 with potassium hydroxide, and make up to 1 L; Wolin's vitamin solution (10×) contains: biotin 20.00 mg, folic acid 20.00 mg, pyridoxine hydrochloride 100.00 mg, thiamine hydrochloride 50.00 mg, riboflavin 50.00 mg, niacin 50.00 mg, D-calcium pantothenate 50.00 mg, vitamin B12 1.00 mg, p-aminobenzoic acid 50.00 mg, (DL)-α-lipoic acid 50.00 mg, make up to 1 L). A mixed gas containing 50% CO, 10% CO2, 30% H2, 9.5% N2 and 0.5% O2 was simultaneously pressurized and sealed in a 200 mL serum bottle, and the gas in the bottle was replaced every 24 hours. The anaerobic bottle was placed in a 37°C shaker for anaerobic shaking culture, and the OD600 was sampled every 24 hours to calculate the biomass of the strain. After 72 hours of fermentation, the levels of metabolites (ethanol and acetic acid) were sampled and tested.

[0249] The results are as follows Figure 7 As shown, the test results were compared with those of the wild strain of Clostridium ljungdahlii DSM13528. The engineered bacteria of Clostridium ljungdahlii DSM13528 co-overexpressing CaDnaK-CaAcsA_*401L-W556H and CaFdhF-CaHydA2_C183D-A311L can efficiently convert CO, CO2 and H2 in the syngas into acetic acid and ethanol in the presence of O2 with a volume fraction of up to 0.5%, and maintain a high growth rate.

[0250] In summary, a recombinant expression vector provided in an embodiment of the present application is split into a first recombinant expression vector and a second recombinant expression vector, and the first recombinant expression vector and the second recombinant expression vector include multiple functional elements derived from ethanol Clostridium; since ethanol Clostridium is the strain currently used in industrial syngas fermentation and has undergone a long period of stress-resistant evolution under an industrial fermentation environment, its related functional elements will show better effects under adverse fermentation environments. Among these functional elements, the molecular chaperone CaDnaK can improve the tolerance of engineered Clostridium aerogenes to oxygen stress, so as to assist in the correct folding of the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H protein; and the carbon monoxide dehydrogenase CaAcsA and hydrogenase CaHydA2 obtained by protein engineering and enzyme activity test screening in a microaerobic environment further improve the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H and hydrogenase mutant CaHydA2_C183D-A311L, respectively. The tolerance of dehydrogenase and hydrogenase to oxygen and the catalytic activity in a micro-oxygen environment; then the formate dehydrogenase CaFdhF and the hydrogenase mutant CaHydA2_C183D-A311L are expressed in tandem to construct a second recombinant expression vector, and the second recombinant expression vector can improve the expression of formate dehydrogenase CaFdhF while shortening the distance of electron transfer of the hydrogenase mutant CaHydA2_C183D-A311L, thereby improving the energy metabolism level of the engineered Clostridium aerovorum bacteria, so as to enhance the growth and metabolic activity of the engineered Clostridium aerovorum bacteria. Therefore, these functional elements in the first recombinant expression vector and the second recombinant expression vector can comprehensively improve the tolerance of the engineered Clostridium aerovorum bacteria to oxygen and the conversion efficiency of the syngas fermentation gas substrate, thereby improving the yield and production efficiency of the fermentation product.

[0251] In addition, an embodiment of the present application provides an engineered bacterium of Clostridium aerovorum with micro-oxygen tolerance. The engineered bacterium of Clostridium aerovorum can tolerate oxygen at a volume concentration of up to 0.5%. Therefore, the engineered bacterium is used for synthesis gas fermentation, which can improve the survival of the bacteria in a micro-aerobic fermentation environment, reduce the cost of oxygen pretreatment in biomass gas or industrial synthesis gas and the need for adding inorganic or organic deoxygenators to the fermentation medium, reduce the cost of industrial fermentation deoxygenation, and ultimately increase the income from synthesis gas fermentation.

[0252] The above is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features applied for by the present application.

Claims

1. A recombinant expression vector, characterized in that: The recombinant expression vector comprises a first recombinant expression vector and a second recombinant expression vector, wherein the first recombinant expression vector and the second recombinant expression vector comprise a plurality of functional elements derived from ethanolic Clostridium, wherein the functional elements of the first recombinant expression vector comprise a tandemly expressed molecular chaperone CaDnaK and a carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H; and the functional elements of the second recombinant expression vector comprise a tandemly expressed formate dehydrogenase CaFdhF and a hydrogenase mutant CaHydA2_C183D-A311L; The gene fragment encoding the molecular chaperone CaDnaK has a nucleotide sequence as shown in SEQ ID NO.1; The gene fragment encoding the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H has a nucleotide sequence as shown in SEQ ID NO.2; The gene fragment encoding the formate dehydrogenase CaFdhF has a nucleotide sequence as shown in SEQ ID NO.3; The gene fragment encoding the hydrogenase mutant CaHydA2_C183D-A311L has a nucleotide sequence as shown in SEQ ID NO.

4.

2. The recombinant expression vector according to claim 1, characterized in that The expression product of the molecular chaperone CaDnaK has an amino acid sequence as shown in SEQ ID NO.5; The carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H includes a *401L mutation site and a W556H mutation site, and the expression product of the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H has an amino acid sequence as shown in SEQ ID NO.6; The expression product of the formate dehydrogenase CaFdhF has an amino acid sequence as shown in SEQ ID NO.7; The hydrogenase mutant CaHydA2_C183D-A311L includes a C183D mutation site and an A311L mutation site, and the expression product of the hydrogenase mutant CaHydA2_C183D-A311L has an amino acid sequence as shown in SEQ ID NO.

8.

3. A method for constructing the recombinant expression vector according to claim 1 or 2, characterized in that: The method comprises: Amplifying a first gene fragment encoding a molecular chaperone CaDnaK and a second gene fragment encoding a carbon monoxide dehydrogenase CaAcsA using a first amplification primer pair and a second amplification primer respectively, to obtain a first amplification product encoding a molecular chaperone CaDnaK and a second amplification product encoding a carbon monoxide dehydrogenase CaAcsA; The first amplification product encoding the molecular chaperone CaDnaK and the second amplification product encoding the carbon monoxide dehydrogenase CaAcsA are sequentially connected to the expression vector to obtain a first construction vector containing CaDnaK-CaAcsA; Constructing a mutant vector of carbon monoxide dehydrogenase CaAcsA of the first construction vector, and then screening carbon monoxide dehydrogenase activity to obtain a first recombinant expression vector containing the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H; Amplifying a third gene fragment encoding formate dehydrogenase CaFdhF and a fourth gene fragment encoding hydrogenase CaHydA2 using a third amplification primer pair and a fourth amplification primer respectively, to obtain a third amplification product encoding formate dehydrogenase CaFdhF and a fourth amplification product encoding hydrogenase CaHydA2; The third amplification product encoding formate dehydrogenase CaFdhF and the fourth amplification product encoding hydrogenase CaHydA2 are sequentially connected to the expression vector to obtain a second construction vector containing CaFdhF-CaHydA2; Constructing a mutation vector of the hydrogenase CaHydA2 of the second construction vector, and then performing hydrogenase activity screening to obtain a second recombinant expression vector containing the hydrogenase mutant CaHydA2_C183D-A311L; Collecting the first recombinant expression vector and the second recombinant expression vector to obtain a recombinant expression vector; Wherein, the first amplification primer pair is used to amplify the first gene fragment, the upstream primer of the first amplification primer pair is shown as SEQ ID NO.9, and the downstream primer of the first amplification primer pair is shown as SEQ ID NO.10; The second amplification primer pair is used to amplify the second gene fragment, the upstream primer of the second amplification primer pair is shown as SEQ ID NO.11, and the downstream primer of the second amplification primer pair is shown as SEQ ID NO.12; The third amplification primer pair is used to amplify the third gene fragment, the upstream primer of the third amplification primer pair is shown as SEQ ID NO.13, and the downstream primer of the third amplification primer pair is shown as SEQ ID NO.14; The fourth amplification primer pair is used to amplify the fourth gene fragment, the upstream primer of the fourth amplification primer pair is shown as SEQ ID NO.15, and the downstream primer of the fourth amplification primer pair is shown as SEQ ID NO.

16.

4. The method according to claim 3, characterized in that The method of constructing a mutant vector of carbon monoxide dehydrogenase CaAcsA of the first construction vector, and then screening carbon monoxide dehydrogenase activity to obtain a first recombinant expression vector containing the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H, comprises the steps of: In the case where the first construction vector is used as a parent, a mutation vector of the carbon monoxide dehydrogenase CaAcsA of the first construction vector is constructed using a site-directed mutagenesis method; The carbon monoxide dehydrogenase activity of the mutant vector of the carbon monoxide dehydrogenase CaAcsA is screened using a gas environment containing a first preset oxygen volume content to obtain a first recombinant expression vector containing the carbon monoxide dehydrogenase mutant CaAcsA_*401L-W556H.

5. The method according to claim 4, characterized in that The volume content V1 of the first preset oxygen and the total gas volume V2 of the gas environment satisfy the relationship: V1:V2≤0.5%.

6. The method according to claim 3, characterized in that The method of constructing a mutation vector of hydrogenase CaHydA2 of the second construction vector, and then screening hydrogenase activity to obtain a second recombinant expression vector containing hydrogenase mutant CaHydA2_C183D-A311L, comprises the steps of: In the case where the second construction vector is used as a parent, a mutation vector of the hydrogenase CaHydA2 of the second construction vector is constructed using a site-directed mutagenesis method; The hydrogenase mutant vector of the second construction vector is screened for hydrogenase activity using a gas environment containing a second preset oxygen volume content to obtain a second recombinant expression vector containing the hydrogenase mutant CaHydA2_C183D-A311L.

7. The method according to claim 6, characterized in that The second preset oxygen volume content V3 and the total gas volume V2 of the gas environment satisfy the relationship: V3:V2≤0.5%.

8. An engineered bacterium of Clostridium aerogenes having microaerobic tolerance, characterized in that: The engineered Clostridium aerovorum bacteria comprises the recombinant expression vector according to claim 1 or 2 or the recombinant expression vector obtained by the method according to any one of claims 3 to 7; wherein the engineered Clostridium aerovorum bacteria comprises at least one of the following: Clostridium alcoholicum, Clostridium ljungdahlii, Clostridium carboxyvorans, Clostridium kluyveri, and Clostridium ragsdalei.

9. A method for constructing the engineered bacterium Clostridium aerovorum according to claim 8, characterized in that: The method comprises: Synchronously introducing the first recombinant expression vector and the second recombinant expression vector in the recombinant expression vector of claim 1 or 2 into Clostridium aeruginosa to obtain an engineered Clostridium aeruginosa bacterium with microaerobic tolerance; The engineered Clostridium aerovorum bacteria were verified by sequencing positive clones to obtain an engineered bacteria that co-expressed the first recombinant expression vector and the second recombinant expression vector.

10. An application of an engineered bacterium of Clostridium aerogenes having microaerobic tolerance, characterized in that: The engineered Clostridium aerovorum of claim 8 is applied to anaerobic syngas fermentation in an atmosphere containing an oxygen concentration of ≤0.5% by volume.

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