Recombinant vector, high-temperature-resistant clostridium aerovorans engineering bacteria, construction method and application

Abstract

The invention provides a recombinant vector, a high-temperature-resistant clostridium aerovorans engineering bacterium as well as a construction method and application thereof, and belongs to the technical field of biology. A functional element of the recombinant vector is derived from Moorella thermoacetica, and the functional element comprises a molecular chaperone MtGroEL gene segment, a molecular chaperone MtGroES gene segment, a carbon monoxide dehydrogenase mutant gene segment and a formate dehydrogenase mutant gene segment which are sequentially connected in series. The recombinant vector is introduced into the clostridium aerovorans, so that the conversion rate of the strain to a carbon gas and the tolerance to high temperature and product stress in a high-temperature environment can be improved, and finally, the yield of a high-temperature anaerobic synthesis gas fermentation product is improved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a recombinant vector, a thermostable Clostridium aerovorum engineered bacterium, and a construction method and application thereof. Background Art

[0002] Syngas fermentation is a biotechnological process that uses anaerobic microorganisms to grow and metabolize syngas (mainly composed of carbon monoxide, carbon dioxide and hydrogen) as a carbon source and energy source to produce compounds under suitable temperature conditions. Syngas fermentation includes medium temperature fermentation (30℃~40℃) and high temperature fermentation. Medium temperature fermentation requires precise temperature control of the fermentation system, which increases the complexity of operation. During high temperature fermentation, although the non-nutritional bodies such as nutrient bodies and spores of miscellaneous bacteria cannot be completely killed, they cannot reproduce. Therefore, high temperature fermentation helps to inhibit the growth of some unwanted microorganisms, better solve the problem of miscellaneous bacteria contamination, and even realize the sterilization-free open fermentation system. In addition, higher fermentation temperature can not only speed up the biochemical reaction rate, save cooling energy consumption, operate extensively, and reduce the heating costs associated with separation of products such as ethanol and butanol by distillation, so high temperature fermentation has unique advantages over medium temperature fermentation and has great development potential.

[0003] So far, although there are many reports on natural and engineered syngas fermentation under medium temperature conditions, there are fewer reports on syngas fermentation under high temperature conditions, and most syngas fermentation under high temperature conditions is only carried out using mixed bacterial communities. The components of the fermentation products obtained in this way are complex and the yield is unstable. Clostridium aerogenes is an industrial strain widely used in syngas fermentation in recent years. The optimum temperature for fermentation is usually 30℃~40℃, but when the fermentation temperature exceeds 42℃, the ability to produce chemicals such as ethanol by fermentation of Clostridium aerogenes will be greatly reduced. In addition, when the fermentation temperature is lower than 30℃, it is easy to be contaminated by miscellaneous bacteria, resulting in a decrease in fermentation capacity. In addition, when the fermentation products of Clostridium aerogenes accumulate to a certain concentration (>10g / L), it is easy to inhibit the growth and metabolic activity of Clostridium aerogenes. Therefore, how to simultaneously improve the conversion rate of one-carbon gas and the tolerance to product stress of Clostridium aerogenes under high temperature environment is the key to ultimately increase the yield of fermentation products. Summary of the invention

[0004] The present application provides a recombinant vector, a thermostable Clostridium aerovorum engineered bacterium, a construction method and an application thereof, in order to solve the following technical problem: how to simultaneously improve the stress resistance of Clostridium aerovorum in a high temperature environment and the conversion efficiency of one-carbon gas.

[0005] In a first aspect, the present application provides a recombinant vector, wherein the functional elements of the recombinant vector are derived from Moorella thermoacetica, and the functional elements include: a molecular chaperone MtGroEL gene fragment, a molecular chaperone MtGroES gene fragment, a carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W gene fragment, and a formate dehydrogenase mutant MtFdhF_G142A-D253V gene fragment connected in series; wherein,

[0006] The nucleotide sequence of the molecular chaperone MtGroEL gene fragment is shown in SEQ ID NO: 1;

[0007] The nucleotide sequence of the molecular chaperone MtGroES gene fragment is shown in SEQ ID NO: 2;

[0008] The nucleotide sequence of the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W gene fragment is shown in SEQ ID NO: 3;

[0009] The nucleotide sequence of the formate dehydrogenase mutant MtFdhF_G142A-D253V gene fragment is shown in SEQ ID NO:4.

[0010] Optionally, the amino acid sequence of the expression product of the molecular chaperone MtGroEL gene fragment is shown in SEQ ID NO: 5;

[0011] The amino acid sequence of the expression product of the molecular chaperone MtGroES gene fragment is shown in SEQ ID NO: 6;

[0012] The carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W includes an R77G mutation site, a F232A mutation site and an A583W mutation site, and the amino acid sequence of the expression product of the gene fragment of the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W is shown in SEQ ID NO: 7;

[0013] The formate dehydrogenase mutant MtFdhF_G142A-D253V includes a G142A mutation site and a D253V mutation site. The amino acid sequence of the expression product of the gene fragment of the formate dehydrogenase mutant MtFdhF_G142A-D253V is shown in SEQ ID NO:8.

[0014] In a second aspect, the present application provides a thermostable engineered bacterium of Clostridium aerovorum, wherein the engineered bacterium of Clostridium aerovorum contains the recombinant vector described in the embodiment of the first aspect.

[0015] Optionally, the aerotrophic Clostridium includes one or more of: ethanolic Clostridium, young's Clostridium, carbon monoxide-eating Clostridium, kluyveromyces and ragsobacteria.

[0016] In a third aspect, the present application provides a method for constructing the thermostable Clostridium aerovorum engineered bacteria described in the embodiment of the second aspect, the method comprising:

[0017] The molecular chaperone MtGroEL gene fragment, the molecular chaperone MtGroES gene fragment, the carbon monoxide dehydrogenase MtAcsA gene fragment and the formate dehydrogenase MtFdhF gene fragment were sequentially connected to the vector to obtain the connection product MtGroEL / MtGroES-MtAcsA-MtFdhF;

[0018] The ligation product is added to competent cells for transformation, followed by sequencing verification to obtain a first recombinant tandem expression vector containing MtGroEL / MtGroES-MtAcsA-MtFdhF;

[0019] A mutant vector of the carbon monoxide dehydrogenase MtAcsA gene fragment and the formate dehydrogenase MtFdhF gene fragment in the first recombinant tandem expression vector is constructed by using a site-directed mutagenesis method, and then positive clones are sequenced and verified and enzyme activity tested and screened to obtain a second recombinant tandem expression vector containing both the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W and the formate dehydrogenase mutant MtFdhF_G142A-D253V; and

[0020] The second recombinant tandem expression vector is introduced into Clostridium aerovorum, and then sequenced and verified to obtain engineered Clostridium aerovorum.

[0021] In a fourth aspect, the present application provides an application of the thermostable Clostridium aerovorum engineered bacteria described in the embodiment of the second aspect, characterized in that the application comprises:

[0022] The engineered Clostridium aerovorum bacteria is used as a bioengineered strain for high-temperature anaerobic syngas fermentation.

[0023] Optionally, the fermentation temperature is >42°C.

[0024] Optionally, the fermentation products include: one or more of formic acid, acetic acid, butyric acid, lactic acid, ethanol, butanol, hexanol, 2,3-butanediol and isopropanol.

[0025] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0026] The present application firstly digs out functional elements from the thermoacetic bacteria, and the functional elements can be a molecular chaperone MtGroEL gene fragment, a molecular chaperone MtGroES gene fragment, a carbon monoxide dehydrogenase MtAcsA gene fragment, and a formate dehydrogenase MtFdhF gene fragment. Then, the four are designed to be expressed in series, and the carbon monoxide dehydrogenase MtAcsA and the formate dehydrogenase MtFdhF are designed and screened for protein engineering, and finally introduced into Clostridium aerovorum to create a thermostable Clostridium aerovorum engineered bacterium. Among them, by expressing the molecular chaperone MtGroEL gene fragment, the molecular chaperone MtGroES gene fragment, the gene fragment of the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W, and the gene fragment of the formate dehydrogenase mutant MtFdhF_G142A-D253V in series, the expression levels of the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W and the formate dehydrogenase mutant MtFdhF_G142A-D253V can be increased, and the tolerance of the engineered Clostridium aerovorum to high temperature environment and product stress can be improved. In addition, the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W and the formate dehydrogenase mutant MtFdhF_G142A-D253V obtained through protein engineering design can further improve the conversion efficiency of one-carbon gas by Clostridium aerovorum engineering bacteria on the basis of improving the stress resistance of recombinant Clostridium aerovorum engineering bacteria by the two thermophilic bacteria-related stress resistance elements MtGroEL and MtGroES, thereby ultimately increasing the yield of fermentation products under high temperature conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] 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.

[0029] Figure 1 A schematic diagram of the structure of the recombinant vector provided in the examples of the present application;

[0030] Figure 2 A schematic diagram of a method for constructing a thermostable Clostridium aerovorum engineered bacterium provided in an embodiment of the present application;

[0031] Figure 3Comparison of growth and metabolic changes of wild-type Clostridium ethanolatum DSM10061 (WT) and MtGroEL / MtGroES-MtAcsA_R77G-F232A-A583W-MtFdhF_G142A-D253V overexpressing engineered bacteria at different fermentation temperatures provided in Example 3 of the present application. (A) Changes in strain biomass after 72 hours of fermentation; (B) Metabolite (ethanol and acetic acid) levels after 72 hours of fermentation. DETAILED DESCRIPTION

[0032] 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.

[0033] 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, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have 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 apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0034] In addition, in the description of the specification of the present application, the terms "including", "comprising", etc. mean "including but not limited to". In this article, relational terms such as "first" and "second", etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A and B can be singular or plural. In this article, "at least one" means one or more, and "plurality" means two or more. "At least one", "the following at least one item (items)" or similar expressions refer to any combination of these items, including any combination of single items (items) or plural items (items). For example, "at least one of a, b, or c", or "at least one of a, b and c", can both mean: a, b, c, ab (i.e. a and b), ac, bc or abc, where a, b, c can be single or plural, respectively.

[0035] 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.

[0036] The creative ideas of this application are:

[0037] One-carbon gas (C1 gas) is a type of gas with a single carbon atom. It has huge reserves and is an important free carbon resource. It mainly comes from low-quality natural gas, tail gas emitted by petrochemical enterprises, and synthesis gas produced after biomass processing. It has the characteristics of wide sources, easy preparation, and low price. The conversion and utilization of one-carbon gas has great application prospects in the field of biomanufacturing.

[0038] At present, all syngas Clostridium are autotrophic acetogenic bacteria. Although they can directly use CO, CO2 and H2 to produce various high-value chemicals, their own metabolic rate is low and requires a high energy supply. The output and production intensity are difficult to meet the needs of industrialization. Using autotrophic syngas Clostridium as the chassis cell, the integration and optimization of carbon fixation pathways based on synthetic biology strategies can achieve efficient utilization of carbon gas and industrial production of fine chemicals. The Wood-Ljugdahl pathway (WLP) is a metabolic pathway for syngas Clostridium to fix CO and CO2 to synthesize organic acids and alcohols such as acetic acid and ethanol. It is also the most effective carbon fixation pathway known to date with the fewest reaction steps. Among them, carbon monoxide dehydrogenase (CODH) and formate dehydrogenase (FDH) are key enzymes in WLP. Strengthening the activity of these two enzymes through synthetic biology technology can improve the carbon fixation efficiency of the strain and the synthesis intensity of the product.

[0039] In order to obtain higher economic benefits, industrial fermentation must maintain a high yield and production intensity of fermentation products. At present, the products of syngas fermentation are mostly ethanol, acetone, butanol and higher alcohols. When they accumulate to a certain concentration (>10g / L), they are easy to inhibit the growth and metabolism of bacteria. The most effective way to improve the robustness of bacteria is to introduce stress-resistant elements using molecular biological technology to strengthen the stress resistance of bacteria. Heat shock proteins (HSPs) are mainly involved in the initial folding of proteins, transmembrane transport, the combination and decomposition of oligomeric proteins, and the degradation of unstable proteins. When the bacteria are faced with adverse fermentation conditions, the expression level of heat shock protein encoding genes is significantly increased, inducing the production of a large number of heat shock proteins, thereby ensuring the correct folding of proteins and assisting the refolding of damaged proteins. Molecular chaperones GroEL / GroES belong to Class I heat shock proteins, which are composed of about 60kDa GroEL and about 10kDa GroES. Studies have shown that GroEL / GroES of Escherichia coli are involved in the folding and refolding of about 300 proteins. Studies have shown that overexpression of endogenous GroEL / GroES can not only improve the tolerance of Clostridium tyrobutyricum to the product butyric acid, but also improve the tolerance of Clostridium beijerinckii to fermentation inhibitors.

[0040] Temperature is an important factor affecting the growth and metabolic activities of microorganisms. Different types of microorganisms have their optimal growth temperatures. Appropriate temperature can promote the growth and metabolism of microorganisms and improve the yield and quality of products. If the temperature is too high, the metabolic rate of microorganisms may be too fast, resulting in insufficient energy and material supply, which will hinder the growth of microorganisms or even cause death. Therefore, fermentation temperature control is an important factor in fermentation regulation. Accurate fermentation temperature control is crucial to ensure the smooth progress of the fermentation process and the quality and yield of the final product.

[0041] Syngas fermentation is a biotechnological process that uses anaerobic microorganisms to grow and metabolize syngas (mainly composed of carbon monoxide, carbon dioxide and hydrogen) as a carbon source and energy source to produce compounds under suitable temperature conditions. At present, the production of organic alcohols such as ethanol, butanol and hexanol by syngas fermentation has good market prospects and strategic significance, attracting a large number of domestic and foreign researchers, and has made some research progress. However, all known research reports on syngas fermentation so far use medium-temperature fermentation (30℃~40℃), and the most typical temperature is 37℃. In order to maintain a stable medium-temperature environment, the fermentation system needs to be precisely controlled, which increases the complexity of the operation. For closed fermentation systems, in order to prevent contamination of bacteria during the fermentation process, the culture medium must be sterilized by high-temperature steam, which inevitably involves a series of sterilization equipment, which is cumbersome to operate and consumes a lot of steam and cooling water. In addition, high-temperature sterilization is prone to cause a large amount of trace elements and vitamins. Loss, while producing many harmful chemicals that affect normal fermentation. For open syngas fermentation systems, since the fermentation container is open to the external environment, the fermentation is easily affected by external pollutants, and its fermentation process is more difficult to accurately control. Dust, bacteria, and changes in gas composition and concentration in the environmental medium may affect the fermentation process, leading to failure of the fermentation process or a decrease in product quality. In addition, the fermentation process is an exothermic process. Microbial metabolism releases energy, part of which is dissipated in the form of heat, causing the temperature of the fermentation system to rise. During the fermentation process, if the temperature rises too quickly, it is necessary to start a cooling system, such as a cold water circulation or air cooling system, to reduce the temperature in the fermenter, which will inevitably consume more energy media.

[0042] High-temperature anaerobic fermentation refers to the process of biological fermentation by specific anaerobic microorganisms under high temperature (usually above 37°C, sometimes even up to 55°C or higher). Generally speaking, it is difficult for bacteria in the environment to grow above 45°C. During high-temperature fermentation, although the non-nutrient bodies such as vegetative bodies and spores of the bacteria cannot be completely killed, they cannot reproduce. Therefore, high-temperature fermentation helps to inhibit the growth of some unwanted microorganisms, better solve the problem of bacterial contamination, and even realize the sterilization-free open fermentation system. In addition, higher fermentation temperatures can not only accelerate the biochemical reaction rate, save cooling energy consumption, operate extensively, and reduce the heating costs associated with the separation of products such as ethanol and butanol by distillation, so high-temperature fermentation has unique advantages over medium-temperature fermentation and has great development potential. So far, although there are many research reports on natural and engineered syngas fermentation under medium-temperature conditions, research reports on syngas fermentation under high-temperature conditions only use mixed bacterial communities for fermentation, and the products are complex and the yield is unstable. There are no reports on high-temperature syngas fermentation using single bacteria.

[0043] In view of the above shortcomings, the present invention provides a recombinant vector, a thermostable Clostridium aerogenes engineered bacterium, a construction method and an application thereof. Clostridium aerogenes is an industrial strain widely used in syngas fermentation in recent years. Its optimum temperature is usually 30°C to 40°C. Generally, when it exceeds 42°C, its ability to produce ethanol by fermentation will be greatly reduced. When the fermentation temperature is low, it is easy to contaminate miscellaneous bacteria, resulting in a decrease in fermentation capacity. Moorella thermoacetica (formerly known as Clostridium thermoaceticum) is a thermophilic acetogenic bacterium isolated from the soil. Its optimum growth temperature is 55°C and its optimum growth pH is 6.5 to 6.8. Compared with functional elements endogenous to industrial strains or derived from ordinary microorganisms, elements derived from extreme microorganisms will show better effects in adverse fermentation environments because they have undergone long-term stress-resistant evolution under harsh environments. By introducing the molecular chaperones MtGroEL / MtGroES expressed in series and the carbon monoxide dehydrogenase mutants MtAcsA_R77G-F232A-A583W and formate dehydrogenase mutants MtFdhF_G142A-D253V with enhanced activity, the upper temperature limit of the fermentation of Clostridium aeruginosa is increased. The engineered Clostridium aeruginosa bacteria constructed by the method provided by the present invention has the characteristics of high temperature resistance, can still maintain a good growth state at high temperature, and is an effective strain suitable for high-temperature syngas fermentation. The strain is used for short-term or long-term high-temperature fermentation, and the operation is extensive, and the open fermentation system can be sterilized, which effectively solves the problem of contamination by foreign bacteria, reduces the energy consumption of fermentation cooling and the heating cost of product distillation separation, improves production efficiency, is suitable for industrial promotion, and has good application value. Therefore, the present application introduces thermophilic bacteria-related functional elements and stress-resistant elements into Clostridium aeruginosa, while improving the conversion rate of the strain to carbon gas and the tolerance to high temperature and product stress in a high-temperature environment, and ultimately increases the yield of the fermentation product.

[0044] Figure 1 This is a schematic diagram of the structure of the recombinant vector provided in the examples of the present application.

[0045] See also Figure 1 The present application provides a recombinant vector, wherein the functional elements of the recombinant vector are derived from Moorella thermoacetica, and the functional elements include: a molecular chaperone MtGroEL gene fragment, a molecular chaperone MtGroES gene fragment, a carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W gene fragment, and a formate dehydrogenase mutant MtFdhF_G142A-D253V gene fragment connected in series; wherein,

[0046] The nucleotide sequence of the molecular chaperone MtGroEL gene fragment is shown in SEQ ID NO: 1;

[0047] The nucleotide sequence of the molecular chaperone MtGroES gene fragment is shown in SEQ ID NO: 2;

[0048] The nucleotide sequence of the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W gene fragment is shown in SEQ ID NO: 3;

[0049] The nucleotide sequence of the formate dehydrogenase mutant MtFdhF_G142A-D253V gene fragment is shown in SEQ ID NO:4.

[0050] The present application obtains carbon monoxide dehydrogenase mutants MtAcsA_R77G-F232A-A583W and formate dehydrogenase mutants MtFdhF_G142A-D253V by site-directed mutagenesis of MtAcsA and MtFdhF of thermoacetic Moorella thermoacetica, and has excellent performance. The mutants are introduced into Clostridium aerogenes in series with molecular chaperones MtGroEL / MtGroES, and stable and efficient expression of the two can be achieved under high temperature environment. The use of the engineered Clostridium aerogenes bacteria for syngas fermentation can expand the suitable fermentation temperature range, making the strain more tolerant to fermentation temperature changes and achieving high temperature fermentation.

[0051] In some embodiments, the amino acid sequence of the expression product of the molecular chaperone MtGroEL gene fragment is shown in SEQ ID NO: 5;

[0052] The amino acid sequence of the expression product of the molecular chaperone MtGroES gene fragment is shown in SEQ ID NO: 6;

[0053] The carbon monoxide dehydrogenase mutant MtAcsAMtAcsA_R77G-F232A-A583W includes an R77G mutation site, a F232A mutation site and an A583W mutation site, and the amino acid sequence of the expression product of the gene fragment of the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W is shown in SEQ ID NO: 7;

[0054] The formate dehydrogenase mutant MtFdhF_G142A-D253V includes a G142A mutation site and a D253V mutation site. The amino acid sequence of the expression product of the gene fragment of the formate dehydrogenase mutant MtFdhF_G142A-D253V is shown in SEQ ID NO:8.

[0055] Based on a general inventive concept, the present application provides a thermostable engineered bacterium of Clostridium aerovorum, wherein the engineered bacterium of Clostridium aerovorum contains the recombinant vector described in the above embodiment.

[0056] In some embodiments, the Clostridium aerovorans includes one or more of Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium carboxidivorans, Clostridium kluyveri, and Clostridium ragsdalei.

[0057] Figure 2 A schematic flow chart of a method for constructing a thermostable Clostridium aerovorum engineered bacterium provided in an embodiment of the present application.

[0058] See also Figure 2 The present application provides a method for constructing the thermostable Clostridium aerovorum engineered bacteria of the above embodiment, the method comprising:

[0059] S1, respectively connecting the molecular chaperone MtGroEL gene fragment, the molecular chaperone MtGroES gene fragment, the carbon monoxide dehydrogenase MtAcsA gene fragment and the formate dehydrogenase MtFdhF gene fragment to the vector in sequence to obtain the connection product MtGroEL / MtGroES-MtAcsA-MtFdhF;

[0060] S2, adding the ligation product to the competent cells for transformation, followed by sequencing verification, to obtain the first recombinant tandem expression vector containing MtGroEL / MtGroES-MtAcsA-MtFdhF;

[0061] S3, constructing a mutant vector of the carbon monoxide dehydrogenase MtAcsA gene fragment and the formate dehydrogenase MtFdhF gene fragment in the first recombinant tandem expression vector by a site-directed mutagenesis method, and then performing sequencing verification and enzyme activity test screening of positive clones to obtain a second recombinant tandem expression vector containing both the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W and the formate dehydrogenase mutant MtFdhF_G142A-D253V; and

[0062] S4. Introducing the second recombinant tandem expression vector into Clostridium aerovorum, and then performing sequencing verification to obtain engineered Clostridium aerovorum.

[0063] Based on a general inventive concept, the present application provides an application of the thermostable Clostridium aerovorum engineered bacteria described in the embodiment of the second aspect, characterized in that the application comprises:

[0064] The engineered Clostridium aerovorum bacteria is used as a bioengineered strain for high-temperature anaerobic syngas fermentation.

[0065] In some embodiments, the fermentation temperature is >42°C.

[0066] In some embodiments, the fermentation products include: one or more of formic acid, acetic acid, butyric acid, lactic acid, ethanol, butanol, hexanol, 2,3-butanediol and isopropanol.

[0067] The present application introduces the molecular chaperone MtGroEL / MtGroES expressed in series from Moorella thermoacetoacetica and the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W and formate dehydrogenase mutant MtFdhF_G142A-D253V with enhanced activity into Clostridium aerogenes through genetic engineering methods, so that the strain can efficiently utilize CO, CO2 and H2 in syngas to synthesize organic acids, organic alcohols and other compounds at higher temperatures (>42°C). The strain is used for short-term or long-term high-temperature fermentation, and the operation is extensive, and the open fermentation system can be sterilized-free, which effectively solves the problem of contamination by foreign bacteria and reduces the energy consumption of fermentation cooling and the heating cost of product distillation separation.

[0068] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to industry standards. If there is no corresponding industry standard, then the conditions recommended by the manufacturer are followed.

[0069] Example 1

[0070] This embodiment provides a method for constructing a recombinant vector, comprising the following steps:

[0071] The invention relates to constructing a recombinant tandem expression vector containing a molecular chaperone MtGroEL, a molecular chaperone MtGroES, a carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W and a formate dehydrogenase mutant MtFdhF_G142A-D253V of Moorella thermoacetica: extracting the genome of Moorella thermoacetica, and obtaining a molecular chaperone MtGroEL (MOTHA_c05830) gene fragment, a molecular chaperone MtGroES (MOTHA_c05820) gene fragment, a carbon monoxide dehydrogenase MtAcsA (MOTHA_c12660) gene fragment and a formate dehydrogenase MtFdhF (MOTHA_c24590) gene fragment by PCR amplification; using the nucleotide sequence of an upstream primer such as SEQ ID NO:9 and the nucleotide sequence of a downstream primer such as SEQ ID NO:10, respectively. A first amplification primer set of SEQ ID NO:10 is used to perform a PCR amplification reaction on the molecular chaperone MtGroEL to obtain an amplified molecular chaperone MtGroES gene fragment; a second amplification primer set of an upstream primer having a nucleotide sequence such as SEQ ID NO:11 and a downstream primer having a nucleotide sequence such as SEQ ID NO:12 is used to perform a PCR amplification reaction on the molecular chaperone MtGroES to obtain an amplified molecular chaperone MtGroES gene fragment; a third amplification primer set of an upstream primer having a nucleotide sequence such as SEQ ID NO:13 and a downstream primer having a nucleotide sequence such as SEQ ID NO:14 is used to perform a PCR amplification reaction on the carbon monoxide dehydrogenase MtAcsA to obtain an amplified carbon monoxide dehydrogenase MtAcsA gene fragment; an upstream primer having a nucleotide sequence such as SEQ ID NO:15 and a downstream primer having a nucleotide sequence such as SEQ ID NO:16 is used to perform a PCR amplification reaction on the carbon monoxide dehydrogenase MtAcsA gene fragment; an upstream primer having a nucleotide sequence such as SEQ ID NO:17 and a downstream primer having a nucleotide sequence such as SEQ ID NO:18 is used to perform a PCR amplification reaction on the carbon monoxide dehydrogenase MtAcsA gene fragment; an upstream primer having a nucleotide sequence such as SEQ ID NO:19 and a downstream primer having a nucleotide sequence such as SEQ ID NO:20 is used to perform a PCR amplification reaction on the carbon monoxide dehydrogenase MtAcsA gene fragment; a second amplification primer set of an upstream primer having a nucleotide sequence such as SEQ ID NO:11 and a downstream primer having a nucleotide sequence such as SEQ ID NO:21 is used to perform a PCR amplification reaction on the carbon monoxide dehydrogenase MtAcsA gene fragment; a second amplification primer set of an upstream primer having a The fourth amplification primer set of NO:16 carried out PCR amplification reaction on formate dehydrogenase MtFdhF to obtain the amplified formate dehydrogenase MtFdhF gene fragment; the molecular chaperone MtGroEL gene fragment, the molecular chaperone MtGroES gene fragment, the carbon monoxide dehydrogenase MtAcsA gene fragment and the formate dehydrogenase MtFdhF gene fragment were sequentially connected to the pMTL83152-Pthl vector using the seamless cloning method.Escherichia coli Top10 competent cells were transformed, positive clones were screened and sequenced, and the recombinant tandem expression vector pMTL83152-Pthl-MtGroEL / MtGroES-MtAcsA-MtFdhF was obtained. The carbon monoxide dehydrogenase MtAcsA and formate dehydrogenase MtFdhF mutant vectors were constructed by site-directed mutagenesis. The positive clones were sequenced and verified and the enzyme activity test was performed to obtain the recombinant tandem expression vector pMTL83152-Pthl-MtGroEL / MtGroES-MtAcsA_R77G-F232A-A583W-MtFdhF_G142A-D253V with excellent performance. The nucleotide sequence of the molecular chaperone MtGroEL gene fragment is such as SEQ ID NO: 1, the nucleotide sequence of the molecular chaperone MtGroES gene fragment is such as SEQ ID NO: 2, the nucleotide sequence of the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W gene fragment is such as SEQ ID NO: 3, and the nucleotide sequence of the formate dehydrogenase mutant MtFdhF_G142A-D253V gene fragment is such as SEQ ID NO: 4.

[0072] Example 2

[0073] Based on the recombinant vector of Example 1, this example provides a method for constructing a thermostable Clostridium aerovorum engineered bacterium, comprising the following steps:

[0074] Wild-type Clostridium autoethanogenum DSM 10061 was purchased from DSMZ (https: / / www.dsmz.de / collection / catalogue / details / culture / DSM-10061). The recombinant tandem expression vector pMTL83152-Pthl-MtGroEL / MtGroES-MtAcsA_R77G-F232A-A583W-MtFdhF_G142A-D253V was introduced into wild-type Clostridium autoethanogenum DSM10061 by conjugation transfer, and the specific steps are as follows. The recombinant tandem expression vector pMTL83152-Pthl-MtGroEL / MtGroES-MtAcsA_R77G-F232A-A583W-MtFdhF_G142A-D253V was transformed into Escherichia coli sExpress competent cells, and the positive clones were screened and sequenced to obtain Escherichia coli sExpress donor bacteria overexpressing the recombinant tandem expression vector. The frozen wild strain of Clostridium ethanolum DSM10061 was quickly placed in an anaerobic operation box, inoculated with YTF liquid culture medium without antibiotics at a 10% inoculation rate, and placed in a 37°C shaker for anaerobic shaking culture. The formula of YTF medium is 1g ammonium chloride, 0.2g magnesium sulfate heptahydrate, 0.8g sodium chloride, 0.1g potassium dihydrogen phosphate, 0.02g calcium chloride dihydrate, 10g yeast extract powder, 16g peptone, 10g fructose, 0.61g potassium sulfate, 0.3g L-cysteine ​​hydrochloride, 0.50mL resazurin sodium (0.1% w / v), adjust pH to 5.5 with phosphoric acid, and dilute to 1L. At the same time, take LB liquid culture medium and add the prepared chloramphenicol and kanamycin respectively, shake well and divide into shaking tubes. The Escherichia coli sExpress donor bacteria overexpressing the recombinant vector were inoculated into LB liquid culture medium containing chloramphenicol and kanamycin, and cultured at 37°C. When the overnight OD600 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, centrifuge and discard the supernatant to obtain donor cells, and transfer them to an anaerobic operation box. Take the overnight cultured wild strain DSM10061 of ethanol Clostridium wild strain and quickly transfer it to an anaerobic operation box, resuspend the donor cells with 200μL of ethanol Clostridium wild strain, and mix them by inversion. Spread the cell mixture on a YTF solid culture medium plate without antibiotics and anaerobically conjugate at 37°C overnight. On the second day, use 500μL PBS to elute the mixed bacteria from the overnight conjugation plate and collect the transduction conjugate. Spread the transduction conjugate on a YTF solid culture medium plate containing thiamphenicol and D-cycloserine, and culture anaerobically at 37°C for 72h.The E. coli sExpress overexpressing the recombinant vector was conjugated with wild strains of Clostridium ethanologenum DSM10061 to transfer the transformants, and single clones were picked out respectively, and cultured anaerobically at 37°C for 72 hours using liquid YTF medium. The positive clones were screened and sequenced to obtain the engineered strain of Clostridium ethanologenum DSM10061 overexpressing MtGroEL / MtGroES-MtAcsA_R77G-F232A-A583W-MtFdhF_G142A-D253V.

[0075] Example 3

[0076] Based on the thermotolerant engineered Clostridium aerovorum of Example 2, this example provides an application of the thermotolerant engineered Clostridium aerovorum, comprising the following steps:

[0077] High-temperature anaerobic shake flask syngas fermentation test was carried out using the engineered bacteria of ethanol Clostridium DSM10061 overexpressing MtGroEL / MtGroES-MtAcsA_R77G-F232A-A583W-MtFdhF_G142A-D253V. The fermentation experiment was carried out in a 200mL serum bottle containing 50mL of culture medium, and the serum bottle was sealed with a rubber stopper. The wild ethanol Clostridium DSM10061 and the engineered bacteria overexpressing MtGroEL / MtGroES-MtAcsA_R77G-F232A-A583W-MtFdhF_G142A-D253V were cultured in liquid YTF medium, and when the strain concentration reached 0.5g / L, they were transferred to 50mL syngas fermentation medium at a 10% inoculation rate. 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, and 1 mL of Wolin's vitamin solution (10×), adjusted to pH 5.5, and fixed to 1 L. The modified Wolin's mineral salt contains 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, 0.1 g of calcium chloride dihydrate, 0.18 g of zinc sulfate heptahydrate, 0.01 g of copper sulfate pentahydrate, 0.02 g of potassium aluminum sulfate dodecahydrate, 0.01 g of boric acid, 0.01 g of sodium molybdate dihydrate, 0.03 g of nickel chloride hexahydrate, 0.30 mg of sodium selenate pentahydrate, and 0.40 mg of sodium tungstate dihydrate. The pH is adjusted to 5.5 with potassium hydroxide and the volume is fixed to 1 L. Wolin's vitamin solution (10×) contains 20.00 mg of biotin, 20.00 mg of folic acid, 100.00 mg of pyridoxine hydrochloride, 50.00 mg of thiamine hydrochloride, 50.00 mg of riboflavin, 50.00 mg of niacin, 50.00 mg of D-calcium pantothenate, 1.00 mg of vitamin B12, 50.00 mg of p-aminobenzoic acid, and 50.00 mg of (DL)-α-lipoic acid, and the volume is fixed to 1L. The bottle is simultaneously pressurized and sealed with a mixed gas containing 50% CO, 20% CO2, and 30% H2, and the gas in the bottle is replaced every 24 hours. The anaerobic bottle is placed in a shaker at 32, 37, 42, and 50°C for anaerobic shaking culture. Samples are taken every 24 hours to detect OD600 to calculate the biomass of the strain. Samples are taken after 72 hours of fermentation to detect the levels of metabolites (ethanol and acetic acid). Figure 3As shown, the results showed that compared with the wild strain of ethanol Clostridium DSM10061, the engineered bacteria of ethanol Clostridium DSM10061 overexpressing MtGroEL / MtGroES-MtAcsA_R77G-F232A-A583W-MtFdhF_G142A-D253V can tolerate the fermentation temperature of 50°C and have the highest ethanol yield at 42°C. Overexpressing MtGroEL / MtGroES-MtAcsA_R77G-F232A-A583W-MtFdhF_G142A-D253V can effectively increase the fermentation temperature of ethanol Clostridium wild strain DSM10061.

[0078] One or more technical solutions in the embodiments of the present application also have at least the following technical effects or advantages:

[0079] In the examples of the present application, the upper temperature limit of Clostridium syngas fermentation was increased by introducing tandemly expressed molecular chaperones MtGroEL / MtGroES and carbon monoxide dehydrogenase mutants MtAcsA_R77G-F232A-A583W and formate dehydrogenase mutants MtFdhF_G142A-D253V with enhanced activity.

[0080] In the embodiment of the present application, the engineered Clostridium aerogenes bacteria constructed has the characteristics of high temperature resistance, can still maintain a good growth state at high temperature, and is an effective strain suitable for high temperature syngas fermentation. The strain is used for short-term or long-term high temperature fermentation, and the operation is extensive, and the open fermentation system can be sterilized, which effectively solves the problem of contamination by miscellaneous bacteria, reduces the energy consumption of fermentation cooling and the heating cost of product distillation separation, improves production efficiency, is suitable for industrial promotion, and has good application value.

[0081] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein 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 the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A recombinant vector, characterized in that: The functional elements of the recombinant vector are derived from Moorella thermoacetica, and the functional elements include: a molecular chaperone MtGroEL gene fragment, a molecular chaperone MtGroES gene fragment, a carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W gene fragment, and a formate dehydrogenase mutant MtFdhF_G142A-D253V gene fragment connected in series; wherein, The nucleotide sequence of the molecular chaperone MtGroEL gene fragment is shown in SEQ ID NO: 1; The nucleotide sequence of the molecular chaperone MtGroES gene fragment is shown in SEQ ID NO: 2; The nucleotide sequence of the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W gene fragment is shown in SEQ ID NO: 3; The nucleotide sequence of the formate dehydrogenase mutant MtFdhF_G142A-D253V gene fragment is shown in SEQ ID NO:

4.

2. The recombinant vector according to claim 1, characterized in that The amino acid sequence of the expression product of the molecular chaperone MtGroEL gene fragment is shown in SEQ ID NO: 5; The amino acid sequence of the expression product of the molecular chaperone MtGroES gene fragment is shown in SEQ ID NO: 6; The carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W includes an R77G mutation site, a F232A mutation site and an A583W mutation site, and the amino acid sequence of the expression product of the gene fragment of the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W is shown in SEQ ID NO: 7; The formate dehydrogenase mutant MtFdhF_G142A-D253V includes a G142A mutation site and a D253V mutation site. The amino acid sequence of the expression product of the gene fragment of the formate dehydrogenase mutant MtFdhF_G142A-D253V is shown in SEQ ID NO:

8.

3. A thermostable Clostridium aerovorum engineered bacterium, characterized in that: The engineered bacterium of Clostridium aerovorum contains the recombinant vector according to claim 1 or 2.

4. The engineered Clostridium aerovorum according to claim 3, characterized in that: The types of the aerotrophic Clostridium include: one or more of: ethanolic Clostridium, young's Clostridium, carbon monoxide-eating Clostridium, kluyveromyces and ragsobacteria.

5. A method for constructing the thermostable Clostridium aerovorum engineered bacterium of claim 3 or 4, characterized in that: The method comprises: The molecular chaperone MtGroEL gene fragment, the molecular chaperone MtGroES gene fragment, the carbon monoxide dehydrogenase MtAcsA gene fragment and the formate dehydrogenase MtFdhF gene fragment were sequentially connected to the vector to obtain the connection product MtGroEL / MtGroES-MtAcsA-MtFdhF; Adding the ligation product to competent cells for transformation, followed by sequencing verification, to obtain a first recombinant tandem expression vector containing MtGroEL / MtGroES-MtAcsA-MtFdhF; A mutant vector of the carbon monoxide dehydrogenase MtAcsA gene fragment and the formate dehydrogenase MtFdhF gene fragment in the first recombinant tandem expression vector is constructed by using a site-directed mutagenesis method, and then positive clones are sequenced and verified and enzyme activity tested and screened to obtain a second recombinant tandem expression vector containing both the carbon monoxide dehydrogenase mutant MtAcsA_R77G-F232A-A583W and the formate dehydrogenase mutant MtFdhF_G142A-D253V; and The second recombinant tandem expression vector is introduced into Clostridium aerovorum, and then sequenced and verified to obtain engineered Clostridium aerovorum.

6. An application of the thermostable Clostridium aerovorum engineered bacteria according to claim 3 or 4, characterized in that: The applications include: The engineered Clostridium aerovorum bacteria is used as a bioengineered strain for high-temperature anaerobic syngas fermentation.

7. The use according to claim 6, characterized in that: The temperature of the fermentation is >42°C.

8. The use according to claim 6, characterized in that: The fermentation products include: one or more of formic acid, acetic acid, butyric acid, lactic acid, ethanol, butanol, hexanol, 2,3-butanediol and isopropanol.

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