Clostridium butyricum engineering strain for improving butyric acid synthesis through coenzyme a transferase pathway and construction method and application thereof

Abstract

The application belongs to the technical field of biology and relates to a Clostridium butyricum engineering strain for improving butyric acid synthesis through a coenzyme A transferase pathway as well as a construction method and application of the Clostridium butyricum engineering strain. Clostridium tyrobutyricum The application expresses a butyryl coenzyme A:acetic acid-coenzyme A transferase gene from Clostridium tyrobutyricum Ctcat1 A coenzyme A transferase (CoAT) pathway is constructed, the pathway synthesizes butyric acid by taking acetic acid as a substrate, therefore, the content of acetic acid in a fermentation product is greatly reduced, and the butyric acid yield is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an engineered strain of Clostridium butyricum that enhances butyric acid synthesis through the coenzyme A transferase pathway, its construction method, and its application. Background Technology

[0002] Clostridium butyricum ( Clostridium butyricum It is a strictly anaerobic spore-forming probiotic that promotes intestinal health in animals, inhibits the growth of pathogens, and enhances the body's immunity.

[0003] The main functional substance responsible for the probiotic effects of Clostridium butyricum is butyrate. Clostridium butyricum primarily synthesizes butyrate via the phosphobutyryltransferase-butyrate kinase (PTB-BK) pathway. In this pathway, glucose first undergoes carbohydrate metabolism to produce acetyl-CoA. Acetyl-CoA can be converted into acetic acid, and then, under the catalysis of thiolase (Thl), β-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonylase (Crt), and butyryl-CoA dehydrogenase (Bcd), it is converted into butyryl-CoA. Finally, butyryl-CoA is converted into butyrate by phosphobutyryltransferase (Ptb) and butyrate kinase (Bk). When synthesizing butyrate using this pathway, the fermentation product typically contains a high amount of acetic acid. Besides the PTB-BK pathway, microorganisms in nature can also synthesize butyrate using the coenzyme A transferase (CoAT) pathway. In this pathway, acetyl-CoA is first used to synthesize acetic acid and butyryl-CoA. Then, under the catalysis of CoAT, acetic acid and butyryl-CoA undergo a coenzyme A transfer reaction to produce butyric acid and acetyl-CoA. Since the CoAT pathway uses acetic acid as a substrate to produce butyric acid, the yield of acetic acid is significantly reduced, while the proportion of butyric acid in the fermentation products is greatly increased. Therefore, establishing a synthetic pathway for butyric acid production using the CoAT pathway in Clostridium butyricum is crucial for increasing its butyric acid yield and thus enhancing its beneficial growth capacity. Summary of the Invention

[0004] The purpose of this invention is to provide an improved Clostridium butyricum engineered strain that synthesizes butyric acid via the CoA transferase CoAT pathway.

[0005] Another object of the present invention is to provide a method for increasing the butyric acid synthesis of Clostridium butyricum.

[0006] The improved Clostridium butyricum engineered strain according to the present invention, which synthesizes butyric acid via the CoAT pathway of coenzyme A transferase, has the following characteristics:

[0007] Overexpression of butyryl-CoA:acetyl-CoA transferase gene, wherein the butyryl-CoA:acetyl-CoA transferase gene is from Ruminococcus bacteria CPB6 ( RuminococcaceaeButyryl-CoA:acetyl-CoA transferase gene derived from bacterium CPB6 Rbcat1 Its nucleotide sequence is shown in SEQ ID NO: 17, Clostridium coccidioides ( Clostridium kluyveri Butyryl-CoA: Acetyl-CoA transferase gene from [source missing] Ckcat3 Its nucleotide sequence is shown in SEQ ID NO: 18, or the butyryl-CoA:acetyl-CoA transferase gene from Clostridium butyricum. Ctcat1 The nucleotide sequence is shown in SEQ ID NO: 2.

[0008] Further, preferably, the butyryl-CoA:acetic acid-CoA transferase gene is integrated into an antibiotic-free expression vector stably inherited in Clostridium butyricum, wherein the antibiotic-free expression vector stably inherited in Clostridium butyricum has a replicon derived from an endogenous plasmid of Clostridium butyricum, and the nucleotide sequence of the replicon is shown in SEQ ID NO: 1.

[0009] Further, preferably, the promoter regulating the butyryl-CoA:acetyl-CoA transferase gene is the butyryl-CoA:acetyl-CoA transferase gene promoter P from Clostridium butyricum. cat (Its nucleotide sequence is shown in SEQ ID NO: 10), or the promoter P of the thiolysis enzyme gene. thl, The thiolase gene promoter P thl The nucleotide sequence is shown in SEQ ID NO: 3.

[0010] Further, preferably, the encoding gene of a CoAT pathway-related enzyme is overexpressed on the antibiotic-free expression vector stably inherited in Clostridium butyricum, wherein the CoAT pathway-related enzyme gene includes one or more of the following genes:

[0011] Thiolase gene thl β-hydroxybutyryl coenzyme A dehydrogenase gene hbd Crotonic acidase gene crt Butyryl-CoA dehydrogenase gene bcd ,

[0012] Among them, the thiolysis enzyme gene thl β-hydroxybutyryl coenzyme A dehydrogenase gene hbd Crotonic acidase gene crt Butyryl-CoA dehydrogenase gene bcd All originated from Clostridium butyricum ( Clostridium butyricum ), thl The nucleotide sequence is shown in SEQ ID NO: 4; hbd The nucleotide sequence is shown in SEQ ID NO: 5; crtThe nucleotide sequence is shown in SEQ ID NO: 6; bcd The nucleotide sequence is shown in SEQ ID NO: 7.

[0013] The method for increasing butyric acid synthesis by Clostridium butyricum according to the present invention comprises the following steps:

[0014] Overexpression of the butyryl-CoA:acetyl-CoA transferase gene in Clostridium butyricum, wherein the butyryl-CoA:acetyl-CoA transferase gene is from CPB6 of Ruminococcus family (Ruminococci). Ruminococcaceae Butyryl-CoA:acetyl-CoA transferase gene derived from bacterium CPB6 Rbcat1 Its nucleotide sequence is shown in SEQ ID NO: 17, Clostridium coccidioides ( Clostridium kluyveri Butyryl-CoA: Acetyl-CoA transferase gene from [source missing] Ckcat3 Its nucleotide sequence is shown in SEQ ID NO: 18, or the butyryl-CoA:acetyl-CoA transferase gene from Clostridium butyricum. Ctcat1 The nucleotide sequence is shown in SEQ ID NO: 2.

[0015] The method for increasing butyric acid synthesis by Clostridium butyricum according to the present invention further includes the steps of:

[0016] The butyryl-CoA:acetyl-CoA transferase gene was integrated into an antibiotic-free expression vector stably inherited in Clostridium butyricum, wherein the antibiotic-free expression vector stably inherited in Clostridium butyricum has a replicon derived from an endogenous plasmid of Clostridium butyricum, and the nucleotide sequence of the replicon is shown in SEQ ID NO: 1.

[0017] According to the method for increasing butyrate synthesis in Clostridium butyricum according to the present invention, the promoter regulating the butyryl-CoA:acetyl-CoA transferase gene is the butyryl-CoA:acetyl-CoA transferase gene promoter P from Clostridium butyricum. cat Its nucleotide sequence is shown in SEQ ID NO: 10, or the promoter P of the thiolase gene. thl, The thiolase gene promoter P thl The nucleotide sequence is shown in SEQ ID NO: 3.

[0018] According to the technical solution of this application, more preferably, in Clostridium butyricum, the promoter P of the thiolytic enzyme gene in a stable, heritable antibiotic-free expression vector in Clostridium butyricum is overexpressed. thl Regulated Clostridium butyricum (Clocas luteolinii) Clostridium tyrobutyricum Butyryl-CoA: Acetyl-CoA transferase gene from [source missing] Ctcat1The butyryl-CoA:acetyl-CoA transferase gene is mentioned. Ctcat1 The nucleotide sequence is shown in SEQ ID NO: 2; the promoter P of the thiolytic enzyme gene is... thl The nucleotide sequence is shown in SEQ ID NO: 3.

[0019] The method for increasing butyric acid synthesis by Clostridium butyricum according to the present invention, wherein the method further comprises:

[0020] The step of overexpressing a gene encoding a CoAT pathway-related enzyme, wherein the CoAT pathway-related enzyme gene includes one or more of the following genes:

[0021] Thiolase gene thl β-hydroxybutyryl coenzyme A dehydrogenase gene hbd Crotonic acidase gene crt Butyryl-CoA dehydrogenase gene bcd ,

[0022] Among them, the thiolysis enzyme gene thl β-hydroxybutyryl coenzyme A dehydrogenase gene hbd Crotonic acidase gene crt Butyryl-CoA dehydrogenase gene bcd All originated from Clostridium butyricum ( Clostridium butyricum ).

[0023] According to the method for preparing butyric acid by fermentation of the present invention, the method comprises the following steps: fermenting Clostridium butyricum engineered strain containing the CoA transferase CoAT pathway in a culture medium without the addition of antibiotics.

[0024] Advantages of the technical solution of the present invention:

[0025] 1. Clostridium butyricum synthesizes butyrate via the phosphobutyryltransferase-butyrate kinase (PTB-BK) pathway. The intermediate product acetyl-CoA in this pathway is readily catalyzed to produce the byproduct acetic acid, resulting in a high acetic acid content in the fermentation product. This invention expresses Clostridium butyricum (… Clostridium tyrobutyricum Butyryl-CoA: Acetyl-CoA transferase gene from [source missing] Ctcat1 A coenzyme A transferase (CoAT) pathway was constructed that synthesizes butyric acid using acetic acid as a substrate, thus significantly reducing the acetic acid content in the fermentation product and significantly increasing the butyric acid yield.

[0026] 2. The plasmid pCB_2 was constructed as a high-efficiency expression vector to establish the CoAT pathway. The replicons of the pCB_2 vector originate from... Clostridium butyricumThe expression vector is an endogenous plasmid of DSM 10702, so it does not require the addition of antibiotics to maintain its presence in Clostridium butyricum, thus ensuring the expression of CoAT pathway-related genes while avoiding the use of antibiotics. Attached Figure Description

[0027] Figure 1 A comparison of butyrate biosynthesis pathways based on the phosphobutyryltransferase-butyrate kinase (PTB-BK) pathway and the coenzyme A transferase (CoAT) pathway;

[0028] Figure 2 The plasmid structures of Clostridium butyricum expression vectors pCB_1 and pCB_2 provided in Example 1 of this invention are shown.

[0029] Figure 3 The *Clostridium butyricum* engineered strain RH-2 (pCB_1-P) provided in Example 1 of this invention. thl -g usA ) and RH-2(pCB_2-P thl -g usA The results of the GusA enzyme activity assay;

[0030] Figure 4 The *Clostridium butyricum* engineered strain RH-2 (pCB_2-P) provided in Example 2 of this invention. thl -g usA ), RH-2(pCB_2-P cat -g usA ), RH-2(pCB_2-P pgi -g usA ), RH-2(pCB_2-P pta -g usA ), RH-2(pCB_2-P ptb -g usA ), RH-2(pCB_2-P adhE -g usA The results of the GusA enzyme activity assay;

[0031] Figure 5 The fermentation results of the wild-type Clostridium butyricum RH-2 and the engineered strains RH-2(pCB_2) and RH-2(pCB_2-PTB-BK) provided in Example 3 of the present invention are shown. In this example, A represents the yield of ethanol, acetic acid and butyric acid; B represents the proportion of ethanol, acetic acid and butyric acid in the total product.

[0032] Figure 6The fermentation results of the engineered strains RH-2(pCB_2-CoAT1), RH-2(pCB_2-CoAT2), RH-2(pCB_2-CoAT3), RH-2(pCB_2-CoAT4), RH-2(pCB_2-CoAT5) and RH-2(pCB_2-CoAT6) provided in Example 4 of this invention are shown. In this figure, A represents the yield of ethanol, acetic acid and butyric acid; B represents the percentage of ethanol, acetic acid and butyric acid in the total product. Detailed Implementation

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0034] Table 1. List of engineered Clostridium butyricum strains constructed in each embodiment of the present invention.

[0035] strain Build process Clostridium butyricum RH-2 wild strain RH-2(pCB_1) Derived from the RH-2 wild-type strain, it contains plasmid pCB_1 (containing replion 1). RH-2(pCB_2) Derived from the RH-2 wild-type strain, it contains plasmid pCB_2 (containing replicon 2). RH-2(pCB_1-Pg) Derived from RH-2 (pCB_1), the pCB_1 plasmid contains a Pg overexpression module. RH-2(pCB_2-Pg) Derived from RH-2 (pCB_2), the pCB_2 plasmid contains a Pg overexpression module. RH-2(pCB_2-Pg) Derived from RH-2 (pCB_2-Pg), the P promoter has been replaced with P RH-2(pCB_2-Pg) Derived from RH-2 (pCB_2-Pg), the P promoter has been replaced with P RH-2(pCB_2-Pg) Derived from RH-2 (pCB_2-Pg), the P promoter has been replaced with P RH-2(pCB_2-Pg) Derived from RH-2 (pCB_2-Pg), the P promoter has been replaced with P RH-2(pCB_2-Pg) Derived from RH-2 (pCB_2-Pg), the P promoter has been replaced with P RH-2(pCB_2-PTB-BK) Derived from RH-2 (pCB_2), the pCB_2 plasmid contains P- and P- overexpression modules. RH-2(pCB_2-CoAT1) Derived from RH-2 (pCB_2), the pCB_2 plasmid contains P- and P-overexpression modules. RH-2(pCB_2-CoAT2) Derived from RH-2 (pCB_2), the pCB_2 plasmid contains P- and P-overexpression modules. RH-2(pCB_2-CoAT3) Derived from RH-2 (pCB_2), the pCB_2 plasmid contains P- and P-overexpression modules. RH-2(pCB_2-CoAT4) Derived from RH-2 (pCB_2), the pCB_2 plasmid contains P- and P-overexpression modules. RH-2(pCB_2-CoAT5) Derived from RH-2 (pCB_2), the pCB_2 plasmid contains P- and P-overexpression modules. RH-2(pCB_2-CoAT6) Derived from RH-2 (pCB_2), the pCB_2 plasmid contains P- and P-overexpression modules. .

[0036] Among them, Clostridium butyricum RH-2 was isolated from a commercially available probiotic product and is a wild fungus isolated from nature.

[0037] As shown in Table 1 above, specific embodiments of this application improve the butyric acid yield and its proportion in the total fermentation product of Clostridium butyricum through the following measures:

[0038] First, based on Clostridium butyricum The replicons of two endogenous plasmids from DSM 10702 were used to construct Clostridium butyricum expression vectors pCB_1 and pCB_2, respectively. Among them, the selected pCB_2 plasmid showed higher genetic stability and gene expression intensity than pCB_1, and could maintain the genetic stability of the plasmid without the addition of antibiotics.

[0039] Second, using the expression vector pCB_2 and the reporter gene g usA Promoters with different expression intensities in Clostridium butyricum were obtained through screening.

[0040] Third, by overexpressing the PTB-BK pathway and the CoAT pathway on the pCB_2 vector, respectively. Figure 1 Related genes were used to increase butyric acid yield and its proportion in total fermentation products, including the use of P thl Promoter overexpression from Clostridium butyricum (Clocas luteolinum) Clostridium tyrobutyricum Butyryl-CoA: Acetyl-CoA transferase gene Ctcat1 At that time, the production level of butyric acid was the highest.

[0041] Example 1: Construction of an expression vector suitable for Clostridium butyricum

[0042] Plasmids are excellent vectors for gene overexpression, but ordinary plasmids require antibiotics to maintain their stable presence in cells. The addition of antibiotics increases fermentation costs, easily leads to the emergence of drug-resistant bacteria, and causes environmental pollution. Therefore, constructing antibiotic-free expression vectors that can be stably inherited in Clostridium butyricum is crucial for establishing an efficient CoAT pathway. Since many Clostridium butyricum strains contain stably inherited endogenous plasmids, those derived from... Clostridium butyricum Expression vectors pCB_1 and pCB_2 were constructed from replicons of two endogenous plasmids from strain DSM 10702. The results showed that pCB_2 exhibited superior genetic stability and gene expression intensity. The specific steps are as follows:

[0043] I. Construction of Clostridium butyricum expression vectors pCB_1 and pCB_2 plasmids

[0044] Clostridium butyricum The DSM 10702 strain contained three endogenous plasmids with sizes of 770.2 kb, 6.1 kb, and 8.1 kb, respectively. Two of the smaller plasmids were suitable for expression vectors, so primers were designed to amplify the replicons of these two plasmids. The nucleotide sequence of replicon 1 of the 6.1 kb endogenous plasmid is shown in SEQ ID NO: 8; the nucleotide sequence of replicon 2 of the 8.1 kb endogenous plasmid is shown in SEQ ID NO: 1. To transform the plasmids into Clostridium butyricum, a traJ module was provided for plasmid transformation via conjugation transfer. The nucleotide sequence of the traJ module is shown in SEQ ID NO: 9. Replicons 1 and 2 were ligated to the conjugation transfer module traJ and then assembled into the plasmid pACYCDuet-1 to obtain plasmids pCB_1 and pCB_2. Figure 2 The specific construction method is as follows:

[0045] Using artificially synthesized DNA fragments of replicon 1 and replicon 2, and the pMTL82151 plasmid as templates, two fragments, replicon 1-traJ and replicon 2-traJ, were obtained by extension PCR amplification. After purification by agarose gel electrophoresis, the two fusion fragments were ligated to the pACYCDuet-1 plasmid and then subjected to thermal shock chemical transformation. E. coli Trans1 competent cells were recovered by shaking at 37°C for 1 h and then plated on LB plates containing 25 µg / mL chloramphenicol. After culturing for 16 h, positive clones were screened and verified by colony PCR and sequencing to obtain plasmids pCB_1 and pCB_2.

[0046] II. Constructing a system containing reporter genes gusA plasmid pCB_1-P thl -g usA and pCB_2-P thl -g usA

[0047] Design primers to amplify the reporter gene. gusA (β-glucuronidase gene), and then using the Clostridium butyricum thiolactic acid enzyme gene promoter P thl The expression was controlled, and finally the expression cassette was assembled onto plasmids pCB_1 and pCB_2, respectively, to obtain plasmid pCB_1-P. thl -g usA and pCB_2-P thl -g usA The specific construction method is as follows:

[0048] Using plasmid pARA-GusA and Clostridium butyricum genome as templates, P was obtained by extension PCR amplification. thl -g usA The fragments were purified by agarose gel electrophoresis, and the obtained fusion fragments were ligated to pCB_1 and pCB_2 plasmids, respectively, and then subjected to thermal chemotransformation. E. coli Trans1 competent cells were recovered by shaking at 37°C for 1 h, then plated on LB agar plates containing 25 µg / mL chloramphenicol and cultured for 16 h. After colonies appeared, positive clones were screened and verified by colony PCR and sequencing to obtain plasmid pCB_1-P. thl - gusA and pCB_2-P thl - gusA .

[0049] III. Construction of engineered Clostridium butyricum strains RH-2(pCB_1), RH-2(pCB_2), and RH-2(pCB_1-P thl -g usA ), RH-2(pCB_2-P thl -g usA )

[0050] (1) Preparation of Escherichia coli CA434 electrotransformation competent cells

[0051] 200 μL of *E. coli* CA434 glycerol stock culture was inoculated into 50 mL LB medium containing 50 µg / mL kanamycin and incubated overnight at 37°C with shaking. The inoculum was then transferred at a 1% inoculation rate to 200 mL LB medium containing 50 µg / mL kanamycin and incubated at 37°C with shaking at 250 rpm until OD (out of control) was reached. 600The culture medium was kept at approximately 0.7. After being placed on ice for 15 min, the cells were collected by centrifugation at 4 °C and 5000 rpm for 10 min. The cells were resuspended in 100 mL of pre-chilled sterile water, and then centrifuged again under the same conditions. The cells were then resuspended in pre-chilled 10% glycerol. Finally, the cells were collected by centrifugation at 4 °C and 5000 rpm for 20 min. The cells were resuspended in 1 mL of pre-chilled 10% glycerol, aliquoted into 100 μL vials, and stored at -80 °C.

[0052] (2) Electroporation of Escherichia coli CA434 and screening of transformants

[0053] E. coli CA434 competent cells stored at -80℃ were thawed on ice. 1 μg of plasmid was added to 100 μL of competent cells, mixed thoroughly by pipetting, and transferred to a 0.1 cm electroporation cuvette. Electroporation was performed at 1800 V, 25 μF, and 200 Ω. The electroporated bacterial culture was then plated onto LB agar plates containing 50 µg / mL kanamycin and 25 µg / mL chloramphenicol and incubated at 37℃ for 16 h. After colony formation, colony PCR was used to confirm pCB_1, pCB_2, and pCB_1-P. thl -g usA pCB_2-P thl -g usA All plasmids were successfully transformed into E. coli CA434 cells.

[0054] (3) Construct strains RH-2(pCB_1), RH-2(pCB_2), and RH-2(pCB_1-P thl -g usA ), RH-2(pCB_2-P thl -g usA )

[0055] Select those containing pCB_1, pCB_2, and pCB_1-P thl -g usA pCB_2-P thl -g usA A single clone of E. coli CA434 containing the plasmid was inoculated into LB liquid medium containing 50 µg / mL kanamycin and 25 µg / mL chloramphenicol and cultured. When OD 600When the culture medium reaches approximately 1.0, 1 mL of culture medium is centrifuged at 5000 rpm for 10 min at room temperature to collect the cells. The cells are resuspended in 1 mL of antibiotic-free LB liquid medium, and centrifuged again under the same conditions. Finally, the cells are resuspended in 100 μL of antibiotic-free LB liquid medium. Clostridium butyricum RH-2 is inoculated into RCM liquid medium, the formulation of which is: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 3.0 g / L, glucose 5.0 g / L, soluble starch 1.0 g / L, sodium chloride 5.0 g / L, sodium acetate 3.0 g / L, L-cysteine ​​hydrochloride 0.5 g / L; pH is controlled at approximately 6.8. The inoculated culture medium is cultured anaerobically at 37°C until the OD reaches... 600 To achieve a pH of 1.0-1.5, mix 100 μL of *Clostridium butyricum* culture with 100 μL of *Escherichia coli* CA434 cell resuspension, then spot the mixture onto an RCM agar plate and incubate at 37°C for 12 h. Next, wash the bacterial cells from the agar plate surface with 500 μL of fresh RCM liquid medium and spread the mixture onto an RCM agar plate containing 250 µg / mL D-cyclic serine and 15 µg / mL thiamphenicol. Incubate at 37°C for 2-3 days until colonies appear. Verify the colony count using colony PCR to confirm pCB_1, pCB_2, and pCB_1-P. thl -g usA pCB_2-P thl -g usA All plasmids were successfully transformed into Clostridium butyricum RH2, yielding the corresponding engineered strains RH-2(pCB_1), RH-2(pCB_2), and RH-2(pCB_1-P, respectively). thl -g usA ), RH-2(pCB_2-P thl -g usA ).

[0056] IV. Verification of plasmid genetic stability of engineered strains RH-2(pCB_1) and RH-2(pCB_2)

[0057] Engineered strains RH-2 (pCB_1) and RH-2 (pCB_2) were inoculated into 50 mL of RCM liquid medium, with 15 µg / mL thiamphenicol added. The cultures were then incubated overnight at 37°C under anaerobic conditions to obtain seed cultures. Subsequently, the inoculum was transferred at a 1% inoculation rate to 100 mL of antibiotic-free RCM liquid medium and incubated anaerobically at 37°C for 12 h. Following the same method, engineered strains RH-2 (pCB_1) and RH-2 (pCB_2) were passaged 10 times consecutively in antibiotic-free medium. The resulting culture media were serially diluted and plated onto RCM solid plates containing thiamphenicol and those without antibiotics, respectively. The plasmid loss rate (1 - number of clones on the antibiotic-treated plate / number of clones on the antibiotic-free plate) was calculated based on the number of clones grown on each type of plate. The results are shown in Table 2.

[0058] Table 2. Results of plasmid passage genetic stability of engineered strains RH-2(pCB_1) and RH-2(pCB_2).

[0059] strain Number of generations Plasmid loss rate (%) RH-2(pCB_1) 0 0 RH-2(pCB_1) 10 47.9 RH-2(pCB_2) 0 0 RH-2(pCB_2) 10 0 .

[0060] As shown in Table 2 above, after 10 consecutive passages in the absence of antibiotics, 47.9% of cells in strain RH-2 (pCB_1) lost the pCB_1 plasmid. Under the same passage conditions, no plasmid loss was observed in strain RH-2 (pCB_2), indicating that pCB_2 has strong plasmid genetic stability in Clostridium butyricum.

[0061] V. Engineered strain RH-2 (pCB_1-P) thl -g usA ), RH-2(pCB_2-P thl -g usA ) gene expression intensity verification

[0062] The engineered strain RH-2(pCB_1-P) thl -g usA ) and RH-2(pCB_2-P thl -g usA The inoculum was separately inoculated into 50 mL of RCM liquid medium, with 15 µg / mL thiamphenicol added to the medium, and cultured overnight at 37 °C under anaerobic conditions to obtain the seed culture. Subsequently, the inoculum was transferred at a 1% inoculation rate to 100 mL of antibiotic-free RCM liquid medium and cultured at 37 °C under anaerobic conditions until OD (dose elapsed). 600 The value reached approximately 1.5. Following the method described above, the engineered strain RH-2(pCB_1-P) was... thl -g usA ) and RH-2(pCB_2-P thl -g usA The cells were passaged 10 times consecutively in antibiotic-free culture medium. The resulting culture was centrifuged to collect the cells, which were then resuspended in GusA buffer. The GusA buffer formulation was: 50 mM Na3PO4, 1 mM EDTA, pH 7.0. Cells were disrupted using an ultrasonic disruptor after resuspension, followed by centrifugation at 8000 rpm for 15 min at 4 °C to remove cell debris. The supernatant was used as the crude enzyme solution. Enzyme activity was verified using 4-methylumbelliferone-D-glucuronide (MUG) as a substrate. The specific detection method was as follows: 0.2 mL of the diluted crude enzyme solution was thoroughly mixed with 1.8 mL of GusA buffer containing 4 mM MUG. This reaction mixture was then added to a quartz fluorescence cuvette (optical path = 1 cm), and the fluorescence intensity was measured using a fluorescence spectrophotometer at 37 °C. The fluorescence kinetic curve was recorded using time-scan mode (excitation wavelength set to 365 nm, emission wavelength set to 455 nm, photomultiplier tube voltage set to 700 V, data recorded every 30 s for a total of 10 min). Finally, the GusA activity (U / mg) was calculated by comparing the slope of the curve with the protein concentration of the sample. Figure 3 As shown, the GusA activity results indicated that after one passage, the GusA activity using pCB_1 as the expression vector was 77.5% of that using pCB_2 as the expression vector; after 10 passages, due to the loss of the pCB_1 plasmid, the GusA activity of strain RH-2(pCB_1-P) was significantly reduced. thl -g usA The GusA activity of strain RH-2 (pCB_2-P) decreased by 51.1% compared to the time of one subculture, while that of strain RH-2 (pCB_2-P) decreased by 51.1%. thl -g usA The GusA activity of pCB_2 remained largely unchanged, indicating that pCB_2 has strong gene expression intensity and stability in Clostridium butyricum.

[0063] Example 2: Screening for promoters suitable for Clostridium butyricum gene expression

[0064] Promoters control gene expression, and strong promoters are crucial for establishing efficient butyrate synthesis pathways. Therefore, we selected promoters of key genes involved in glycolysis in *Clostridium butyricum*, promoters of key genes involved in the synthesis of butyrate / acetic acid / ethanol products, and strong promoters from exogenous *Clostridium butyricum* to... gusA As a reporter gene, its expression intensity in Clostridium butyricum was verified. The results showed that the expression intensity of Clostridium butyricum's own thiolactic enzyme gene promoter P... thl The expression intensity is relatively good. The specific steps are as follows:

[0065] I. Constructing a system containing reporter genes gusA plasmid pCB_2-P cat -g usA pCB_2-P pgi -g usA pCB_2-P pta -g usA pCB_2-P ptb -g usA and pCB_2-P adhE -g usA

[0066] Design primers to amplify the reporter gene. gusA (β-glucuronidase gene), and then using the butyryl-CoA:acetyl-CoA transferase gene promoter P from Clostridium butyricum. cat (Its nucleotide sequence is shown in SEQ ID NO: 10), the promoter P of the 6-phosphate glucose isomerase gene derived from Clostridium butyricum. pgi (Its nucleotide sequence is shown in SEQ ID NO: 11), Phosphoacetyltransferase gene promoter P pta (Its nucleotide sequence is shown in SEQ ID NO: 12), Phosphobutyryltransferase gene promoter P ptb (Its nucleotide sequence is shown in SEQ ID NO: 13), acetaldehyde-ethanol dehydrogenase gene promoter P adhE (Its nucleotide sequence is shown in SEQ ID NO: 14) to control its expression, and finally the expression cassette was assembled onto plasmid pCB_2 to obtain plasmid pCB_2-P. cat -g usA pCB_2-P pgi -g usA pCB_2-P pta -g usA pCB_2-P ptb -g usA and pCB_2-P adhE -g usA The specific construction method is as follows:

[0067] Using plasmid pARA-GusA, Clostridium butyricum genome, and Clostridium butyricum genome as templates, P was obtained by extension PCR amplification. cat -g usA P pgi -g usA P pta -g usA P ptb -g usA and P adhE -g usAThe fragments were purified by agarose gel electrophoresis, and the obtained fusion fragments were ligated to pCB_2 plasmids, followed by thermal chemitransformation. E. coli Trans1 competent cells were recovered by shaking at 37°C for 1 h, then plated on LB agar plates containing 25 µg / mL chloramphenicol and cultured for 16 h. After colonies appeared, positive clones were screened and verified by colony PCR and sequencing to obtain plasmid pCB_2-P. cat -g usA pCB_2-P pgi -g usA pCB_2-P pta -g usA pCB_2-P ptb -g usA and pCB_2-P adhE -g usA .

[0068] II. Construction of strain RH-2(pCB_2-P) cat -g usA ), RH-2(pCB_2-P pgi -g usA ), RH-2(pCB_2-P pta -g usA ), RH-2(pCB_2-P ptb -g usA ), RH-2(pCB_2-P adhE -g usA )

[0069] Following the Escherichia coli electroporation and Clostridium butyricum conjugation transfer experimental method described in Example 1, the constructed plasmid pCB_2-P cat -g usA pCB_2-P pgi -g usA pCB_2-P pta -g usA pCB_2-P ptb -g usA and pCB_2-P adhE -g usA The engineered strain RH-2 (pCB_2-P) was obtained by transforming Escherichia coli CA434 into Clostridium butyricum RH-2. cat -g usA ), RH-2(pCB_2-P pgi -g usA ), RH-2(pCB_2-P pta -g usA ), RH-2(pCB_2-P ptb -g usA ), RH-2(pCB_2-P adhE -g usA ).

[0070] III. Engineered strain RH-2 (pCB_2-P) thl -g usA ), RH-2(pCB_2-P cat -g usA ), RH-2(pCB_2-P pgi -g usA ), RH-2(pCB_2-P pta -g usA ), RH-2(pCB_2-P ptb -g usA ), RH-2(pCB_2-P adhE -g usA ) gene expression intensity verification

[0071] The engineered strain RH-2(pCB_2-P) cat -g usA ), RH-2(pCB_2-P pgi -g usA ), RH-2(pCB_2-P pta -g usA ), RH-2(pCB_2-P ptb -g usA ), RH-2(pCB_2-P adhE -g usA ) and the engineered strain RH-2 (pCB_2-P) constructed in Example 1 thl -g usA The inoculum was separately inoculated into 50 mL of RCM liquid medium containing 15 µg / mL thiamphenicol and cultured overnight at 37 °C under anaerobic conditions to obtain the seed culture. Subsequently, the inoculum was transferred at a 1% inoculation rate to 100 mL of antibiotic-free RCM liquid medium and cultured at 37 °C under anaerobic conditions until OD (dose elapsed). 600 The concentration reached approximately 1.5. The obtained culture medium was centrifuged to collect bacterial cells, and crude enzyme solution was prepared and GusA enzyme activity was measured.

[0072] The preparation of crude enzyme solution and the determination of GusA enzyme activity are the same as those described in Example 1.

[0073] like Figure 4 As shown in the GusA activity results, the expression intensity of the six promoters was ranked as follows: P thl >P cat >P pgi >P ptb >P pta >P adhE With P thl The GusA enzyme activity was highest when it was the promoter, indicating that its expression effect was good and it is suitable for the expression of genes in the butyrate efficient synthesis pathway.

[0074] Example 3: Enhancing Butyric Acid Production by Enhancing the PTB-BK Pathway in Clostridium butyricum

[0075] The main beneficial substance of Clostridium butyricum is butyric acid, but in addition to butyric acid, Clostridium butyricum also synthesizes large amounts of byproducts such as acetic acid and ethanol. Therefore, overexpression of the thiolase gene is crucial for its development. thl β-hydroxybutyryl coenzyme A dehydrogenase gene hbd Crotonic acidase gene crt Butyryl-CoA dehydrogenase gene bcd Phosphobutyryltransferase gene ptb Butyrate kinase gene bk Strengthen its own PTB-BK pathway ( Figure 1 This can introduce more carbon metabolic flux into the butyrate synthesis pathway, thereby increasing butyrate production.

[0076] I. Construction of pCB_2-PTB-BK plasmid

[0077] Design primers to amplify the thiolytic enzyme gene of Clostridium butyricum. thl β-hydroxybutyryl coenzyme A dehydrogenase gene hbd Crotonic acidase gene crt Butyryl-CoA dehydrogenase gene bcd Phosphobutyryltransferase gene ptb Butyrate kinase gene bk Among them, the phosphobutyryltransferase gene derived from Clostridium butyricum ptb The nucleotide sequence is shown in SEQ ID NO: 15; butyrate kinase gene from Clostridium butyricum. bk The nucleotide sequence is shown in SEQ ID NO: 16, and each gene is transmitted via P. thl The promoter controls its expression, and finally the expression cassette is assembled onto plasmid pCB_2 to obtain plasmid pCB_2-PTB-BK; the specific construction method is as follows:

[0078] Using the Clostridium butyricum genome as a template, P was obtained by extended PCR amplification. thl - thl P thl - hbd P thl - crt P thl - bcd P thl - ptb and P thl - bk The fragments were purified by agarose gel electrophoresis, and the obtained fusion fragments were ligated to the pCB_2 plasmid, followed by thermal chemical transformation. E. coli Trans1 competent cells were recovered by shaking at 37°C for 1 h and then plated on LB plates containing 25 µg / mL chloramphenicol. After culturing for 16 h, positive clones were screened and verified by colony PCR and sequencing to obtain plasmid pCB_2-PTB-BK.

[0079] II. Construction of strain RH-2(pCB_2-PTB-BK)

[0080] Following the electroporation of Escherichia coli and the conjugation transfer experiment of Clostridium butyricum described in Example 1, the constructed plasmid pCB_2-PTB-BK was transformed into Clostridium butyricum RH-2 using Escherichia coli CA434 to obtain the engineered strain RH-2 (pCB_2-PTB-BK).

[0081] III. Fermentation experiments of Clostridium butyricum RH-2, engineered strains RH-2 (pCB_2) and RH-2 (pCB_2-PTB-BK)

[0082] Wild-type Clostridium butyricum strain RH-2, engineered strains RH-2 (pCB_2) and RH-2 (pCB_2-PTB-BK) were inoculated into 50 mL of RCM liquid medium and cultured overnight at 37 °C under anaerobic conditions to serve as seed culture. Subsequently, the inoculum was transferred to 100 mL of RCM liquid medium at a 5% inoculation rate and fermented under anaerobic conditions at 37 °C with shaking for 24 h. Samples were then taken to determine the content of butyric acid, acetic acid, and ethanol in the fermentation broth. Figure 5 As shown, Figure 5 Figure A shows the yields of ethanol, acetic acid, and butyric acid. Figure 5 Figure B shows the proportions of ethanol, acetic acid, and butyric acid in the total products. In the wild-type strain *Clostridium butyricum* RH-2, butyric acid accounts for 60% of the total product yield, making it the main metabolite. Compared to the wild-type strain, the yields and proportions of butyric acid, acetic acid, and ethanol in the engineered strain RH-2 (pCB_2) remained largely unchanged, indicating that the empty vector pCB_2 has a relatively small impact on the metabolic network of *Clostridium butyricum*. After enhancing the PTB-BK pathway, the butyric acid yield of the engineered strain RH-2 (pCB_2-PTB-BK) increased by 26.6% compared to the wild-type strain, and the proportion of butyric acid in the total products also increased from 60% to 71%, while the proportions of byproducts acetic acid and ethanol in the total products decreased by 5% and 6%, respectively. This indicates that enhancing the PTB-BK pathway can improve the butyric acid synthesis capacity of *Clostridium butyricum*.

[0083] Example 4: Improving butyric acid production by Clostridium butyricum through the construction of the CoAT pathway

[0084] The PTB-BK pathway inherent in Clostridium butyricum is relatively inefficient in synthesizing butyrate, and the intermediate product acetyl-CoA is easily converted into byproducts such as acetic acid. In contrast, the CoAT pathway synthesizes butyrate using acetic acid and butyryl-CoA as precursors. Figure 1 This can significantly reduce the synthesis of byproducts such as acetic acid and increase butyric acid yield. Therefore, overexpressing the thiolase gene can significantly reduce the synthesis of byproducts such as acetic acid and increase butyric acid yield. thl β-hydroxybutyryl coenzyme A dehydrogenase gene hbd Crotonic acidase gene crt Butyryl-CoA dehydrogenase gene bcd Butyryl-CoA: Acetyl-CoA transferase gene cat The CoAT pathway was introduced into Clostridium butyricum.

[0085] I. Construction of plasmids pCB_2-CoAT1, pCB_2-CoAT2, pCB_2-CoAT3, pCB_2-CoAT4, pCB_2-CoAT5, and pCB_2-CoAT6

[0086] Design primers to amplify the butyryl-CoA:acetyl-CoA transferase gene from Clostridium butyricum. Ctcat1 （ SEQ ID NO: 2 ） Ruminococcal bacteria CPB6 ( Ruminococcaceae Butyryl-CoA:acetyl-CoA transferase gene derived from bacterium CPB6 Rbcat1 (Its nucleotide sequence is shown in SEQ ID NO: 17), Clostridium coccidioides ( Clostridium kluyveri Butyryl-CoA: Acetyl-CoA transferase gene from [source missing] Ckcat3 (Its nucleotide sequence is shown in SEQ ID NO: 18), each gene is transmitted via P thl or P cat The promoter controls its expression, and finally, according to the combination shown in Table 1, it is integrated into the plasmid pCB_2-PTB-BK and replaces P. thl - ptb - bk The overexpression module is constructed as follows:

[0087] Ctcat1 , Rbcat1 and Ckcat3 The gene was obtained through artificial synthesis, and other construction methods were the same as those for the pCB_2-PTB-BK plasmid construction.

[0088] II. Construction of strains RH-2(pCB_2-CoAT1), RH-2(pCB_2-CoAT2), RH-2(pCB_2-CoAT3), RH-2(pCB_2-CoAT4), RH-2(pCB_2-CoAT5) and RH-2(pCB_2-CoAT6)

[0089] Following the Escherichia coli electroporation and Clostridium butyricum conjugation transfer experimental method described in Example 1, the constructed plasmids pCB_2-CoAT1, pCB_2-CoAT2, pCB_2-CoAT3, pCB_2-CoAT4, pCB_2-CoAT5, and pCB_2-CoAT6 were transformed into Clostridium butyricum RH-2 using Escherichia coli CA434, respectively, to obtain engineered strains RH-2(pCB_2-CoAT1), RH-2(pCB_2-CoAT2), RH-2(pCB_2-CoAT3), RH-2(pCB_2-CoAT4), RH-2(pCB_2-CoAT5), and RH-2(pCB_2-CoAT6).

[0090] III. Fermentation experiments of engineered strains RH-2(pCB_2-CoAT1), RH-2(pCB_2-CoAT2), RH-2(pCB_2-CoAT3), RH-2(pCB_2-CoAT4), RH-2(pCB_2-CoAT5), and RH-2(pCB_2-CoAT6)

[0091] The constructed RH-2(pCB_2-CoAT1), RH-2(pCB_2-CoAT2), RH-2(pCB_2-CoAT3), RH-2(pCB_2-CoAT4), RH-2(pCB_2-CoAT5), and RH-2(pCB_2-CoAT6) strains were fermented under anaerobic conditions, as follows:

[0092] The fermentation medium is RCM liquid medium.

[0093] The fermentation method described herein is the same as that described in Example 3.

[0094] The method for detecting fermentation products is the same as that described in Example 3.

[0095] Combination Figure 5 and Figure 6 , Figure 6 Figure A shows the yields of ethanol, acetic acid, and butyric acid. Figure 6 Figure B shows the percentages of ethanol, acetic acid, and butyric acid in the total products. Constructing the CoAT pathway in *Clostridium butyricum* significantly increases butyric acid production and reduces the production of byproducts acetic acid and ethanol. Overexpression of the same source... cat When using genes, use P thl Promoter than using P cat The promoter increased butyrate production by 5%-9%. However, when using the same promoter, but from different sources... cat The gene's performance in butyrate production is as follows: Ctcat1 > Ckcat3 > Rbcat1 The above results indicate that overexpression of Clostridium butyricum-derived... Ctcat1 and using P thl The promoter controls the highest butyric acid production level when it is expressed. Compared with the wild-type strain RH-2 and the engineered strain RH-2(pCB_2-PTB-BK), the butyric acid production of strain RH-2(pCB_2-CoAT1) was increased by 55.8% and 23.1%, respectively, and the proportion of butyric acid in the total product was increased by 26% and 15%, respectively, indicating that the CoAT pathway is superior to the PTB-BK pathway in improving the butyric acid production level of Clostridium butyricum.

[0096] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. An improved Clostridium butyricum engineered strain for synthesis of butyric acid via the Coenzyme A transferase, CoAT, pathway, characterized in that, The Clostridium butyricum engineered strain is a Clostridium butyricum having the following characteristics: overexpressing a butyryl-CoA:acetate-CoA transferase gene, wherein the nucleotide sequence of the butyryl-CoA:acetate-CoA transferase gene is shown as SEQ ID NO: 2, and the nucleotide sequence of the promoter regulating the butyryl-CoA:acetate-CoA transferase gene is shown as SEQ ID NO:

3.

2. The Clostridium butyricum engineered strain for improved synthesis of butyric acid via the Coenzyme A transferase (CoAT) pathway of claim 1, characterized in that, The butyryl-CoA:acetate-CoA transferase gene is integrated into an antibiotic-free expression vector that is stably inherited in Clostridium butyricum, wherein the nucleotide sequence of the replicon of the antibiotic-free expression vector that is stably inherited in Clostridium butyricum is shown as SEQ ID NO:

1.

3. The Clostridium butyricum engineered strain for improved synthesis of butyric acid via the Coenzyme A transferase (CoAT) pathway of claim 2, characterized in that, On the antibiotic-free expression vector that is stably inherited in Clostridium butyricum, the coding genes of CoAT pathway-related enzymes are overexpressed, and the CoAT pathway-related enzyme genes include one or more of the following genes: sulfolyase gene thl , beta-hydroxybutyryl-CoA dehydrogenase gene hbd , crotonase gene crt , butyryl-CoA dehydrogenase gene bcd , Among them, the thiolysis enzyme gene thl The nucleotide sequence is shown in SEQ ID NO: 4, β-hydroxybutyryl coenzyme A dehydrogenase gene. hbd The nucleotide sequence is shown in SEQ ID NO: 5, crotonase gene. crt The nucleotide sequence is shown in SEQ ID NO: 6, butyryl-CoA dehydrogenase gene. bcd The nucleotide sequence of the homogeneous nucleotide is shown in SEQ ID NO:

7.

4. A method for increasing the amount of butyric acid synthesis by Clostridium butyricum, characterized by, The method comprises the following steps: overexpressing a butyryl-CoA:acetate-CoA transferase gene in Clostridium butyricum, wherein the nucleotide sequence of the butyryl-CoA:acetate-CoA transferase gene is shown as SEQ ID NO: 2, and the nucleotide sequence of the promoter regulating the butyryl-CoA:acetate-CoA transferase gene is shown as SEQ ID NO:

3.

5. The method of increasing the amount of butyric acid synthesis by Clostridium butyricum according to claim 4, characterized by, The method further comprises the step of integrating the butyryl-CoA:acetate-CoA transferase gene into an antibiotic-free expression vector that is stably inherited in Clostridium butyricum, wherein the nucleotide sequence of the replicon of the antibiotic-free expression vector that is stably inherited in Clostridium butyricum is shown as SEQ ID NO:

1.

6. The method of increasing the amount of butyric acid synthesis by Clostridium butyricum according to claim 5, characterized by, The method further comprises the step of overexpressing the coding genes of CoAT pathway-related enzymes, and the CoAT pathway-related enzyme genes include one or more of the following genes: sulfolyase gene thl , beta-hydroxybutyryl-CoA dehydrogenase gene hbd , crotonase gene crt , butyryl-CoA dehydrogenase gene bcd , Among them, the thiolysis enzyme gene thl The nucleotide sequence is shown in SEQ ID NO: 4, β-hydroxybutyryl coenzyme A dehydrogenase gene. hbd The nucleotide sequence is shown in SEQ ID NO: 5, crotonase gene. crt The nucleotide sequence is shown in SEQ ID NO: 6, butyryl-CoA dehydrogenase gene. bcd The nucleotide sequence of the homogeneous nucleotide is shown in SEQ ID NO: 7.

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