A genetically engineered bacterium with high butyric acid production and a construction method and application thereof

Abstract

This invention discloses a genetically engineered bacterium producing high levels of butyric acid, its construction method, and its applications. The invention constructs a genetically engineered bacterium producing high levels of butyric acid by overexpressing a ferrous ion transporter protein in Clostridium. Compared to wild-type strains, the genetically engineered bacterium of this invention exhibits enhanced metabolic flux in reactions related to butyric acid synthesis, inhibits the allocation of carbon to byproduct pathways such as acetic acid and lactic acid, and maintains good growth and metabolic activity even under butyric acid stress, thereby prolonging the effective acid production time and improving the stability and final accumulation of butyric acid in the later stages of fermentation. In other words, the genetically engineered bacterium of this invention can increase the proportion of butyric acid in fermentation products under the same substrate conditions, reduce byproduct formation, and lower the difficulty and cost of downstream separation and purification. Furthermore, the genetically engineered bacterium of this invention is robust, suitable for scale-up and industrial applications, and beneficial for the biosynthesis and widespread application of butyric acid.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and fermentation engineering technology. More specifically, it relates to a genetically engineered bacterium that produces high levels of butyric acid, its construction method, and its applications. Background Technology

[0002] Butyric acid, also known as butyric acid, is a four-carbon, straight-chain saturated monocarboxylic fatty acid that plays a vital role in maintaining physiological homeostasis. It provides a primary energy source for colonic epithelial cells, maintains intestinal homeostasis, and participates in the regulation of inflammatory responses. Studies have found that butyric acid levels in patients with various chronic non-communicable diseases are typically lower than in healthy individuals, suggesting that butyric acid has potential physiological and medical significance in disease prevention and treatment. In addition to its physiological functions, butyric acid and its derivatives are widely used in chemical, food, pharmaceutical, and animal feed industries, and demand is steadily increasing.

[0003] The industrial production of butyric acid mainly relies on two pathways: chemical synthesis and microbial fermentation. Compared with chemical synthesis, microbial fermentation for butyric acid production is more sustainable and has greater application potential. On the one hand, this pathway can utilize renewable biomass or agricultural byproducts as substrates, achieving resource recycling and reducing production costs. On the other hand, butyric acid produced by microbial fermentation has high purity and biosafety, making it more suitable for high-value-added fields such as food and pharmaceuticals. In contrast, the chemical synthesis pathway relies excessively on fossil resources and generates toxic byproducts, posing potential threats to both the environment and safety. Although microbial fermentation has significant advantages in terms of raw material renewability and environmental friendliness, the current biosynthesis of butyric acid still faces key challenges such as low yield, insufficient substrate conversion efficiency, and high costs for subsequent separation and purification, severely limiting its application and promotion in industrial production.

[0004] Clostridium butyricum (Clocas luteolinum) Clostridium tyrobutyricum Clostridium butyricum (C. butyricum) is one of the most representative butyric acid-producing strains, considered a core microorganism for butyric acid biosynthesis due to its stable fermentation performance and clear metabolic pathway. However, the butyric acid production capacity of *C. butyricum* is limited by substrate utilization efficiency and metabolic regulation mechanisms, resulting in a low butyric acid yield. For example, a large amount of carbon in the fermentation system flows to byproducts such as acetic acid and lactic acid, leading to insufficient butyric acid yield and selective metabolic flow towards butyric acid when glucose is used as a substrate, reducing the economic viability and technological attractiveness of butyric acid biosynthesis. In addition, although *C. butyricum* has strong robustness, its growth is inhibited as the concentration of organic acids in the fermentation broth increases. When the butyric acid concentration in the fermentation broth exceeds 40 g / L, wild-type *C. butyricum* begins to lose its growth ability.

[0005] Currently, research on improving the butyrate production capacity of Clostridium butyricum mainly focuses on two aspects: metabolic pathway regulation and fermentation condition optimization. For example, adjusting the pH, temperature, and carbon source ratio of the fermentation system can improve the physiological state and redox balance of the bacteria, thereby increasing butyrate yield to some extent. Alternatively, knocking out genes related to acetic acid or lactic acid synthesis pathways can reduce byproduct formation and promote butyrate carbon flux enrichment, thus increasing butyrate yield. However, such modifications often lead to decreased bacterial growth rate or redox imbalance, making it difficult to achieve stable high yields in high-density fermentation systems, resulting in insufficient system stability. Furthermore, existing strategies mostly focus on metabolic pathway regulation, failing to simultaneously improve the strain's tolerance to acid stress caused by butyrate accumulation. This leads to inhibited bacterial growth or even premature death under high butyrate production conditions, making it difficult to maintain stable and efficient butyrate synthesis in the later stages of fermentation. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a genetically engineered bacterium that produces high levels of butyric acid, its construction method, and its applications.

[0007] The first objective of this invention is to provide a genetically engineered bacterium that produces high levels of butyric acid.

[0008] A second objective of this invention is to provide a method for constructing the genetically engineered bacteria.

[0009] A third objective of this invention is to provide the application of the genetically engineered bacteria in the biosynthesis of butyric acid.

[0010] A fourth object of the present invention is to provide the use of the genetically engineered bacteria in the preparation of products for the biosynthesis of butyric acid.

[0011] The fifth object of this invention is to provide a method for the biosynthesis of butyric acid.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution:

[0013] Ferrous ion transporter protein (FeoB) is a major ferrous ion transporter in prokaryotes. This invention discovers that overexpression of FeoB protein in *Clostridium butyricum* can enhance its fermentation performance, improve its tolerance to butyric acid stress, and achieve efficient butyric acid production. Therefore, this invention provides a genetically engineered bacterium with high butyric acid production and its construction method.

[0014] This invention provides a genetically engineered bacterium that produces high levels of butyric acid. Specifically, the genetically engineered bacterium overexpresses a ferrous ion transporter protein in Clostridium butyricum, which has butyric acid synthesis capabilities and is strictly anaerobic; the ferrous ion transporter protein is derived from Clostridium butyricum.

[0015] In a specific embodiment of the present invention, the Clostridium with butyric acid synthesis ability and strict anaerobic properties is Clostridium butyricum.

[0016] Specifically, the ferrous ion transporter protein is FeoB1 protein and / or FeoB2 protein derived from Clostridium butyricum; the amino acid sequence of the FeoB1 protein is shown in SEQ ID NO.2; and the amino acid sequence of the FeoB2 protein is shown in SEQ ID NO.4.

[0017] Specifically, the gene encoding the FeoB1 protein feoB1 The nucleotide sequence is shown in SEQ ID NO.1; the gene encoding the FeoB2 protein. feoB2 The nucleotide sequence is shown in SEQ ID NO.3.

[0018] The present invention also provides a method for constructing the genetically engineered bacteria, wherein the method comprises: transferring the encoding gene of the ferrous ion transporter protein derived from Clostridium butyricum into Clostridium butyricum, which has butyric acid synthesis ability and is strictly anaerobic, and overexpressing the ferrous ion transporter protein.

[0019] Specifically, the ferrous ion transporter protein is FeoB1 protein and / or FeoB2 protein derived from Clostridium butyricum; the amino acid sequence of the FeoB1 protein is shown in SEQ ID NO.2; and the amino acid sequence of the FeoB2 protein is shown in SEQ ID NO.4.

[0020] Specifically, the gene encoding the FeoB1 protein feoB1 The nucleotide sequence is shown in SEQ ID NO.1; the gene encoding the FeoB2 protein. feoB2 The nucleotide sequence is shown in SEQ ID NO.3.

[0021] Optionally, the genetically engineered bacteria can be constructed by constructing an overexpression recombinant plasmid containing the encoding gene and transferring it into the Clostridium.

[0022] Without using free plasmids, the coding gene can be integrated into the Clostridium chromosome for stable expression through gene editing techniques such as homologous recombination and CRISPR / Cas, replacing plasmid overexpression and obtaining a long-term stable and heritable high-butyrate phenotype. Furthermore, butyrate synthesis capacity can be enhanced to varying degrees by adjusting the copy number or transcriptional intensity of the coding gene within the bacterial cell.

[0023] In a specific embodiment of the present invention, the method for constructing the genetically engineered bacteria includes the following steps: S1. The recombinant plasmid is constructed by PCR amplification or artificial synthesis of the coding gene and insertion into the multiple cloning site of the overexpression plasmid; S2. The recombinant plasmid obtained in S1 is transferred into Clostridium that has butyrate synthesis ability and is strictly anaerobic.

[0024] In a specific embodiment of the present invention, the Clostridium with butyric acid synthesis ability and strict anaerobic properties is Clostridium butyricum.

[0025] In a specific embodiment of the present invention, the overexpression plasmid is pMTL82151 plasmid.

[0026] More specifically, the plasmid is pMTL82151 carrying a constitutive promoter; the constitutive promoter is the PCAT1 promoter.

[0027] In addition to constitutive promoters, inductive promoters, staged promoters, or stress-response promoters related to the fermentation process can also be used.

[0028] In a specific embodiment of the present invention, the recombinant plasmid is transferred into the Clostridium via conjugation transformation.

[0029] Specifically, the donor bacteria used for conjugation transformation is Escherichia coli.

[0030] More specifically, the *E. coli* is E coli Strain CA434.

[0031] Compared to wild-type strains, the butyric acid yield is significantly increased when using the genetically engineered bacteria described in this invention for butyric acid biosynthesis. Therefore, this invention seeks protection for the application of the genetically engineered bacteria in butyric acid biosynthesis.

[0032] The present invention also seeks protection for the use of the genetically engineered bacteria in the preparation of products for the biosynthesis of butyric acid.

[0033] Optionally, the product is a microbial preparation containing the genetically engineered bacteria.

[0034] The present invention also provides a method for biosynthesizing butyric acid, wherein the method comprises fermenting and culturing the genetically engineered bacteria using glucose or glucose derivatives as a carbon source.

[0035] Specifically, during fermentation, the pH of the fermentation system is controlled within the range of 5.5 to 6.5.

[0036] Preferably, the pH of the fermentation system is controlled within the range of 5.8 to 6.2.

[0037] More preferably, the pH of the fermentation system is controlled at 6.

[0038] In a specific embodiment of the present invention, the culture medium used for fermentation is a glucose-based fermentation medium.

[0039] In addition to glucose, the fermentation medium also contains yeast extract, peptone, NaCl, (NH4)2SO4, K2HPO4, MgSO4·7H2O and FeSO4·7H2O, with a pH of 6.0±0.2.

[0040] The present invention has the following beneficial effects: This invention constructs a genetically engineered bacterium that produces high levels of butyric acid by overexpressing a ferrous ion transporter protein in Clostridium. Compared with wild-type Clostridium, the genetically engineered bacterium of this invention has the following advantages: 1. Unlike existing metabolic engineering strategies that knock out byproduct pathways or forcibly enhance the activity of key enzymes in butyrate synthesis, this invention enhances the endogenous ferrous ion transport system of Clostridium butyricum through genetic means to improve butyrate yield. This avoids direct intervention in the main butyrate synthesis pathway, thereby reducing the risks of growth inhibition, energy imbalance and system instability caused by metabolic pathway reconstruction.

[0041] 2. Under conditions of high butyrate accumulation, wild-type Clostridium butyricum is prone to growth stagnation or even inactivation, limiting the butyrate concentration at the fermentation endpoint. However, the genetically engineered bacteria described in this invention can maintain good growth and metabolic activity under butyrate stress, thereby prolonging the effective acid production time and improving the stability and final butyrate accumulation in the later stages of fermentation.

[0042] 3. In the genetically engineered bacteria described in this invention, the metabolic flux of butyric acid synthesis-related reactions is enhanced, while the allocation of carbon to byproduct pathways such as acetic acid and lactic acid is inhibited. Compared with existing technologies, under the same substrate conditions, this invention can increase the proportion of butyric acid in fermentation products and reduce byproduct formation, thereby reducing the difficulty and cost of downstream separation and purification.

[0043] 4. Unlike existing strategies that improve butyric acid yield by optimizing fermentation conditions, this invention reduces the reliance on precise control of culture conditions, improves the repeatability and stability of butyric acid production under different fermentation scales, and has good potential for industrial scale-up. Attached Figure Description

[0044] Figure 1 The figures show the fermentation results of wild-type and recombinant *Clostridium butyricum* strains under the same conditions for different durations in shake flasks; Figure A represents the fermentation result of wild-type *Clostridium butyricum*; Figure B represents the fermentation result of recombinant strain 25755 / cat1-. feoB1 Fermentation results; C in the figure represents the recombinant Clostridium butyricum strain 25755 / cat1- cofeoB The fermentation results.

[0045] Figure 2The figures show the fermentation results of wild-type and recombinant *Clostridium butyricum* under the same conditions for different fermentation times; Figure A represents the fermentation result of wild-type *Clostridium butyricum*; Figure B represents the fermentation result of recombinant strain 25755 / cat1- feoB1 Fermentation results; C in the figure represents the recombinant Clostridium butyricum strain 25755 / cat1- cofeoB The fermentation results. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0047] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0048] The Clostridium butyricum used in the embodiments of this invention ( Clostridium tyrobutyricum () is Clostridium butyricum strain ATCC25755.

[0049] 1 L Clostridium perfringens medium (RCM): 5 g glucose, 5 g NaCl, 10 g peptone, 10 g beef meal, 3 g yeast extract, 3 g anhydrous sodium acetate, 1 g soluble starch, 0.5 g cysteine ​​hydrochloride, pH 6.8±0.2, solid medium supplemented with 1.5% (m / v) agar powder.

[0050] 1 L fermentation medium (CGM): 5 g yeast extract, 5 g peptone, 5 g NaCl, 3 g (NH4)2SO4, 1.5 g K2HPO4, 0.6 g MgSO4·7H2O, 0.03 g FeSO4·7H2O. The amount of carbon source added depends on the experimental requirements and should be sterilized separately. pH value 6.0±0.2.

[0051] Example 1: Clostridium butyricum ferrous ion transporter gene ( feoB PCR amplification primer design This invention targets the ferrous ion transporter gene of Clostridium butyricum. feoB1 and feoB2 Corresponding PCR amplification primers (with restriction enzyme sites introduced into the primers) were designed, and the gene was obtained by PCR amplification. feoB1 and feoB2 The gene sequences were obtained and constructs were made containing the gene sequences of the gene sequences. feoB1 Gene sequence and feoB1 and feoB2 Recombinant plasmids containing gene sequences.

[0052] The gene feoB1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the ferrous ion transporter FeoB1 encoded by it is shown in SEQ ID NO.2; feoB2 The nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence of the ferrous ion transporter FeoB1 it encodes is shown in SEQ ID NO.4. A design was developed for gene amplification. feoB1 and feoB2 The sequences and PCR amplification primers used to construct recombinant plasmids are shown in Table 1.

[0053] Table 1 PCR amplification primers

[0054] Note: Underlined areas indicate primer restriction sites; lowercase bases indicate homologous fragments.

[0055] Example 2: Clostridium butyricum recombinant strain 25755 / cat1- feoB1 and 25755 / cat1- cofeoB Construction 1. PCR amplification of the target fragment (1) Extraction of Clostridium butyricum genomic DNA Genomic DNA of Clostridium butyricum was extracted using a bacterial genomic DNA extraction kit produced by Beijing Tiangen Biotech Co., Ltd. Specific procedures were performed according to the kit's instruction manual.

[0056] (2) PCR to obtain the target fragment Using extracted genomic DNA from *Clostridium butyricum* as a template, PCR amplification was performed using primers feoB1Pcat1-F / feoB1Pcat1-R. The PCR products were purified and recovered after gel electrophoresis to obtain the target fragment feoB1. Separately, PCR amplification was performed using primers co-feoB1-F / co-feoB1-R and co-feoB2-F / co-feoB2-R, respectively. The PCR products were purified and recovered after gel electrophoresis to obtain the target fragments co-feoB1 and co-feoB2, respectively.

[0057] The reaction system and procedure used for PCR amplification are shown below: Table 2 Reaction system used for PCR amplification

[0058] The reaction program used for PCR amplification was as follows: 98℃ for 2 min; 98℃ for 10 s, 51℃ for 10 s, 72℃ for 1–2 min (1 min / kb), 35 cycles; extension at 72℃ for 5 min.

[0059] 2. Construction of overexpression and co-expression recombinant plasmids (1) Enzyme digestion and ligation and transformation of ligation products Using restriction endonucleases Xba I and Sac II. The pMTL82151 plasmid carrying the PCAT1 promoter and the obtained target fragment feoB1 were double-digested (5 μL of 10× restriction endonuclease buffer, 1 μL of restriction endonuclease). Xba I and Sac II. Add 2 μL of each of the target fragment and plasmid, and bring the volume to 50 μL with sterile water. Digest at 37°C for 5 h. After purification and recovery, the digested products are ligated using a T4 DNA Ligase kit (molar ratio of linearized plasmid to target fragment feoB1 is 1:5, ligation at 16°C for 12 h). The ligation product is then transformed into E. coli. E. coli After reviving and culturing DH5α competent cells at 37°C for 1 h, the culture was spread onto LB solid medium plates containing chloramphenicol and cultured at 37°C for 12–20 h.

[0060] The co-expression recombinant plasmid was constructed using In-Fusion cloning technology. Restriction endonucleases were used. Xba I and Kpn I. Linearize the pMTL82151 plasmid carrying the PCAT1 promoter by double enzyme digestion (digestion system and conditions as above); prepare the reaction system according to the In-Fusion cloning kit instructions, and then co-... feoB1 With co- feoB2 The linearized plasmid and the target fragment were added in an equimolar ratio, with the molar ratio set at 3:1 (i.e., linearized plasmid:co-). feoB1 :co- feoB2 The reaction system was incubated at 50°C for 25 min (3:1:1 ratio) to achieve directional seamless ligation of the fragments. The ligation product was then transformed into *E. coli*. E. coli After reviving and culturing DH5α competent cells at 37°C for 1 h, the culture was spread onto LB solid medium plates containing chloramphenicol and cultured at 37°C for 12–20 h.

[0061] (2) Screening of positive clones and extraction and verification of recombinant plasmids Single colonies growing on LB solid agar plates containing chloramphenicol were randomly selected and inoculated into 500 μL of LB liquid agar containing chloramphenicol. The culture was incubated at 37°C for 6–8 h. One μL of the bacterial culture was used as a template for colony PCR identification to screen for positive clones containing the target fragment. The positive clones selected by colony PCR were further inoculated into LB liquid agar containing chloramphenicol for amplification. Recombinant plasmids were extracted using a plasmid extraction kit, and a portion was sent to a sequencing company for sequencing verification. Sequencing verification confirmed the overexpression of the gene. feoB1 Overexpression of recombinant plasmid pfeoB1 and overexpression of gene feoB1 and feoB2 The co-expression recombinant plasmid pco-feoB.

[0062] 3. Clostridium butyricum recombinant strain 25755 / cat1- feoB1 and 25755 / cat1- cofeoB Construction Clostridium butyricum is a strictly anaerobic, Gram-positive bacterium. Therefore, this invention employs conjugation transformation to introduce the obtained recombinant plasmid into Clostridium butyricum. The basic principle is as follows: [The text abruptly shifts to a different topic:] *Escherichia coli* E coli CA434, as a donor bacterium, can undergo cell-cell conjugation with the recipient bacterium Clostridium butyricum, thereby transferring the intracellular recombinant plasmid of the donor bacterium to the recipient bacterium.

[0063] The specific operating steps are as follows: (1) The recombinant plasmid (pfeoB1 / pco-feoB) that has been verified by sequencing is transformed into the target plasmid using the heat shock method. E. coli Positive clones were selected from CA434 competent cells and inoculated into LB liquid medium containing 25 μg / mL chloramphenicol. The cells were cultured at 37°C with shaking at 200 rpm until OD500 was reached. 600 It is around 1.5; (2) Take 3 mL of the above E. coli CA434 bacterial culture was centrifuged at 4200 g for 2 min to collect bacterial cells. The cells were washed with sterile PBS buffer and centrifuged again. The mixture was then analyzed with 200 μL OD. 600 Resuspension of Clostridium butyricum culture at 2.0 g / L; (3) Spread the resuspended mixed bacterial solution evenly on antibiotic-free RCM solid medium and place it in an anaerobic incubator at 37°C for conjugation culture. (4) After 24 h of conjugation culture, the bacterial cells on the surface of the RCM plate were washed with 1 mL of PBS buffer. The eluent was spread on RCM solid medium containing 25 μg / mL thiamphenicol and 200 μg / mL D-cycloserine and cultured at 37℃ for 36-48 h. Transconjugators were screened to obtain transconjugators. (5) Select suspected positive clones from the screening plate and inoculate them into RCM liquid medium containing 25 μg / mL thiamphenicol. After culturing at 37°C for 8–12 h, select positive clones for colony PCR (using KOD FX DNA polymerase with high amplification success rate and sensitivity) to confirm whether the target gene exists in Clostridium butyricum and is stably inherited, thereby obtaining Clostridium butyricum genetically engineered bacteria that overexpress the target gene. After full activation, add glycerol to a final concentration of 20% (v / v) and store at -80°C.

[0064] Colony PCR identification confirmed that the present invention successfully obtained the recombinant strain 25755 / cat1- of Clostridium butyricum. feoB1 and Clostridium butyricum recombinant strain 25755 / cat1- cofeoB。

[0065] Example 3 Fermentation culture of recombinant Clostridium butyricum strain This invention targets the wild-type Clostridium butyricum (ATCC strain 25755) and the recombinant strain 25755 / cat1- of Clostridium butyricum. feoB1 and 25755 / cat1- cofeoB Fermentation culture was conducted to test the butyric acid production, selectivity (carbon flow), and tolerance to butyric acid stress of the obtained recombinant Clostridium butyricum strain.

[0066] 1. Shake-flask fermentation culture of recombinant Clostridium butyricum strain (1) Activation of Clostridium butyricum strains and preparation of seed culture In an anaerobic incubator, use an inoculation loop to collect the preserved wild-type and recombinant strains of Clostridium butyricum (25755 / cat1-). feoB1 / 25755 / cat1- cofeoB The bacterial culture was streaked onto RCM plates and anaerobically incubated at 37°C for 24–36 h. After single colonies formed, a single colony was picked and inoculated into a 100 mL anaerobic culture flask containing 50 mL of liquid RCM medium. The flask was then incubated at 37°C with low-speed shaking until the bacterial culture reached its OD value. 600 Approximately 2.0, yielding activated bacterial solution.

[0067] The resulting activated bacterial solution can be stored for a short period at 4°C. When large-scale culture is required, the activated bacterial solution stored at 4°C is inoculated at a rate of 5% (v / v) into a 100 mL anaerobic culture flask containing 50 mL of liquid RCM medium and cultured at 37°C until the bacterial cell OD reaches its maximum. 600 The bacterial culture is approximately 2.0, and the resulting culture can be used as a seed culture for subsequent shake-flask fermentation or fermenter culture.

[0068] (2) Shake flask fermentation culture The prepared wild-type Clostridium butyricum and the recombinant strain of Clostridium butyricum were inoculated at a 5% (v / v) inoculation rate into 100 mL serum bottles containing 50 mL of liquid CGM medium (with 60 g / L glucose as the sole carbon source) and 50 g / L calcium carbonate powder, respectively. Fermentation was carried out at 37℃ and 150 rpm for 60 h. During the fermentation, samples were taken every 6 h, with each sample being 1 mL. The changes in glucose, acetic acid, and butyrate during the fermentation process were monitored by HPLC. The experiment was performed in triplicate.

[0069] Clostridium butyricum wild-type and Clostridium butyricum recombinant strain 25755 / cat1- feoB1 and 25755 / cat1- cofeoB The fermentation results after shaking flask fermentation for different times under the same conditions are as follows: Figure 1 As shown in A to C. Figure 1 It can be seen that, compared with the wild-type strain, the recombinant strain 25755 / cat1- feoB1 and 25755 / cat1- cofeoB There was no significant difference in acetic acid production between the recombinant strain 25755 / cat1- feoB1 The butyric acid production of the recombinant strain 25755 / cat1- increased by 18% compared to the wild-type strain. cofeoB The butyric acid yield increased by 44% compared to the wild-type strain, showing a significant difference. These results indicate that the recombinant strain described in this invention can increase the proportion of butyric acid in the fermentation product and reduce byproduct formation under the same substrate conditions.

[0070] 2. Fermentation culture of recombinant Clostridium butyricum strain This invention utilizes CGM medium with glucose as a substrate to separately process wild-type and recombinant strain 25755 / cat1- of Clostridium butyricum in a 3 L fully automated mechanically stirred fermenter. feoB1 and 25755 / cat1- cofeoB A continuous fed-batch fermentation culture was carried out with the pH adjusted to 6 using 30% ammonia.

[0071] (1) Activation of Clostridium butyricum strains and preparation of seed culture Same as above.

[0072] (2) Fermentation culture Before fermentation, the fermentation apparatus was sterilized at 121°C for 20 min. 1 L of liquid CGM medium (with 70 g / L glucose as the sole carbon source) was added, and the apparatus was sterilized at 115°C for 20 min. After cooling, air was introduced into the sterile nitrogen purging tank to achieve an anaerobic environment. Seed cultures of wild-type Clostridium butyricum and the recombinant strain of Clostridium butyricum were inoculated into the fermentation tank at a 5% (v / v) inoculation rate. The stirring speed and temperature were set to 150 rpm and 37°C, respectively. The pH of the fermentation broth was controlled at 6.0 by adding 30% (v / v) ammonia. When the carbon source in the fermentation system was about to be depleted, a high concentration (700 g / L) of glucose was added to maintain the initial sugar concentration of 70 g / L until the Clostridium butyricum cells stopped metabolic activity due to excessive butyric acid concentration. During fermentation, samples were taken every 6 h to detect cell density, substrate consumption, and product formation.

[0073] Clostridium butyricum wild-type and Clostridium butyricum recombinant strain 25755 / cat1- feoB1 and 25755 / cat1- cofeoB Fermentation results after different fermentation times under the same conditions are as follows: Figure 2 As shown in A to C. Figure 2 It can be seen that, compared with the wild-type strain, the recombinant strain 25755 / cat1- feoB1 and 25755 / cat1- cofeoB There was no significant difference in acetic acid production between the recombinant strain 25755 / cat1- feoB1 The butyric acid production of the recombinant strain 25755 / cat1- increased by 37% compared to the wild-type strain. cofeoB The butyric acid yield of the recombinant strain increased by 73% compared to the wild-type strain, showing a significant difference. Furthermore, the cell density of the recombinant strain was significantly higher than that of the wild-type strain. These results indicate that the recombinant strain of this invention can maintain good growth and metabolic activity under butyric acid stress conditions, reduce byproduct formation, and improve stability and final butyric acid accumulation in the later stages of fermentation.

[0074] The results above show that the present invention has successfully constructed a genetically engineered bacterium that produces high levels of butyric acid, namely the Clostridium butyricum recombinant strain 25755 / cat1- described in this invention. feoB1 and 25755 / cat1- cofeoB .

[0075] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A genetically engineered bacterium that produces high levels of butyric acid, characterized in that, Overexpression of a ferrous ion transporter protein in Clostridium butyrate synthesizing and strictly anaerobic bacteria; the ferrous ion transporter protein is derived from Clostridium butyrate.

2. The genetically engineered bacterium according to claim 1, characterized in that, The Clostridium that possesses butyric acid synthesis ability and is strictly anaerobic is Clostridium butyricum.

3. The genetically engineered bacterium according to claim 2, characterized in that, The ferrous ion transporter protein is FeoB1 protein and / or FeoB2 protein derived from Clostridium butyricum; the amino acid sequence of the FeoB1 protein is shown in SEQ ID NO.2; the amino acid sequence of the FeoB2 protein is shown in SEQ ID NO.

4.

4. The genetically engineered bacterium according to claim 3, characterized in that, The gene encoding the FeoB1 protein feoB1 The nucleotide sequence is shown in SEQ ID NO.1; the gene encoding the FeoB2 protein. feoB2 The nucleotide sequence is shown in SEQ ID NO.

3.

5. The method for constructing the genetically engineered bacteria according to claim 1, characterized in that, The gene encoding a ferrous ion transporter from Clostridium butyricum was transferred into a strictly anaerobic Clostridium species capable of butyrate synthesis, thereby overexpressing the ferrous ion transporter.

6. The construction method according to claim 5, characterized in that, Includes the following steps: S1. The recombinant plasmid is constructed by PCR amplification or artificial synthesis of the coding gene and insertion into the multiple cloning site of the overexpression plasmid; S2. The recombinant plasmid obtained in S1 is transferred into Clostridium that has butyrate synthesis ability and is strictly anaerobic.

7. The construction method according to claim 5 or 6, characterized in that, The Clostridium that possesses butyric acid synthesis ability and is strictly anaerobic is Clostridium butyricum.

8. The use of the genetically engineered bacteria according to any one of claims 1 to 4 in the biosynthesis of butyric acid.

9. The use of the genetically engineered bacteria according to any one of claims 1 to 4 in the preparation of products for the biosynthesis of butyric acid.

10. A method for biosynthesizing butyric acid, characterized in that, The genetically engineered bacteria described in any one of claims 1 to 4 are fermented and cultured using glucose or glucose derivatives as a carbon source.

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