Method for preparing 1, 4-butanediol through synergistic fermentation of multiple strains

Abstract

The invention relates to the field of fermentation processes, in particular to a method for preparing 1, 4-butanediol through multi-strain synergistic fermentation, which comprises the following steps: inoculating a first strain and a second strain into a culture medium containing glucose and / or xylose for synergistic fermentation, producing ornithine by the first strain by taking glucose and / or xylose as a substrate, and converting ornithine into 1, 4-butanediol by the second strain. The invention discloses 1, 4-butanediol. Glucose is converted into ornithine through the first strain, ornithine is converted into 1, 4-butanediol through the second strain, the first strain and the second strain are subjected to synergistic fermentation, preparation of 1, 4-butanediol through microbial synergistic fermentation is achieved, and higher metabolic capacity and higher production efficiency are achieved. The mixed sugar of xylose and glucose prepared from biomass raw materials is used as a substrate for fermentation, so that the method has the advantages that the raw materials are easy to obtain, renewable and low in cost, and the production cost can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of fermentation technology, and in particular to a method for preparing 1,4-butanediol through cooperative fermentation of multiple strains. Background Art

[0002] 1,4-Butanediol (1,4-BDO) is widely used in the chemical industry as a solvent, polymer intermediate, and fine chemical intermediate. Traditional 1,4-BDO production methods rely primarily on chemical synthesis, such as through the hydrogenation of acetylene and formaldehyde, or from maleic anhydride and propylene oxide. However, these chemical methods have numerous drawbacks and generally rely on fossil feedstocks, hindering the stable production of 1,4-BDO.

[0003] The preparation of compounds through microbial fermentation has the advantages of low cost, low pollution and mild reaction conditions, and has been widely used in the chemical industry and other fields. In the existing technology, the preparation of 1,4-butanediol through microbial fermentation is often done by a single strain using glucose and synthesizing it through the α-ketoglutarate or succinyl-CoA pathway. However, this technology has obvious disadvantages: on the one hand, the metabolic pathway is prone to produce by-products, which reduces the production yield of 1,4-butanediol; on the other hand, the lengthy metabolic pathway causes a heavy metabolic burden on the strain, and the microorganism's own metabolic regulation mechanism will also limit the supply of precursor substances. In addition, the introduced exogenous rate-limiting enzymes are prone to poor compatibility with the host metabolic system, and their expression efficiency and stability are insufficient, which aggravates the blockage of metabolic flow and further affects the efficiency of product synthesis. Summary of the Invention

[0004] The object of the present invention is to provide a method for preparing 1,4-butanediol by cooperative fermentation of multiple strains with higher yield.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing 1,4-butanediol by cooperative fermentation of multiple strains, comprising:

[0007] A first strain and a second strain are inoculated into a culture medium containing glucose and / or xylose for collaborative fermentation. The first strain produces ornithine using glucose and / or xylose as substrates, and the second strain converts ornithine into 1,4-butanediol.

[0008] Optionally, the first strain has the following enzyme reaction:

[0009] The enzymatic reaction that converts glucose into α-ketoglutarate,

[0010] The enzymatic reaction that converts α-ketoglutarate into glutamate,

[0011] The enzymatic reaction that converts glutamate into N-acetylglutamate,

[0012] The enzymatic reaction that converts N-acetylglutamate into N-acetylglutamate phosphate,

[0013] The enzymatic reaction that converts N-acetylglutamate phosphate into N-acetylglutamate semialdehyde,

[0014] The enzymatic reaction that converts N-acetylglutamate semialdehyde to N-acetylornithine,

[0015] Enzyme reaction that converts N-acetylornithine and glutamate into ornithine and N-acetylglutamate.

[0016] Optionally, the first strain is obtained by gene editing W3110 Escherichia coli, wherein the gene editing includes knocking out or weakening the expression of the argE gene and inserting a gene encoding ornithine acetyltransferase into the genome.

[0017] Optionally, the gene editing also includes weakening the expression of the sucA gene, knocking out or weakening the expression of one or more of the speC gene, speF gene, argF gene and argI gene, and inserting the lysE gene from Corynebacterium glutamicum into the genome.

[0018] Optionally, the gene editing further comprises inserting one or more of the argB gene, argC gene, and argD gene derived from Corynebacterium glutamicum into the genome;

[0019] The gene editing also includes inserting the argB gene from Escherichia coli into the genome and knocking out or weakening the expression of the argR gene.

[0020] Optionally, the gene editing further comprises inserting the pntAB gene from Escherichia coli into the genome.

[0021] Optionally, the pH value of the culture medium is any one of 6.5 to 7.5.

[0022] Optionally, the inoculation mass ratio of the first strain to the second strain is 1: any value between (1 and 5).

[0023] Optionally, the first strain is inoculated and cultured for any time between 6 hours and 18 hours, and then the second strain is inoculated.

[0024] Optionally, the mass ratio of glucose to xylose in the culture medium is any value between (3.5 and 5.5):1, the xylA gene of the first strain is knocked out or weakened before inoculation, and the gene for transporting glucose into the second strain is knocked out or weakened before inoculation.

[0025] The beneficial effect of the present invention is that glucose is converted into ornithine by the first strain, and ornithine is converted into 1,4-butanediol by the second strain, thereby achieving the production of 1,4-butanediol by microbial fermentation. In the prior art, single strain fermentation is usually used to achieve the production of the target product. This is because the metabolic pathway of a single strain is clear, the products and pathways are relatively simple, and it is easy to accurately control through experiments or industrial parameters. The by-products are also controllable, thereby achieving stable production of the product. However, when multiple strains are fermented together, it is difficult to control, and fermentation delays or product abnormalities may occur due to imbalance of the bacterial flora. In the present invention, two strains are used for mixed fermentation. The two strains respectively realize two reactions: substrate to intermediate product and intermediate product to final product. Since the reaction pathways of the two strains are relatively clear and mutually synergistic, it is not easy for the two strains to be unbalanced during the fermentation process, thereby overcoming the technical prejudice in the field. The first strain and the second strain are co-cultured, using glucose and / or xylose as substrates to obtain energy, and glucose as the initial raw material to synergistically produce 1,4-butanediol, thereby obtaining 1,4-butanediol at low cost and high efficiency.

[0026] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram showing the principle of the method for preparing 1,4-butanediol by cooperative fermentation of multiple strains as shown in Example 1 of the present invention;

[0028] Figure 2 This is a fermentation analysis diagram of the recombinant strain Orn-12 shown in Example 1 of the present invention when fermented alone. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0030] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0031] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0032] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] The present invention claims a method for preparing 1,4-butanediol by cooperative fermentation of multiple strains, comprising:

[0034] The first strain and the second strain are inoculated into a culture medium containing glucose and / or xylose for cooperative fermentation. The first strain uses glucose and / or xylose as substrates to produce ornithine, and the second strain converts ornithine into 1,4-butanediol.

[0035] The first strain is used to convert glucose into ornithine, and the second strain is used to convert ornithine into 1,4-butanediol, thereby achieving microbial cooperative fermentation to produce 1,4-butanediol. In the prior art, a single strain fermentation is usually used to achieve the production of the target product. This is because the metabolic pathway of a single strain is clear, the products and pathways are relatively simple, and it is easy to accurately control through experiments or industrial parameters, and the by-products are controllable, thereby achieving stable production of the product. However, it is difficult to control when multiple strains are fermented together, and fermentation delays or product abnormalities may occur due to imbalance of the bacterial flora. In the present invention, two strains are used for mixed fermentation. The two strains respectively realize two reactions from substrate to intermediate product and from intermediate product to final product. Since the reaction pathways of the two are relatively clear and mutually coordinated, it is not easy for the two to be unbalanced during the fermentation process, thereby overcoming the technical prejudice in this field. The first strain and the second strain are co-cultured, using glucose and / or xylose as substrates to obtain energy, and glucose as the initial raw material to cooperatively produce 1,4-butanediol, thereby obtaining 1,4-butanediol at low cost and high efficiency.

[0036] In some embodiments, the first strain and the second strain are obtained by editing and adjusting the genes of the same strain differently, so that the optimal culture environments of the first strain and the second strain are close, preventing bacterial imbalance caused by mismatch in the growth states of the two strains.

[0037] In some embodiments, the first strain has the following enzyme reactions:

[0038] The enzymatic reaction that converts glucose into α-ketoglutarate,

[0039] The enzymatic reaction that converts α-ketoglutarate into glutamate,

[0040] The enzymatic reaction that converts glutamate into N-acetylglutamate,

[0041] The enzymatic reaction that converts N-acetylglutamate into N-acetylglutamate phosphate,

[0042] The enzymatic reaction that converts N-acetylglutamate phosphate into N-acetylglutamate semialdehyde,

[0043] The enzymatic reaction that converts N-acetylglutamate semialdehyde to N-acetylornithine,

[0044] Enzyme reaction that converts N-acetylornithine and glutamate into ornithine and N-acetylglutamate.

[0045] After glucose is transported into the first strain, it is converted into α-ketoglutarate (α-KG) through the TCA reaction. Under the action of multiple enzymes expressed by the first strain, α-KG is sequentially converted into glutamate, N-acetylglutamate, N-acetylglutamate phosphate, N-acetylglutamate semialdehyde, and N-acetylornithine. Through the enzymatic reaction that converts N-acetylornithine and glutamate into ornithine and N-acetylglutamate, N-acetylornithine is further recycled to produce N-acetylglutamate, achieving a reaction cycle and maximizing the utilization of reactants while suppressing the formation of byproducts. This helps to increase the yield of ornithine, and therefore the yield of 1,4-butanediol.

[0046] In some embodiments, the first strain is obtained by gene editing W3110 Escherichia coli, and the gene editing includes knocking out or weakening the expression of the argE gene, and also includes inserting a gene encoding ornithine acetyltransferase into the genome.

[0047] In E. coli, the enzyme expressed by the argE gene converts N-acetylornithine into ornithine, producing acetic acid as a byproduct. This results in low purity of the ornithine produced and affects the pH of the internal and external bacterial environment, hindering the growth of the strain. Therefore, it is necessary to knock out or weaken the expression of the argE gene to inhibit the formation of byproducts and insert an exogenous gene to express ornithine acetyltransferase, thereby converting N-acetylornithine into ornithine. This helps create a suitable growth environment for the second strain and thus increases the yield of 1,4-butanediol.

[0048] In some embodiments, the gene encoding ornithine acetyltransferase is the argJ gene from Corynebacterium glutamicum, and the ornithine acetyltransferase WP_011014333.1 encoded by the argJ gene has strong activity and is less likely to generate by-products.

[0049] In some embodiments, gene editing further includes weakening the expression of the sucA gene, knocking out or weakening the expression of one or more of the speC gene, speF gene, argF gene and argI gene, and inserting the lysE gene from Corynebacterium glutamicum into the genome.

[0050] Weakening the expression of the sucA gene inhibits the conversion of α-KG to acetyl-CoA in the tricarboxylic acid (TCA) cycle. Since the efficiency of converting glucose to α-KG in the TCA cycle remains unchanged, while the efficiency of converting α-KG to acetyl-CoA decreases, a large amount of α-KG is converted to glutamate and then to ornithine, which helps increase ornithine yield. The enzymes produced by the speC and speF genes convert ornithine to putrescine, while the enzymes produced by the argF and argI genes convert ornithine to citrulline, which is then converted to arginine. Knocking out or weakening the expression of these four genes inhibits the conversion of ornithine to other substances, helping to increase ornithine yield and suppress the formation of byproducts, thereby increasing the yield of 1,4-butanediol.

[0051] In some embodiments, gene editing further comprises inserting one or more of the argB gene, argC gene, and argD gene derived from Corynebacterium glutamicum into the genome. The enzyme obtained by expressing the argB gene from Corynebacterium glutamicum can convert N-acetylglutamate into N-acetylglutamate phosphate, the enzyme obtained by expressing the argC gene from Corynebacterium glutamicum can convert N-acetylglutamate phosphate into N-acetylglutamate semialdehyde, and the enzyme obtained by expressing the argD gene from Corynebacterium glutamicum can convert N-acetylglutamate semialdehyde into N-acetylornithine. By inserting exogenous genes, enzymes that promote the above-mentioned conversion process are generated in the bacteria, thereby generating a large amount of N-acetylornithine, a precursor of ornithine, which helps to increase the yield of ornithine and thereby increase the yield of 1,4-butanediol.

[0052] In some embodiments, multiple copies of the argJ gene from Corynebacterium glutamicum are inserted into the genome of the W3110 Escherichia coli, which helps to further promote the reaction of converting N-acetylornithine to ornithine, thereby increasing the yield of ornithine and further increasing the yield of 1,4-butanediol.

[0053] In some embodiments, gene editing also includes inserting the argB gene from Escherichia coli into the genome and knocking out or weakening the expression of the argR gene. The enzyme obtained by expressing the argB gene from Escherichia coli promotes the conversion of N-acetylglutamate to N-acetylglutamate phosphate. By inserting homologous genes, the expression of the enzyme is increased, promoting conversion, thereby increasing the yield of N-acetylglutamate phosphate, and then increasing the yield of ornithine. The enzyme obtained by expressing the argR gene in the Escherichia coli genome inhibits the expression of the homologous argA gene, argB gene, argC gene, argD gene, and argE gene. Therefore, knocking out or weakening the expression of the argR gene helps to promote the conversion of glutamate to N-acetylornithine, thereby increasing the yield of ornithine, and then increasing the yield of 1,4-butanediol.

[0054] In some embodiments, gene editing further comprises inserting the pntAB gene from E. coli into the genome. Overexpression of the pntAB gene can increase the sugar uptake rate of the bacteria and promote the conversion of NADH to NADPH, thereby increasing the reducing power supply within the strain cells.

[0055] In some embodiments, the pH value of the culture medium is any value between 6.5 and 7.5, for example, any value between 6.5, 6.7, 6.9, 7.1, 7.3 and 7.5, which is conducive to the growth of the strain and improves the yield.

[0056] In some embodiments, the inoculum mass ratio of the first strain to the second strain is 1: any value in the range of (1 to 5), for example, any value in the range of (1:1), (1:2), (1:3), (1:4), and (1:5). By regulating the ratio of the first strain to the second strain, it is helpful for the second strain to fully utilize the ornithine obtained by fermentation of the first strain and convert it into 1,4-butanediol. The appropriate inoculation ratio can accurately balance the metabolic division of labor of the two strains in the mixed bacterial system, give full play to the advantages of co-cultivation, help reduce the residual ornithine in the product, improve the purity of 1,4-butanediol in the product, and increase the yield of 1,4-butanediol.

[0057] In some embodiments, the first strain is inoculated and cultured for any time between 6 hours and 18 hours, for example, any time between 6 hours, 9 hours, 12 hours, 15 hours and 18 hours, and then the second strain is inoculated. The second strain relies on the ornithine produced by the fermentation of the first strain to produce 1,4-butanediol. If the second strain is inoculated too early, the first strain will not grow fully and ornithine will be insufficient; if it is inoculated too late, the first strain will enter the stable phase and some metabolic activity will decrease, which is not conducive to the efficient synthesis of 1,4-butanediol in the co-culture system and will increase the culture time. Inoculating the second strain at the right time helps to reduce time costs and improve yields.

[0058] In some embodiments, the mass ratio of glucose to xylose in the culture medium is any value in the range of (3.5 to 5.5):1, for example, it can be any value in the range of (3.5:1), (4:1), (4.5:1), (5:1) and (5.5:1). The first strain has its xylA gene knocked out or weakened before inoculation, and the second strain has its gene for transporting glucose into the second strain knocked out or weakened before inoculation. By adjusting the ratio of glucose to xylose in the culture medium to make it close to the ratio in the biomass raw material, it is convenient to use renewable biomass raw materials as the initial material for the reaction, pre-treat them and produce 1,4-butanediol through microbial fermentation, which helps to reduce costs and does not require reliance on non-renewable energy sources such as petroleum. By treating the first and second strains before inoculation, the transport of xylose into the first strain is inhibited, and the transport of glucose into the second strain is inhibited, thereby preventing the two from competing for raw materials, helping to improve reaction efficiency and increase the yield of 1,4-butanediol.

[0059] Please refer to the following examples for details.

[0060] Example 1:

[0061] A method for preparing 1,4-butanediol by cooperative fermentation of multiple strains as shown in a preferred embodiment of the present application includes:

[0062] The first strain and the second strain are inoculated into a culture medium containing glucose and / or xylose for cooperative fermentation. The first strain uses glucose and / or xylose as substrates to produce ornithine, and the second strain converts ornithine into 1,4-butanediol.

[0063] In this example, the first and second strains were both obtained by using CRISPR Cas9 gene editing technology to edit W3110 Escherichia coli. Figure 1 , the first strain carried out the knockout of argE gene, speC gene, speF gene, argF gene, argI gene, argR gene, weakening of sucA gene, insertion of argB gene and pntAB gene from W3110 Escherichia coli, and insertion of argB gene, argC gene, argD gene, argJ gene and lysE gene from Corynebacterium glutamicum 13032 (Corynebacterium glutamicum). Since the argB gene, argC gene, argD and argJ gene from Corynebacterium glutamicum are connected gene fragments, they together constitute the ornithine synthesis operon argCJBD and are inserted into the genome together. Since the ornithine acetyltransferase encoded by the argJ gene from Corynebacterium glutamicum plays an important role in the reaction and needs to be expressed in large quantities, double copies were also inserted in addition to the ornithine synthesis operon argCJBD. Figure 1In the figure, the superscript Eco in the gene name indicates that it is an endogenous gene of Escherichia coli, and the superscript cgl in the gene name indicates that it is an exogenous gene derived from Corynebacterium glutamicum.

[0064] The insertion sites of the target genes to be inserted into the W3110 genome are shown in Table 1 below.

[0065] Table 1:

[0066]

[0067]

[0068] In this example, the expression of the sucA gene was suppressed by replacing the sucA gene promoter with PrpsL instead of PsucA. Since the specific sequences of each target gene and insertion site can be obtained through simple query, and the methods for inserting and knocking out gene fragments are all existing technologies, the specific procedures for knocking out, inserting, and attenuating genes are not detailed in this example. The resulting engineered strain is named recombinant strain Orn-12.

[0069] In this example, the gene editing performed on the second strain included the insertion of the davB, davA, gabT, yahK, car, and sfp genes from Corynebacterium glutamicum. The resulting engineered strain was named recombinant strain B-5. Since the construction of the second strain is prior art, its metabolic pathway will not be described in detail here.

[0070] Since all recombinant strains in this embodiment are obtained by gene editing based on Escherichia coli, the expanded culture and fermentation environment of each recombinant strain are the same. The specific method includes: inoculating the glycerol-preserved strain into a test tube containing LB liquid culture medium at a 1% inoculation rate, culturing and activating it in a shaker at 37°C and 220 rpm for 12 hours, then transferring it to a shake flask containing 100 mL of LB liquid culture medium at a 1% inoculation rate, and continuing to culture it in a shaker at 37°C and 220 rpm for 6 hours to 12 hours, and detecting the biomass of the bacterial solution (OD 600) to the preset value, culture was stopped and the cells were quantitatively transferred to M9 medium for fermentation at 37°C and 220 rpm in a shaker. The components of LB liquid medium for a total volume of 1 L include 10 g tryptone, 10 g sodium chloride, and 5 g yeast extract. After adding water to the volume, the pH of the medium was adjusted to approximately 7.0 using 10 M NaOH solution and sterilized at 121°C and 0.1 MPa for 20 min. When preparing LB solid medium, 1.5% (w / v) agar powder was added. To prepare a total volume of 1 L of M9 medium, 17.1 g Na2PO4·12H2O, 3 g KH2PO4, 0.5 g NaCl, 1 g NH4Cl, and an appropriate amount of carbon source were weighed. After adding water to the volume to 1 L, the pH of the medium was adjusted to the preset value using 10 M NaOH solution. Aliquot 50 mL of the solution into a 250 mL conical flask and sterilize at 121°C, 0.1 MPa pressure for 20 min. Carbon sources, including substrates for fermentation products and carbohydrates for energy, should be added according to the experimental requirements and concentration. After sterilization, add 250 μL of a 1 M sterile MgSO₄·7H₂O solution and 5 μL of a 1 M sterile CaCl₂·2H₂O solution.

[0071] The recombinant strain Orn-12 was cultured and fermented separately. During the fermentation, the carbon source added to the M9 medium was glucose at a concentration of 80 g / L. After the start of fermentation, samples were taken at intervals of 12 hours for a total of 48 hours, and the OD values ​​of the samples were measured. 600 , residual glucose content and ornithine content. Figure 2 After 48 h of culture, the ornithine accumulation of the recombinant strain Orn-12 was 26.30 g / L, and the sugar-acid conversion rate was 38.55%.

[0072] Recombinant strain B-5 was cultured and fermented separately. During fermentation, the carbon sources added to M9 medium included 10 g / L glucose and 30 g / L ornithine. After 48 hours of co-cultivation, the 1,4-butanediol concentration was measured. The results showed that strain B-5 could produce 5.16 g / L of 1,4-butanediol.

[0073] The recombinant strain Orn-12 and the recombinant strain B-5 were cultured separately and then fermented in synergy. When the two strains were fermented in synergy, the OD600 of the bacterial solution of the two strains needed to be adjusted to the same value before the cells were transferred to M9 medium for fermentation, and then inoculated according to the required inoculation ratio. The two strains were cultured separately until the OD600 of the bacterial solution was 0. 600When the pH value was 0.2, the culture was stopped and transferred to M9 medium for co-fermentation, with the two strains added in a 1:1 ratio. Glucose at a concentration of 80 g / L was added to the M9 medium as the carbon source, and the initial pH of the M9 medium was adjusted to 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. After 48 hours of co-fermentation, samples were collected and determined for ornithine and 1,4-butanediol content. The results are shown in Table 2.

[0074] Table 2:

[0075] pH Ornithine (g / L) 1,4-Butanediol (g / L) 6.0 15.29 0.56 6.5 9.66 3.08 7.0 5.16 4.24 7.5 8.24 3.65 8.0 9.53 0.60

[0076] As can be seen from Table 2, when the initial pH value of the M9 medium was 7.0, the 1,4-butanediol yield was the highest, reaching 4.24 g / L, which was significantly higher than other pH conditions. This is because this pH value is close to the optimal pH range of the key metabolic enzymes of the two strains, promoting the activity of each enzyme in the metabolic pathway from glucose to 1,4-butanediol, thereby improving the overall conversion efficiency. When the pH value is lower or higher than 7.0, the 1,4-butanediol yield decreases significantly. This may be because the pH affects the permeability of the cell membrane and the structure and function of the enzymes, hindering the smooth flow of metabolic fluid.

[0077] The ratios of recombinant strain Orn-12 and recombinant strain B-5 were adjusted to (1:10), (1:5), (1:3), (1:2), (2:1), (3:1), (5:1), and (10:1), respectively. The initial pH of the M9 medium was adjusted to 7.0. Other reaction conditions remained unchanged. After 48 hours of co-fermentation, samples were collected and the ornithine and 1,4-butanediol contents were determined. The results are shown in Table 3.

[0078] Table 3:

[0079] pH Ornithine (g / L) 1,4-Butanediol (g / L) 1:10 1.07 1.36 1:5 3.55 4.22 1:3 3.62 5.02 1:2 4.81 4.47 1:1 5.16 4.24 2:1 9.11 2.91 3:1 11.33 1.03 5:1 12.21 0.65 10:1 16.21 0.15

[0080] Recombinant strain Orn-12 efficiently synthesizes precursors for the 1,4-butanediol biosynthesis pathway, while recombinant strain B-5 efficiently converts these precursors into 1,4-butanediol. When the Orn-12 ratio is too high, precursor synthesis is excessive, but subsequent conversion is insufficient. Conversely, when the B-5 ratio is too high, insufficient precursor supply limits 1,4-butanediol production. An appropriate inoculation ratio precisely balances the metabolic division of labor between the two strains in a mixed culture, fully leveraging the advantages of co-culture. As shown in Table 3, 1,4-butanediol production peaked at 5.02 g / L at an inoculation ratio of 1:3. At this ratio, the two strains achieve optimal metabolic complementarity.

[0081] Because recombinant strain B-5 relies on ornithine produced by recombinant strain Orn-12 for 1,4-butanediol production, it was hypothesized that inoculating recombinant strain B-5 after recombinant strain Orn-12 has grown for a certain period of time would increase 1,4-butanediol production. This hypothesis was verified experimentally.

[0082] Recombinant strain Orn-12 was first cultured alone in M9 medium with an initial pH of 7.0. Recombinant strain B-5 was then inoculated after 0, 6, 12, 18, and 24 hours of culture, with the ratio of recombinant strain Orn-12 to recombinant strain B-5 being 1:3. Other reaction conditions remained unchanged. Culture was continued for 48 hours, and samples were collected to determine the ornithine and 1,4-butanediol contents. The results are shown in Table 4.

[0083] Table 4:

[0084] Inoculation time of recombinant strain B-5 Ornithine (g / L) 1,4-Butanediol (g / L) 0 3.62 5.02 6 3.55 5.21 12 3.01 6.09 18 6.75 5.31 24 7.18 4.08

[0085] As shown in Table 4, the highest 1,4-butanediol yield, reaching 13.1 g / L, was achieved when recombinant strain B-5 was inoculated 12 hours after Orn-12 was cultured. Initially, recombinant strain Orn-12 requires time for growth and metabolic adaptation, accumulating essential metabolic intermediates. By 12 hours, recombinant strain Orn-12 has reached the late logarithmic growth phase, at which point its cell density and metabolic activity are optimal. Inoculation with recombinant strain B-5 allows it to rapidly utilize the metabolite ornithine produced by recombinant strain Orn-12, initiating and efficiently promoting the 1,4-butanediol synthesis pathway. Inoculation with recombinant strain B-5 too early results in inadequate growth of recombinant strain Orn-12 and insufficient ornithine accumulation, preventing B-5 from achieving its maximum production efficiency. Inoculation with recombinant strain B-5 too late results in Orn-12 entering a stationary phase, with some metabolic activity declining. This makes it difficult to efficiently and stably provide ornithine during subsequent fermentation, hindering efficient 1,4-butanediol synthesis in the co-culture system and increasing the culture time.

[0086] In summary, in the synergistic fermentation system of recombinant strain Orn-12 and recombinant strain B-5, the initial pH of the M9 culture medium was controlled at 7.0, the inoculation ratio of the recombinant strain Orn-12 to the recombinant strain B-5 was set to 1:3, and the recombinant strain B-5 was inoculated after the recombinant strain Orn-12 was cultured alone for 12 hours. This can significantly improve the conversion efficiency from glucose to 1,4-butanediol, providing an important reference basis for subsequent fermentation process optimization and industrial application.

[0087] Combining the yields of recombinant strains Orn-12 and B-5 fermented separately, we concluded that the yield of ornithine obtained by Orn-12 fermentation, followed by inoculation of recombinant strain B-5 with the obtained ornithine and sufficient glucose as substrates, was approximately 4.5 g / L. This yield is lower than the yield of co-fermentation with the two strains, demonstrating that co-fermentation can effectively increase 1,4-butanediol production, simplify operations, and improve production efficiency.

[0088] Since the recombinant strain Orn-12 and the recombinant strain B-5 use the same carbon source for conversion during the collaborative fermentation process, there is competition for carbon sources between the two strains, which will have an adverse effect on the stability of the bacterial community. In order to circumvent this competition, the xylA gene in the genome of the recombinant strain Orn-12 was further knocked out to block its xylose utilization pathway, thereby obtaining the recombinant strain Orn-13. The manZ gene and ptsG gene in the genome of the recombinant strain B-5 were knocked out to block its glucose utilization pathway, thereby obtaining the recombinant strain B-7. During fermentation, the carbon source added to the M9 medium included glucose at a concentration of 80 g / L and xylose at a concentration of 16 g / L, so that the content ratio of the two sugars was close to the ratio in the biomass raw material, thereby reducing the cost of obtaining the carbon source. The initial pH of the M9 culture medium was controlled at 7.0, and the inoculation ratio of the recombinant strain Orn-13 to the recombinant strain B-7 was set to 1:3. After the recombinant strain Orn-13 was cultured alone for 12 hours, the recombinant strain B-7 was inoculated. After 48 hours of fermentation, the production of 1,4-butanediol was significantly increased to 7.62 g / L.

[0089] Example 2:

[0090] The only difference between this embodiment and Example 1 is that straw sugar purchased from Suzhou Juwei Yuanchuang Biotechnology Co., Ltd. is used as a carbon source. Since straw sugar is one of the products obtained by straw processing, it is low in cost and renewable. Straw sugar includes glucose with a concentration of 494g / L and xylose with a concentration of 95.6g / L. Straw sugar is added to M9 culture medium and diluted so that the concentration of glucose in M9 culture medium is 80g / L. The initial pH of M9 culture medium is controlled at 7.0, and the inoculation ratio of recombinant strain Orn-13 and recombinant strain B-7 is set to 1:3. After the recombinant strain Orn-13 is cultured alone for 12h, the recombinant strain B-7 is inoculated. After fermentation for 48h, the yield of 1,4-butanediol is significantly improved, reaching 7.55g / L.

[0091] The beneficial effects of the present invention lie in leveraging the advantages of the two strains through a collaborative fermentation system, achieving efficient conversion from mixed sugars to ornithine and then to 1,4-butanediol. Compared to traditional single-strain fermentation, this system offers enhanced metabolic capacity and higher production efficiency. Because the two strains are engineered from the same strain with different gene editing techniques, their optimal environments are identical, making imbalances less likely to occur, thereby achieving stable multi-strain collaborative fermentation.

[0092] By constructing the ornithine-producing strain Orn-13 that weakens the xylose utilization pathway and the butanediol-producing strain B-7 that weakens the glucose utilization pathway, the competition between the two strains in carbon source utilization was reduced, and the metabolic flow was more concentrated towards the synthesis of the target product, effectively reducing the generation of by-products and improving the yield and production efficiency of 1,4-butanediol.

[0093] The use of a mixture of glucose and xylose as a carbon source fully utilizes the various sugars in the lignocellulose hydrolysate, broadens the source of raw materials, reduces production costs, and improves resource utilization.

[0094] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0095] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing 1,4-butanediol by cooperative fermentation of multiple strains, characterized in that: include: A first strain and a second strain are inoculated into a culture medium containing glucose and / or xylose for collaborative fermentation. The first strain produces ornithine using glucose and / or xylose as substrates, and the second strain converts ornithine into 1,4-butanediol.

2. The method for preparing 1,4-butanediol by cooperative fermentation of multiple strains according to claim 1, characterized in that: The first strain has the following enzyme reactions: The enzymatic reaction that converts glucose into α-ketoglutarate, The enzymatic reaction that converts α-ketoglutarate into glutamate, The enzymatic reaction that converts glutamate into N-acetylglutamate, The enzymatic reaction that converts N-acetylglutamate into N-acetylglutamate phosphate, The enzymatic reaction that converts N-acetylglutamate phosphate into N-acetylglutamate semialdehyde, The enzymatic reaction that converts N-acetylglutamate semialdehyde to N-acetylornithine, and Enzyme reaction that converts N-acetylornithine and glutamate into ornithine and N-acetylglutamate.

3. The method for preparing 1,4-butanediol by cooperative fermentation of multiple strains according to claim 2, characterized in that: The first strain is obtained by performing gene editing on W3110 Escherichia coli, wherein the gene editing includes knocking out or weakening the expression of the argE gene and inserting a gene encoding ornithine acetyltransferase into the genome.

4. The method for preparing 1,4-butanediol by cooperative fermentation of multiple strains according to claim 3, characterized in that: The gene editing also includes weakening the expression of the sucA gene, knocking out or weakening the expression of one or more of the speC gene, speF gene, argF gene and argI gene, and inserting the lysE gene from Corynebacterium glutamicum into the genome.

5. The method for preparing 1,4-butanediol by cooperative fermentation of multiple strains according to claim 3, characterized in that: The gene editing further comprises inserting one or more of the argB gene, argC gene, and argD gene derived from Corynebacterium glutamicum into the genome; The gene editing also includes inserting the argB gene from Escherichia coli into the genome and knocking out or weakening the expression of the argR gene.

6. The method for preparing 1,4-butanediol by cooperative fermentation of multiple strains according to claim 3, characterized in that: The gene editing also includes inserting the pntAB gene from Escherichia coli into the genome.

7. The method for preparing 1,4-butanediol by cooperative fermentation of multiple strains according to claim 1, characterized in that: The pH value of the culture medium is any one of 6.5 to 7.

5.

8. The method for preparing 1,4-butanediol by cooperative fermentation of multiple strains according to claim 1, characterized in that: The inoculum mass ratio of the first strain to the second strain is 1:(1-5) or any value thereof.

9. The method for preparing 1,4-butanediol by cooperative fermentation of multiple strains according to claim 1, characterized in that: The first strain is inoculated and cultured for any time between 6 hours and 18 hours, and then the second strain is inoculated.

10. The method for preparing 1,4-butanediol by cooperative fermentation of multiple strains according to claim 1, characterized in that: The mass ratio of glucose to xylose in the culture medium is any value between (3.5 and 5.5):1, the xylA gene of the first strain is knocked out or weakened before inoculation, and the gene for transporting glucose into the second strain is knocked out or weakened before inoculation.

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